DISSOCIATION OF BIOLOGICAL SAMPLES

Abstract

A method of processing a biological sample to obtain single cells and/or groups of cells comprises: fixing cells of the biological sample using a fixative; contacting the biological sample with a dissociation medium; dissociating the biological sample in the presence of the dissociation medium to obtain the single cells and/or groups of cells; wherein the fixative comprises a deep eutectic solvent. Fixing cells of the biological sample using a deep eutectic solvent facilitates dissociation of the biological sample into single cells, while at the same time inhibiting the degradation of biomolecules such as DNA and RNA in the cells. Also provided is an apparatus useful in the method, deep eutectic solvents and uses thereof, and a method of extracting cell nuclei.

Claims

1. A method of processing a biological sample to obtain single cells and/or groups of cells, the method comprising: fixing cells of the biological sample using a fixative; contacting the biological sample with a dissociation medium; dissociating the biological sample in the presence of the dissociation medium to obtain the single cells and/or groups of cells; wherein the fixative comprises a deep eutectic solvent.

2. The method according to claim 1, wherein dissociating the biological sample comprises sonication of the biological sample using a sonicator, and wherein the method further comprises constraining at least a part of the biological sample to a cavitation zone during the sonication.

3-4. (canceled)

5. The method according to claim 2, wherein the constraining comprises feeding the biological sample into the cavitation zone as the biological sample is reduced in size.

6-12. (canceled)

13. The method according to claim 1, wherein the fixing comprises sonicating the biological sample.

14-23. (canceled)

24. The method according to claim 1, wherein the sonicator has a liquid bath containing an oil, a hydrocolloid, an echography gel, or a salt solution.

25-26. (canceled)

27. The method according to claim 1, wherein the deep eutectic solvent of the fixative comprises a first component and a second component, wherein first component is a compound of Formula I: ##STR00032## wherein: R.sub.6 is H or OH; R.sub.7 is selected from H, CH.sub.3, Cl, Br, a carbonyl oxygen, and ##STR00033## Z is selected from —CH.sub.2—, O and S; R.sub.8 is R.sub.11 or OH; and wherein the second component comprises a compound of Formula II or a salt thereof: ##STR00034## wherein: A is selected from O, S, and NH; R.sub.1 is selected from H, an alkene group having 1 to 6 carbon atoms, R.sub.9, —NH.sub.2, —NH—(CH.sub.2).sub.nCH.sub.3, and —C(R.sub.3)(R.sub.4)(R.sub.5); wherein n is 0 or an integer from 1 to 5; R.sub.2 is selected from H and linear alkyl group having 1 to 3 carbon atoms; R.sub.3 is an optionally substituted 5- or 6-membered aliphatic or aromatic ring, wherein the substituent is R.sub.10; R.sub.4 and R.sub.5 are each independently H or F; and wherein R.sub.9, R.sub.10, and R.sub.11 are each independently selected from alkyl groups having one to three carbon atoms, monochloroalkyl groups having one to three carbon atoms, and mono-, di- or tri-fluoroalkyl groups having one to three carbon atoms.

28. The method according to claim 27, wherein: i) the first component is trimethylglycine and the second component is trifluoroacetamide; or ii) the first component is trimethylglycine and the second component is urea; or iii) the first component is choline chloride and the second component is urea.

29. The method according to claim 1, wherein the deep eutectic solvent of the fixative comprises a first component and a second component, wherein the first component comprises a compound of Formula III: ##STR00035## wherein: R.sub.6 is H or OH; R.sub.7 is selected from H, CH.sub.3, Cl, Br, a carbonyl oxygen, and ##STR00036## Z is selected from —CH.sub.2—, O and S; wherein R.sub.8 is selected from OH, an alkyl group having one to three carbon atoms, a monochloroalkyl group having one to three carbon atoms, and a mono-, di- or tri-fluoroalkyl group having one to three carbon atoms; and wherein the second component is a sugar or a sugar alcohol having at least 3 carbon atoms.

30-31. (canceled)

32. The method according to claim 1, wherein the fixative further comprises: i) formic acid at a concentration in the range 4 to 9 M; or ii) formic acid at a concentration in the range 50 to 500 mM; or iii) acetic acid at a concentration of at least 2 M.

33-37. (canceled)

38. The method according to claim 1, wherein the dissociation medium comprises a deep eutectic solvent, wherein the deep eutectic solvent of the dissociation medium comprises a first component and a second component, wherein first component is a compound of Formula I: ##STR00037## wherein: R.sub.6 is H or OH; R.sub.7 is selected from H, CH.sub.3, Cl, Br, a carbonyl oxygen, and ##STR00038## Z is selected from —CH.sub.2—, O and S; R.sub.8 is R.sub.11 or OH; and wherein the second component comprises a compound of Formula II or a salt thereof: ##STR00039## wherein: A is selected from O, S, and NH; R.sub.1 is selected from H, an alkene group having 1 to 6 carbon atoms, R.sub.9, —NH.sub.2, —NH—(CH.sub.2).sub.nCH.sub.3, and —C(R.sub.3)(R.sub.4)(R.sub.5); wherein n is 0 or an integer from 1 to 5; R.sub.2 is selected from H and linear alkyl group having 1 to 3 carbon atoms; R.sub.3 is an optionally substituted 5- or 6-membered aliphatic or aromatic ring, wherein the substituent is R.sub.10; R.sub.4 and Rs are each independently H or F; and wherein R.sub.9, R.sub.10, and R.sub.11 are each independently selected from alkyl groups having one to three carbon atoms, monochloroalkyl groups having one to three carbon atoms, and mono-, di- or tri-fluoroalkyl groups having one to three carbon atoms.

39. The method according to claim 38, wherein: i) the first component of the deep eutectic solvent of the dissociation medium is trimethylglycine and the second component is trifluoroacetamide; or ii) the first component of the deep eutectic solvent of the dissociation medium is trimethylglycine and the second component is urea; or iii) the first component of the deep eutectic solvent of the dissociation medium is choline chloride and the second component is urea.

40. The method according to claim 1, wherein the dissociation medium comprises a deep eutectic solvent, wherein the deep eutectic solvent of the dissociation medium comprises a first component and a second component, wherein the first component comprises a compound of Formula III: ##STR00040## wherein: R.sub.6 is H or OH; R.sub.7 is selected from H, CH.sub.3, Cl, Br, a carbonyl oxygen, and ##STR00041## Z is selected from —CH.sub.2—, O and S; wherein R.sub.8 is selected from OH, an alkyl group having one to three carbon atoms, a monochloroalkyl group having one to three carbon atoms, and a mono-, di- or tri-fluoroalkyl group having one to three carbon atoms; and wherein the second component is a sugar or a sugar alcohol having at least 3 carbon atoms.

41-45. (canceled)

46. The method according to claim 1, wherein the dissociation medium comprises: i) acetic acid at a concentration of at least 2 M; or ii) formic acid at a concentration in the range 4 to 9 M; or iii) formic acid at a concentration in the range 50 to 500 mM.

47-53. (canceled)

54. The method according to claim 1, wherein the dissociation medium comprises a glycol selected from ethylene glycol, glycerol, diethylene glycol, triethylene glycol, and a polyethylene glycol, PEG, having a molecular weight in the range 200 to 800.

55-64. (canceled)

65. The method according to claim 1, further comprising: freezing the biological sample before the fixing; and thawing the biological sample during the fixing.

66. (canceled)

67. The method according to claim 1, further comprising, after the fixing and before the dissociating, softening the biological sample using a glycol.

68-70. (canceled)

71. The method according to claim 1, further comprising, after the dissociating, lysing the single cells and/or groups of cells, and isolating cell nuclei after the lysing.

72-75. (canceled)

76. The method according to claim 1, further comprising, at one or more time points during the dissociating, isolating groups of cells from the dissociation medium.

77-78. (canceled)

79. The method according to claim 1, further comprising adding dimethyl sulfoxide to the dissociation medium before, during or after the dissociating.

80-129. (canceled)

130. A method of processing a fixed biological sample to obtain single cells and/or groups of cells, the method comprising: contacting the fixed biological sample with composition comprising a deep eutectic solvent; subsequently contacting the biological sample with a dissociation medium; and dissociating the biological sample in the presence of the dissociation medium to obtain the single cells and/or groups of cells.

131-133. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0225] The present invention will become more fully understood from the detailed description and the accompanying drawings, in which:

[0226] FIG. 1 shows RNA quality from single-cells obtained from mouse liver treated with

[0227] Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) containing 300 mM Formic acid for 24 hours, then dissociated according to Example 15 (35 kHz), for 15 minutes at room temperature. Lane 1, 85% Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) Dissociation buffer, Lane 2. 85% Trimethylglycine:Glycerol (1:2 mol:mol), Lane 3. Postive control, sample not subjected to ultrasound. It can be seen that the ultrasound treatment of the liver sample in either Dissociation buffer had no effect on RNA quality.

[0228] FIG. 2 shows Frozen-Thawed Mouse Intestine Thawed in RNAssist and Dissociated Mechanically (DAPI Stain).

[0229] FIG. 3 shows typical cellular debris obtained when dissociating fixed mouse liver in water.

[0230] FIG. 4 shows loss of cellular integrity and membrane loss when dissociating fixed mouse intestine in PBS.

[0231] FIG. 5 shows loss of cellular integrity and membrane loss when dissociating RNAlater fixed mouse intestine. RNAlater is a commercially-available composition for inhibiting the degradation of RNA, which does not include a DES.

[0232] FIG. 6 shows loss of cellular integrity and membrane loss when dissociating NBF fixed mouse intestine;

[0233] FIG. 7 shows single cells dissociated from human skin using 85% Trimethylglycine:Trifluoroacetamide (2:1 mol:mol), 26 kHz/70% Pulse/70% Amplitude/5 minutes at 4° C.

[0234] FIG. 8 shows Single cells dissociated from frozen-thawed mouse intestine using 85% Trimethylglycine:Trifluoroacetamide (2:1 mol:mol), 26 kHz/70% Pulse/70% Amplitude/5 minutes at 4° C.;

[0235] FIG. 9 shows single cells dissociated from frozen-thawed mouse nasal epithelium (brush border) using 85% Trimethylglycine:Trifluoroacetamide (2:1 mol:mol), 26 kHz/70% Pulse/70% Amplitude/5 minutes at 4° C.;

[0236] FIG. 10 shows Cells dissociated from frozen-thawed mouse kidney (DAPI stained nuclei) using 85% Trimethylglycine:Trifluoroacetamide (2:1 mol:mol), 26 kHz/70% Pulse/70% Amplitude/5 minutes at 4° C.;

[0237] FIG. 11 shows frozen-thawed mouse liver undergoing dissociation using 85% Trimethylglycine:Trifluoroacetamide (2:1 mol:mol), 37 kHz waterbath for 5 minutes at 4° C.;

[0238] FIG. 12 shows individual nuclei derived from Trimethylglycine:Trifluoroacetamide (2:1 mol:mol) 300 mM Formic acid fixed mouse intestine dissociated in 100% DMSO 37 kHz water bath sonicator for 5 minutes at 24° C.;

[0239] FIG. 13 shows dissolution of dextran blue (2000 Kd) in DESs with varying water content and Dissociation buffers 30 or 60 seconds sonication 35 kHz;

[0240] FIG. 14. shows dissolution of dextran blue (2000 Kd) in Trimethylglycine:Trifluoroacetamide (2:1 mol:mol) with varying water content using 30 seconds sonication at 35 kHz;

[0241] FIG. 15 shows dissolution of carbon blue paper (duplicate strips) at varying temperatures, using 30 seconds sonication in 85% Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) using a 40 kHz water bath;

[0242] FIG. 16 shows single cancer cells obtained from Dissociated human sarcoma, 4 minutes sonication in 95% Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) using 80% Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) 80% pulse/80% Amplitude, 26 kHz according to Example 1;

[0243] FIG. 17 is a flow chart showing an example method for processing a biological sample;

[0244] FIG. 18 is a schematic diagram of an example apparatus.

DETAILED DESCRIPTION

[0245] The terms “dissociation medium” and “dissociation buffer” are used interchangeably herein. A dissociation medium or dissociation buffer may, but does not necessarily, provide pH buffering.

[0246] The content of all documents cited herein is hereby incorporated by reference in its entirety.

[0247] As used herein, the verb ‘to comprise’ is used as shorthand for ‘to include or to consist of’. In other words, although the verb ‘to comprise’ is intended to be an open term, the replacement of this term with the closed term ‘to consist of’ is explicitly contemplated.

[0248] The word “about” in connection with a numeric value may mean that the value may vary by, for example, ±10%.

[0249] It will be appreciated that some compounds disclosed herein may be ionisable, i.e. some compounds may be weak acids, weak bases, or ampholytes. Representations of the free forms of ionisable compounds are intended to encompass the corresponding ionised forms. For example, representations of carboxylic acid groups encompass the corresponding carboxylate groups.

[0250] A multicellular biological sample can be either (i) fixed in a Deep Eutectic Solvent (DES) composition as set out in U.S. Pat. No. 9,696,247 followed by tissue dissociation, or alternatively, (ii) dissociated into single-cells in the presence of a DES composition so that fixation and dissociation occur at the same time to provide a fixed single-cell sample, or (iii) dissociated into single-cells using well-known methods such as proteases and/or mechanical means followed by fixation of the produced single cells as a suspension or pellet.

[0251] Most preferably the biomolecules found in and around the cells are stabilized before dissociation, less preferably during dissociation, or least preferably after sample dissociation. Most preferably the biomolecules are stabilized prior to dissociation in order to limit changes to their quantity, quality and distribution during the pre-analytical period. It is also important to limit pre-analytical changes to the cell morphology that may occur during tissue processing. Furthermore it is preferable that the amount, quantity and distribution of biomolecules in and around the cell remains unaltered before, during and after processing. Ideally both the cell morphology (phenotype) and biomolecules resemble, as closely as possible, their quantity, quality and molecular structure in vivo.

[0252] The specific biological, biophysical, physical and chemical properties of the DES are beneficial for the process of biomolecule stabilization and tissue dissociation. The particular type or combination of DES used for (i) sample fixation, (ii) tissue dissociation and (iii) downstream processing, can be the same, different or a combination of DES at each stage. There can also be more than two DESs used in the process, for example a separate DES to serve as a (i) pre-fixative, (ii) a tissue softener and (iii) a tissue Dissociation buffer, (iv) a secondary Dissociation buffer, and/or (v) a post-Dissociation stabilization buffer and/or (vi) a post-Dissociation solution for single-cell analysis such as for FACS or other types of cell sorting, protein analysis, phosphoprotein analysis, single-cell or single-nuclei sequencing of the RNA and/or DNA.

[0253] It will be evident to one skilled in the art that it is possible to supplement the DES with one or more additives to provide additional specific desired properties. The DES containing the additive can then be used for example, as a tissue pre-fixative, tissue fixation, tissue softener, tissue dissociation, post-Dissociation buffer and/or a buffer for downstream analysis of single or groups of cells.

[0254] Also provided therefore is the use of an additive in combination with a DES composition to improve tissue dissociation into single-cells or groups of cells for subsequent analysis such as single-cell RNA sequencing (scRNAseq). The purpose of the additive is to improve the properties of the DES composition, for example tissue dissociation into single-cells or groups of cells. By way of example only, the additive could be a (i) colourant or dye to aid in handling or staining or processing the tissue, (ii) an ionic or non-ionic surfactant or detergent, quaternary ammonium salt or Saponin to improve penetration of the cell plasma membrane with the DES mixture and improve tissue dissociation, (iii) anti-microbial such as an antibiotic or antiseptic, (iv) protein precipitant, (v) desiccant to remove excess water from the DES mixture to improve biomolecule stability, (vi) a probe, a hybridising complementary nucleic acid, an aptamer, a peptide, protein, nucleic acid or labelled molecule, an internal control, a blocking sequence or a carrier nucleic acid such as a nucleic acid sequence, (vii) a peptide, enzyme or other protein such as an antibody, (viii) molecules for detecting an analyte, (ix) an anti-oxidant to remove oxygen from the sample and reduce damaging oxidative effects, (x) a ribonuclease inhibitor such as Vanadate ions, RNasin, DTT, DTE, b-mercaptoethanol, a chaotrope such as Thiorurea, Urea, Sodium perchlorate, Lithium perchlorate, Sodium iodide, Potassium iodide, Guanidine thiocyanate, Guanidine HCl, an acid, Sodium trichloroacetate, (xi) buffer to stabilise the pH between 5-8 or more preferably between 6 and 7, used in the range 0.5-20 mM such as, (g) a chelator to remove divalent metal cations, used in the range 0.005-20 mM, (xii) dissolved gas such as Carbon dioxide or Nitrogen (in the final concentration range of 0.01-5%, a non-reactive gas such as Argon (in the final concentration range of 1-20%) and or a buffer, (xiii) an alcohol in the range of 1-20% (wt:wt) to reduce DES viscosity such as a tert-butanol and/or (xiv) a glycol such as Glycerol, Ethylene glycol, Propylene glycol, Diethyleneglycol monoethylether acetate (DEGMEA), Polyethylene glycol in the Molecular weight range 100-6000 Daltons, more preferably 150-600 Daltons and most preferably 200-400 Daltons.

[0255] A non-exhaustive list of possible Dissociation buffer additives are, but are not limited to, one or more of the following; aqueous solvents, organic solvents, enzymes, antibodies, stains, dyes, markers, salts and chelators. Specific examples of components are water, DMSO, DMF, Urea, Thiourea, Acetamide, Acetone, Dihydroxyacetone, Formamide, Dimethylformamide, Dimethylourea, 1,3-Dimethylurea, 1,1-Dimethylurea, 1,3-Diamino-acetone, Trifluoroacetamide, 1,1,1,3,3,3-Hexafluoro-2-propanol, 1,1,1,3,3,3-Hexafluoro-2-phenyl-2-propanol, 1,1,1,3,3,3-Hexafluoro-2-methyl-2-propanol, 2,2-Difluoropropanediamine, Tetrafluorosuccinamide, 2,3,3,3-Tetrafluoropropianamide, 2,2,3,3-Tetrafluoropropanamide, Trifluoropyruvamide, Difluoropropanamide, Chlorodifluoroacetamide, Trifluoropyruvic acid, Trifluoropyruvamide, N-Hydroxymethyl trifluoroacetamide, 3,3,3-Trifluoropropionamide, Malonamide, Glycinamide, Lactamide, Formaldehyde, Glutaraldehyde, Ammonium p-toluenesulphonic acid, Sodium p-toluenesulphonic acid, Ammonium sulphate, Ammonium chloride, Ammonium thiosulphate, Sodium dodecyl sulphate, Sodium lauryl sulphate, Sodium Benzoate, Dodecyldimethyl(3-sulphopropyl)ammonium hydroxide, Dimethylbenzene sulphonic acid, Congo Red, Giemsa, DAPI, Ethidium bromide, Mallory's stain, Orcein, Aldehyde fuchin, Osmium tetroxide, Chromium trioxide, Chromic acid, Feulgen, Dichromate, Mercuric chloride, Haematoxylin and Eosin stain (H&E), Coomassie Blue, Methylene Blue, Xylene cyanol, Crystal violet, Fuchsin, Acridine orange, DAPI, Carmine, Eosin, Ethidium bromide, Bismarck brown, Hoechst, Malachite green, Methyl green, Neutral blue, Nile blue, Rhodamine or Safranin, Formaldehyde, Glutaraldehyde, Pentanol, Butanol including tert-butanol, Propanol, Ethanol, Methanol, Methyltriphenylphosphonium bromide, Cetyltetrabutylammonium bromide (CTAB), Tetrabutylammonium bromide (TBAB), Tetramethylammonium oxide (TMAO), Sodium deoxycholate, IGEPAL-CA630, Brij-35, Brij-58, NP-40, Triton X-100, Triton X-114, Tween-20, Tween-80, Octyl beta glucoside, CHAPS, Solanine, Sodium dodecyl sulphate (SDS), Sodium lauryl sulphate (SLS), kanomycin, streptomycin or penicillin, Sodium nitrate, Sodium nitrite or Sodium benzoate, kanomycin, streptomycin or penicillin, single-stranded or double-stranded RNA or DNA sequences, including labelled sequences, an aptamer specific to a cellular epitope and containing 5′ and 3′ polymerase amplification sites, a peptide, enzyme, antibody, biotin, biotin labelled molecule, horseradish peroxidase, avidin, streptavidin, GFP or variant thereof, Luciferase, fluorescent labelled molecule such as Fluorescein, Rhodamine, Texas Red, Alexa Fluor™, LNA, labelled LNA, a Molecular Beacon™, a Scorpion probe™, FISH probe, bDNA, PCR primer, oligo (dT), PNA, anti-oxidant, RNasin, SUPERase.IN™, RNaseOUT™, phenylmethylsulfonyl fluoride (PMSF), diisopropyl fluorophosphate (DFP), aprotinin or Pefabloc SC™, RNasin, SUPERase.IN™, RNaseOUT™, or a protease inhibitor such as phenylmethylsulfonyl fluoride (PMSF), DEPC, diisopropyl fluorophosphate (DFP), aprotinin or Pefabloc SCTM, Tris-HCl, PIPES, MES, HEPES, MOPS, MOPSO, CAPS, CAPSO, BIPES, phosphate, imidazole, BAPTA, EDTA, EGTA, D-Penicillamine, a dissolved gas such as a Argon, Nitrogen, Xenon, Air, Carbon dioxide, Trichloroacetic acid, Sulphosalicyclic acid, Tetramethyl urea, Imidazole, 1-Methylimidazole, 1-Ethylimidazole, 1-Benzylimidazole, 4-Methylimidazole, N-Methylpyrrolidone, N-Ethylpyrrolidone, N-Benzylpyrrolidone, Guanidine, Guanidine HCl, Guanidine isothiocyanate, Ribitol, Rhamnose, Trehalose, D-Sorbitol, L-Sorbitol, Sorbose, Xylitol, Glucose, Sucrose, Lactose, Fructose, Maltose, Mannose, Mannitol, Arabinose, Galactose, Raffinose, Inositol, Erythritol, Xylose, Zinc acetate, Zinc EDTA, Zinc phosphate, Zinc Trifluoroacetate, Zinc citrate, Zinc PTSA, Zinc gluconate, Zinc chloride and/or Zinc sulphate, an oil, a wax, liquid Paraffin, Silica gel, Polyacrylamide, Polyacrylic acid, a Carboxylic acid such as Formic, Acetic, Propanoic, Butanoic, Lactic, Citric acids and halogenated derivatives thereof, Chloroform, Phenol, Glycerol, Ethylene glycol, Diethylene glycol, Triethylene glycol, Polyethylene glycol (PEG) with a MW of 400 to 400,000 Daltons, Beta-mercaptoethanol, DTT, DTE, TCEP, Cysteine, Cystine, a salt such as Sodium chloride, Sodium phosphate, Sodium acetate, Sodium formate, a gel containing a high molecular weight polysaccharide such as Glucomannan, Cellulose, Starch, Inulin, Amylose, a gum such as Xanthan, Arabic or Guar, a protein such as Albumin, Casein or Haemoglobin, an antibody, an enzyme, a dessicant such as Molecular sieves 4A, silica gel, Sodium acrylate, CaCl2 and/or LiCl, hard abrasive particles such as ground glass, hydroxylapatite, magnetic hydroxylapatite or silica, or ceramic, synthetic diamonds of no more than 5 μm, preferably no more than hum, even more preferably no more than 100 nm in diameter to serve as enhancers of tissue dissociation during sonication. The dissociation buffer may be degassed.

