HANDLING OF BIOLOGICAL SAMPLES

Abstract

The present invention relates to the handling of biological samples, for example, the holding, manipulating and culturing of biological samples. In one form the invention provides an overlay encapsulant for an in vitro cell culture comprising a synthetic compound and in another aspect the invention provides methods of temporarily encapsulating an in vitro cell culture comprising a synthetic compound. The invention has use in relation to the culturing and more particularly the encapsulation of biological samples, such as for example zygotes, embryos, oocytes, stem cells, sperm located in a culturing space, relevant pluripotent derivative(s) and/or differentiated progeny, intact or dispersed tissue and/or intact organism(s).

Claims

1. An overlay encapsulant for an in vitro cell culture comprising a synthetic compound.

2. An overlay encapsulant according to claim 1 wherein the cell culture comprises one or more cells in a culture media.

3. An overlay encapsulant according to claim 2 wherein the one or more cells comprises at least one or a combination of: ovum; zygote; embryo; animal/human-derived embryonic stem cell(s); relevant pluripotent derivative(s) and/or differentiated progeny; intact or dispersed tissue and/or intact organism.

4. An overlay encapsulant according to claim 1 wherein the synthetic compound is a synthetic small molecule composition exhibiting unequivocal chemical composition as identified via conventional analytical techniques within limits of detection and comprising one or a combination of. synthetic monomer(s); oligomers or polymers; chemical derivatives and/or copolymers of polyalphaolefins, each exhibiting specific chemical, biophysical and spectroscopic properties.

5. An overlay encapsulant according to claim 1 wherein the synthetic compound comprises at least one hydrocarbon.

6. An overlay encapsulant according to claim 1 wherein the synthetic compound comprises a modified hydrocarbon.

7. An overlay encapsulant according to claim 6 wherein the modified hydrocarbon comprises a fluorinated hydrocarbon.

8. An overlay encapsulant according to claim 1 wherein the synthetic compound comprises one or a combination of long-chained, short-chained and cyclic hydrocarbons.

9. An overlay encapsulant according to claim 8 wherein the synthetic compound comprises a combination of long-chained, short-chained and cyclic hydrocarbons in the mixture of 45% long-chained, 38% short-chained and 17% cyclic, respectively.

10. A method for temporary encapsulation of an in vitro cell culture comprising the step of overlaying the cell culture with a synthetic compound.

11. A method for temporary encapsulation of at least one of protein(s), DNA, RNA sequence(s), relevant construct(s) and/or derivative(s), chemically-modified or derived analogues thereof for in vitro, ex vivo and/or in vivo manipulation thereof, the method comprising the step of: overlaying a manipulation and/or screening media utilised in the in vitro, ex vivo and/or in vivo manipulation with a synthetic compound.

12. An overlay encapsulant according to claim 1 wherein the synthetic compound comprises a well-defined chemical compound as described by conventional analytical techniques comprising one of NMR, HPLC, LCMS within the limit of detection wherein the compound is exemplified by one of: i) regimented polymer with well-defined chemical and/or biophysical properties, ii) small molecule, iii) inert gas heavier than air.

13. An overlay encapsulant according to claim 1 which is adapted to monitor deviations in the properties and composition of media encapsulated thereby.

14. An overlay encapsulant according to claim 13 wherein the monitored properties and composition of encapsulated media comprise one or a combination of: pH, ammonia concentration, osmolarity, presence of reactive oxygen species, and presence or volatile organic compounds.

15. An overlay encapsulant according to claim 1 in which the overlay encapsulant is adapted to be used as a supplement source comprising one or a combination of: vitamins, hormones, growth factors, nutrients, protectants, RedOx traps, amino acids and their derivatives, peptoids, peptides, proteins, antibodies and relevant derivatives, fragments and full length oligonucleotides and their synthetic derivatives.

16. An overlay encapsulant according to claim 1 in which the overlay encapsulant is adapted to be used for screening or biological manipulations involving one or more of: element-sensitive proteins, cells or cell cultures, multi-origin tissues or tissue cultures, and intact organisms.

