CORNEAL TISSUE

Abstract

The invention provides an isolated dehydrated corneal tissue, comprising a full thickness corneal stroma, and substantially all, or all, of the Bowman's membrane, wherein the stroma contains cellular material. Also provided is a method to product the tissue and uses of the tissue.

Claims

1. An isolated dehydrated corneal tissue, comprising a full thickness corneal stroma, and substantially all, or all, of the Bowman's membrane, wherein the stroma contains cellular material.

2. The isolated dehydrated corneal tissue of claim 1 wherein the tissue comprises some or substantially all, or all, of the Descemet's membrane.

3. The isolated dehydrated corneal tissue of claim 1 or claim 2 wherein the tissue does not comprise an epithelium or an endothelium.

4. The isolated dehydrated corneal tissue of any of claims 1 to 3 wherein the tissue consists of an entire corneal stroma, and substantially all or all of the Bowman's membrane.

5. The isolated dehydrated corneal tissue of any of claims 1 to 3 wherein the tissue consists of an entire corneal stroma, substantially all, or all, of the Bowman's membrane and some or substantially all, or all, of the Descemet's membrane.

6. The isolated dehydrated corneal tissue of any preceding claim wherein in the stroma of the dehydrated corneal tissue at least some of the corneal keratocytes are retained.

7. The isolated dehydrated corneal tissue of any preceding claim wherein the tissue does not comprise any viable/living cells or live cellular layers from the donor.

8. The isolated dehydrated corneal tissue of any preceding claim wherein the tissue is not prepared by freeze-drying.

9. The isolated dehydrated corneal tissue of any preceding claim wherein the tissue has not been lathed.

10. A method of producing dehydrated corneal tissue, the method comprising: a) providing corneal tissue that has been obtained from a donor; b) suspending the corneal tissue in a solution comprising an osmotic agent; c) agitating the solution in which the corneal tissue is suspended in order to remove the endothelial cells and epithelial cells, whilst the stroma retains cellular material; and d) vacuum drying the corneal tissue from c) in order to produce a dehydrated corneal tissue.

11. The method of claim 10 wherein the corneal tissue provided in step (a) comprises substantially all, or all, of the epithelium; substantially all, or all, of the Bowman's membrane; substantially all, or all, of the stroma; substantially all, or all, of the Descemet's membrane; and substantially all, or all, of the endothelium.

12. The method of claim 9 or 10 wherein the corneal tissue after step (c) comprises substantially all, or all, of the Bowman's membrane; substantially all, or all, of the stroma; and substantially all, or all, of the Descemet's membrane.

13. The method of claim 12 wherein after step (c) the corneal tissue does not comprise the epithelial layer (the epithelium) and/or the endothelial layer (endothelium).

14. The method of any of claims 10 to 13 wherein the dehydrated corneal tissue produced contains at least some keratocytes in the stroma of the tissue.

15. The method of any of claims 10 to 14 wherein the corneal tissue after step (c) comprises all or substantially all of the Bowman's membrane and the stroma.

16. The method of claim 15 wherein after step (c) the corneal tissue further comprises all or substantially all of the Descemet's membrane.

17. The method of any of claims 10 to 16 wherein the corneal tissue after step (c) consists of all or substantially all of the Bowman's membrane and all or substantially all of the stroma.

18. The method of any of claims 10 to 16 wherein the corneal tissue after step (c) consists of all or substantially all of the Bowman's membrane, all or substantially all of the stroma, and all or substantially all of the Descemet's membrane.

19. The method of claim 10 wherein the Descemet's membrane and/or the Bowman's membrane of the donor are not intentionally removed from the corneal tissue.

20. The method of any of claims 10 to 19 wherein the corneal tissue comprises substantially the entire diameter of the donor's cornea.

21. The method of any of claims 10 to 20 wherein the stroma of the corneal tissue is of full thickness.

22. The method of any of claims 10 to 21 wherein the osmotic agent is a salt.

23. The method of any of claims 10 to 22 wherein the osmotic agent is a glucose polymer, such as dextran.

24. The method of any of claims 10 to 23 wherein the solution further comprises a drying protectant.

25. The method of any of claims 10 to 24 wherein the agitation in step (c) is intended to remove all or substantially all the cells in the epithelial layer and all the cells in the endothelial layer.