[0256] One particularly preferred type of additive to the DES are acids, preferably carboxylic acids, even more preferably short-chain carboxylic acids such as Propanoic, Acetic acid or most preferably Formic acid, such carboxylic acids can have 1, 2 or more carboxylic groups and contain 1, 2 or more halogen atoms such as in Trifluoroacetic acid. The preferred concentration range of the acid is set out in Example 24. The time that the DES is in contact with the sample is of particular importance and that sustained exposure of the sample to a low pH will damage the DNA, such as gDNA, and to a lesser extent the RNA. It is therefore important to add sufficient acidity to provide the desired property of the DES whilst not so much that the biomolecule is measurably damaged during the period of contact. The damage can be determined by gel electrophoresis as set out extensively in U.S. Pat. No. 9,696,247.

[0257] Generally as set out in U.S. Pat. No. 9,696,247 in order to maintain good quality RNA in mammalian samples for a week or more at 37° C. it is often necessary to keep the final water content in the DES at 10% or less. It should be noted that in the present methods the RNA and other biomolecules may only need to be stabilized for a few hours or less during the dissociation and therefore the DES composition can contain significant amounts of water or other additives, for example 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or 75% final concentration of water that would otherwise, over the course of several days lead to RNA degradation.

[0258] Also provided is a combination of a DES for fixation and biomolecule stabilization followed by the use of a liquid that is not a DES such as a Glycerol for subsequent dissociation. The DES can also be mixed with varying amounts of a solvent such as water, DMSO, an Alcohol, a Glycol, N-Methylpyrrolidone, Tetramethylurea, or 1-Methylimidazole to give for example a secondary solvent concentration of 99, 90, 80, 70, 60, 50, 40, 30, 20, 10, 1 or less than 1% in the DES. The DES composition can also be composed of more than one DES, for example a mixture of one part of Trimethylglycine:Trifluoroacetamide and one part of Trimethylglycine:Glycerol.

[0259] The dissociation step is not particularly limited to one process or procedure and can include mechanical, enzymatic, chemical or a combination of each. Mechanical dissociation is preferred and sonication in particular.

[0260] It will be evident to one skilled in the art that it may be necessary to empirically test the most appropriate combination of solvents, including the use of a DES with the most appropriate dissociation process for a specific tissue type. One particular preferred DES composition is Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) with Formic acid as an additive in the range of 50-1000 mM, optionally 100-500 mM, further optionally 200-400 mM, further optionally 250-350 mM or 300 mM final concentration; or in the range of 1-12M, more preferably 4-9M, more preferably 6-8M and most preferably 7-8M acid.

[0261] Other preferred compositions are: (i) Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) with Acetic acid as an additive in the range of 2-17.39M, more preferably 8-17.39M, and most preferably 11-17.39M, or (ii) an aqueous or alcoholic (such as methanol, ethanol or propanol) dilution of Acetic acid in the range of 2-17.39M, more preferably 6-17.39M, and most preferably 9-17.39M, or (iii) pure glacial Acetic acid.

[0262] DES compositions containing carboxylic acids such as Formic acid and Acetic acid can be used either as (i) the initial primary fixative of the fresh or the fresh-frozen tissue and prior to dissociation, (ii) subsequent to fixation with a DES that does not contain carboxylic acid, such as Trimethylglycine:Trifluoroacetamide (1:2 mol:mol), or (iii) after dissociation of the DES fixed tissue, such as a secondary fixative of the single-cells produced from a dissociated tissue and preferably carried out either on (a) the cell suspension, (b) on the cells located on a filter, or (c) as a cell pellet.

[0263] DES compositions containing carboxylic acids such as Formic acid and Acetic acid can be used either as (i) the initial primary fixative of the fresh or the fresh-frozen tissue and prior to dissociation, (ii) subsequent to fixation with a DES that does not contain carboxylic acid, such as Trimethylglycine:Trifluoroacetamide (1:2 mol:mol), or (iii) after dissociation of the DES fixed tissue, such as a secondary fixative of the single-cells produced from a dissociated tissue and preferably carried out either on (a) the cell suspension, (b) on the cells located on a filter, or (c) as a cell pellet.

[0264] Solubilisation of DES fixed tissues and cells is generally an important step in the analysis of the biomolecule composition of a sample. Non-fixed fresh or frozen-thawed cell samples are generally easy to solubilise, meaning a partial or complete lysis of the cell membrane, cytoplasm, cytoplasmic contents with or without lysis of the nuclei. Many protocols require cell lysis to, for example to analyse the protein content (such as Western blotting), the DNA (such as whole genome sequencing), and the RNA (such as for RT-qPCR and scRNAseq).

[0265] Common methods to solubilise such samples include using a mild non-ionic detergent, such as Tween-20 or Triton-X100; a hypotonic solution; or stronger methods such as anionic, cationic, zwitterionic detergents; a chaotrope such as urea or guanidine, phenol, and often combined with a physical process such as shaking, bead-beating, vortexing and/or heating.

[0266] In contrast to live or frozen-thawed cells, fixed cells such as those fixed with Methanol, Acetone, Formaldehyde or a DES are much more difficult to lyse and may require harsher conditions to do so. As one example Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) fixed samples and cells generally require harsher conditions to lyse them than fresh samples, which can be problematic for protocols such as Drop-Seq scRNAseq (e.g. Chromium, 10× Genomics, USA) which are not compatible with harsh cell lysis conditions (such as strong detergents, solvents and chaotropes) that may lead to both disruption of the oil-water interface of the droplets and inhibit reverse transcriptases. However other methods of scRNAseq such as Seq-Well (Gierahn et al., Nature Methods (2017) 14:395) usefully has a strong chaotrope lysis step for the input single-cells which separates the cell lysis from the sensitive reverse transcription step allowing stronger reagents to be used prior to scRNAseq.

[0267] It has been found that the commonly used chaotropes Potassium iodide, Urea, Guanidine thiocyanate and Guanidine HCl are most efficient when combined with physical disruption such as shaking. It has surprisingly been found that the chaotropes 5M Sodium perchlorate, 5M Potassium iodide and 5M Thiourea are efficient at lysing Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) fixed samples. Furthermore the organic solvents beta-mercaptoethanol, 1,1,1,3,3,3-Hexafluoro-2-propanol, 1,1,1,3,3,3-Hexafluoro-2-phenyl-2-propanol, 1,1,1,3,3,3-Hexafluoro-2-methyl-2-propanol, DMSO and phenol, when used in the range of 80-100% final concentration, are particularly effective at effectuating cell lysis. Of note is that whilst DMSO very efficiently solubilises the Trimethylglycine:Trifluoroacetamide fixed cell's cytoplasm but not the nuclei, 1,1,1,3,3,3-Hexafluoro-2-propanol very efficiently solubilises both the cytoplasm and nuclei. This is important for example if studying the RNA component of the cytoplasm, nuclei or the entire cell contents as a selection of the source of the RNA can be made based on the solvent used.

[0268] Sonication

[0269] It has been found that tissues that have been fixed with Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) as set out in U.S. Pat. No. 9,696,247 are particularly well suited to dissociation of samples into smaller collections of cells or single cells.

[0270] Due to the importance of dissociating tissues or groups of cells into single-cells, there are many different protocols that have been developed but most generally rely on either (i) mechanical, (Lima et al., J Vis Exp. (2017) 125 10.3791/55913), (ii) enzymatic (Baron et al; Cell Systems, (2016) 3, 346-360, Muraro et al., Cell Systems, (2016) 3, 385-394) or (iii) a combination of both for dissociation (Martignani et al., Methods Mol Biol. (2018); 1817:137-144).

[0271] Mechanical methods for dissociating non-fixed tissues (ie fresh or fresh-frozen) include using devices such as a Dounce homogenizer, dicing with a knife, polytron, ultrathorax, Waring blender, mortar and pestle, trituration with a glass Pasteur pipette, vortexing, specialised devices such as GentleMACS (Miltenyi, Germany) and have been described extensively in the literature (Adams. L. et al., J. Immun. Methods (2015) 426, 42-49). These rely on a variety of physical forces such as shearing, slicing, cutting, vibration, pressure, crushing to pull apart or tear or otherwise separate the tissue sample into smaller pieces such as clumps of cells or individual cells. Such methods are extensively covered in the literature for example; Wiesmann U.N. (1999) Segregating Cells—Proteases in Tissue Culture/Proteolytic Enzymes. Springer Lab Manual., Biofiles (2006) Issue 2, Sigma-Aldrich (available online).

[0272] Mechanical methods such as those discussed above may be useful in the dissociating performed in the methods provided herein.

[0273] The products of tissue dissociation can be not only single cells but also organelles, cell membranes, nuclei, molecules such as peptides, proteins, phosphoproteins, lipids and fats, RNA and DNA and small molecules of less than 500 Daltons such as sugars, amino-acids and nucleotides. The products can also be multicellular such as clumps of 2 to 99 cells, 100 to 999 cells, 1000 to 9,999 cells, 10,000 to 99,999 cells, 100,000 to 999,999, or more than a million cells. The number of cells in a sample can be determined by using a nuclear stain such as DAPI, or using a cell counter for example the TC20™ Automated Cell Counter (Bio-Rad Inc.), Flow cytometry, FACS, weighing the sample and dividing by the average weight of a cell etc.

[0274] Dissociation of a tissue can result in not only clumps of cells but also cell matrix, extracellular matrix, connective tissue, collagen rich material, fat, bone, cartilage and cell debris. Whilst cells are of principle interest, other components can also have utility for the diagnostic interpretation of the sample. One example is observing the density, formation, structure and shape of the extracellular matrix of the ascending thoracic aorta of Marfan patients. Another example would be to study the extracellular matrix in a cancer biopsy. Such studies can be accompanied by additional tests such as staining for the presence of collagen, for elastin, for fibrillin, an antibody or a nucleic probe. Staining of cells and tissues is extensively covered in the literature (Ross M.H. and Wojciech P. (2011) Histology. sixth edition, Walters Kluwer).

[0275] The sample is biological in origin, such as plant; fungal; animal; bacterial; algal; a parasite such as plasmodium, schistosome, trypanosome; a human blood clot; a mammalian tissue and/or a human clinical sample. The sample is not necessarily a tissue but could be other sample types such as a predominately non-cellular sample such as an egg, cornea, sclera, hair, wool, finger nails, bone, teeth, or wood.

[0276] The sample can also be sub-cellular such as a cell nuclei, an organelle such as a mitochondria or chloroplast, a particle such as a virus or ribosome, or a molecule such as a metabolite, lipid, fat, carbohydrate, protein, phosphoprotein, DNA or RNA.

[0277] Definitions

[0278] ‘Dissociation buffer’ is the medium used to immerse the sample in during dissociation. It can be a liquid, a gel, a wax, an oil, a biphasic or triphasic mixture, a solid or a mixture of a liquid and solid. It can also contain particles such as beads or crystals to aid in the dissociation of the tissue.

[0279] Preferably, Deep Eutectic Solvents (DESs) are used as the Dissociation Buffer. DES are particularly useful as Dissociation buffers because they are mostly viscous solutions and the viscosity can be easily modified by for example changing the composition, and therefore the amount of hydrogen bonding, increasing or decreasing the temperature or adding water. As one example, using a single Dissociation buffer composition, it is possible to reduce the viscosity by 10-100 fold by increasing the temperature by 20-80° C. or adding 3-50% water. Likewise it is possible to increase the viscosity by reducing the temperature or removing water. It will be evident to one skilled in the art that it is possible to use a Dissociation buffer with a wide variety of desired properties by modifying the composition and the external environment. Properties of the Dissociation buffer that are particularly desired include; (i) viscosity, to promote dissociation of tissues into intact cells or groups of cells rather than breaking, damaging and lysing cells thereby transforming them into debris, (ii) a reducing environment to reduce or prevent oxidative damage to the sample, (iii) the pH in the range of 4-7 to reduce hydrolysis of fragile molecules in particular RNA, (iv) gas content to increase or reduce cavitation and therefore the strength of dissociation, (v) liquid convection to increase the circulation of the sample within the focus of the sonication power, (vi) a stabilizer that protects biomolecules in particular proteins, phosphoproteins, RNA and DNA, (vi) an environment that protects the structure, abundance and localization of cellular and extracellular epitopes so that downstream or in situ antibody binding is not compromised.

[0280] The Dissociation buffer can also be a (i) single pure substance such as Glycerol or Polyethylene glycol such as PEG200 or PEG600, (ii) a mixture such as an aqueous or organic solvent containing a buffer or a salt, (iii) a DES composition, or (iv) a DES composition containing one or more additives such as those set out herein.

[0281] ‘Sample’: the sample is generally of biological origin and may comprise one or more living or dead cells or viral particles, bacteria, plant, fungal or animal origin including parasites, nematodes, insects, mollusc, fish, amphibians, reptiles, birds, mammals including human samples and clinical samples such as a cancer biopsy, a sample from an epileptic patient or a post-mortem brain sample.

[0282] The mammalian sample is not particularly limited but can be liver, spleen, brain, spinal cord, muscle, heart, oesophagus, testis, ovaries, thymus, kidneys, skin, pancreas, olfactory organ, adrenal glands, lung, smooth, skeletal or cardiac muscle, aorta, blood vessel, blood, plasma, serum, spleen, thymus, salivary gland, uterus, skin, eye, tongue, retina, oesophagus, stomach, intestine, pancreas, gallbladder, lymph nodes, bone, bone marrow, a connective tissue or cartilage.

[0283] Furthermore the sample can be or can contain a pathogen such as a prion, viroid, virus, bacteria, fungi or parasite. The sample can also include or consist of viruses such as those found in blood, in particular viruses, bacteria or parasites that are trapped, embedded in or present in blood, dried blood, a blood clot, semen, mucus, faeces, saliva, plasma, serum, amniotic fluid, or other body fluid such as cerebral spinal fluid, urine or pleural effusion.

[0284] ‘Dissociation’ is the process of reducing the overall size, weight and/or complexity of a biological sample. One of the primary purposes of dissociation is for separating cells from a tissue into smaller groups of cells such as clumps of cells or individual cells. The dissociation may take place in stages, so that distinct parts of the biological sample are separated together, for example a kidney nephron may be separated during the dissociation process, from other nephrons and blood vessels before, optionally further dissociation into smaller groups of cells and/ single-cells. Dissociation can therefore be the progressive reduction in sample size with distinct steps based on tissue or organ structure, with the possibility of sampling and analysis of the partially dissociated sample at any stage. The progressive dissociation of the sample provides a means to collect groups of cells based on their original in situ localisation so that single-cells are subsequently obtained that were originally localized in the same part of the sample.

[0285] Provided herein are methods for reducing the complexity of a tissue sample, for example by reducing the number of cells that are attached to one another.

[0286] ‘Cavitation’ is the result of the growth and collapse of a gas filled bubble in a liquid or gel in the presence of sonication energy. The size, growth and energy released when the cavitating bubble collapses is dependent on a large number of parameters including the frequency (kHz), the temperature, the viscosity of the Dissociation buffer, the type of dissolved gas, the ambient pressure in the container, the surface tension of the liquid and the sonicator pulse delay.

[0287] ‘Sonication’ is the application of sound energy to a sample such as a biological tissue in order to dissociate it into smaller parts such as clumps of cells, single cells or even nuclei. Generally the sound energy has a (ultrasonic) frequency greater than 20 kHz and up to 3 MHz. The most useful frequencies have been found to be 20 kHz to 100 kHz, more preferably 20 to 60 kHz, more preferably between 20 to 40 kHz, and most preferably between 20 to 30 kHz. The exact preferred frequency may be dependent on a number of factors such as the tissue type and size to be dissociated, the sample fixative composition and how long it was fixed, the Dissociation buffer composition, the dissociation temperature, the container shape, material and dimensions, and the final size of the dissociated tissue desired (clumps or single-cells). It will be evident to one skilled in the art that the exact frequency preferred has to be determined empirically although the range of frequencies available in commercial sonication devices is somewhat limited, for example 26 kHz, 36 kHz, 46 kHz or 52 kHz. It should be noted that some sonication devices can produce more than one frequency allowing a combination of frequencies to be used during the dissociation process. As examples are Ultrasonic Generator 600W, adjustable multifrequency 20-68 KHz (Cat. No. SMUG600W2068ND2 Steminc, USA), Transonic TI-H 5, multifrequency 25/45 kHz or 35/130 kHz, and xtra ST 25/45 kHz (Elma Schmidbauer GmbH).

[0288] ‘Additive’ is any substance that can be dissolved, partially dissolved, is miscible or immiscible in a DES composition, and can be present in amounts greater than, equal to or less than the total amount of the DES mixture (weight:weight). The additive such as an oil or wax may form two or more phases with the DES, or an emulsion as with a hydrophobic solvent. A list of examples of Additives is set out herein.

[0289] ‘Metabolite’ is any metabolically derived biological substance with a molecular weight of less than or equal to 1000 Daltons.

[0290] ‘Frequency’ is the number of vibrations created by the transducer per second, also known as Hertz (Hz). Ultrasound commonly refers to frequencies of 20 kHz or more, for example 26 kHz, 36 kHz, 52 kHz etc. and may be as much as 1 MHz, 2 MHz or even 3 MHz. Generally the lower the frequency the greater the mechanical force created, for example a frequency of 20 kHz leads to greater dissociation of a biological sample than 100 kHz or 1 MHz. Higher frequencies such as 1 MHz or more provide a means to dissociate or remove fine particles (<100 nm diameter) from a biological sample such as particles or debris from the outside of a bone or tooth but are generally not suitable for creating sufficient cavitation energy to remove cells or clumps of cells from a biological sample. In general frequencies of 1 MHz or more do not lead to cavitation in any type of Dissociation buffer. Ultrasound is the transmission of pressure waves at frequencies above human hearing, specifically above 20 kHz.

[0291] ‘Power’ is the intensity (also called power density) of ultrasound and is measured in units of Watts/cm.sup.2 or Watts per second.

[0292] ‘Transducer’ is the mechanical device that is the source of ultrasound power, energy for the transducer is provided by the generator. Commonly the transducer is attached to a metal sonotrode or probe that transmits the ultrasound energy either directly or indirectly to the container containing the

[0293] Dissociation buffer and the sample to be dissociated. Generally a transducer produces only a single frequency of ultrasound which is matched with the physical dimensions and materials of the sonotrode to produce maximum energy transfer.