17-18. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0096] Further disclosure, objects, advantages and aspects of preferred and other embodiments of the present invention may be better understood by those skilled in the relevant art by reference to the following description of embodiments taken in conjunction with the accompanying drawings, which are given by way of illustration only, and thus are not limitative of the disclosure herein, and in which:

[0097] FIG. 1 illustrates an analysis of pluripotency markers Genea018, overlaid with Sage IVF Oil (fused image) in accordance with a preferred embodiment of the invention.

[0098] FIG. 2 illustrates an analysis of pluripotency markers in Genea018, overlaid with Compound 1 (fused image) in accordance with a preferred embodiment of the invention.

[0099] FIG. 3 illustrates as analysis of pluripotency markers in Genea018, overlaid with Compound 2 (fused image) in accordance with a preferred embodiment of the invention.

[0100] FIG. 4 illustrates an analysis of pluripotency markers in Genea018, overlaid with Compound 3 (fused image) in accordance with a preferred embodiment of the invention.

DETAILED DESCRIPTION

[0101] In contrast to the use of mineral oils in the prior art, with preferred embodiments, the inventor proposes to use well-characterized synthetic polymer(s), synthetic or natural monomeric small molecule organic compound(s) or appropriate mixtures thereof with additional components including but not limited to other small molecules, polymers, antioxidants, nutrients, biomolecules including but not limited to nucleotide and nucleotide sequences, oligomers (ex., DNA, RNAs, their fragments and/or synthetic analogues), amino acids, peptides, proteins, antibodies and other favorable biomolecules displaying well-defined and controlled chemical composition, embryo-compatible (bio)physical and (bio)chemical properties, stability and easily available commercially as food-grade or medical device-grade (H-1 or higher, as per National Sanitation Foundation categorization) inert silent media component for embryo or general in vitro/ex vivo protein and cell biology. Furthermore, physical, chemical and biological properties of these and related compounds could be further optimized synthetically or via additives in order to attain the desired physiological and clinical outcome appropriate for biological samples.

[0102] In a first stage of a representative calibration approach, the inventor has identified several polyalpha- or related polymers and relevant (co)polymers from commercial sources. Representative examples comprise the following:

[0103] Food Grade Synthetic Oil ISO 220, 55 Gal (http://www.grainger.com/product/CRC-Food-Grade-Synthetic-Oil-ISO-12G564) [0104] Food Grade Silicon Spray (Weston Brand, http://www.schaefferoil.com/276-food-grade-lube.html) [0105] Summit Syngear Food Grade (FG) fully synthetic lubricants (http://www.klsummit.com/products/lubricant/syngear-fg-series) [0106] Sprayon LU209 Food Grade Synthetic Oil (http://www.sprayon.com/product-categories/industrial-lubricants/food-grade-synthetic-oil-aerosol-lu209) [0107] Lubriplate NSF H1 Registered Food Machinery Lubricants (https://www.lubriplate.com/Products/NSF-H-1-Registered-Food-Machinery-Lubricants.aspx)

[0108] For embodiments, the key selection criteria include: [0109] 1. A true organic small-molecule oil with tentative molecular weight MW<5,000D. The preferred candidate is a well characterized inert monomer or polymer as exemplified, including but not limited to, long chain alkanes, cycloalkanes, long chain aliphatic alcohols, ethers, esters, amides, lactones, lactams, etc. [0110] 2. A specific set of biophysical, chemical, stability, toxicity criteria including density, viscosity (kinetic and dynamic), surface tension, etc [0111] 3. Synthetic or well defined naturally originating oils, which are generally recognized as safe, ie GRAS; [0112] 4. Well-defined chemical composition and (micro)impurities including both organic and inorganic substances; [0113] 5. Chemical stability and inertness, such as to sunlight, air/oxygen and, temperature. Biological stability/inertness, embryo and/or relevant oligonucleotide, protein, cell, tissue, intact organism-isolation/encapsulation potential; [0114] 6. Physical properties compatible with objects defined in the selection criterion 5. These comprise volatility, melting point, boiling point, standalone safety, flashpoint, molecular weight, viscosity range, surface tension, gas/liquid diffusion/miscibility potential, etc.; [0115] 7. Physical properties which allow the compound to prevent evaporation, therefore allowing it to be used as an overlay for culture media to prevent osmolality, temperature and pH deviations; [0116] 8. Feasibility of access, modification, synergistic potential with favorable additives and ease to use/operate; [0117] 9. Commercial feasibility.