26. The method of any of claims 10 to 25 wherein the method does not include a step in which the tissue is lathed.

27. The method of any of claims 10 to 26 wherein the vacuum drying step is performed by vacuum evaporation.

28. An isolated dehydrated corneal tissue produced according to the method of any of claims 10 to 27.

29. An isolated dehydrated corneal tissue according to any of claims 1 to 9 or claim 28 for use in therapy.

30. An isolated dehydrated corneal tissue according to any of claims 1 to 9 or claim 28 for use in treating or preventing corneal blindness in a subject.

31. An isolated dehydrated corneal tissue according to any of claims 1 to 9 or claim 28 for use in treating a damaged, diseased or infected eye in a subject.

32. The dehydrated corneal tissue for the use of claim 31 wherein the tissue if for use in treating keratoconus, corneal melts, corneal ulcers and/or corneal perforation, and/or in tectonic support or anterior lamellar keratoplasty applications.

33. A method of treating a subject suffering from corneal blindness, the method comprising: obtaining isolated dehydrated corneal tissue according to any of claims 1 to 9 or claim 28; rehydrating the corneal tissue and transplanting the rehydrated corneal tissue into a subject in need thereof.

34. A method of treating a subject with a damaged, diseased or infected eye, the method comprising: obtaining isolated dehydrated corneal tissue according to any of claims 1 to 9 or claim 28; rehydrating the corneal tissue and transplanting the rehydrated corneal tissue into a subject in need thereof.

35. A kit comprising dehydrated corneal tissue according to any of claims 1 to 9 or claim 28, and a rehydration solution.

Description

[0074] For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying drawings, in which:

[0075] FIG. 1 shows the structure of an eye. FIG. 1A shows the location of the cornea, limbus and sclera within the eye. FIG. 1B shows the structure of the cornea in cross section including the epithelial layer (the epithelium), Bowman's membrane, the stroma, Descemet's membrane and the endothelial layer (the endothelium).

[0076] FIG. 2 shows the effect of low temperature vacuum evaporation on weight, transparency and metabolic activity of human corneal buttons. Human corneal buttons were dried after agitation in PBS, 5% dextran and compared to non-dried controls that had remained static or were agitated. Dried corneal buttons were rehydrated in PBS. FIG. 2A shows the change in weight of corneal buttons upon drying and rehydration for 3 hr. Data displayed as % of initial weight. FIG. 2B shows the change in transparency of corneal buttons after drying and rehydration. Data shown as percentage change in transparency from initial, represented as mean±SEM (n=5). FIG. 2C illustrates the metabolic activity of cells within corneal buttons measured over time after drying and rehydration. FIG. 2D shows LDH released from sample after rehydration versus as a percentage of the SDS-lysed control. Data for FIGS. 2A-2D are represented by mean±SEM (n=5). Statistical significance vs. non-dried static: *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001. FIG. 2E shows images of (i) Organ culture cornea prior to drying, (ii) 8.5 mm corneal button before drying, (iii) corneal button after drying, (iv) corneal button after drying and rehydration;

[0077] FIG. 3 shows the effect of low temperature vacuum evaporation on structure of human corneal buttons. Human corneal buttons were dried after agitation in PBS or in 5% dextran and compared to non-dried controls that had remained static or were agitated. Dried corneal buttons were rehydrated in PBS. FIG. 3A shows representative images of haematoxylin and eosin staining (upper row) and alcian blue and fast red staining (lower row) of sections of treated corneal buttons. Scale bar=200 μm. FIG. 3B shows the approximate collagen content of corneal buttons with and without drying and rehydration measured by hydroxyproline assay. FIG. 3C shows the approximate sGAG content of corneal buttons with and without drying and rehydration measured by DMMB assay. Data represented by mean±SEM (n=5);