[0294] ‘Container’ is the enclosure which contains the Dissociation buffer and biological sample. The container may be in contact with the ultrasound energy source which can be for example a sonotrode, a block holding the container and in contact with a sonotrode, an ultrasound probe or an ultrasonic liquid filled bath. The container may have dimensions such that the ultrasound energy is sufficiently powerful to dissociate the sample, in general smaller internal volumes are preferred for optimal energy intensity, for example no more than 50 times greater, more preferably no more than 25 times and most preferably no more than 15 times the volume of the sample to be dissociated. The container is preferably made of a hard material such as polycarbonate which efficiently transmits sonication energy and is sufficiently transparent to allow the dissociation of the sample to be observed or monitored. Other hard materials include shatter-proof glass (eg. Duran), stainless steel, tungsten alloys, titanium alloys, copper alloys, aluminium alloys, polystyrene, polyamide, polymethylmethacrylate, TPX plastic and/or ceramics. The container can also be composed of 2 or more materials for example a hardwall for transmitting the sonication energy and an inlet and outlet made from flexible tubing. Thickwall carbonate centrifuge tubes manufactured by Beckman Inc., have been found to be suitable containers for sonication as they are available in a variety of sizes and wall thicknesses. Container materials are preferred that are transparent, have a hardness of at least 100 MPa (N/mm2) but are not prone to shattering.

[0295] The container can have numerous features including one or more of: an observation and/or measurement window composed of transparent material, an inlet for introducing fresh Dissociation buffer, an outlet for removing Dissociation buffer including single-cells and clumps of cells, a filter and/or membrane for removing cellular and tissue debris, cells and/or clumps of cells, a sealable cap for allowing introduction of the sample, an insert for limiting movement of the sample and/or Dissociation buffer, a mesh cassette with hole sizes of less than 1 mm for maintain the sample within an optimal sonication energy field but allowing cells and/or clumps of cells to escape from the sonication energy to protect the liberated cells from excessive damage such as cell membrane breakage, whereby the cassette fits into a holder within the container and is submerged in the Dissociation buffer, an inlet port for allowing injection or addition of a substance such as an additive, a colourant, a fluorophore, a stain, an antibody, a nucleic acid and/or a sample. The container can include a means to count, quantify the size, volume and 3D shape of a cell, specific morphological features such as brush borders, number of nuclei, mitotic index, localization of a fluorescent dye such as those for ISH and FISH analysis or antibody staining, identify, distinguish and measure organelles such as the golgi and mitochondria, or nuclei. This can be by way of a probe in contact with the Dissociation buffer, an electrode, a means to measure conductivity, a microcapillary, a capillary, an array of nanowells sufficiently proportioned that each well allows entry of a single cell for analysis, a window that is optically transparent to a laser, visible light, ultraviolet wavelengths or infrared wavelengths. The container can include a window for the detection of transmitted, emitted, absorption, reflected and/or generated wavelength frequencies.

[0296] ‘Sonication Device’ is composed of one or more of the following; an ultrasound generator, a transducer, a means to transfer sonication energy to a container such as a metal block, a probe or, a liquid bath, an orifice of suitable dimensions to allow the entry of a container or vessel containing the Dissociation buffer and sample, a heating and cooling unit such as a Peltier device or water cooler, a tube holder, a pump to circulate and/or filter the Dissociation buffer and single-cells in the container capable of circulating 0.1-100 ml per minute, a tube connected from the pump to the inlet of the container, and a tube connected from the outlet of the container to the pump, preferably with a cell filter such as a 1-50 um mesh size nylon CellMicroSieve (BioDesign Inc, USA), PluriStrainer (PluriSelect, Germany), or a 1-50 um pore size track-etched Isopore polycarbonate filter membranes (EMD Millipore, USA) or the like between the outlet and the pump, a means to quantify the number and size of dissociated material including single cells that are released from the sample into the Dissociation buffer such as lasers and detectors used for flow cytometry quantification, a secondary container where a stain, a colorant, an antibody or nucleic acid probe can be introduced to the single cells, a binding or hybridization reaction occurs, washing steps with a wash buffer, and detection of the reaction product that is detected and quantified such as by the use of a microscope, a laser, multispectral imaging, a spectrophotometer and/or a photometric detector.

[0297] The Sonication device can be portable for bedside use, and may optionally include internal rechargeable batteries. Alternatively, the sonication device may be immobile for laboratory bench use and may include a touch screen for controlling, setting and adjusting power, frequency and amplitude; a screen and/or printer for observing and recording results; a means to connect to a ‘smart-phone’, ‘tablet’ or the like such as a Bluetooth(®) and/or wifi connection; one or more holes or orifices to allow introduction of one or more Containers including a sample and Dissociation buffer, and/or samples; a means to insert and remove a cassette or pouch containing one or more liquid filled reservoirs containing one or more of the following: binding, hybridization, reaction reagents, washing and detection reagents such as a detergent containing buffer and an antibody, nucleic acid probe, colourant and/or stain, the cassette connected to a pump or pumping device to allow delivery, mixing and recovery of the reagents added to the sample, or the sample to the reagents, an automated pipette including disposable plastic pipette tips, a waste container for removal and disposal of used reagents, disposable plastics and/or sample, wherein the sample are single-cells derived from an animal such as a clinical human biopsy such a cancer or biopsy from brain sample undergoing neurodegeneration, and/or prion particles, viral particles, bacterial cells, fungi, parasites and the like.

[0298] Deep Eutectic Solvents

[0299] Various deep eutectic solvents (DES) are useful in the practice of the methods provided herein, for example as a dissociation medium/Dissociation buffer. The DES may be as described in International patent application publication no. WO 2014/131906 A1 to Goldsborough and Bates, the content of which is hereby incorporated by reference in its entirety.

[0300] A particularly preferred DES is Trimethylglycine:Trifluoroacetamide. A preferred molar ratio of Trimethyglycine to Trifluoroacetamide is 1:2.

[0301] By way of example, but without limitation, a DES (in particular, the DES used in the dissociation buffer or dissociation medium) may be made by mixing, or mixing and heating, one or more component(s) which may be chosen from the group: Choline nitrate, Choline tetrafluoroborate, Choline hydroxide, Choline bitartrate, Choline dihydrogen citrate, Choline p-toluenesulfonate, Choline bicarbonate, Choline chloride, Choline bromide, Choline iodide, Choline fluoride, Chlorocholine chloride, Bromocholine bromide, lodocholine iodide, Acetylcholine hydroxide, Acetylcholine bitartrate, Acetylcholine dihydrogen citrate, Acetylcholine p-toluenesulfonate, Acetylcholine bicarbonate, Acetylcholine chloride, Acetylcholine bromide, Acetylcholine iodide, Acetylcholine fluoride, Chloroacetylcholine chloride, Bromoacetylcholine bromide, lodoacetylcholine iodide, Butyrylcholine hydroxide, Butyrylcholine bitartrate, Butyrylcholine dihydrogen citrate, Butyrylcholine p-toluenesulfonate, Butyrylcholine bicarbonate, Butyrylcholine chloride, Butyrylcholine bromide, Butyrylcholine iodide, Butyrylcholine fluoride, ChloroButyrylcholine chloride, BromoButyrylcholine bromide, lodoButyrylcholine iodide, Acetylthiocholine chloride, L-Carnitine, D-Carnitine, Betaine, Sarcosine, Trimethylamine N-oxide, Betaine HCl, Cetyl betaine, Cetyltrimethylammonium fluoride, Cetyltrimethylammonium chloride, Cetyltrimethylammonium bromide, Lauryl betaine, N,N-Dimethylenethanolammonium chloride, N,N-diethyl ethanol ammonium chloride, Beta-methylcholine chloride, Phosphocholine chloride, Choline citrate, Benzoylcholine chloride, Lauryl sulphobetaine, Benzyltrimethylammonium chloride, Methyltriphenylphosphonium chloride, Methyltriphenylphosphonium bromide, Methyltriphenylphosphonium iodide, Methyltriphenylphosphonium fluoride, N,N-diethylenethanol ammonium chloride, ethylammonium chloride, Tetramethylammonium chloride, Tetramethylammonium bromide, Tetramethylammonium iodide, Tetramethylammonium fluoride, Tetraethylammonium chloride, Tetraethylammonium bromide, Tetraethylammonium iodide, Tetraethylammonium fluoride, Tetrabutylammonium chloride, Tetrabutylammonium bromide, Tetrabutylammonium iodide, Tetrabutylammonium fluoride, (2-chloroethyl) trimethylammonium chloride, Terbium (III) chloride, Zinc (II) chloride, Zinc (II) bromide, Zirconium (III) chloride, Iron (III) chloride, Tin (II) chloride, Copper (II) chloride, Magnesium (II) chloride; with, one or more other component(s) that can also form a DES, including for example, but without limitation, one or more of the following chemicals chosen from the group: Urea, Formamide, Thiourea, 1-Methylurea, 1,1-Dimethylurea, 1,3-Dimethylurea, Carbohydrazide, Tetramethylurea, 1,3-bis(hydroxymethyl)urea, Benzamide, Girards Reagent T, Lactamide, Acetamide, Fluoroacetamide, Difluoroacetamide, Trifluoroacetamide, Chlorofluoroacetamide, Chlorodifluoroacetamide, Chloroacetamide, Dichloroacetamide, Dichlorofluoroacetamide, Trichloroacetamide, Bromoacetamide, Dibromoacetamide, Tribromoacetamide, Bromofluoroacetamide, Bromodifluoroacetamide, Bromochlorofluoroacetamide, Iodoacetamide, Diiodoacetamide, Triiodoacetamide, 2-Methyl-2,2-difluoroacetamide, 2-Methyl-2-fluoroacetamide, 2,2-Dimethyl-2-fluoroacetamide, 2-Ethyl-2,2-difluoroacetamide, 2-Ethyl-2-fluoroacetamide, 2,2-Diethyl-2-fluoroacetamide, 2-Propyl-2,2-difluoroacetamide, 2-Propyl-2-fluoroacetamide, 2,2-Propyl-2-fluoroacetamide, 2-Fluoropropionamide, 3-Fluoropropionamide, 2,2-Difluoropropionamide, 2,3-Difluoropropionamide, 3,3-Difluoropropionamide, 3,3,3-Trifluoropropionamide, 2-Fluoro-3,3,3-trifluoropropionamide, 2-Chloro-3,3,3-trifluoropropionamide, 2,2-Chloro-3,3,3-trifluoropropionamide, 2-bromo-3,3,3-trifluoropropionamide, 2,2-Bromo-3,3,3-trifluoropropionamide, Pentafluoropropionamide, Heptafluorobutyramide, Trimethylacetamide, 1-(Trifluoroacetyl)imidazole, N,O-Bis(trifluoroacetyl)hydroxylamine, Bistrifluoroacetamide, N-Methyl-fluoroacetamide, N-Methyl-difluoroacetamide, N-Methyl-trifluoroacetamide, N-Methyl-chlorofluoroacetamide, N-Methyl-chlorodifluoroacetamide, N-Methyl-chloroacetamide, N-Methyl-dichloroacetamide, D N-Methyl-dichlorofluoroacetamide, N-Methyl-trichloroacetamide, N-Methyl-bromoacetamide, N-Methyl-dibromoacetamide, N-Methyl-tribromoacetamide, N-Methyl-bromofluoroacetamide, N-Methyl-bromodifluoroacetamide, N-Methyl-bromochlorofluoroacetamide, N-Methyl-iodoacetamide, N-Methyl-diiodoacetamide, N-Methyl-triiodoacetamide, N-Methyl-2-methyl-2,2-difluoroacetamide, N-Methyl-2-methyl-2-fluoroacetamide, N-Methyl-2,2-dimethyl-2-fluoroacetamide, N-Methyl-2-ethyl-2,2-difluoroacetamide, N-Methyl-2-ethyl-2-fluoroacetamide, N-Methyl-2,2-diethyl-2-fluoroacetamide, N-Methyl-2-propyl-2,2-difluoroacetamide, N-Methyl-2-propyl-2-fluoroacetamide, N-Methyl-2,2-propyl-2-fluoroacetamide, N-Methyl-2-fluoropropionamide, N-Methyl-3-fluoropropionamide, N-Methyl-2,2-difluoropropionamide, N-Methyl-2,3-difluoropropionamide, N-Methyl-3,3-difluoropropionamide, N-Methyl-3,3,3-trifluoropropionamide, N-Methyl-2-fluoro-3,3,3-trifluoropropionamide, N-Methyl-2-chloro-3,3,3-trifluoropropionamide, N-Methyl-2,2-chloro-3,3,3-trifluoropropionamide, N-Methyl-2-bromo-3,3,3-trifluoropropionamide, N-Methyl-2,2-bromo-3,3,3-trifluoropropionamide, N-Methyl-pentafluoropropionamide, N-Methyl-heptafluorobutyramide, N,N-Dimethyl-2,2,2-trifluoroacetamide, N-Ethyl-2,2,2-trifluoroacetamide, N,N-Diethyl-2,2,2-trifluoroacetamide, N-(Hydroxymethyl)Trifluoroacetamide, Ethyltrifluoroacetate, Dithiothreitol, Dithioerythritol, Beta-mercaptoethanol, Penicillamine, Tiopronin, Acrylamide, Methanol, Ethanol, Propanol, Butanol, Formaldehyde, Glutaraldehyde, Taurine, Aconitic acid, Adipic acid, Benzoic acid, Citric acid, Malonic acid, Malic acid, DL-Maleic acid, Oxalic acid, Phenylacetic acid, Phenylpropionic acid, Succinic acid, Levulinic acid, Tartaric acid, Gallic acid, p-Toluenesulphonic acid, Glycine, Alanine, Valine, Leucine, Isoleucine, Serine, Threonine, Tyrosine, Cysteine, Methionine, Aspartic acid, Asparagine, Glutamic acid, Glutamine, Arginine, Lysine, Histidine, Phenylalanine, Tryptophan, Proline, Ethylene glycol, Triethyleneglycol, Glycerol, Resorcinol, Phenol, 1,2-propanediol, 1,3-propanediol, 1,4-Butanediol, 1,5-Pentanediol, 1,6-Hexanediol, 1,8-Octanediol, 1,12-Dodecanediol, m-Cresol, Imidazole, 1-Methylimidazole, 4-Methylimidazole, N-Methylpyrrolidone, N-Ethylpyrrolidone, N-Benzylpyrrolidone, 2-imidazolindone, tetrahydro-2-pyrimidione, Guanidine, Guanidine HCl, Guanidine isothiocyanate, Guanidine sulphate, Ammonium acetate, Ammonium bicarbonate, Ammonium chloride, Ammonium citrate dibasic, Ammonium formate, Ammonium iodide, Ammonium nitrate, Ammonium phosphate monobasic, Ammonium phosphate dibasic, Ammonium sulfamate, Ammonium sulfate, Ammonium tartrate dibasic, Ammonium isothiocyanate, Ammonium benzoate, Ammonium bromide, Ammonium fluoride, Ammonium hydrogensulphate, Ammonium trifluoroacetate, Ammonium thiosulphate, Adonitol, Ribitol, Rhamnose, Trehalose, D-Sorbitol, L-Sorbitol, Sorbose, Xylitol, Glucose, Sucrose, Lactose, Fructose, Maltose, Mannose, Mannitol, Arabinose, Galactose, Raffinose, Inositol, Erythritol or Xylose.

[0302] It should be noted that certain components such as Zinc chloride can form a DES with not only Choline Chloride but also Urea; both Choline chloride and Zinc chloride appear in the same component group above, therefore certain DES mixtures can be prepared from chemicals within the same group.

[0303] It should also be noted that the lists above are non-exhaustive, and that the DES components are not particularly limited to any type of molecule or property except hydrogen-bonding between DES components is frequent but not an absolute requirement.

[0304] In other arrangements, a deep eutectic solvent may comprise a metal salt and a sugar or sugar alcohol.

[0305] It should also be noted that there is no particular maximum number of DES components in the DES mixture, for example 2, 3, 4, 5, 6, 7, 8, 9, 10 or more components can be mixed to produce a DES mixture, usually but necessarily, in integer molar ratios for example in a two component DES mixture, component 1 and component 2 can be mixed in the following ratios: 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10 (mol:mol) or for example in a three component DES mixture, component 1, component 2 and component 3 can be mixed in these ratios: 10:1:1, 9:1:1, 8:1:2, 7:1:3, 6:1:4, 5:1:5, 4:1:6, 3:1:7, 2:1:8, 1:1:9, 1:2:8, 1:3:7, 1:4:6, 1:5:5, 1:6:4, 1:7:3, 1:8:2, 1:9:1, 1:10:1 (mol:mol:mol).

[0306] The skilled artisan will understand that other ratios of mixing the components are also possible, for example 20:1 or 1:30 (mol:mol) and that the particular molar ratio leading to the eutectic point is not necessarily the optimum or desired molar ratio for the particular purpose of, for example, stabilising RNA and/or fixing cell morphology. The stabilisation activity of the DES may be related to the hydrogen bonding properties of the DES mixture. There is no particular restriction or limitation to the number of components or the ratio of mixing to make a useful DES mixture for a particular application. Additional physical properties of the DES for example viscosity or density can be modified by either adding a further component, or alternatively, by adding an ‘additive’ as set-out below. Some DES mixtures can be prepared using a fractional molar ratio, for example ZnCl2:urea has been prepared at 1:3.5 (mol:mol) ratio and there are again no particular restrictions to the molar ratios of component 1 and component 2, or component 1, component 2 and component 3 etc. that are used, and the optimum ratio of the components comprising the DES mixture to be used for the application, such as cell fixation, is most accurately determined empirically. By way of example only, Choline chloride/Trifluoroacetamide can be mixed in a 2:1, 1.75:1, 1.5:1, 1.25:1, 1:1, 1:1.25, 1:1.5, 1:1.75 or a 1:2 mol:mol ratio, or further fractional amounts thereof, and subsequently individually tested to determine which has the optimum RNA stabilization and/or cell fixation property.

[0307] Other factors such as the cost or toxicity may also lead the artisan to reduce the proportion of a particular component and to partially replace it with a cheaper alternative. For example only, an Acetylcholine chloride:urea (1:2 mol:mol) DES mixture can, for certain applications be partially replaced with the cheaper Choline chloride as in Acetylcholine chloride:Choline chloride:urea (1:1:4 mol:mol:mol).

[0308] It will also be apparent to the artisan that due to, for example, the presence of contaminants in the components used to make the DES, such as water, oxidation, absorption of CO2, contaminating by-products during synthesis or break-down products of one of the components, and that the desired exact ratio, for example only, of a 1:1 (mol:mol) mixture may practically mean +/−10%, but more preferably 5% and even more preferably 1% difference in the exact amount of either component 1 or component 2. As way of illustration, a 5% error could potentially give the following final molar ratios: 0.95:1, 1:0.95, 1.05:0.95 or 0.95:1.05 (mol:mol) and the effect of this error can generally only be determined empirically, for example its effect on RNA quality as set-out in Example 1. Generally in order to remove contaminants including water, the well-known procedure of recrystallisation in ethanol can be carried out followed by extensive vacuum and/or chemical drying. Methods are set-out in ‘Purification of Laboratory Chemicals’ Butterworth-Heinemann (2012).

[0309] Typically, but certainly not exclusively, a DES is prepared by mixing a hydrogen bond acceptor from one of the following classes of chemicals; (i) a nitrogen salt with a positively charged cation such as primary, secondary, tertiary or quaternary nitrogen, one example of a nitrogen salt is one with a halide, such as Choline chloride, (ii) a metal salt such as a transition metal salt halide, with, a hydrogen bond donor which may include (iii) an amine, (iv) a hydroxyl, (v) an aldehyde, (vi) an amide, or (vii) carboxylic acid, hydrogen bond donors such as sugars, carboxylic acids, ureas such as Trifluoroacetamide, and alcohols would thus be included.

[0310] In one arrangement, the deep eutectic solvent is a type III or a type IV deep eutectic solvent, wherein the RNA extracted from 10 mg of a rat liver sample incubated at 24° C. for 20 days with 400 mg of the deep eutectic solvent has an RNA integrity number (RIN) of at least 4.0 as measured using an Agilent Bioanalyser 2100.

[0311] RNA integrity number may be measured using an RNA 6000 Nano total RNA Kit (Cat. No. 5067-1511, Agilent Technologies, USA) and a Bioanalyser 2100 instrument (Cat. No. G2939AA, Agilent Technologies, USA).

[0312] As used herein, the term “type III deep eutectic solvent” refers to a deep eutectic solvent having at least a first component and a second component, wherein the first component is a quaternary ammonium or phosphonium compound such as choline chloride or N,N,N-trimethylglycine (betaine), and wherein the second component is a hydrogen bond donor, such as urea or trifluoroacetamide. A type IV deep eutectic solvent is a deep eutectic solvent comprising at least a first component and a second component, wherein the first component is a metal salt such as zinc chloride and wherein the second component is a hydrogen bond donor, such as urea.