[0118] The identified lead candidates for an encapsulating overlay that satisfy the abovementioned criteria may be further evaluated in stem cell and embryonic development assays as per standard protocol described for paraffin/mineral oil(s) to further select candidates. In addition, adding chemically/biologically inert additives to the encapsulating overlay comprising small molecule-based monomer compounds and/or related fully synthetic compound(s) in order to further optimize their physical/biological properties is envisaged. These additives include but are not limited to respective surfactants, reactive oxygen species/metabolite scavengers and/or nutrients, gene-altering antisense DNA or RNA sequences, peptides, proteins, peptides, peptoids and other favorable molecules.

[0119] Lead candidate compounds which include additives could also be used for other screening and biological manipulations involving element-sensitive proteins, cells, cell cultures, multi-origin tissues, tissue cultures and intact organisms.

[0120] Numerous single small molecule-based compounds are readily available commercially and may be further customized to match specific embryo culture specifications via a variety of synthetic procedures. Chemical classes which could be utilised include hydrocarbons of various lengths, both branched, linear and cyclic, as well as modified hydrocarbons (including, but not limited to, fluorocarbons).

[0121] In one embodiment, Polyalphaolefins (PAOs) may be utilised. PAOs are readily available commercially and may be further customized to match specific embryo culture specifications via a variety of synthetic procedures. In this respect, the following listed references may be utilised for such procedures: [0122] 1. Rudnick, L. R. Polyalphaolefins, Chemical Industries (Boca Raton, Fla., United States) (2013), 135 (Synthetics, Mineral Oils, and Bio-Based Lubricants), 3-40. [0123] 2. Gee, J. C. et al. Behavior of protonated cyclopropyl intermediates during polyalphaolefin synthesis: Mechanism and predicted product distribution, Journal of Physical Organic Chemistry (2012), 25(12), 1409-1417. [0124] 3. Yu, X. et. al Synthesis of polyalphaolefins on AlCl3/TiCl4 catalyst, China Petroleum Processing and Petrochemical Technology (2012), 14(2), 55-59. [0125] 4. Azizov, A. H., et al Advancement in the synthesis &production of polyalphaolefin synthetic oils: I. synthesis of poly--olefin synthetic oils by catalytic oligomerization of -olefins with acidic & complex catalysts, Neft Kimyasi va Neft E'mali Proseslari (2010), 11(1), 53-78. [0126] 5. Azizov, A. H., et al Advancement in the synthesis and production of polyalphaolefin synthetic oils: II. Synthesis of polyalphaolefin synthetic oils by catalytic oligomerization of alpha-olefins in the presence of ionic liquid catalysts, eft Kimyasi va Neft E'mali Proseslari (2010), 11(2), 163-182. [0127] 6. Tsvetkov, O. N. Catalytic processes in the manufacture of poly -olefins, Kataliz v Promyshlennosti (2002), (6), 33-40. [0128] 7. Shubkin, R. L. Polyalphaolefins, Chemical Industries (Dekker) (1993), 48 (Synthetic Lubricants and High-Performance Functional Fluids), 1-40. Galli, R. D. A New Synthetic Food Grade White Oil, Lubrication Engineering (1982), 38(6), 365-72.

[0129] It is worth noting that multiple publications describe utility of polyalphaolefins (PAOs) as food-grade (H-1, as designated by the National Sanitation Foundation) in the last 2 decades. Hence, there is now a clear indication that PAO's are a safe material in the food industry and, by the inventor's inference and investigation, PAO's may be safely and validly synthesised as candidates for the synthetic compound utilised in embodiments of the present invention.