[0078] FIG. 4 shows the effect of dextran on corneal swelling during washing and agitation before drying. Whole corneas with sclera were agitated with 100 mM Amaranth Red dye with and without 5% dextran, at 20° C. or 37° C., for 10 s, 5 min, 30 min, 1 h or 2 h. FIG. 4A shows the total absorbed Amaranth dye during treatment. FIG. 4B shows the change in weight of corneas after treatment, represented as percentage of initial weight. FIG. 4C shows the change in thickness of corneas after treatment, represented as a percentage of initial thickness. Data represented mean±SEM (n=3). Statistical significance vs. same timepoint in dextran at 20° C.: *p≤0.05, ***p≤0.001, ****p≤0.0001. FIG. 4D shows stereo microscope images of Amaranth red dyed corneas, incubated at 20° C. (upper rows). Same images manipulated to better show levels of swelling (lower rows). Scale bar=2 mm;

[0079] FIG. 5 concerns the optimisation of the low temperature vacuum evaporation drying time. Whole human corneas were agitated in 5% dextran in NaCl for 60 min before drying. Drying timepoints of 1 h, 2 h, 3 h, 4 h, 5 h, 9 h and 24 h were analysed with separate corneas. FIG. 5A shows stereo microscope images of corneas at different drying points. Scale bar=2 mm. FIG. 5B shows representative images of haematoxylin and eosin staining and FIG. 5C shows Alcian Blue and Fast Red staining. Scale bar=200 μm. FIG. 5D shows representative fluorescent immunohistochemistry images of sections of human corneas either non-dried or dried for 4 hours. Scale bar=100 μm. FIG. 5E shows the change in weight of corneas upon drying. Data displayed as % of initial weight. FIG. 5F shows the change in thickness of corneas upon drying. Data displayed as % of initial thickness. Data for FIGS. 5E and 5F represent mean±SEM (n=5). Statistical significance vs. initial: *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001;

[0080] FIG. 6 shows the effect of different rehydration solutions on final properties of dried corneas. Whole human corneas were agitated in 5% dextran for 60 min before drying for 4 hours. Rehydration was performed with different solutions for up to 24 h. FIG. 6A shows representative images of haematoxylin and eosin staining and FIG. 6B shows Alcian Blue and Fast Red staining after rehydration for 24 h. Scale bar=200 μm. FIG. 6C shows the change in dried cornea weight over time in different rehydration solutions. Data displayed as pre-drying weight. FIG. 6D shows the dried cornea weight after 24 h rehydration. Data displayed as % pre-drying weight. FIG. 6E shows the change in dried cornea thickness over time in different rehydration solutions. Data displayed as % pre-drying thickness. FIG. 6F shows dried cornea thickness after 24 h rehydration. Data displayed as % pre-drying thickness. FIG. 6G shows the effect of rehydration solution on dried cornea transparency. Data is shown as percentage change in transparency from initial transparency. Data for FIGS. 6C-6G represent mean±SEM (n=3). Statistical significance vs. pre-drying: *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001;

[0081] FIG. 7 shows the effect of prolonged storage prior to drying, and final structure of dried corneas. Whole corneas were stored in Optisol for up to 3 months before analysis. FIG. 7A is representative images of haematoxylin and eosin staining and FIG. 7B shows Alcian Blue and Fast Red staining after storage. Scale bar=200 μm. FIG. 7C shows representative images of collagen-I fluorescent immunohistochemistry after storage. FIG. 7D shows representative images of laminin fluorescent immunohistochemistry after storage. FIG. 7E shows merged images of collagen-I, laminin and DAPI. In FIGS. 7D-7E) the scale bar=100 μm. FIG. 7F shows the effect of storage prior to drying on cornea cell viability prior to drying. The data is represented as percentage of stored for <1 month. FIG. 7G shows the effect of storage prior to drying on cornea transparency. Data represented by change in transparency compared to corneas stored for <1 month. Data for FIG. 7G and FIG. 7H are represented by mean±SEM (n=4). Statistical significance vs. <1 month: *p≤0.05, **p≤0.01, ***p≤0.001. FIG. 7H shows macro-images of human corneas pre-drying, dried and rehydrated. FIG. 7I shows representative TEM images of cornea structure pre-drying and after drying and rehydration using optimized protocol. Scale bars: first column=5000 nm, second column=2000 nm; and