[0313] Optionally, the deep eutectic solvent is a type III deep eutectic solvent, wherein the deep eutectic solvent comprises a compound comprising a trifluoromethyl group. The compound comprising the trifluoromethyl group may be present in the deep eutectic solvent in an amount of at least 5% by weight of the deep eutectic solvent.

[0314] In one arrangement, the deep eutectic solvent comprises a first component and a second component, wherein first component is a compound of Formula I:

##STR00019## [0315] wherein: [0316] R.sub.6 is H or OH; [0317] R.sub.7 is selected from H, CH.sub.3, Cl, Br, a carbonyl oxygen, and

##STR00020## [0318] Z is selected from —CH.sub.2—, O and S; [0319] R.sub.8 is R.sub.11 or OH; and [0320] wherein the second component comprises a compound of Formula II or a salt thereof:

##STR00021## [0321] wherein: [0322] A is selected from O, S, and NH; [0323] R.sub.1 is selected from H, an alkene group having 1 to 6 carbon atoms, R.sub.9, —NH.sub.2, —NH—(CH.sub.2).sub.aCH.sub.3, and —C(R.sub.3)(R.sub.4)(R.sub.5); [0324] wherein a is 0 or an integer from 1 to 5; [0325] R.sub.2 is selected from H and linear alkyl group having 1 to 3 carbon atoms; [0326] R.sub.3 is an optionally substituted 5- or 6-membered aliphatic or aromatic ring, wherein the substituent is R.sub.10; [0327] R.sub.4 and R.sub.5 are each independently H or F; and

[0328] wherein R.sub.9, R.sub.10, and R.sub.11 are each independently selected from alkyl groups having one to three carbon atoms, monochloroalkyl groups having one to three carbon atoms, and mono-, di- or tri-fluoroalkyl groups having one to three carbon atoms.

[0329] Preferably, in this arrangement, A is selected from O, S, and NH; R1 is selected from H, —CH═CH2, R9, —NH2, —NHCH3, and —C(R3)(R4)(R5); R2 is selected from H and —CH3; R3 is an optionally substituted 5- or 6-membered aliphatic or aromatic ring, wherein the substituent is R10; R4 and R5 are each independently H or F; and R9, R10, and R11 are each independently selected from alkyl groups having one to three carbon atoms, monochloroalkyl groups having one to three carbon atoms, and mono-, di- or tri-fluoroalkyl groups having one to three carbon atoms. Optionally, R7 is selected from H, Br, a carbonyl oxygen, and

##STR00022##

[0330] A may be O or S.

[0331] R.sub.2 may be H.

[0332] R.sub.1 may be selected from H, R.sub.9, —CH═CH.sub.2, and C(R.sub.3)(R.sub.4)(R.sub.5). In this arrangement, it is preferred that R.sub.1 is R.sub.9, wherein R.sub.9 preferably has one carbon atom.

[0333] Preferably, the second component is acetamide, or 2-chloroacetamide.

[0334] In another arrangement, R.sub.9 is a mono-, di- or tri-fluoromethyl group.

[0335] The second component is preferably trifluoroacetamide, trifluorothioacetamide or N-methyltrifluoroacetamide.

[0336] In another arrangement, R.sub.9 has two carbon atoms, preferably wherein R.sub.9 is a mono-, di- or trifluoroethyl group.

[0337] It is preferred that the second component is 2,2-difluoropropanamide or 3,3,3-trifluoropropanamide.

[0338] In another arrangement, the second component is formamide or acrylamide.

[0339] In a further arrangement, R.sub.1 is C(R.sub.3)(R.sub.4)(R.sub.5), wherein R.sub.4 and R5 are preferably F.

[0340] R.sub.3 may be an optionally substituted 6-membered aromatic ring, such as an optionally substituted phenyl group. Preferably, the first component is 2,2-difluoro-2-phenylacetamide. Alternatively, R3 comprises a substituent, preferably at the 2-position of the phenyl group.

[0341] The substituent is preferably a mono-, di- or tri-fluoromethyl group. Thus, in one arrangement the second component is 2-(trifluoromethyl)phenyl acetamide.

[0342] In a further arrangement R.sub.1 is selected from —NH.sub.2 and —NHCH.sub.3. Preferably, the second component is urea, thiourea or 1,3-dimethylurea.

[0343] In a further arrangement, A is NH. In this arrangement the second component may be guanidine, optionally wherein the guanidine is present in the form of a hydroisothiocyanate salt.

[0344] In a further arrangement, R.sub.7 is H. In this arrangement the first component preferably comprises choline.

[0345] In another arrangement, the first component comprises bromocholine.

[0346] In another arrangement, the first component is N,N,N-trimethylglycine, optionally wherein the second component is selected from trifluoroacetamide and urea.

[0347] In another arrangement R.sub.7 is

##STR00023##

[0348] In this arrangement Z may be O or S and R.sub.8 may be R.sub.11, wherein R.sub.11 preferably has one carbon atom.

[0349] In this arrangement the first component preferably comprises acetylcholine or acetylthiocholine.

[0350] Alternatively R.sub.11 may have three carbon atoms, wherein the first component preferably comprises butyrylcholine.

[0351] In a further arrangement Z may be CH.sub.2. In this arrangement, R.sub.8 may be OH. In this arrangement, the second component is preferably carnitine.

[0352] The first component may comprise a counterion, which counterion is typically a halide anion but can be another anion such as nitrate (NO3-) or tetrafluoroborate (BF4-). The halide anion may be selected from fluoride, chloride, bromide, and iodide, preferably chloride. In this arrangement it is preferred that the first component is choline chloride, optionally wherein the second component is selected from trifluoroacetamide, trifluorothioacetamide; 3,3,3-trifluoropropanamide; 2,2-difluoro-2-phenylacetamide; thiourea and urea, preferably trifluoroacetamide.

[0353] The molar ratio of the first component to the second component is in the range 1:3 to 2:1, preferably in the range 1:1.5 to 1:2.5, more preferably 1:2.

[0354] In a further arrangement, the deep eutectic solvent comprises a first component and a second component, wherein the first component is a halide salt of choline; and wherein the second component is an optionally-substituted imidazole, wherein the or each substituent is an alkyl group having 1 to 3 carbon atoms. In this arrangement, the molar ratio of the first component to the second component is preferably in the range 2.8:1 to 2:1.

[0355] The halide salt of choline is preferably choline chloride and the substituted imidazole is preferably a methyl imidazole, such as N-methylimidazole or 4-methylimidazole. The use of benzyl-substituted imidazoles such as 1-benzylimidazole is also contemplated. Alternatively, the imidazole is unsubstituted.

[0356] In a further arrangement, the deep eutectic solvent comprises a first component and a second component, wherein the first component comprises a compound of Formula III:

##STR00024## [0357] wherein: [0358] R.sub.6 is H or OH; [0359] R.sub.7 is selected from H, CH.sub.3, Cl, Br, a carbonyl oxygen, and

##STR00025## [0360] Z is selected from —CH.sub.2—, O and S; [0361] wherein R.sub.8 is selected from OH, an alkyl group having one to three carbon atoms, a monochloroalkyl group having one to three carbon atoms, and a mono-, di- or trifluoroalkyl group having one to three carbon atoms; and [0362] wherein the second component is a sugar or a sugar alcohol having at least 3 carbon atoms.

[0363] R.sub.7 is optionally selected from H, Br, a carbonyl oxygen, and

##STR00026##

[0364] The sugar alcohol may be selected from glycerol, thrietol, xylitol, sorbitol and volemitol, preferably from glycerol, xylitol and sorbitol, and is most preferably sorbitol. Alternatively, the sugar may be trehalose.

[0365] In this arrangement, the second component may comprise choline, such as choline chloride. The molar ratio of the first component to the second component may be in the range 1:2 to 2:1, preferably in the range 1:0.8 to 1:1.2.

[0366] In a further arrangement, the deep eutectic solvent comprises a first component and a second component, wherein the first component is a zinc (II) halide or zirconium (IV) halide, and wherein the second component is a compound of Formula IV:

##STR00027## [0367] wherein:

[0368] A is selected from O, S, and NH;

[0369] R.sub.1 is selected from H, an alkene group having 1 to 6 carbon atoms, R.sub.9, —NH.sub.2, —NH—(CH.sub.2).sub.nCH.sub.3, and —C(R.sub.3)(R.sub.4)(R.sub.5);

[0370] wherein n is 0 or an integer from 1 to 5;

[0371] R.sub.2 is selected from H and linear alkyl group having 1 to 3 carbon atoms;

[0372] R.sub.3 is an optionally substituted 5- or 6-membered aliphatic or aromatic ring, wherein the substituent is R.sub.10;

[0373] R.sub.4 and R.sub.5 are each independently H or F; and

[0374] wherein R.sub.9 is selected from alkyl groups having one to three carbon atoms, monochloroalkyl groups having one to three carbon atoms, and mono-, di- or tri-fluoroalkyl groups having one to three carbon atoms.

[0375] The preferred zinc (II) halide is ZnCl.sub.2. The preferred zirconium (IV) halide is ZrCl.sub.4. The second component is preferably urea. The molar ratio of the first component to the second component may be in the range 1:3 to 1:4.

[0376] The second component is preferably urea.

[0377] In a further arrangement, the deep eutectic solvent comprises a first component and a second component, wherein the first component is a compound of Formula V:

##STR00028## [0378] wherein: [0379] Y.sup.− is Cl.sup.− or Br.sup.−; [0380] X is N or P; [0381] R.sub.12, R.sub.13, R.sub.14 and R.sub.15 are each independently a linear alkyl group having 1 to 16 carbon atoms, a linear alcohol group having 1 to 16 carbon atoms, a benzyl group, or a phenyl group; [0382] wherein the second component is a compound of Formula I or a salt thereof:

##STR00029## [0383] wherein: [0384] A is selected from O, S, and NH; [0385] R.sub.1 is selected from H, —CH═CH.sub.2, R.sub.9, —NH.sub.2, —NHCH.sub.3, and —C(R.sub.3)(R.sub.4)(R.sub.5); [0386] R.sub.2 is selected from H and —CH.sub.3; [0387] R.sub.3 is an optionally substituted 5- or 6-membered aliphatic or aromatic ring, wherein the substituent is R.sub.10; and [0388] R.sub.4 and R.sub.5 are each independently H or F; [0389] wherein R.sub.9 and R.sub.10 are each independently selected from alkyl groups having one to three carbon atoms, monochloroalkyl groups having one to three carbon atoms, and mono-, di- or tri-fluoroalkyl groups having one to three carbon atoms.

[0390] Y.sup.− may be Cl.sup.−. In alternative arrangements, Y.sup.− may be any suitable counterion, such as a halide, nitrate, or tetrafluoroborate.

[0391] X may be N; in this arrangement, the compound of Formula V is a quaternary ammonium salt.

[0392] Optionally, R.sub.12, R.sub.13, R.sub.14 and R.sub.15 are each independently a linear alkyl group having 1 to 16 carbon atoms, a benzyl group, or a phenyl group

[0393] R.sub.12, R.sub.13, R.sub.14, and R.sub.15 may be each independently selected from linear alkyl groups having 1 to 4 carbon atoms, and are preferably each methyl groups. In this arrangement the first component is preferably tetramethylammonium chloride.

[0394] Alternatively, R.sub.12, R.sub.13, R.sub.14, and R.sub.15 are each ethyl groups. In this arrangement the first component is preferably tetraethylammonium chloride.

[0395] Alternatively R.sub.12, R.sub.13, R.sub.14, and R.sub.15 are each butyl groups. In this arrangement the second component is tetrabutylammonium chloride or tetrabutylammonium bromide.

[0396] Alternatively, the first component comprises:

##STR00030##

[0397] Alternatively, the first component comprises:

##STR00031##

[0398] In another arrangement X may be P.

[0399] At least one of R.sub.12, R.sub.13, R.sub.14, and R.sub.15 may be a phenyl group. In this arrangement the first component preferably comprises methyltriphenylphosphonium and may be methyltriphenylphosphonium bromide.

[0400] In another arrangement A may be O or S.

[0401] R.sub.1 may be selected from —NH.sub.2 and —NHCH.sub.3and the second component is preferably urea. Alternatively the second component may be trifluorothioacetamide. Alternatively the second component may be trifluoroacetamide.

[0402] The molar ratio of the first component to the second component may be 1:1.5 to 1:2.5, preferably 1:1.8 to 1:2.2.

[0403] In a further arrangement the deep eutectic solvent comprises a first component and a second component, wherein the first component comprises choline and wherein the second component is an alkanediol having 5 to 7 carbon atoms. The alkanediol is preferably hexanediol.

[0404] In a still further arrangement the deep eutectic solvent comprises a first component and a second component, wherein the first component comprises choline and wherein the second component comprises an N-alkyl pyrrolidone, wherein the N-alkyl group has 1 to 5 carbon atoms. Preferably, the N-alkyl pyrrolidone is N-methylpyrrolidone. Preferably, the choline is choline chloride. In this arrangement, the molar ratio of the first component to the second component may be 1:2.

[0405] In another arrangement, the deep eutectic solvent comprises a first component and a second component, wherein the first component comprises choline, and wherein the second component comprises beta-mercaptoethanol. In this arrangement, the choline may be choline chloride and/or the molar ratio of the first component to the second component may be about 1:2.

[0406] In another arrangement, the deep eutectic solvent comprises a first component and a second component, wherein the first component comprises choline, and wherein the second component comprises dithiothreitol. The choline may be choline chloride. The ratio of the first component to the second component may be about 1:2.

[0407] The eutectic point of a two or more component DES mixture is the point where the proportion of component 1 relative to the other component(s) leads to the lowest melting temperature. For example the eutectic point of a Choline chloride:Urea DES mixture is (1:2 mol:mol) with a reported freezing point (Fp) of 12° C. From a practical stand-point this means that the DES can be handled as a liquid at room-temperature but will slowly solidify in the fridge. Other DES mixtures, particularly those containing Choline chloride mixed with Glycerol, Ethylene glycol and Trifluoroacetamide or Phenylacetic acid have much lower Fp, remaining liquid below 0° C. or even −20° C. Although handling liquids provides certain advantages such as ease of measuring exact volumes and mixing, the present methods are not limited to DES mixtures that are liquid at room-temperature of any other particular temperature. Heating and mixing the components of a DES provides a means for individual atoms and molecules to establish hydrogen bonds or other interactions with other atoms and molecules and therefore produce the particular property of the DES mixture. Once this has occurred, whether the DES mixture is a liquid, gel or solid does not necessarily change its capacity to, for example, stabilise RNA in a tissue sample. Simple contact between the tissue sample and the DES mixture will occur even when the DES is a solid at room temperature and rapid stabilisation can occur. Although traditionally RNA stabilisation reagents are liquids such as RNAlater, AllProtect, PAXgene Tissue or formalin, the methods described here can make use of the liquid, gel or solid DES RNA stabilisation and/ or cell fixation properties. Indeed the use of a solid form of a DES can be advantageous when handling tissue samples; a solid block of DES can be formed by heating the DES solid above its eutectic point so that it melts and then pouring it into a suitable receptacle to cool and solidify or, molded or cut to fit a specific container and the tissue sample placed on the solid DES surface to bring about stabilisation. Conveniently wells, depressions or grooves can be made in the solid DES surface to serve as specific storage locations for each sample, one well receiving one sample etc, advantageously with the use of a well, the surface area in contact between the solid DES and the sample is increased, whilst an additional layer of a DES solid or liquid above the sample located in the well will further increase the total amount of DES available and surface area of contact with the sample.

[0408] It may be useful to use an additive in combination with a DES mixture. The purpose of the additive is to improve the properties of the DES mixture, for example, RNA, DNA and/or protein stabilisation and/or cell and tissue fixation or another application. The effects of various additives on cell morphology for example are set out in Table A. The purpose of the additive is to enhance the property of the DES mixture for the particular application, for example RNA stabilisation, storage, transport, and/or cell or tissue fixation. By way of example only, the additive could be a (i) colourant or dye to aid in handling or staining or processing the tissue such as H&E stain, Coomasie Blue, Methylene Blue, Xylene cyanol, Crystal violet, fuchsin, Acridine orange, DAPI, Carmine, Eosin, Ethidium bromide, Bismarck brown, Hoechst, Malachite green, Methyl green, Neutral blue, Nile blue, Osmium tetroxide, Rhodamine or Safranin, (ii) detergent, quaternary ammonium salt or saponin to improve penetration of the cell plasma membrane with the DES mixture, such as SDS, sodium lauryl sulphate (SLS), cetyltetrabutylammonium bromide (CTAB), tetrabutylammonium bromide (TBAB), sodium deoxycholate, Brij-35, Brij-58, NP-40, Triton X-100, Triton X-114, Tween-20, Tween-80, Octyl beta glucoside, CHAPS, Solanine, (iii) anti-microbial such as an antibiotic or antiseptic, such as kanomycin, streptomycin or penicillin, sodium nitrate, sodium nitrite or sodium benzoate, (iv) protein precipitant such as Trichloroacetic acid, Ammonium sulphate, Sulphosalicyclic acid, Zinc salt such as ZnCl2 or ZnSO4., (v) desiccant to remove excess water from the DES mixture such as Molecular sieves 4A, silica gel, CaCl2 or LiCl, (vi) a probe, a hybridising complementary nucleic acid, a peptide, protein, nucleic acid or labelled molecule, an internal control, a blocking sequence or a carrier nucleic acid such as (a) ss or ds RNA or DNA sequences, (b) a peptide, enzyme or other protein such as an antibody, (c) molecules for detecting an analyte such as a biotin, horseradish peroxidase, avidin, streptavidin, fluorescent labelled molecule such as fluorescein, Texas Red, Alexa Fluor™ labelled LNA, (d) a Molecular Beacon™, a Scorpion probe™, (vii) anti-oxidant to remove oxygen from the sample and reduce damaging oxidative effects during storage on for example the RNA or DNA nucleobases, such as Vitamin C or glutathionine, (viii) ribonuclease inhibitor such as RNasin, SUPERase.IN™, RNaseOUT™, or a protease inhibitor such as phenylmethylsulfonyl fluoride (PMSF), diisopropyl fluorophosphate (DFP), aprotinin or Pefabloc SC™, (ix) buffer to stabilise the pH between 5-8 or more preferably between 6 and 7, used in the range 0.5-20 mM such as Tris-HCl, PIPES, MES, HEPES, MOPS, MOPSO, CAPS, CAPSO, BIPES, phosphate, imidazole, (x) chelator to remove divalent metal cations, used in the range 0.5-20 mM such as BAPTA, EDTA, EGTA, citric acid, D-Penicillamine, (xi) dissolved oxygen (in the final concentration range of 2-20%), and/or CO2 (in the final concentration range of 0.01-5%, a non-reactive gas such as Argon (in the final concentration range of 1-20%) and or a buffer, nutrients (such as glucose and amino-acids) to enhance cell, tissue and organism viability, and/or (xii) an alcohol in the range of 1-20% (wt:wt) to reduce DES viscosity such as a tert-butanol.