[0130] It is further anticipated that the disclosed embodiments of the invention could be used in a broader array of in-vitro, cell-based, tissue culture and in-vivo assays involving intact organisms. Specifically, the aforementioned inert compounds may be applied directly to insulate the actual screening media (including, but not limited to (micro)drop(s) in the screening well of 96-, 384-, 1536-well or any alternative plate, open or closed channel microfluidics devices, etc) from exposure to the environment and/or to maintain key screening parameters including volume, composition, osmolarity, nutrient content, etc. The invention is of particular benefit to screening biological objects, cells, tissues, and organisms that may be sensitive to elements using any conventional, medium- or high throughput dispensing technique. Additional benefit(s) provided by the disclosed inert compounds used as overlaying encapsulants for biological samples may also include complete transparency to the common non-intrusive light-absorbance, emission, scattering detection techniques including UV-vis, near-IR, far-IR spectroscopy, electron paramagnetic resonance and biophysical platforms including but not limited to surface plasmon resonance (SPR), thermal melt and other assay techniques. Representative examples include, but are not limited to: [0131] i) in-vitro manipulation (storage, dispensing, screening) of air/oxygen, UV light-sensitive, osmolarity, pH proteins. By way of example, biomolecules comprising multiple SH and/or SS bonds as exemplified by the family of cytokine and chemokine proteins; proteins/enzymes featuring coordinated metal(s) including but not limited to Zn, Mg, Mn, Cu, Fe as exemplified by the epigenetics targets including but not limited to histone deacetylases, histone demethylases, histone acetylases, metalloproteinases, hydrolases, etc; [0132] ii) in-vitro manipulation of any nucleotide sequences including but not limited to endogenous, intact, fragmented, chemically modified DNA, mRNA, shRNA, siRNA, miRNAs as exemplified by q-PCR, transfection and gene editing techniques; [0133] iii) cell-based screening including but not limited to any manipulations of stem cell(s) or relevant derivative(s) thereof as exemplified by human/animal-derived embryonic stem cells, induced pluripotent stem cells, immediate or advanced (differentiated) derivatives of these, genetically manipulated derivatives of stem cells, etc; [0134] iv) any cell culture in a relevant treatment receptacle including but not limited to microtiter, midi- or macro-plates, microfluidics devices, stationary, suspended drop, flow systems or similar. These cell cultures include but are not limited to human/animal embryos/cells, specific differentiated human/animal cells as exemplified by an organ/tissue derived neurons, cardiomyocytes, fibroblasts, hepatocytes, renal cells; stem cells/primary cells/cancer cells/otherwise immortalized cells, genetically altered/engineered cells, stably and/or transiently transfected cells, cells labelled with fluorescent, radio, radical and/or other detection functionalities, etc. [0135] v) functional/phenotypic screening using relevant healthy, diseased, modified or transfected cell lines as exemplified by differentiation, proliferation, migration, adhesion, motility, chemotaxis and other cellular assays; [0136] vi) screening using intact or suspended tissue (for example, matrigel-based clonogenic assay(s)) of interest and/or intact organisms as exemplified by the sea urchin embryo, zebrafish and other in-vivo assay(s) where maintenance of homeostasis is of critical importance.

Preliminary Experimental Data on Use of Synthetic Compounds as Overlay for Cell and Embryo Culture

[0137] Experimental results from trials conducted by the inventor involving embodiments of the invention are as follows:

Aims

[0138] To perform preliminary tests about the feasibility of three synthetic compounds in cell and embryo culture.

1. Experiment 1Stem Cells

[0139] 1.1. Experimental Procedure

[0140] Materials:

[0141] Test compounds:

[0142] Compound 1

[0143] Compound 2

[0144] Compound 3

[0145] According to brochures available from the manufacturer, the selected test compounds are hydraulic and lubricating compounds based on high-purity hydrocarbons with paraffinic synthetic oil. They are a combination of basic oils and additives, which can be used in the food processing industry. In particular, Compound 1 is a mixture of short, long and branched, fully saturated hydrocarbons with no presence of aromatic groups. An example of a suitable candidate that would fall within the scope of Compound 1 is found in the source: TURMOSYNTH VG series Technical Information and technical information is presented in the following Table 1.