[0082] FIG. 8 shows the recellularisation of dried and rehydrated human corneas. Corneas were dried and rehydrated using the optimized protocol. Corneal buttons of 8 mm were recellularised with ihCEC, CSC or B4G12 corneal endothelial cells (Endo) for 7 days. FIG. 8A shows representative images of haematoxylin and eosin staining and FIG. 8B shows Alcian Blue and Fast Red staining after recellularisation. Scale bar=200 μm. FIG. 8C shows the relative proliferation and viability of cells grown on/in dried corneas. Data represented as % day 1 viability. FIG. 8D shows the effect of recellularisation on corneal opacity. Data represented as % non-cellularised control. Data for FIGS. 8C and 8D are represented by mean±SEM (n=5). Statistical significance vs. day 1: ***p≤0.001.

EXAMPLES

[0083] Methods

[0084] Human Tissue

[0085] Anonymised human corneas were obtained from SightLife (now CorneaGen, WA, USA) under a materials transfer agreement. All work was performed in a laboratory under a research license from the Human Tissue Authority. Informed consent was obtained from donors/relative prior to collection.

[0086] Preparation and Drying of Corneas

[0087] Human corneas were delivered in Optisol™ (Bausch+Lomb, NJ, USA) and stored at 4° C. until use. Corneal buttons were prepared by punching out the middle of a cornea using an 8.5 mm trephine. Whole corneas with sclera were processed for drying as received from the eye bank with no further trimming. Corneal samples were transferred aseptically to a scintillation vial containing the sterile agitation media of either: Phosphate Buffered Saline (PBS) or 5% (w/v) dextran 70 (Sigma-Aldrich, Dorset, UK) in 0.9% NaCl. Scintillation vials containing corneas were agitated on a rotator (Grant PTR-60) at 60 rpm for up to 120 minutes, at either room temperature or 37° C. depending on experiment. Post-agitation, corneal samples were aseptically transferred to 10 mL glass vials (Schott via Adelphi Healthcare Packaging, West Sussex, UK) and a bromobutyl lyophilisation stopper (West Pharmaceutical Services via Adelphi) placed loosely on each vial. Vials were transferred to a Alpha 1-4 LSC, Advanced Freeze Dryer, Christ Osterode and dried at a shelf temperature of 25° C., pressure of 1.5 mbar, condenser temp −55° C. to −80° C. for different lengths of time depending on experiments. Once dried vial stoppers were pushed down and aluminium flip-up, tear-off crimp seal applied (West via Adelphi). Rehydration of corneal buttons or whole dried corneas was performed by injecting 3 mL sterile rehydration solution into the vial.

[0088] Unless otherwise stated, the standard, optimised procedure for drying the corneas was agitation in 5% (w/v) dextran in NaCl at room temperature for 60 minutes, drying for 4 hours and rehydration in 5% (w/v) dextran in NaCl.

[0089] Measurement of Corneal Weight and Thickness

[0090] Corneas weights were taken using an Ohaus Adventurer Balance. Corneas were place on pre-weighed weighboats and the weight of weighboats subtracted. Cornea thickness was measured using digital callipers.

[0091] Transparency Measurement

[0092] Light transmittance through the corneas was measured using a CLARIOstar plate reader (BMG LABTECH, Buckinghamshire, UK) at 492 nm with 12 readings taken across each cornea and averaged.

[0093] Metabolic Activity Assay

[0094] Metabolic activity was measured using PrestoBlue™ Cell Viability Reagent (Invitrogen, ThermoFisher, UK). Samples were placed in a 24-well plate and covered in 10% (v/v) Presto Blue reagent in Hank's Balanced Salt Solution (HBSS, Gibco, ThermoFisher). The plate was immediately transferred to a CLARIOstar plate reader pre-set at 37° C. and fluorescence readings at excitation 560 nm/emission 590 nm were taken every 30 minutes for 150 min.

[0095] Lactate Dehydrogenase (LDH) Release Assay

[0096] The Pierce lactate dehydrogenase (LDH) assay kit was used to quantify levels of LDH released into the rehydration media of dried corneas, to estimate levels of cell membrane lysis. The assay was performed according to the manufacturer's protocol. Briefly, 50 μL of rehydration media and 50 μL of reaction mix were transferred to a 96-well plate and incubated at room temperature for 30 min. The optical absorbance was read on the plate reader at 490 nm with background correction at 690 nm. The maximum levels of LDH that could be released from a cornea was assessed using non-dried corneas that had been agitated in 1% (v/v) sodium dodecyl sulphate detergent at 37° C. for 24 hours.