TABLE-US-00001 TABLE A Effects of Various DES Mixtures with or without additives on Cell Morphology. Effect on Ratio Additive HeLa Component 1 Component 2 (mol:mol) Final % Morphology 1 Choline chloride Trifluoroacetamide 1:1.8 — ++++ 2 Choline chloride Trifluoroacetamide 1:2 — ++++ 3 Choline chloride Trifluoroacetamide 1:2.25 — ++ 4 Choline chloride Trifluoroacetamide 1:2.5 — ++++ 5 Choline chloride Trifluoroacetamide 1:2.75 — ++++ 6 Choline chloride Trifluoroacetamide 1:3 — +++++ 7 Choline chloride Trifluoroacetamide 1:2 H2O (17%) +++++ 8 Choline chloride Trifluoroacetamide 1:2 H2O (13%) +++ 9 Choline chloride Trifluoroacetamide 1:2 H2O (10%) +++ 10 Choline chloride Trifluoroacetamide 1:2 H2O (5%) ++++ 11 Choline chloride Trifluoroacetamide 1:2 H2O (2.5%) +++ 12 Choline chloride Trifluoroacetamide 1:2 Ethylene glycol (17%) ++ 13 Choline chloride Trifluoroacetamide 1:2 1,6-Hexanediol (17%) +++ 14 Choline chloride Trifluoroacetamide 1:2 Ethanol (17%) ++++ 15 Choline chloride Trifluoroacetamide 1:2 Methanol (17%) +++ 16 Choline chloride Trifluoroacetamide 1:2 Dimethylformamide (17%) + 17 Choline chloride Trifluoroacetamide 1:2 Dimethylsulphoxide (17%) +++ 18 Choline chloride Trifluoroacetamide 1:2 N-Methyl pyrrolidone (17%) +++++ 19 Choline chloride Trifluoroacetamide 1:2 N-Ethyl pyrrolidone (5%) +++++ 20 Choline chloride Trifluoroacetamide 1:2 Ethyleneurea (5%) ++ 21 Choline chloride Trifluoroacetamide 1:2 Pivalamide (5%) +++ 22 Choline chloride Trifluoroacetamide 1:2 1,3-Dimethylurea (5%) ++ 23 Choline chloride Trifluoroacetamide 1:2 N,N′-Dimethylourea (5%) ++ 24 Choline chloride Trifluoroacetamide 1:2 Isopropanol (17%) ++++ 25 Choline chloride Trifluoroacetamide 1:2 Butanol (17%) ++++ 26 Choline chloride Trifluoroacetamide 1:2 Glycerol (17%) ++ 27 Choline chloride Trifluoroacetamide 1:2 1-Methylimidazole (33%) +++++ 28 Choline chloride Trifluoroacetamide 1:2 1-Methylimidazole (5%) ++ 29 Choline chloride Trifluoroacetamide 1:2 1-Ethylimidazole (5%) ++ 30 Choline chloride Trifluoroacetamide 1:2 1-Benzylimidazole (2.5%) +++++ 31 Choline chloride Trifluoroacetamide 1:2 1-Benzylimidazole (5%) +++++ 32 Choline chloride Trifluoroacetamide 1:2 Tetramethylurea (1%) +++++ 33 Choline chloride Trifluoroacetamide 1:2 Tetramethylurea (5%) +++++ 34 Choline chloride Trifluoroacetamide 1:2 Ethylene carbonate (33%) ++ 35 Choline chloride Trifluoroacetamide 1:2 Imidazole 33%) ++++ 36 Choline chloride Trifluoroacetamide 1:2 Lithium acetate (33%) ++ 37 Choline chloride Trifluoroacetamide 1:2 4-Formyl morpholine (33%) +++++ 38 Choline chloride Trifluoroacetamide 1:2 Acetonyl acetone (20%) ++ 39 Choline chloride Trifluoroacetamide 1:2 Guanidine HCl (3.4%) ++ 40 Choline chloride Acrylamide 1:2 — ++ 41 Choline chloride 2-Chloroacetamide 1:2 — ++ 42 Choline chloride Bistrifluoroacetamide 1:2 — ++++ 43 Choline chloride 2,2- 1:2 — +++ Difluoropropanamide 44 Choline chloride 2,2,2- 1:2 — ++ Trifluorothioacetamide 45 Choline chloride Formamide 1:2 — ++ 46 Choline chloride Methanol 1:2 — ++ 47 Choline chloride Ethanol 1:2 — ++ 48 Choline chloride Trifluoroacetamide 1:2 Sorbitol (5%) ++ 49 Choline chloride Trifluoroacetamide 1:2 Xylitol (5%) ++ 50 Choline chloride Urea 1:2 — ++ 51 Choline chloride Urea 1:2 Na cacodylate (10%) +++ 52 Choline chloride Urea 1:2 SDS (5%) ++ 53 Choline chloride Urea 1:2 Na p-Toluene sulphonic acid (6%) + 54 Choline chloride Urea 1:2 Triton TX-45 (12%) + 55 Choline chloride Urea 1:2 Na benzoate (8%) +++ 56 Choline chloride Urea 1:2 Guanidine isothiocyanate (7%) + 57 Choline chloride Urea 1:2 Sulpho salicylic acid (10%) + 58 Choline chloride Urea 1:2 CTAB (8%) ++ 59 Choline chloride Urea 1:2 Zinc chloride (11%) +++ 60 Choline chloride Urea 1:2 Methyl p-toluenesulphonate (25%) ++ 61 ZnCl2 Ethylene glycol 1:4 — + 62 ZnCl2 Ethylene 1:3:1 — + glycol:Trifluoroacetamide 63 ZnCl2 Urea 1:3.5 — + 64 ZnCl2 Trifluoroacetamide 1:3.5 — ++ 65 Choline chloride Sorbitol 1:1 — ++++ 66 Choline chloride Guanidine 2:1 — + isothiocyanate 67 Choline chloride Phenylacetic acid 1:2 — + 68 Choline chloride Malonic acid 1:2 — + 69 Choline chloride Boric acid 1:1.5 — +++ 70 Acetylcholine Urea 1:2 — + chloride 71 Acetylcholine Trifluoroacetamide 1:2 — ++ chloride 72 Choline bromide Urea 1:2 — + 73 Choline bromide Trifluoroacetamide 1:2 — ++++ 74 Beta- Trifluoroacetamide 1:2 — + methylcholine chloride 75 Carnitine Trifluoroacetamide 1:2 — ++ 76 Taurine Trifluoroacetamide 1:2 — + 77 Methyltriphenylph Trifluoroacetamide 1:3 — + osphonium bromide 78 Grignard Reagent Trifluoroacetamide 1:2 — +++ T 79 Chloroethyltrimethyl Trifluoroacetamide 1:2 — +++ ammonium chloride 80 Cetyltrimethyl- Trifluoroacetamide 1:2 — ++ ammonium chloride 81 Tetramethyl Trifluoroacetamide 1:2 — + ammonium oxide 82 Choline chloride Trichloroacetamide 1:2 — ++ 83 Benzyltrimethyl- Trifluoroacetamide 1:2 — +++ ammonium chloride 84 Betaine Trifluoroacetamide 1:2 — +++ Effect on HeLa cell morphology, scale +(worst) to +++++ (best).

[0409] An ‘additive’ is defined here as any substance that can be dissolved in a particular DES mixture, and can be present in amounts greater than, equal to or less than the total amount of the DES mixture (weight:weight). By way of example only, Choline chloride:Urea:Zinc chloride (1:2:0.5 mol:mol:mol), the ZnCl2 is an additive, or Choline chloride:Trifluoracetamide:Urea (1:2:0.1 mol:mol:mol), the Urea is an additive. It should also be noted that the additive does not necessarily have any particular effect on the freezing point of the DES mixture or form hydrogen bonds with any of the DES components but endows the DES mixture with a unique property.

[0410] A non-exhaustive list of possible additives to a DES mixture are: Ammonium p-toluenesulphonic acid, Sodium p-toluenesulphonic acid, Ammonium sulphate, Ammonium chloride, Ammonium thiosulphate, Sodium dodecyl sulphate, Sodium lauryl sulphate, Sodium Benzoate, Dodecyldimethyl(3-sulphopropyl)ammonium hydroxide, Dimethylbenzene sulphonic acid, Congo Red, Giemsa, DAPI, Ethidium bromide, Mallory's stain, Orcein, Aldehyde fuchin, Osmium tetroxide, Chromium trioxide, Chromic acid, Feulgen, Dichromate, Mercuric chloride, Haematoxylin and Eosin stain (H&E), Formaldehyde, Glutaraldehyde, Acetone, Ethanol, Methanol, Methyltriphenylphosphonium bromide, Cetyltetrabutylammonium bromide (CTAB), tetrabutylammonium bromide (TBAB), sodium deoxycholate, Brij-35, Brij-58, NP-40, Triton X-100, Triton X-114, Tween-20, Tween-80, Octyl beta glucoside, CHAPS, Solanine, kanomycin, streptomycin or penicillin, sodium nitrate, sodium nitrite or sodium benzoate, ss or ds RNA or DNA sequences, ss or ds RNA or DNA labelled sequences, an aptamer, a peptide, enzyme, antibody, biotin, biotin labelled molecule, horseradish peroxidase, avidin, streptavidin, GFP or variant thereof, Luciferase, Fluorescein, Rhodamine, Texas Red, Alexa Fluor™, LNA, labelled LNA, a Molecular Beacon™, a Scorpion probe™, FISH probe, bDNA, PCR primer, oligo (dT), PNA, anti-oxidant, RNasin, SUPERase.IN™, RNaseOUT™, phenylmethylsulfonyl fluoride (PMSF), diisopropyl fluorophosphate (DFP), aprotinin or Pefabloc SC™, Tris-HCl, PIPES, MES, HEPES, MOPS, MOPSO, CAPS, CAPSO, BIPES, phosphate, imidazole BAPTA, EDTA, EGTA, citric acid, D-Penicillamine, O2, CO2, N2, Argon, propanol, tert-butanol, Trichloroacetic acid, sulphosalicyclic acid, Water, Methanol, Ethanol, Propanol, Butanol, Tetramethyl urea, Imidazole, 1-Methylimidazole, 1-Ethylimidazole, 1-Benzylimidazole, 4-Methylimidazole, N-Methylpyrrolidone, N-Ethylpyrrolidone, N-Benzylpyrrolidone, Guanidine, Guanidine HCl, Guanidine isothiocyanate, Ribitol, Rhamnose, Trehalose, D-Sorbitol, L-Sorbitol, Sorbose, Xylitol, Glucose, Sucrose, Lactose, Fructose, Maltose, Mannose, Mannitol, Arabinose, Galactose, Raffinose, Inositol, Erythritol, Xylose, Zinc acetate, Zinc EDTA, Zinc phosphate, Zinc Trifluoroacetate, Zinc citrate, Zinc PTSA, Zinc gluconate, Zinc chloride and/or Zinc sulphate. Non-dissolvable additives to DES mixtures include but not limited to: Paraffin, Silica gel, Sodium sulphate or Molecular Sieves™, Polyacrylamide, Aerogel, Polyacrylic acid and/or a Quantum Dot.

[0411] It will be apparent that there are many types of DES mixture that can be made and the choice of a particular DES for the purpose of preserving RNA in a sample will be determined by one or more of the following preferable features of the DES: (1) compatibility with RNA, preferably it should have a pH between pH 4.5 and 8.5, more preferably between pH 5 and 8, more preferably between pH 5.5 and 7.5 and most preferably between pH 6 and 7 when mixed with the sample, (2) compatibility with the RNA purification reagents and protocol, for example it should not interfere with the binding between the RNA and silica spin column such as RNeasy (RNeasy Mini Kit, Cat. No. 74106, Qiagen, Germany) or alter the partitioning of RNA in phenol containing reagents such as TRIzol (Cat. No. 15596018 Life Technologies, USA), (3) stabilising RNA in the tissue sample sufficiently quickly as to substantially alter the activity of ribonucleases and/or accessibility of RNA to hydrolysis. Alternatively RNA is stabilised in the tissue sufficiently quickly that the RNA Integrity Number (RIN) is not reduced by more than 2 RIN units, more preferably not more than 1 RIN units, even more preferably not more than 0.5 RIN units and most preferably less than 0.1 RIN units during the initial 18-24 hours storage with the DES mixture at room temperature compared with RNA extracted from a fresh tissue sample, without storage, of the same type, (4) have a capacity to stabilise RNA when the tissue represents at least 10%, more preferably 20% and even more preferably at least 50% of the weight of the DES mixture (weight:weight), (5) not reduce RNA yield by more than 20% compared with fresh tissue, (6) provide stabilisation of DNA, proteins and the phosphate groups of phospho-proteins, (7) be chemically stable, non-flammable, non-toxic to the user and the environment, biodegradable, not react with bleach or reagents used during RNA purification to form toxic compounds, (8) have a shelf-life of at least 6 months at room temperature, (9) fix and stabilise native cell morphology and histology including antibody epitopes and sub-cellular organisation and organelles, whilst stabilising RNA and other biomolecules, (10) fix and stabilise cells in such a way as to subsequently allow histological and immunohistochemistry applications for example with antibodies and stains such as Hoechst or H&E stain, (11) stabilise RNA in FFPE samples to protect and enhance the reversal of formalin cross linking at temperatures greater than 50° C. and to allow the melting and therefore the easy removal of paraffin from the fixed sample, (12) fix and stabilise circulating tumour cells in whole blood to allow their purification, detection and molecular analysis.

[0412] It will be evident to one skilled in the art that various DES can be tested for their suitability by adding them to the sample at a ratio of at least 10:1 (wt:wt). Comparisons of the relative RNA stabilisation can be made using a control solution of RNAlater (10:1 (vol:wt) used according to the manufacturer's instructions (Cat. No. 76106, Qiagen, Germany). Following incubation at, for example 37° C. for one day or more, the RNA is extracted according to a standard protocol such as RNeasy (RNeasy Mini Kit, Cat. No. 74106, Qiagen, Germany) and its intactness analysed by gel electrophoresis or by using a a RNA 6000 Nano total RNA Kit (Cat. No. 50674511, Agilent Technologies, USA) and Bioanalyser 2100 or other suitable method such as RT-qPCR as described. RNA yields can be determined by OD260 nm uv spectrometry and purity by the well-known OD260/230 and OD260/280 ratios. A Nanodrop ND2000 (ThermoScientific Inc., USA) is one example of a suitable spectrometer for such determination. Once suitable DES mixtures have been identified their optimum amount, purity, amount of contaminating water and use can be determined by further such tests.

[0413] Generally the most important single factor for optimisation is the relative molar ratios of the individual components in the DES mixture. In the first instance mixing only two components of the DES mixture is the most straightforward means to identify approximately, and by empirical means as described above, components that result in the desired effect, such as RNA stabilisation, yield, purity and suitability for downstream applications such as RT-PCR or hybridisation. However it will also be apparent to one skilled in the art that a blend of more than two components may be desirable to further enhance or add novel properties to the DES mixture. There are obviously a very large number of potential mixtures of components that can lead to a DES mixture, and although it is easiest to start with the preparation of, in the first instance simple stoichiometric molar ratios of the DES components such as, by way of example only, a 2:1, 1:1 or 1:2 molar ratio (mol:mol) of choline chloride and urea. It is thought that the depression in freezing point of two component DES mixtures is dictated, at least in part, by the hydrogen bonding between the available hydrogen bond donor (e.g. choline chloride) and acceptor (e.g. urea). However it should be noted that the preferred components of the DES mixture and their optimal ratio of mixing for a particular application such as RNA preservation or cell fixation is not necessarily related to the extent of the observed freezing point depression. Identifying the optimum DES mixture for an application can therefore be determined empirically and consequently trial and error testing may be involved. It will be apparent that identifying the best blend for a particular purpose of a greater than two component DES mixture may involve significant effort. Therefore, in the first instance and in the interest of rapidly defining suitable molar ratios of components, stoichiometric ratios may be used as described above. Once approximate molar ratios have been identified, further refinement involving fixing the molar amount of one component whilst varying the other two or more can be made. It should be noted that the molarity of the various DES components is variable and depends on the density, molar ratios and molecular weight of the individual components. By way of example, the Choline chloride concentration in a Choline chloride:Urea (1:2) mixture is approximately 5M and the Urea 10M, compared with a Choline chloride concentration of 3.6M and a Trifluoroacetamide of 7.2M in a Choline chloride:Trifluoroacetamide (1:2) mixture.

[0414] In one aspect, there is provided a deep eutectic solvent, DES, comprising an additive, wherein the additive is Formic acid. The inclusion of Formic acid may reduce the viscosity of the DES in comparison with a DES not which does not include the Formic acid additive. Reduced viscosity may make the deep eutectic solvent easier to handle. The inclusion of Formic acid as an additive in the DES also helps to maintain cell and tissue volume during cell or tissue fixation. It has also surprisingly been found that, when a biological sample is transferred from such a DES to an aqueous medium, RNA integrity is maintained. Without wishing to be bound by theory, it is believed that Formic acid efficiently and permanently inactivates RNases, thereby protecting the analyte RNA from degradation by RNases, even when the fixed cell is subsequently transferred to an aqueous buffer as is common for many RNA analytical protocols.

[0415] The DES may be any of deep eutectic solvents described hereinabove. Preferably, the DES is trimethylglycine:trifluoroacetamide. For example, the DES may be trimethylglycine:trifluoroacetamide at a molar ratio of 1:2 mol:mol.

[0416] The concentration of Formic acid in the DES is not particularly limited. Typically, the concentration of Formic acid is in the range 50 mM to 12 M.

[0417] For some applications, Formic acid concentrations in the range 4 to 9 M, optionally 6 to 8 M, further optionally 7 to 8 M may be preferred. This is because Formic acid concentrations of about 8 M have been found to be optimal for maintaining RNA stability.

[0418] For other applications, Formic acid concentrations in the range 50 to 500 mM, optionally 150 to 400 mM, and further optionally 250 to 350 mM may be preferred. The use of these lower concentrations of Formic acid may be preferable when the biological sample is in contact for more than 6 hours at room-temperature such as during transport and handling and/or when the biomolecule to be analysed is a protein or DNA molecule, both of which are generally more sensitive to acid hydrolysis than RNA. Additionally, these lower Formic acid concentrations can lead to less sample hardening and therefore improve cell lysis and biomolecule purification yield.

[0419] In one aspect, there is provided a deep eutectic solvent, DES, comprising an additive, wherein the additive is Acetic acid. The Acetic acid may be present at a concentration of at least 8 M. It is been found that an Acetic acid concentration of at least 8 M produces similar benefits to those of Formic acid, as detailed above. Acetic acid is preferred for some applications, because it is safer to handle than Formic acid.

[0420] The DES may be any of deep eutectic solvents described hereinabove. Preferably, the DES is trimethylglycine:trifluoroacetamide. For example, the DES may be trimethylglycine:trifluoroacetamide at a molar ratio of 1:2 mol:mol.

[0421] The concentration of the Acetic acid is at least 8 M, optionally at least 9 M, further optionally at least 11 M. It is believed that there is no upper limit on the useful concentration of Acetic acid, although the concentration of Acetic acid will be less than 17.4 M (i.e., the concentration of pure glacial Acetic acid). For example, the concentration of Acetic acid may be in the range 8 to 17.39 M, 9 to 17.39 M, or 11 to 17.39 M; or 8 to 17 M, 9 to 17 M, or 11 to 17 M.

[0422] Any of the various methods and uses described herein may be practiced using a DES comprising a Formic acid additive or an Acetic acid additive.

[0423] For example, there is provided a method of fixing a biological sample, which method comprises contacting the biological sample with a DES to form a fixed biological sample, wherein the method further comprises contacting the biological sample with an additive, and wherein the additive is Formic acid or Acetic acid.

[0424] The DES may be any of the DESs described herein, for example, trimethylglycine:trifluoroacetamide. The biological sample may be contacted with both the DES and the additive in a single step, in other words contacted with a mixture comprising the DES and the additive.

[0425] More preferably, the method comprises a fixation step comprising fixing the biological sample using the DES and then a post-fixation step of contacting the additive with the biological sample. In arrangements where the additive is Acetic acid, the post-fixation step may comprise contacting the biological sample with glacial Acetic acid, or a solution further comprising a solvent selected from a DES, water, an alcohol, and mixtures thereof. The alcohol may be a C1 to C6 alkanol, such as methanol, ethanol, or isopropanol. The DES may be the same as or different to the DES used in the fixing step. Where a solution of Acetic acid is used, the concentration of Acetic acid in the solution is at least 8 M, preferably at least 9 M, more preferably at least 11 M. The concentration of Acetic acid in the solution will be less than the concentration of glacial Acetic acid (approximately 17.4 M) due to the presence of the solvent, but is otherwise not particularly limited.

[0426] Optionally, the biological sample is contacted with the DES for a period of 6 hours or less for example 1-15, 15-60 minutes, 1-2 or 2-6 hours, typically the sample is contacted for 15-20 minutes at room temperature. It has been found that limiting the contact time to 6 hours or less helps to maintain nucleic acid integrity.

[0427] The biological sample may be any of the various biological samples disclosed hereinabove, e.g. a tissue sample.

[0428] The fixed biological sample may be subjected to further process steps. For example, the fixed biological sample may be dissociated into single cells using any of the techniques taught herein. The further process steps may include contacting the fixed biological sample with an aqueous medium.

[0429] A fixed biological sample may be dissociated by sonication, for example, sonication using a bath sonicator, as discussed above. The liquid present in the bath may be water, an aqueous salt solution (e.g. a sodium chloride solution, such as a 5-15 wt %, optionally 10 wt %, sodium chloride solution based on the total weight of the solution), an oil such as a vegetable seed oil or an echography gel. Preferably, the liquid is a hydrocolloid, such as a 0.1-10% (weight:volume) solution of guar gum, xanthan gum, starch, locust bean gum, gum karaya, gum tragacanth, gum Arabic, cellulose derivatives, alginate, pectin, carrageenan, gelatin, gellan and agar. Using a hydrocolloid in water has the advantage of significantly increasing the transfer of sonication energy from the sonicator to the vial or container containing the Dissociation buffer and the sample to be dissociated. This has the advantage of increasing the rate of the desired tissue dissociation and the production of single cells. An advantage of a water bath sonicator is a wider variety of hard-walled tubes, vessel or containers can be used such as glass which might otherwise shatter and break in a metal block type sonicator as well as simpler temperature control.

[0430] Another aspect provides the use of a DES to inhibit degradation of a biomolecule, wherein the DES comprises an additive, wherein the additive is Formic acid.

[0431] A still further aspect provides the use of a DES as a Dissociation buffer Dissociation buffer as defined herein, wherein the DES comprises an additive, wherein the additive is Formic acid or Acetic acid.