TABLE-US-00001 TABLE 1 TURMOSYNTH VG Technical Data 15 32 46 68 100 150 220 320 460 680 1500 NSF/H1- 127133 132163 139108 127132 127138 127139 132161 132161 127122 132162 127131 registration Colour Clear, nearly colourless Density 0.85 0.86 0.87 0.88 0.88 0.88 0.88 0.88 0.88 0.89 0.89 at +20 C. (g/cm.sup.3) Temperature 10 C. to +100 C., higher viscosities a short time up to +120 C. range Viscosity(mm.sup.2/s) DIN EN ISO 3104 at +40 C. 16.3 33.6 43.5 67.6 100.8 142.7 227.2 323 459.9 692 1504 at +100 C. 3.6 5.7 6.7 9.0 11.7 13.8 23.0 32 42.6 59.1 117.7 Viscosity index 102 109 107 108 104 116 125 138 144 149 174 DIN ISO 2909 Applications Machines in the food industry with oil lubrication, like hydraulics, gears, bearings, chains, spindles, levers and links.

[0146] The manufacturing process for the synthesis of the three selected compounds comprises the combination of specific raw materials within a mixing vessel. This differs from the mineral oil process which involves the fractional distillation of a natural product (crude oil) and purification to reach the finished product.

[0147] Human embryonic stem cell lines (hESC) lines [0148] Manually passaged, cultured on mouse fibroblast feeder layers [0149] Cells cultured on Nunc IVF 1-well dishes, in KnockOut Serum Replacement.sup.16 (KSR)-media in large incubator at +37 C. at 6% CO.sub.2, 5% O.sub.2 and 89% N.sub.2 .sup.16 See https://www.thermofisher.com/order/catalog/product/10828028

[0150] The used hESC lines were manually passaged human embryonic stem cell lines. The dishes for the experiment were dishes remaining after manual cutting and removal of hESC colonies 8 days after previous passaging. The remaining colonies are still able to be cultured, although eventually they start to differentiate and lose pluripotency, and even degenerate if not adequately fed. Each dish contained cells from a different cell line and passage number.

[0151] Control cultures were plated and overlaid with Sage IVF Oil, which is regularly used in embryo culture. This was to allow comparisons to be drawn between the ability of cells to be cultured under Sage IVF Oil and test compounds.

[0152] The KSR media had been changed for passaging, so the experiment started with 1 ml of fresh media in each dish. For the actual experiment all wells were layered with 1 ml of test oil that had been equilibrated overnight in a 20% O.sub.2 and 5% CO.sub.2 incubator at 20% O.sub.2. The dishes were then cultured further in a low oxygen incubator (6% CO.sub.2, 5% O.sub.2 and 89% N.sub.2) for overnight.

[0153] The media on dishes were replaced with fresh KSR media the following day and then left without media change over the next two days. Cell appearance was observed at dish preparation, after overnight culture (at 1 day), after 3 more days of culture (at 4 days) and at day 7, when immunohistochemical staining was also performed using three antibody markers (SSEA-4, Oct-4 and Nanog). These particular molecular markers were used because their presence verifies the pluripotent status of the stem cells. A down-regulation of either SSEA-4, Oct-4 or Nanog would signify that cells are differentiating and are no longer pluripotent, meaning that the hESCs are under stress. All dishes were discarded at that point and the experiment concluded.

[0154] 1.2. Results & Discussion

[0155] hESCs continued to grow under all test compounds and displayed pluripotency on day 7. FIGS. 1 to 4 show the cells cultured under an overlay of the Sage IVF oil, Compound 1, Compound 2 and Compound 3, respectively, after 7 days of culture. The development of cells cultured underneath an overlay of Compound 3 is very similar to those cultured under an overlay of Sage IVF Oil (the control). Cells cultured underneath an overlay of Compound 1 and Compound 2, although not forming a perfect monolayer, did not simply degenerate and therefore, Compound 1 and Compound 2 were not immediately cytotoxic. However, they may not provide an environment for cell proliferation which is as suitable as Compound 3 or Sage IVF oil.

[0156] No detailed information about proliferation rate or cellular differentiation rates were obtained in this experiment, as the intention was only to test preliminary reaction of cells to test compounds.

[0157] The cells used in this experiment were hESC lines that were maintained and passaged as colonies rather than single cells. This method of culturing is still the method used at initial derivation of new lines from human embryos, and is also used for early passages to best maintain the integrity of stem cell lines and to avoid chromosomal deviations that may arise in later passages, especially if passaged enzymatically as single cells.