[0432] Although the above aspects have been described with reference to Formic acid, in variants alternative acids may be used instead of, or in addition to, Formic acid. For example, an acid selected from acetic acid, citric acid, or lactic acid may be used as the additive. Further alternative acids which may be suitable for use as the additive include carboxylic acids selected from pyruvic acid, tartaric acid, propionic acid, oxalic acid, maleic acid, salicylic acid, ascorbic acid, benzoic acid or butanoic acid; and mineral acids selected from hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, and boric acid.

EXAMPLES

Example 1

Dissociation of Mammalian Samples

[0433] To 1 ml of Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) was added 5-50 mg (at least one dimension of the sample has a thickness of no more than 4mm) of a freshly dissected mouse, rat or human clinical sample such as liver and incubated for at least 2 hours at room temperature with occasional mixing at approximately 24° C. Following this fixation and stabilization step, the sample can either be used immediately or stored at −80, −20, 4 or 24° C. for at least one week prior to recovery of the tissue sample and use as follows.

[0434] Excess liquid around the fixed tissue sample is briefly removed using an adsorbent material and then immediately placed in a 300 ul thickwall polycarbonate centrifuge tube (Beckman Cat. No. 343775 (7×20 mm) containing 100-300 ul of 95% Trimethylglycine:Trifluoroacetamide (1:2 mol:mol), 15% water by volume, and the tube sealed using a self-adhesive silicone closure. The tube is then placed in a VialPress fitted to a Vial-Tweeter-Sonotrode (S26d11×10), using an Ultrasonic generator (UP200St-G) 200W, 26 kHz, Ultrasonic Transducer (UP200St-T), run at Constant Power, 70% Pulse, 70% Amplitude (100% amplitude=10 um) for 3 minutes at 4° C. (Hielscher GmbH Germany).

[0435] Alternatively, the 95% Trimethylglycine:Trifluoroacetamide can be replaced with 65, 70, 75, 80, 85, 90, or 100% Trimethylglycine:Trifluoroacetamide. Preferably 85-100% is used with 95% most preferred. The pulse can be varied between 10-100% although it is found that the optimum is be in the range 50-90%, whilst the amplitude can be varied in the range of 20-100% with an optimum in the range 60-100%.

[0436] The temperature of the sample should be maintained in the range of −20 to 37° C. more preferably −5 to +10° C. and most preferably at 4° C. to maintain Dissociation buffer viscosity and biomolecule stabilization.

[0437] The time typically necessary for sufficient sample dissociation is dependent on a number of factors including the amount and type of sample, the tube size, the pause time, the amplitude, the power, temperature, the Dissociation buffer used, the additive(s) used, the percentage water in the Dissociation buffer, fixation time and the fixative type.

[0438] The extent of tissue dissociation can be followed by a variety of means including visual inspection of the opacity of the liquid in the tube and decrease in size of the tissue under white light either by eye or in more detail using a binocular microscope at 20× magnification, by an increase in the optical density (OD600 nm), using a surrogate indicator such as a sonication sensitive dye such as Crystal violet or solid matrix such as carbon blue copy paper.

[0439] A general method for using a waterbath type sonication device for dissociation is as follows; the fixed biological sample in the range of for example, 1-150 mg but preferably less than 50 mg, is placed in a Container such as a thickwall polycarbonate centrifuge tube such as a 8×51 mm (Beckman Coulter™ Cat. No. 355657) containing 25-500 ul of 75-95% Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) containing 5-25% water by volume and the tube sealed using a self-adhesive silicone closure. The tube is then placed in a floating tube holder and floated on the surface of a waterbath type sonicator such as Elmasonic P30H (37/80 kHz) used at 37 kHz, or preferably an Elmasonic TI-H F MF2 (25/45 kHz) used at 25 kHz, containing water equilibrated at 4-24° C. Waterbath type sonicators should be used with a frequency ≤40 Kz and most preferably ≤30 kHz. Initially the sonicator should be used at 50% of the total power setting for 1 minute and the extent of tissue dissocation can be be approximately ascertained by observing the opacity of the Dissociation buffer. An increase in opacity is associated with an increase in the number of the desired cells released from the tissue sample. The exact number of cells can be determined using a 10 microlitre portion of the Dissociation buffer and a cell counter such as Countess™ II Automated Cell Counter (ThermoFisher Inc. Cat. No. AMQAX1000). If sufficient cells have not been dissociated from the sample, the dissociation can be continued for 2, 3, 4, 5 minutes or by increasing the power setting from 50% to 60, 70, 80, 90 100, or even 120% (if the device is equipped with a pulse function), until sufficient cells have been produced. Care should be taken to maintain the temperature at or below 24° C., preferably below 10° C. (ice can be used to maintain the temperature during use). Other waterbath sonicator functions such as ‘degas’ and ‘sweep’ can also be tested to determine if tissue dissociation is more efficient.

[0440] Single cells collected in this manner are suitable for; scRNAseq, scDNAseq, flow cytometry including FACS and cell staining for example immunofluorescence and DAPI nuclear staining applications.

Example 2

Dissociation of Mouse or Rat Tissues

[0441] To 1 ml of Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) was added 10-100 mg (at least one dimension of the sample has a thickness of no more than 4 mm) of a freshly dissected mouse or rat tissue such as, brain, salivary glands, tongue, retina, olfactory organ, skeletal muscle, lungs, blood vessels including the aorta, heart, oesophagus, testis, ovaries, spleen, thymus, kidneys, testis, ovaries, lymph glands, skin, stomach, intestine, gall bladder, pancreas, adrenal glands, bone or bone marrow and incubated for at least 2 hours at room temperature (approximately 24° C.) with mixing.

[0442] Tissue dissociation was carried out by sonication according to Example 1 except that the total time was dependent on the tissue type. As an approximate guide the relative sonication energy necessary to dissociate different tissue types is set out in Table 1.

[0443] One of the important variables in the production of single cells from a sample is the type of tissue used, for example tissues which are rich in fibrous connective tissue and structural proteins such as muscle can require additional sonication energy input to completely dissociate the sample. It has been found that striated muscle such as leg muscle dissociates into myofibrils with individual cells difficult to identify in part because muscle cells are multinucleate.

[0444] The following table is an approximation of the relative energy typically needed for sample dissociation. 1 (easiest) to 10 (most difficult).

TABLE-US-00002 TABLE 1 Relative Energy Typically Tissue Type Required brain 5 salivary glands 4 tongue 9 retina 3 olfactory organ 6 skeletal muscle 8 lungs 10 blood vessels 7 heart 10 oesophagus 10 ovaries 8 spleen 6 thymus 5 kidneys 6 testis 2 lymph glands 3 skin 10 stomach 8 intestine 7 gall bladder 5 pancreas 5 adrenal glands 5 bone marrow 1 bone 10 teeth 10

Example 3

Dissociation of Human Tissues

[0445] To 1 ml of Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) was added 10-100 mg (at least one dimension of the sample has a thickness of no more than 4 mm) of a freshly dissected human tissue such as, brain, salivary glands, tongue, retina, olfactory organ, skeletal muscle, lungs, blood vessels including the aorta, heart, oesophagus, testis, ovaries, spleen, thymus, kidneys, testis, ovaries, lymph glands, skin, stomach, intestine, gall bladder, pancreas, adrenal glands, bone, bone marrow or a biopsy such as a cancer biopsy, and incubated for at least 2 hours at room temperature (approximately 24° C.) with mixing. Dissociation was carried out as set out in Example 1.

Example 4

Dissociation of a Cancer Biopsy

[0446] To 1 ml of Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) was added 10-100 mg (no dimension of the sample has a thickness of more than 4 mm) of a human cancer biopsy and incubated for at least 2 hours at room temperature with mixing.

[0447] The cancer biopsy can be as way of example a: (i) Carcinoma including (a) adenocarcinomas and (b) squamous cell carcinoma, (ii) Sarcoma including (a) Osteosarcoma or osteogenic sarcoma, (b) Chondrosarcoma, (b) Leiomyosarcoma (c) Rhabdomyosarcoma (d) Mesothelial sarcoma or mesothelioma, (e) Fibrosarcoma, (f) Angiosarcoma or hemangioendothelioma, (g) Liposarcoma, (h) Glioma or astrocytoma, (i) Myxosarcoma and Mesenchymous or mixed mesodermal tumor; (iii) Leukemias, (iv) Lymphomas including (a) Hodgkin Lymphoma and, (b) Non-Hodgkin Lymphoma.

[0448] Dissociation was carried out by sonication as set out in Example 1. Following dissociation the single cells were examined by microscopy.

Example 5

Sample collection of Analytes from Human Skin

[0449] 100 μl of Trimethylglycine:Glycerol (1:2 mol:mol) containing 10% water was spotted onto the lower forearm of a patient and gently rubbed into the skin for 1 minute and then a piezoelectric disc such as a 15 mm 30 KHz transducer (Cat. No. SMUN15K12F30 Steminc, USA) or 35 mm Piezo ceramic disc 56 kHz (Cat. No. SMD35T4S311, Steminc, USA) was placed onto the prepared skin zone, maintained in place with an adhesive strip, the disc attached to a suitable electric generator and sonication carried out for 5 minutes with constant gentle pressure applied to the disc, after which the disc was removed and an adsorbent disc was immediately placed on the sonicated skin zone and held, with firm pressure for 1 minute to recuperate the liquid containing the DES composition and any skin cells, bacteria, viruses, particles, blood, plasma, nucleic acids, fats, proteins, phosphoproteins, and/or metabolites that have been extracted from the skin surface. After removal from the skin, to the adsorbent disc bearing the sample was added 150 ul of Trimethylglycine:Glycerol (1:2 mol:mol), saline solution or water and then the disc rolled and placed vertically in a 1.5 ml polypropylene centrifuge tube and centrifuged for 60 seconds at 12,000 g and the recovered liquid used as a source of analyte material for a test such as a glucose test, a therapeutic or doping drug test, a viral or bacterial diagnostic test, a genetic test, an allergy test, a test for the presence of a specific patient antibody, a blood chemistry test such as for cholesterol or haemoglobin, a cancer test for example for CEA or PSA antigens, or as a source of DNA such as genomic DNA, circulating free DNA or circulating foetal DNA for a genetic test. The type of analytical test is not particularly limited to a chemistry, haematology, microbiology or a molecular based analytical test.

[0450] Alternatively the analytical test is carried out within the adsorbent disc that has been in contact with the patients skin, for example specific antibodies are added to adsorbent disc for a lateral flow test or other assay to detect a specific antigen.

Example 6

Dissociation of Varying Tissue Sizes

[0451] The energy transferred from the sonication device to the container or vessel containing the sample is proportional to the mass of the sample. It has been found that larger tissue pieces may require a larger energy input to achieve sufficient dissociation. However, when tissue pieces smaller than 10 mg are placed in the cavitation zone of the Dissociation buffer they tend to be pushed by the cavitation bubbles to parts of the Dissociation buffer where there is less cavitation thereby reducing the rate of sample dissociation. To avoid this issue with smaller tissue pieces, a smaller diameter tube is preferred thereby physically constraining the sample to the cavitation zone. Alternatively, the sample can be attached or restricted to the focal area of the cavitation zone using a tethering structure within the tube. This can be a vertical needle attached to the tube lid and descending into the dissociation liquid with the tissue sample firmly skewered on it thereby limiting its movement. Another alternative is to place the tissue sample in a plastic cage or mesh within the container so that the Dissociation buffer and cavitation energy can freely contact the sample but the sample is held firmly within a defined cavitation zone of the tube.

[0452] Large pieces of tissue of up to 10 g can be dissociated by sonication but this has been found to be most easily accomplished in a continual manner from one edge of the tissue that is presented towards the cavitation zone, and as the tissue is dissociated and therefore reduced in size, the tissue is progressively ‘fed’ into the cavitation zone in a successive manner until the entire sample is dissociated. It will be evident to one skilled in the art that more sonication power is typically important for larger samples and this may also require longer Dissociation buffer cooling periods during the process.

[0453] It has been found that tissue samples of very variable starting weight can be dissociated, as way of example only; 1 to 100 mg, 100 to 1000 mg or 1 g to 10 g.

Example 7

Dissociation of Tissue in Different Dissociation Buffers

[0454] Many different types of Dissociation buffers can be used with the sonication device, preferably these buffers have the following characteristics (i) water content less than 50%, more preferably less than 40% and even more preferably less than 30%, and most preferably less than 15%, (ii) a viscosity of at least 5 centipoise (cP), more preferably 10 cP, even more preferably 25 cP, and most preferably between 50-125 cP, and (iv) protection and stabilization of biomolecules such as RNA, DNA, proteins and phsophoproteins, (v) softening of the sample to improve dissociation, (vi) good transmission of sonication power to the sample, (vii) high cavitation bubble formation to improve the rate of dissociation, (viii) protection of single cells from breaking down into sub-cellular components and debris, and, (ix) high coefficient of heat energy transfer from the container to the surrounding environment. Dissociation buffers composed of DESs have been found to be particularly effective for this purpose.

[0455] Specific examples include 65-100% Trimethylglycine:Trifluoroacetamide (1:1.5 mol:mol, 1:2 mol:mol or 1:3 mol:mol), 65-100% Trimethylglycine:Glycerol (1:2 mol:mol), 65-100% Choline chloride:Urea (1:2 mol:mol), 65-100% Trimethylglycine:Urea (1:2 mol:mol). As set out herein, the use of additives to the DES can provide improved dissociation properties such as by the addition of a detergent.

[0456] Although protecting and stabilizing biomolecules such as RNA less well than DESs, glycols may serve as useful Dissociation buffers, for example Ethylene glycol, Glycerol, Diethylene glycol, Triethylene glycol, PEG 200, PEG 300, PEG 400, PEG 600 and PEGs with a MW in excess of 800 with 0-60% water to provide a room temperature liquid with a viscosity of at least 5 cP.

Example 8

Softening Tissue Using Glycols Prior to Dissociation

[0457] It has been found that fixed tissues that are difficult to dissociate can be rendered softened and therefore facilitate the dissociation by sonication into single cells by soaking the sample in at least 20 volumes of a 20-100% solution of a glycol for 1 or more hours at room-temperature. The following room temperature liquid glycols with a molecular weight of less than 1000 Daltons have been found to be effective for tissue softening; ethylene glycol, glycerol, diethylene glycol, triethylene glycol, PEG 200, PEG 300, PEG 400 or PEG 600.

[0458] A 20 mg sample of mouse liver was treated for 1 week with 100% Trimethylglycine:Trifluoroacetamide (1:2 mol:mol), in order to fix and stabilize the RNA, DNA and protein content, the fixed sample was then removed, excess fixative removed and then placed in 500 μl of glycerol (TCI France) and softened overnight at room-temperature. The sample was then removed and added to 300 μl of 95% Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) in a 1 ml (8×51 mm) thickwall Polycarbonate centrifuge tube (Beckman Cat. No. 355657), the tube was then placed in the VialPress attached to a VialTweeter as described in Example 1 and run at 70% Pulse with 70% amplitude for 3 minutes at 4° C.

[0459] An alternative method is to carry out the dissociation step in the presence of a softener. A 20 mg sample of mouse liver was treated for 1 week with 100% Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) in order to fix and stabilize the RNA, DNA and protein content, the fixed sample was then removed, excess fixative removed and then placed in 300 μl of 60% Trimethylglycine:Trifluoroacetamide (1:2 mol:mol), 25% PEG200 (TCI France) and 15% water in a 1 ml (8×51 mm) thickwall Polycarbonate centrifuge tube (Cat. No. 355657 Beckman, USA), the tube was then placed in the VialPress attached to a VialTweeter as described in Example 1 and run at 70% Pulse with 70% amplitude for 3 minutes at 4° C.

Example 9

Different Types of Tubes/Dissociation Containers

[0460] A 20 mg sample of mouse liver was treated for 6 hours with 100% Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) in order to fix and stabilize the RNA, DNA and protein content. The mouse liver sample was then removed and added to 300 μl of 95% Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) in a 1 ml (8×51 mm) thickwall Polycarbonate centrifuge tube (Beckman Cat. No. 355657), the tube was then placed in the VialPress attached to a VialTweeter as described in Example 1 and run at 70% Pulse with 70% amplitude for 3 minutes at 4° C.

[0461] A comparison was made by replacing the Polycarbonate tube with a 1.5 ml ‘Eppendorf’ Polypropylene tube and running the sonication under identical conditions. It was found that the extent of tissue dissociation and therefore the number of cells released, (which is a measure of the efficiency of energy transfer) was approximately 30% less with a Polypropylene tube compared with a Polycarbonate tube. It was also found that on extended sonication the Polypropylene tube tended to become softer and even deform and melt. It was further found that thick walled Polycarbonate tubes were particularly well suited to transferring energy to the sample.

[0462] It was found that when the tube is in direct contact with the metal block of the VialTweeter, and sonication energy applied, that glass, even thick walled glass designed for laboratory use which has excellent sonication energy transmitting properties nevertheless shattered and failed regularly, similarly polystyrene 8 ml tubes (Cat. No. 352027, Corning Inc, USA) tended to form stress and fracture lines in the walls which eventually led to leakage. Therefore polycarbonate was found to be an excellent compromise between the energy transmitting properties of glass and the strength and low cost of a polymer, with the additional benefit that the sample can be easily observed through the transparent walls during the dissociation procedure.

Example 10

Varying the Temperature of the Dissociation Buffer

[0463] The temperature of the dissociation process is of great importance, firstly the viscosity of the Dissociation buffer is highly dependent on the temperature. In the case of 100% Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) or aqueous dilutions of, viscosity is greatly reduced as the temperature increases. As the viscosity has a marked effect on cavitation by altering the growth and collapse of the cavitation bubble, heating the Dissociation buffer increases the bubble size formed and the dissociation effect. However, increasing the temperature also negatively effects the RNA, DNA, protein and phosphoprotein quality therefore it is preferable to keep the temperature during sonication below 24° C., more preferably below 12° C. and even more preferably at about 4° C. or less. The majority of the sonication energy is converted into heat at the site of cavitation, or as friction between the tube holding the sample and the metal VialTweeter block (Hielscher, Germany).

[0464] Maintaining steady temperature is most easily accomplished by pre-chilling equipment such as the metal components, block, water bath, sample and tubes at 4° C., and if possible running the dissociation experiment in a 4° C. ‘cold-room’ so excess container heat is rapidly dissipated. Also by running the sonicator in ‘pulse mode’ such as 20, 40, 60 or 80% (20% pulse means 20/100.sup.th of a second on, 80/100.sup.th of a second off for each cycle), rather than at 100% leads to less heat generation per minute. It will be evident to one skilled in the art that there are various well known methods to remove heat from an object such as by using heat sinks, blowing cool air, water cooling for example with a water jacket, or by Peltier cooling. Preferably the Dissociation buffer and sonication of the sample is carried out at 4° C. Control of the temperature is one means to control viscosity and therefore the rate of dissociation.

Example 11

Varying the Water Content and/or Pressure of the Dissociation Buffer

[0465] The final water content in the Dissociation buffer is one factor which may influence tissue dissociation, the number of single cells to debris, sample heating, cavitation, and the rate of decomposition of the analytes such as RNA.

[0466] Cavitation is dependent on both the amount of dissolved gases but also the viscosity of the Dissociation buffer. It has been found that it in order to maintain RNA quality, final water concentration in the DES should not exceed 35%, more preferably it should be no more than 30%, even more preferably no more than 25%, even more preferably no more than 20%, even more preferably no more than 10% and most preferably no more than 5% final volume. Viscous solvents such as pure glycerol have been found to be ineffective at promoting cavitation without added water present, preferably in the order of 15% final volume.

[0467] It has been found that when water is present at 5-10% final volume in the Dissociation buffer that there is a tendency for larger portions of the tissue sample to dissociated leading to larger clumps of cells than when a higher proportion of water, for example 25%. Increasing the water content to 50% or more leads to a massive increase in the formation of cell debris at the cost of a reduction in the number of single cells. Therefore it will be evident to one skilled in the art that it is possible to fine tune both the rate of tissue dissociation but also the number of single-cells produced during dissociation.

[0468] Pressure can be easily altered using a syringe attached to the Dissociation container, increasing the pressure to 4 atmospheres markedly reduces the amount and size of cavitation bubbles and the amount of dissociation whilst reducing the pressure to 0.8 atmospheres increases the number and size of cavitation bubbles. It will be evident to one skilled in the art that it is possible to vary the pressure in the dissociation container to regulate the degree of sample dissociation either between different samples runs or during a single dissociation experiment.

Example 12

Varying the Viscosity of the Dissociation Buffer

[0469] The viscosity of the Dissociation buffer can be altered in a number of different ways; (i) the solvent type, (ii) percentage of water as described in Example 11 above, (iii) temperature, the latter being particularly marked for solvents with strong hydrogen bonding such as glycerol, ethylene glycol, water and DESs. It has been found that the temperature of the reaction container should preferably be kept at room temperature or more preferably at 4-12° C. both to reduce RNA degradation but also to reduce viscosity. When the viscosity decreases, cavitation increases and may lead to a disequilibrium of the cell debris:single cell product ratio. The viscosity of the Dissociation buffer can either be determined empirically, or for pure solvents, by consulting published online sources such as Pubchem (NIH). It will be understood by one skilled in the art that by careful selection of solvents with a desired viscosity at a set temperature that the extent of dissociation and the type of products can be attained.

Example 13

Varying the Pulse Time and Amplitude of the Sonication Device

[0470] Both the pulse time and the amplitude are important factors for the control of the tissue dissociation. This has an important effect on dissociation as the tissue sample is more effectively dissociated when the pulse time is between 65 to 95% rather than 100%. The pulse of cavitation energy causes the tissue to dissociate faster than with a continual energy input which was unexpected. The amplitude is the distance traveled in micrometres by the sonitrode and therefore also the metal block or device holding the container with the sample in it. As one example, the amplitude of the VialTweeter is 10 μm at 100%. By reducing the amplitude there is a corresponding decrease in the amount of energy applied to the sample. It will be evident to one skilled in the art that it is possible to vary both the pulse time and the amplitude in order to obtain the amount of dissociation appropriate for the sample being dissociated. It has been found that a suitable pulse time is 70% and amplitude 70%.

Example 14

Enzymatic Pre-treatment Prior to Tissue Dissociation

[0471] Protease digestion of tissues to dissociate them into single-cells is well known. Most of the published studies have been carried out with fresh tissues with the goal of using the single cells for tissue culture. There are dozens of proteases that have been used for tissue dissociation which are either general proteases or proteases specific towards proteins such as collagen. By way of reference some of the more widely used proteases are; (i) Liberases (Roche, UK), (ii) Collagenase Type I, II, III or IV, (iii) Trypsin, (iv) Papain, (v) Proteinase K, (vi) Chymotrypsin, (vii) Elastase, (viii) Dispase and/or (ix) Pronase (Sigma-Aldrich, UK). Other types of enzymes can also be used such as Hyaluronidase, DNase and Lipases to facilitate dissociation and reduce sample viscosity.

[0472] One of the advantages of using proteases is that they can separate cells from connective tissue, however there are many disadvantages to using proteases including the removal of cell surface epitopes that would otherwise be used as markers of cell identity (Garg A, et al., Cytotherapy. (2014);16:545-559), or on prolonged incubation the cell integrity can be lost. A particularly negative consequence of using proteases is that they generally require incubation at 37° C. or more for prolonged periods (e.g. one to several hours) in non-physiological conditions that inevitably lead to profound changes to the transcriptome and other analytes which can be radically altered from their original in vivo levels during enzymatic treatment.

[0473] Additionally cells released from fresh tissues treated with proteases fail to maintain the morphology that is characteristic of their original in vivo shape so this information is lost during the procedure despite have great value for cellular identification. The advantage of tissue fixation prior to tissue dissociation to overcome these issues is therefore profound, however few fixatives are capable of maintaining RNA integrity during tissue dissociation.

[0474] To 1 ml of Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) was added 50 mg of a freshly dissected mouse liver sample and incubated for at least 2 hours at room temperature (approximately 24° C.). The sample was removed and any excess fixation solution blotted away on a paper towel, the sample was then placed in a thickwall polycarbonate tube (Cat. No. 343775, Beckman USA) containing 200 ul of a 65-100% DES, preferably at least 85% DES, Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) solution containing 0.5-5 mg Pronase enzyme powder (Cat. No. 10165921001, Roche, UK) and incubated for 20 min to 4 hours at room temperature with constant gentle mixing. The tube was then transferred to the sonication device as set out in Example 1 and dissociation carried out.

[0475] Alternatively, Proteinase K (Cat. No. 70663-4) Trypsin (Cat. No. T4799), Collagenase (Cat. No. C0130) Liberase, Collagenase (Type I, II, III or IV), Papain (Cat. No. 76218), Chymotrypsin (Cat. No. 1.02307), Elastase (Cat. No. E7885), and/or Dispase (Cat. No. D4818), powder (0.05-5 mg Sigma-Aldrich, UK) was used to digest the sample instead of Pronase.

[0476] Alternatively the enzymatic digestion solution can be, but is not limited to 65-100% Choline chloride:Urea (1:2 mol:mol), Trimethylglycine:Urea (1:2 mol:mol) and/or Trimethylglycine:Glycerol (1:2 mol:mol).

Example 15

Dissociation of Tissue Using Indirect Sonication

[0477] To 1 ml of Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) was added 50 mg of a freshly dissected mouse or rat liver sample and incubated for at least 2 hours at room temperature.

[0478] Excess liquid around the fixed tissue sample was briefly removed using an adsorbent material and then immediately placed in a standard polypropylene 1.5 ml Eppendorf centrifuge tube containing 200 μl of 85-100% Trimethylglycine:Trifluoroacetamide (1:2 mol:mol), 15% water, the tube was then transferred to a Banderex 1.5L 35 kHz sonicator water bath and run constantly for 3-30 min depending on the tissue type at 4-24° C. (temperature can be adjusted by adding ice to the bath). Indirect sonication was stopped once sufficient cell dissociation was obtained as determined visually or by cell counting.

[0479] Alternative indirect sonication water baths include the Elmasonic P series Dual frequency (37/80 kHz 2.75L Cat. No. P30H Elma Schmidbauer GmbH) or multi-frequency ultrasonic cleaning unit TI-H-5 MF-2 25/45 kHz, or MF-3 35/130 kHz .

[0480] Instead of tap water in the water bath, a solution of 10% NaCl, vegetable seed oil or echography gel (eg. Aquasonic Clear Ultrasound gel, Parker Inc Cat. No. 03-50) can be used.

[0481] Alternatively a hydrocolloid such as 0.1-10% (weight:volume) solution of guar gum, xanthan gum, starch, locust bean gum, gum karaya, gum tragacanth, gum arabic and cellulose derivatives, alginate, pectin, carrageenan, gelatin, gellan and agar can be dispersed in water and used as a replacement for tap or distilled water in the water bath sonicator, for example the Elma Ultrasonics Model P30H. Using a hydrocolloid in water has the advantage of significantly increasing the transfer of sonication energy from the sonicator to the vial or container containing the Dissociation buffer and the sample to be dissociated. This has the advantage of increasing the rate of the desired tissue dissociation and the production of single cells. An advantage of a waterbath sonicator is a wider variety of hard-walled tubes, vessel or containers can be used such as glass which might otherwise shatter and break in a metal type block sonicator as well as simpler temperature control and on cost grounds.

[0482] Preferably the tube or container has an insert or plunger which has an adjustable height within the container, allowing it to be placed in contact with the Dissociation buffer and thereby limiting the movement, gas exchange and evaporation of Dissociation buffer within the tube, vessel or container. The insert or plunger may contain a means to cool the sample, such as a metallic heat transfer element to equilibrate the Dissociation buffer temperature, or a filter allowing the removal of the Dissociation buffer and sample from the tube, vessel or container. The insert or plunger may also have an abrasive surface to improve sample dissociation which is particularly important for robust samples such as the lung, muscle and eye cornea. The insert or plunger also provides a means to either increase or decrease the ambient pressure above the Dissociation buffer thereby controlling the force of cavitation as set out in Example 11.

[0483] It has been found that a better transmission of the ultrasound energy to the sample tube can be obtained using these alternative liquids and gels. Tap water can also be degassed by boiling for 15 minutes and then cooling before use, or alternatively, it can be degassed under vacuum or using the ‘degassing’ function of certain water baths such as the Elmasonic P series. Degassing improves sonication power transmission to the sample container.

[0484] When using certain water bath sonicators, care should be taken to position the tube containing the sample in the correct part of the bath as indirect sonication water baths can have a very unequal distribution. Sonicators with a sweep function such as the Elmasonic P series (Elma Schmidbauer GmbH) are preferred because there is not one focal point of high energy rather the energy is distributed throughout the bath.

[0485] It is also advantageous to have more than one ultrasound frequency as is available with Elmasonic P series (37 and 80 kHz), generally the low frequency is capable of removing larger blocks of cells from the tissue mass whilst the higher frequency removes cell debris and cleans the cells of broken debris. Other devices with multifrequency output is TI-H5 (Elma Schmidbauer GmbH) operating at either 25 and 45 kHz (Model MF2), or alternatively 35 and 130 kHz (Model MF3). Using more than one frequency provides additional control of the dissociation process.

Example 16

Dissociation of Tissue Using Direct Sonication

[0486] To 1 ml of Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) was added 50 mg of a freshly dissected mouse or rat liver sample and incubated for at least 2 hours at room temperature (approximately 24° C.).

[0487] Excess liquid around the fixed tissue sample was briefly removed using an adsorbent material and then immediately placed in a standard polypropylene 1.5 ml Eppendorf centrifuge tube containing 400 μl of 85-100% Trimethylglycine:Trifluoroacetamide (1:2 mol:mol), 15% water, was placed in an ice bath and a sonication probe (3 mm tip diameter, 180 μm amplitude) Model 120 sonic 20 kHz dismembrator (Cat. No. FB4422, Fisher Scientific, UK) was then placed in direct contact with the tube contents and run constantly for 3-30 min depending on the tissue type. Direct sonication was stopped once sufficient cell dissociation has occurred as determined optically. It was found that dissociation was rapidly achieved using direct sonication but both overheating and contamination of the sample with the probe were significant drawbacks.

Example 17

Device for Holding the Tissue Sample During Dissociation

[0488] It has been observed that the sonication energy in the Dissociation buffer causes the tissue sample to be displaced to a part of the tube with a lower ambient energy and therefore less dissociation results. In order to avoid this displacement, the tissue sample can be physically constrained within the energy field. It was found that a number of different constraints for the sample were suitable such as impaling the sample on a hook or screw like metal wire and suspended in the Dissociation buffer at the focal point of the energy. Alternatively a cassette can also be used such as those used for paraffin embedding tissue PrintMate™ Biopsy Cassettes (Cat. No. A84210111, ThermoFisher Scientific), Micromesh™ (Cat. No. M507-2, Simport, USA) conveniently allows the sample to be dissociated and smaller clumps of cells pass easily through the mesh (0.38 mm mesh size) but not the sample which is held within the cassette. It will be evident to one skilled in the art that various commercially available mesh sizes can be used to enclose the sample so that using a mesh size for example of 8 μm will only allow cells, for example erythrocytes and immune cells, of less than that diameter to pass through and be collected whilst larger mesh sizes for example 50 μm will allow the majority of mammalian single-cells to pass through and be collected whilst maintaining the tissue sample within the cassette. A variety of woven plastic mesh sizes (200, 100, 70, 50, 35, 25, 20, 15, 10, 8 and 5 μm were found to be particularly useful (Cat. No. N50S, BioDesign Inc, USA) at removing large clumps of cells and other large debris from the desired single-cells which pass through the filter and can be collected.

Example 18

Device for Continual Flow

[0489] The device consists of a square or round container with an opening for inserting the tissue sample and addition of a Dissociation buffer, and in addition an inlet and an outlet both of which are connected to the interior of the container and the Dissociation buffer. The Dissociation buffer can thus be circulated out of the container where the single cells from the dissociation process collected for analysis and an inlet feeding fresh Dissociation buffer back into the container in a continual process. The overall interior volume of the container is about 100-500 μl. The container, preferably constructed of polycarbonate is shaped such as to fit into, and make good contact with a metal block attached to a sonotrode, or alternatively, a sonication bath. Preferably the contents of the container are visible to the operator such that the dissociation process can be followed visually, either by the naked eye or using a stereomicroscope (Stereo Discovery.V20 or Smartzoom 5, Carl Zeiss, Germany). The liquid is moved by way of a small pump (e.g. Model 9QX Peristaltic Pump, Boxer GmbH) and tubing (e.g. 9000.714 diameter 1.0 mm, Boxer GmbH, Germany) that can be controlled in its rate of flow. Single cells can be removed from the circulating Dissociation buffer by filtration, centrifugation, flow cytometry, Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) CS etc for subsequent staining with a stain such as Crystal Violet, Methylene Blue, Trypan Blue, antibody or nucleic acid hybridization probe, extraction of proteins, phosphoproteins, DNA or RNA, microscopical observation and digital analysis.

[0490] A variety of woven plastic mesh sizes (200, 100, 70, 50, 35, 25, 20, 15, 10, 8 and 5 μm such as Cat. No. N50S have been found to be particularly useful (BioDesign Inc, USA).

Example 19

Device for Tissue Dissociation and Automatic Separation of Single Cells and Display Results on Monitor

[0491] It is convenient for the dissociation and analysis of multiple samples such as in a clinical setting to reduce the number of manual steps necessary to complete the diagnosis. Ideally the sample, for example a biopsy, such as a prostate or breast cancer biopsy, is removed from the patient and immediately placed in 20 volumes of Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) and the sample dissociated as set out in Example 1 for sufficient time to dissociate the entirety of the biopsy. The cells are then removed from the outlet and separated from the Dissociation buffer by filtration and tissue debris removed by way of filtration or density gradient centrifugation. The filtered cells are transferred by means of a pump into a separate container where, in suspension, they are stained with a colourant, a stain, a primary and secondary labelled antibody and/or nucleic probe to aid in their identification. Alternatively the cells are transferred and attached onto a glass microscope slide where they can be stained with for example the nuclear stain DAPI or an antibody and observed by means of a fluorescent microscope. The presence of abnormal cells as judged by their morphology, their staining pattern or both associated with the disease phenotype can then be analysed by a pathologist, preferably with the aid of an appropriate software such as HALO developed by Indica Labs, UK that can determine the shape, size or abnormal staining pattern.

[0492] Once the tissue sample has been dissociated into single cells, it is then possible to use the single cells for a number of and diverse number of applications. There are a number of ways to select single cells including using antibodies either as fluorescent markers in the FACS procedure, or using antibodies bound to a solid phase such as a bead a fiber or magnetic beads including MACS (Miltenyi GmbH, Germany). It will be evident to one skilled in the art that there are a great number of protocols and products available for binding to single cells.

[0493] The analytical procedure can depend on counting the relative and absolute number of cells of various types such as T cells and B cells CD4 positive and CD8 positive cells. This in itself can be the diagnostic purpose of the experiment or diagnostic essay.

[0494] Following selection the single cells can be used for a number of molecular tests, for example RNAseq with the RNA transcript of each individual cells is determined. Several commercialized RNA-seq platforms exist including 10× Chromium, Fluidigm C1, BioRad ddSEQ and Dolomite RNA-seq system.

[0495] Alternatively the single cells can be used for proteome analysis by extracting protein. As one example, this could be for studying the phosphoprotein component of the cell. Conveniently it is been found that Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) fixation and 85-100% Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) tissue Dissociation buffer protects the phosphate group on the protein including Serine, Threonine and Tyrosine phosphate proteins. The single cells can also be used for genomics sequencing to ascertain the sequence of a genome.

Example 20

Dissociation of Tissue, Separation and Identification of Single Cells Based on Morphology

[0496] To a 100 mg of mouse large intestine sample was added 1 mL of Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) and incubated for 12 hours at ambient temperature to fix the sample. The tissue sample was then removed and placed in 500 μL of 85-100% Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) in a 1 mL polycarbonate tube. Sonication was carried out for 15 seconds as set out in Example 1, 500 μL of the dissociation buffet was then removed and a fresh 500 μL of 85-100% Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) was added and sonication carried out again, for 60 seconds. This process was repeated with a sonication time of 60 seconds per cycle using using 70% pulse and 70% amplitude. The Dissociation buffer was passed through an 8 μm CellSieve mesh filter to remove debris and to capture the single cells. The single-cells were washed off the filter and resuspended in water before FACS analysis and sorting based on cell size or shape. On recovery and examination of the filtered cells using a microscope it was found that the initial dissociation cycles led initially to the dissociation of epithelial cells followed by the intestinal luminal cells and finally muscle cells and connective tissue.

Example 21

Staining the Tissue Sample Prior to, During, or After Dissociation

[0497] It is possible to carryout tissue fixation and staining in a variety of ways: (i) staining followed by fixation, (ii) fixation followed by staining, or (iii) staining and fixation at the same time. It is also possible to combine staining, fixation and dissociation at the same time by using the Dissociation buffer containing a stain as the sample fixative solution. A fresh tissue sample or a fresh sample that has been snap-frozen was placed placed in a 1 ml thick-wall polycarbonate centrifuge tube (Beckman Cat. No. 355657) containing 200 μl of 85-100% Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) and 0.02% Methylene blue, and the tube sealed using a self-adhesive silicone closure. The tube is then placed in a VialPress fitted to a Vial-Tweeter-Sonotrode (S26d11×10), using an Ultrasonic generator (UP200St-G) 200 W, 26 kHz, Ultrasonic Transducer (UP200St-T, Hielscher GmbH, Germany), run at Constant Power, 10% Pulse, 10% Amplitude for 60 minutes at 4° C. It was found that single-cells were both well fixed, intact and stained with Methylene blue.

[0498] Preferably the dissociation and fixation solution is at least 95% Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) and the sonication power is such that the unfixed cells break open and lyse before they are fixed. It is therefore preferred that both the Pulse and Amplitude are limited to a maximum of 20% to favourise initial tissue fixation followed by dissociation. It has been found that gentle sonication in this manner can lead to faster fixation than in its absence, therefore the sonication serves the role of speeding up the fixation as well as causing the tissue to dissociate into single cells.

[0499] It is also possible to include a stain during this process so that tissue fixation, staining and dissociation all occur at the same time. The stain can be a colourant, an antibody or a nucleic acid probe.

Example 22

Dissociation of Tissue into Separated Groups or Clumps of Cells for Subsequent Dissociation into Single Cells

[0500] Partial dissociation of the tissue sample leads to large collections of cells together. The cells making up the clump of cells are by definition, cells that were spatially located in the original tissue sample together. By separating these collections of cells into individual containers by, for example microfluidic means, filtering, density gradient centrifugation, manual sorting or by use of a colourant, a stain such as an antibody or fluorescent probe, and then their subsequent dissociation into single cells allows the analysis of cells that were spatially localized together in the original sample this allows a comparison of, for example, gene expression patterns of neighbouring cells. It will also be apparent that this approach is applicable not only for large collections of cells (greater than a million) but also for very small numbers, for example groups of 10 or less, five or less, or even two or less cells joined together. Such a progressive method of tissue dissociation provides a means to understand the spatial structure of the original biopsy and therefore cell to cell variation of, as one example, mRNA expression patterns.

Example 23

Dissociation of Fixed Tissue into Single Nuclei Using DMSO

[0501] Traditional methods for purifying nuclei require long and arduous protocols involving multiple centrifugation steps and detergents (Kizer, K., Mds (2006) 40:296-302). It has surprisingly been found that the addition of the polar aprotic solvent Dimethylsulphoxide (DMSO) at 25% or more final concentration to a DES fixed sample is particularly efficient at the solubilization and removal of the cell membrane and cytoplasm but not the nucleus, which remains intact. Such intact nuclei can be sorted, pelleted selected, and their contents analysed either in entire nuclei by using, for example, labeled antibodies specific to nuclear epitopes such as Ki-67 or, by using fluorescent nucleic acid probes for detecting RNA and DNA. Alternatively the purified nuclei can be lysed and their contents including proteins, DNA and RNA released for further analysis such as genomic DNA sequencing, protein-protein interactions, intron splicing, CHIP analysis, chromosome analysis and/or RNA sequencing. As an example, single nuclei have been successfully used for RNA-seq (Habib, N. et al., Nat. Methods (2017) 10:955-958) and with the 10× Chromium sequencing platform (10× Genomics, USA).

[0502] DMSO at 25% or more can be added to a sample at one or more steps; (i) after the sample has been fixed with a DES; the sample, for example, tissue culture cells or a tissue is treated with DMSO, and optionally, vortexing, pipetting, or using other mechanical means included to improve cytoplasmic solubilisation, or (ii) DMSO is added to the Dissociation buffer during sonication, or (iii) after tissue dissociation is complete the single cell suspension is treated with DMSO to produce a pure population of nuclei.

[0503] The ability of DMSO (and, to a lesser extent, diethylsulfoxide; ethyl methyl sulfoxide may show similar behavior) to dissolve a DES fixed cell but not the nuclei is unexpected and unusual; no other solvents, alcohols, organic solvents or hydrocarbons have been identified that have this property. The differential solubility of cytoplasm and nuclei in DMSO is particularly unexpected given that DMSO treatment of living cells is well known as a cryoprotectant to maintain cell viability during freezing.

[0504] As an alternative to purifying nuclei, DMSO can also be usefully employed to solubilize the cell cytoplasm in order to efficiently release cytoplasmic biomolecules, for example, cytoplasmic RNA such as mRNA, or proteins and phosphoproteins, without contaminating nuclear protein, RNA and/or genomic DNA (gDNA) which is a serious problem in many purification protocols. As one example, purified RNA from cells and tissues are frequently treated with DNase or another DNA removal process to remove the omnipresent contaminating co-purifying gDNA prior to RT-PCR. In this example therefore, following DMSO treatment of the DES fixed sample, the nuclei can either be filtered out using, for example a 0.25 um filter, or pelleted away from the desired solubilized cytoplasmic fraction of the cell to leave a substantially DNA free sample.