[0158] FIG. 1 illustrates an analysis of pluripotency markers Genea018, overlaid with Sage IVF Oil (fused image). FIG. 2 illustrates an analysis of pluripotency markers in Genea018, overlaid with Compound 1 (fused image). FIG. 3 illustrates as analysis of pluripotency markers in Genea018, overlaid with Compound 2 (fused image). FIG. 4 illustrates an analysis of pluripotency markers in Genea018, overlaid with Compound 3 (fused image).

[0159] 1.3. Conclusions

[0160] The stem cells survived and grew when applied with an overlay of all compounds. Cells cultured under an overlay of Compound 3 displayed proliferation similar to control (Sage IVF Oil). Cells cultured under an overlay of Compound 1 and Compound 2, although not forming a complete monolayer as could be seen for cells under Compound 3 and Sage IVF Oil, still did experience growth and were not dead after 7 days of culture. Therefore, it is clear that none of the compounds were cytotoxic, and that all allowed cell proliferation. In addition, it is possible to see from the images showing the fluorescence of all three pluripotency markers in each of the samples that the hESCs maintained their pluripotency after 7 days of culture under all test and control compounds.

2. ExperimentEmbryos

[0161] 2.1. Experimental Procedure

[0162] Materials:

[0163] Test compounds:

[0164] Compound 1

[0165] Compound 2

[0166] Compound 3

[0167] Sage IVF Oil (CONTROL)

[0168] Single-Step Human Embryo Culture Medium

[0169] Falcon 60 mm dishes

[0170] Mouse embryos at 2PN stage.

[0171] 60 mm Falcon petri dishes were prepared with Single-Step Human Embryo Culture Medium and Sage IVF Oil (control compound), Compound 1, Compound 2 or Compound 3 (test compounds) as per routine culture of mouse embryos. Briefly, 920 l drops were prepared under 6 ml of control or test compound, and left to equilibrate in a Cook MINC incubator.sup.17 at +37 C. at 6% CO.sub.2, 5% O.sub.2 and 89% N.sub.2 overnight. The next day (Day 1), embryos which had been classified as being 2PN stage were placed into the drops following removal of cumulus cells. No more than ten embryos were placed in each drop. Embryos were then assessed for development as per routine mouse embryo assay (MEA) protocol on days 2, 5, 6 and 7. .sup.17 See https://www.cookmedical.com/products/wh_minc_1000_webds/

[0172] 2.2. Results & Discussion

[0173] Embryo development and quality was comparable between the control and Compound 3 at all stages of assessment. Embryos degenerated prior to their first cell division when media was overlaid with Compound 1 or Compound 2. Therefore, although Compound 1 and Compound 2 were not toxic to stem cells, they are both clearly toxic to embryos.

[0174] 2.3. Conclusions

[0175] Overlaying the culture media with Compound 3 enabled embryos to fully develop to blastocyst stage. The amount of embryos which developed and their quality was not statistically different between control and test groups. Overlaying the culture media with Compound 1 or Compound 2 caused almost instant embryo degeneration.

[0176] While this invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modification(s). This application is intended to cover any variations uses or adaptations of the invention following in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth.

[0177] As the present invention may be embodied in several forms without departing from the spirit of the essential characteristics of the invention, it should be understood that the above described embodiments are not to limit the present invention unless otherwise specified, but rather should be construed broadly within the spirit and scope of the invention as defined in the appended claims. The described embodiments are to be considered in all respects as illustrative only and not restrictive.

[0178] Various modifications and equivalent arrangements are intended to be included within the spirit and scope of the invention and appended claims. Therefore, the specific embodiments are to be understood to be illustrative of the many ways in which the principles of the present invention may be practiced. In the following claims, means-plus-function clauses are intended to cover structures as performing the defined function and not only structural equivalents, but also equivalent structures. For example, although a nail and a screw may not be structural equivalents in that a nail employs a cylindrical surface to secure wooden parts together, whereas a screw employs a helical surface to secure wooden parts together, in the environment of fastening wooden parts, a nail and a screw are equivalent structures.

[0179] Comprises/comprising and includes/including when used in this specification is taken to specify the presence of stated features, integers, steps or components but does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof. Thus, unless the context clearly requires otherwise, throughout the description and the claims, the words comprise, comprising, includes, including and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of including, but not limited to.