[0505] As another example, cells and tissues fixed with DES, particularly Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) fixed samples can be resistant to efficient solubilisation with detergents that are used standardly with fresh samples leading to low yields particularly of proteins. By the addition of DMSO at 25% or more to the standard solubilisation buffer, the cytoplasm but not the nuclei are efficiently dissolved and solubilised so that recovery yields of proteins and other biomolecules are greatly improved compared with the non-DMSO treated sample.

[0506] It has also been found that the nuclei that are released from the DES fixed cell maintain a great deal of their morphology, for example muscle nuclei from DES fixed muscle samples have long spindle shapes (FIG. 12), whilst liver nuclei are round and of variable volume as has been observed from Formalin Fixed Paraffin Embedded (FFPE) H&E stained slides of these tissues. Therefore the nuclei maintain their in vivo shape and size which is representative of the cell type of origin. This has clinical applications, for example if a cancer biopsy is fixed with a DES, preferably Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) as set out in U.S. Pat. No. 9,696,247 and then treated with a 25% solution of DMSO or more to dissolve the cytoplasm and recover nuclei by filtration using a 0.25 μm filter or other means, the nuclear shape, diameter, volume, symmetry, density, opacity and/or texture can be observed with a microscope and is highly representative of the type and stage of cancer or other diseases (Carleton N M., J. Cell Biochem. (2018) doi: 10.1002/jcb.27156), and can therefore have a useful diagnostic application. Indeed, changes to the shape of the nucleus may precede the appearance of cancer. It will be evident to one skilled in the art that producing and analyzing such nuclei is also much faster than processing a biopsy with FFPE and cutting sections and staining. Additionally, observation of nuclei in FFPE sections are only in 2D and can be partially obscured by the cytoplasm, whilst when prepared with DMSO, the nuclei can be observed in their entirety in 3D.

[0507] It will be evident to one skilled in the art that DMSO at 25% or more can be used either in aqueous solution such as water, a buffer, a kosmotrope, a chaotrope, a detergent, or alternatively as part of another solvent such as a DES, an alcohol, a phenol or an organic solvent. One advantage of using DMSO dissolved in a DES is that the RNA and other biomolecules are protected from degradation. One preferred DES is Trimethylglycine:Trifluoroacetamide (2:1 mol:mol). One less preferred alternative to DMSO is Diethylsulphoxide.

Example 24

Dissociation of Tissue Fixed with Trimethylglycine:Trifluoroacetamide and Formic Acid

[0508] It has been observed that certain mammalian tissues that have fixed with Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) and then transferred and immersed in 20 volumes or more of Phosphate Buffered Saline (PBS) or other salt solutions such as 20 mM NaCl, KCl, LiCl or NH4Cl have suboptimal RNA stability. The removal of the DES post-fixation and its replacement with an aqueous buffer and/or salt solution may lead to a reactivation of nucleases, or a chemical hydrolysis of the RNA. This issue has not been observed when the sample has first been treated with Choline chloride:urea or Trimethylglycine:urea (1:2 mol:mol) before being transferred to PBS. If the Trimethylglycine:Trifluoroacetamide fixed sample needs to be transferred to an aqueous solution for example for FACS or antibody binding reaction, it is preferred that the solution (i) does not contain a salt or a buffer such as Tris-HCl, or the minimum necessary is used, (ii) the sample is handled at no more than 4° C., (iii) Dithioerythritol, Beta-mercaptoethanol, TCEP or other such reductant is added in the range 10-100 mM, (iv) the aqueous solution has a pH in the range of 3-5, (v) contains a detergent such as Triton X-100, CTAB, NP40, CHAPS, Tween-20 or other detergent in the range 0.01-1%, (vi) contains a PEG such as PEG200 in the range 20-90%, (vii) contains at least 60%, preferably at least 70%, and even more preferably 75-100% Trimethylglycine:Trifluoroacetamide, Choline chloride:urea or Trimethylglycine:urea (1:2 mol:mol), (viii) contains at least 60%, preferably at least 70%, and even more preferably 75-100% alcohol such as Methanol, Ethanol, Propanol or Butanol such as 1-Butanol.

[0509] Alternatively, it has been found that the acidification of the DES fixative in particular Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) before its addition to the sample for fixation, with an acid such as Hydrochloric acid, Sulphuric acid, Phosphoric acid, Nitric acid, Boric acid, but most preferably a Carboxylic acid such as Citric, Lactic, Pyruvic, Butanoic acid, Propanoic, Acetic, and most preferably Formic acid are particularly effective. The acidification of the DES should not lead to a pH of less than 1.5 or the biomolecules may become hydrolysed, therefore, preferably the pH should be in the range pH 2 or more, more preferably in the range pH 2-5.5 and most preferably in the range pH 4-5. Longer term storage of the sample in acidified DES should be avoided and the fixation time should be less than 1 week at room temperature, preferably less than 3 days to avoid DNA hydrolysis. Formic acid is the preferred acid when added in the range 1-12M, more preferably 4-9M, more preferably 6-8M and most preferably 7-8M Formic acid final concentration in Trimethylglycine:Trifluoroacetamide (1:2 mol:mol). Alternatively, Formic acid may be present It has therefore been surprisingly found that Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) containing 7-8M Formic acid has been found to protect RNA, DNA, proteins and phosphoproteins whilst fixing the cells in the fixed sample even when it is subsequently transferred to PBS. Advantageously the addition of Formic acid greatly reduces the viscosity of the DES.

[0510] It has additionally been found that tissues that have been fixed with Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) containing 7-8M Formic acid dissociate easily during sonication as set out in Example 1, providing a means to produce single cells with high quality RNA suitable for single-cell RNA seq applications. Therefore it has surprisingly been found that high concentrations of Formic acid in Trimethylglycine:Trifluoroacetamide are compatible with RNA and DNA integrity if treatment time is limited to 6 hours or less. It has also been found that treatment of the sample with Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) with 1-12M, more preferably 4-9M, more preferably 6-8M and most preferably 7-8M Formic acid leads not only to excellent cell morphology but also leads to permanent protection of RNA even when the cell is transitioned into an aqueous buffer such as water or PBS.

[0511] The use of Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) Formic acid as the fixative has several advantages, in particular (i) permanent protection of RNA when the fixed sample is incubated in an aqueous buffer such as PBS, and (ii) the volume of the fixed sample is retained and does not shrink thereby improving the histological detail of the tissue and morphological detail of the cells.

[0512] It will be evident to one skilled in the art that a variety of acids can be used for acidification of the DES, preferably Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) but acids with a smaller molecular weight are preferred as are low molecular weight Carboxylic acids including halogenated Carboxylic acids.

Example 25

Dissociation of Pre-Frozen Tissue into Single cells

[0513] It has been found that Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) consolidates and binds together cells and tissues that have previously been frozen. It is known that frozen tissues and cells when thawed out show ice-damage, breakage, membrane damage and loss of cell integrity. It was found that individual cells despite having been frozen and thawed, maintained structural integrity and retained their cellular morphology when, during or following thawing they are placed in contact with Trimethylglycine:Trifluoroacetamide (1:2 mol:mol). This has great utility as many clinical samples are routinely frozen and stored in biobanks but cellular morphology and quality suffers greatly as a result of thawing non-fixed cells. Generally cells and tissues are protected from ice-damage by the application of cryoprotectants such as DMSO prior to freezing and thawing but this process is rarely used routinely with clinical samples prior to freezing. One example of the direct consequence of freeze-thawing tissues is the inability to maintain brain cell integrity for scRNAseq, therefore only the RNA component found in the nucleus is currently analysed but not the cytoplasmic RNA which is lost following thawing the sample.

[0514] Therefore, provided herein is a means to maintain cell integrity of previously frozen samples for downstream applications including dissociation, collection, sorting, isolation, manipulation, handling, processing, identification, staining, labeling with a dye, an antibody, an aptamer, a fluorophore, a MassTag, an enzymatic reaction, a nucleic acid probe, sorting, separation, purification, identification, FACS sorting, flow cytometry, centrifugation, gradient centrifugation, microfluidics, handling, manipulation such as those associated with morphological analysis, droplet sequencing (Drop-Seq), Seq-Well and other single-cell analysis platforms used for determining the composition, diversity and/or sequence of RNA and/or DNA, and/or protein and/or phosphoprotein analysis.

[0515] It has been found that Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) is excellent at fixing frozen samples, maintaining individual complete cells.

[0516] 250 mg of mouse liver was frozen at −80° C. for one week, the frozen sample was then removed from the freezer and immediately immersed and allowed to thaw in 1 mL of Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) and fix for 6 hours at room temperature. The fixed mouse liver tissue was then placed in 300 μL of 95% Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) and sonicated to obtain single cells as described in Example 1. Alternatively, the frozen-thawed-Trimethylglycine:Trifluoroacetamide fixed sample can be processed into paraffin and sectioned according to standard paraffin embedding procedures used for FFPE sections.

Example 26

Alternative Tissue Fixation Procedures

[0517] One of the advantages of using Formic acid mixed with Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) is that the mixture can be used in a number of different ways during tissue fixation, dissociation and manipulation of single cells. As representative examples, a tissue sample such as 20 mg of mouse liver was treated with 20 volumes of (i) Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) 8M Formic acid for 20 minutes to 12 hours and then transferred into 50 volumes of Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) to wash out and dilute the Formic acid then left for 1 hour to 1 month at room temperature before dissociation into single-cells as set out for example in Example 1 using 80-95% Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) as the Dissociation buffer. It was found that the RNA was of excellent quality even after the cells were incubated in an aqueous buffer such as PBS for 2 hours; (ii) Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) for 20 minutes to 1 month and then transferred into 20 volumes of Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) 8 M Formic acid and left for 1 to 12 hours at room temperature before dissociation into single-cells as set out for example in Example 1 using 80-95% Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) as the Dissociation buffer; (iii) Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) 8M Formic acid for 20 minutes to 12 hours and then transferred directly into 80-95% Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) as the sonication Dissociation buffer as set out in Example 1; or (iv) Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) 8 M Formic acid for 20 minutes to 12 hours and then transferred into 100 mM Tris, 10 mM EDTA buffer pH 7.4 to neutralize the acidity prior to transfer into 80-95% Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) as the sonication Dissociation buffer as set out in Example 1. It will be evident that single-cells such as tissue culture cells can also be treated in a similar sequence but without the dissociation step.

Example 27

Alternative Tissue Dissociation Procedures

[0518] It has been found that either Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) or Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) 8 M Formic acid 2 hour fixed mouse liver (20 mg) can be dissociated into single cells by first washing the fixed tissue in 500 volumes of water, removing excess water and then placing in 100 μl of water on for example an aluminium foil support and then freezing either in a freezer at −20° C., −80° C. or dry ice, then allowing the frozen sample to thaw at room temperature and repeating the freeze-thaw five times. Whilst this method was found to be efficient for liver samples, it was not useful for brain samples which underwent general lysis. When using Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) 8M Formic acid fixed tissues, the water can be replaced with a number of other aqueous buffers such as PBS, 1× TE or SSC. RNA quality as determined by RNA purification and gel electrophoresis was found to be excellent following such dissociated mouse liver.

Example 28

Storage of Fixed Cell in PBS

[0519] It has been found that Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) 8M Formic acid 2 hour fixed mouse liver (20 mg) was washed in 500 volumes of PBS, excess PBS removed by blotting with a tissue then placed in 500 μl of PBS and incubated for 1, 4, or 12 hours in PBS at room temperature before RNA purification (RNeasy, Qiagen) and analysis by gel electrophoresis showed no detectable RNA degradation. RNA stability in fixed cells and tissues is important for protocols and applications using aqueous buffers for extended periods such as FACS analysis, cell sorting, antibody binding and scRNAseq.

Example 29

Lysis of Fixed Cells

[0520] 20 mg of mouse liver was fixed in 20 volumes of Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) 8 M for 2 hour at room-temperature and dissociated into single-cells according to Example 1. The dissociated cells were then briefly stained using 0.02% bromophenol blue and 500 cells were placed in individual wells of a 96-well plate and treated with various solvents and aqueous solutions and the effect on cell lysis observed after 5 min, 2 hours or 4 days incubation. The lysis results are shown in the following table:

TABLE-US-00003 Reagent Cell lysis effect (4 day treatment) Pronase (30 mg/ml) Complete Lysis 2% SDS Complete Lysis 0.1% SDS >15% cell lysis 8M Urea >95% cell lysis (not nuclei) 1% CHAPS >80% cell lysis 1% Triton X100 >60% cell lysis 1% NP40 >50% cell lysis 1% CTAB 100% cell lysis (not nuclei) 1% Tween-20 >60% cell lysis 10% IGEPAL-CA630 No lysis 30% IGEPAL-CA630 No lysis 50% IGEPAL-CA630 100% cell lysis 4M Guanidine thiocyanate >50% cell lysis 100% PEG200 No lysis 10 mg/ml BSA (acetylated) No lysis 100% Acetone No lysis 10% Beta-mercaptoethanol No lysis 20% Beta-mercaptoethanol No lysis 40% Beta-mercaptoethanol No lysis 60% Beta-mercaptoethanol >50% cell lysis 80% Beta-mercaptoethanol >80% cell lysis 100% Beta-mercaptoethanol 100% cell lysis 1M LiCl No lysis 100% Ethanol No lysis 100% Glycerol No lysis 100% 1-Methylimidazole No lysis 100% 1-Ethylimidazole No lysis 100% TRIzol 100% cell lysis 100% N-ethyl pyrrolidone No lysis 50% N-methyl pyrrolidone No lysis 100% Formyl morpholine No lysis 100% Formamide No lysis 100% Tetramethylurea No lysis 100% DMSO >70% cell lysis 100% 1,1,1,3,3,3-Hexafluoro-2-propanol Complete lysis including nuclei 90% 1,1,1,3,3,3-Hexafluoro-2-propanol Complete lysis including nuclei 50% 1,1,1,3,3,3-Hexafluoro-2-propanol >40% cell lysis 10% 1,1,1,3,3,3-Hexafluoro-2-propanol No lysis 50% 1,1,1,3,3,3-Hexafluoro-2-propanol >80% Lysed 50% DMSO Saturated Trifluoroacteamide No lysis Saturated Tetrafluoropropionamide No lysis Saturated N,N-Diethyl-2,2,2- No lysis Trifluoroacetamide Saturated N,N-Methyl-2,2,2- No lysis Trifluoroacetamide 50% Tetramethyl ammonium oxide 100% cell lysis 5M Tetramethyl ammonium chloride No lysis 5M Tetrabutyl ammonium chloride No lysis 5M Sodium perchlorate 100% cell lysis 5M Guanidine thiocyanate >50% cell lysis 5M Guanidine HCl >50% cell lysis 5M Potassium iodide >50% cell lysis 1M Potassium iodide No lysis Saturated Thiourea 100% cell lysis

Example 30

Post-Tissue Fixation Procedures using Either Pure Acetic acid (17.4M) or dilutions of Acetic acid in Trimethylglycine:Trifluoroacetamide or Water

[0521] 20 mg of mouse liver was treated with 20 volumes of Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) for 20 minutes to 12 hours at ambient temperature and then transferred into 50 volumes of a secondary post-fixative containing either (i) pure glacial Acetic acid, or (ii) an aqueous or alcoholic dilution to give a final concentration of 9-17.39M Acetic acid, or (iii) a Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) dilution to give a final concentration of 11-17.39 M Acetic acid then incubated for 10 minutes to 1 hour at room temperature before dissociation into single-cells as set out for example in Example 1 using 75-95% Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) as the Dissociation buffer. The Acetic acid component can then be neutralized using Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) as set-out for Formic acid in Example 26.

[0522] Alternatively and conveniently, dissociated single cells generated as set-out in Example 1, in the Dissociation buffer can be poured over and captured on a filter such as a Pluriselect 1-4 μm filter, and then the cells on the filter are treated with either (i) pure Acetic acid, or (ii) a 9-17.39M dilution of Acetic acid in water or alcohol, or, (iii) a 11-17.39M dilution of Acetic acid in Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) for 10-60 minutes before removal of the acid by filtration and neutralization with 50 volumes of Trimethylglycine:Trifluoroacetamide (1:2 mol:mol). Alternatively, the cells can be recovered by the addition of 1 volume of ethanol and centrifugation at 600 g for 5 minutes to pellet the cells.

[0523] It was found that the RNA was of excellent quality even after the Acetic acid treated cells were incubated in an aqueous buffer such as PBS for 2 hours.

Example 31

Dissociation using Ultrasound Transmission Gel

[0524] To 1 ml of Trimethylglycine:Trifluoroacetamide (1:2 mol:mol) was added 10-100 mg of a freshly dissected mammalian tissue such as liver and incubated for at least 30 minutes at room temperature (approximately 24° C.).

[0525] Tissue dissociation was then carried out by sonication according to Example 1 except the Dissociation buffer was 50-500 μl of Aquasonic® 100 Ultrasound Transmission Gel (Parker Laboratories Inc., USA) with a viscosity of 130,000-185,000 centipoise.

[0526] It was found that the Transmission gel was an efficient medium for transferring ultrasound energy to the sample and led to good tissue dissociation into single cells.

Example 32

Dissociation in Small Volumes of Dissociation Buffer

[0527] Fixation of the biological sample was carried out according to Example 1 except the volume of Dissociation buffer was reduced to between 5-100 μl, with a preference of 10-20 μl. With such small volumes the sample was not completely covered with the Dissociation buffer but was in contact with the container such as the polycarbonate tube and therefore the sonication energy via the thin layer of Dissociation buffer. It was found that Dissociation was rapid and effective and provided a simple means to concentrate the single-cells in a small volume for subsequent manipulation such as pelleting or filtration, and was particularly useful for very small amounts of sample.

Example 33

Illustrative method of processing a biological sample

[0528] FIG. 17 is a flow chart illustrating general steps of an example method for processing a biological sample.

[0529] The method includes obtaining (1) a biological sample such as a fresh or frozen human or animal tissue. The sample is fixed (2) using a fixative by placing the sample in contact with the fixative. The sample is then optionally stored (3) in the fixative.

[0530] The sample is then transferred to a dissociation medium and dissociated (4). Larger dissociated pieces of tissue are optionally removed by filtering (5).

[0531] Single cells are collected (6) by for example pelleting the cells or by filtration. The collected cells are optionally treated (7) with a secondary fixative such as an acid.

[0532] The single cells may be stored (8), resuspended (9) in an aqueous medium, and then analysed (10).

Example 34

Illustrative Apparatus

[0533] FIG. 18 is a schematic diagram of one example of an apparatus 100 useful for processing a biological sample 102 to obtain single cells and/or groups of cells.

[0534] Apparatus 100 includes a container 110 for containing a biological sample 102 and a dissociation medium 104. The apparatus also includes a conduit 120, for example a tube. Conduit 120 has an inlet 122 for removing liquid dissociation medium from container 110, and an outlet 126 downstream of inlet 122. In this example, outlet 126 is for returning liquid dissociation medium to the container. This allows for recycling of dissociation medium.

[0535] First and second filters 132, 134 are arranged in the conduit. First filter 132 is a 40 to 70 μm filter, in other words, apertures in the filter have sizes in the range 40 to 70 μm. In this way, the first filter is configured to trap larger pieces of dissociated material such as fragments of extracellular matrix 133. Second filter 134 is downstream of first filter 132. Second filter 132 is a 1 to 12 μm filter. Filter sizes in this range are useful for isolating single cells 135. Cell sizes may vary, and the filter size may be selected as desired within this range.

[0536] A pump 124 for pumping liquid dissociation medium from the inlet 122 of the conduit 120 to the outlet 126 of the conduit 120.

[0537] In use, a biological sample 102 to be dissociated and an aliquot of a dissociation medium 104 such as a deep eutectic solvent are placed in the container 110. Sonication energy provided by a sonication source 140 is applied to the container, and is transmitted to the biological sample 102 via the dissociation medium. The sonication causes cavitation of the dissociation medium 104. The cavitation causes the biological sample 102 to be dissociated, producing single cells and/or groups of cells along with pieces of extracellular matrix. The single cells and/or groups of cells are suspended in the dissociation medium.

[0538] Pump 124 is activated, drawing dissociation medium and the single cells, groups of cells and extracellular matrix into the inlet 122 of conduit 120. The dissociation medium flows through the conduit in downstream direction 123. Pieces of extracellular matrix 133 are too large to fit through first filter 132, and are separated from the stream of dissociation medium. The dissociation medium then reaches second filter 134, where single cells 135 are separated from the dissociation medium. After passing through second filter 134, dissociation medium is returned to the container via outlet 126 of the conduit.

[0539] The biological sample is contacted with a deep eutectic solvent during the processing. This could be before or during the dissociating. The nature of the biological sample is not particularly limited. It may be fresh, frozen, or fixed. For fixed samples, fixation may be with a deep eutectic solvent or a deep eutectic solvent may be applied post-fixation either as dissociation medium or in an intermediate step.

[0540] Single cells obtained using the apparatus may be used in, for example, single cell DNA sequencing or single cell RNA sequencing, amongst other applications.