[0097] Histology

[0098] Samples were fixed in 4% paraformaldehyde overnight, before washing and storage in PBS. Samples were prepared for sectioning in a Tissue Processor (Leica TP1020) through a series of graded ethanol solutions, then paraffin embedded. Sections (7 μm) were cut using a Leica 2245 microtome and transferred to adherent glass slides (SuperFrost Plus, ThermoScientific). Samples were de-paraffinised in xylene and rehydrated in a series a graded ethanol solutions. Regressive haematoxylin and eosin staining was performed using Harris Haematoxylin and 1% eosin. Alcian blue and fast red staining was performed to visualise acid mucosubstances and red cell nuclei. Slides were mounted in DPX after staining and imaging was performed on a Leica DM1000 upright microscope and MC170 Camera.

[0099] Hydroxyproline Assay

[0100] Hydroxyproline assays were performed as described previously (Edwards and O'Brien 1980) to estimate the levels of collagen within the corneas. Corneal buttons were digested in a 0.1 mg/mL papain solution in 0.2 M sodium phosphate buffer containing 8 mg/mL sodium acetate, 4 mg/mL ethylenediaminetetraacetic acid, and 0.8 mg/mL L-cysteine hydrochloride agitated at 65° C. overnight. Briefly, acid hydrolysis of papain digested samples was achieved by heating samples with concentrated hydrochloric acid to 120° C. for 5 hours. Subsequently, samples were dried at 80° C. until only residue remained, which was dissolved in 0.2 M sodium phosphate buffer. Samples were transferred in triplicate to a 96-well plate, an equal volume of 70 mM chloramine T solution was added and incubated at room temperature for 20 minutes. Subsequently, an equal volume of 1.16 M dimethylaminobenzaldehyde solution was added and samples incubated at 60° C. for 30 minutes. Colour change was assessed by absorbance at 540 nm. Hydroxyproline concentration was calculated using a standard curve. Collagen concentration was estimated using a conversion factor of 7.6. Collagen readings were corrected for the original weight of the corneal button.

[0101] Sulphated Glycosaminoglycans (sGAG) Assay

[0102] Corneal buttons were digested in papain as described above. The Blyscan™ 1,9 dimethyl methylene blue (DMMB) assay (Biocolor Ltd., Belfast, UK) assay was performed on samples according to manufacturer's instructions. Briefly, 200 μL of papain digest was added to 1 mL DMMB dye solution and agitated for 30 minutes, before centrifugation at 10,000×g for 10 minutes. The pellet was dissolved in 0.5 mL dissociation reagent and 200 μL transferred to each well of a 96-well plate. Absorbance was measured at 656 nm. sGAG concentration was determined using a standard curve. sGAG readings were corrected for original weight of the corneal button.

[0103] Amaranth Dye Absorption Assay

[0104] Amaranth Red dye was used to assess swelling of corneas before drying during pre-treatment with and without dextran at different times and temperatures. Solutions of 100 mM Amaranth Red (Sigma-Aldrich) in 0.9% NaCl and 100 mM Amaranth Red with 5% (w/v) dextran 70 in 0.9% NaCl were prepared. Whole corneas with sclera were place in the Amaranth Red solutions. Samples were incubated at either room temperature (20° C.) or 37° C. on a rotator set at 60 rpm. At required timepoints samples were removed from rotator, imaged using a Leica S6 D stereo microscope and with MC170 Camera, weighed, thickness measured and the Amaranth dye washed back out into 3 mL 0.9% NaCl on a roller. Total amount of Amaranth Dye was washed out was measured by reading absorbance at 520 nm and comparing to an Amaranth Dye standard curve.

[0105] Images of Amaranth Red dyed corneas were analysed using image J version 1.52a. Images were split into separate RGB channels, and the red channel image adjusted for brightness and contrast identically in all images.

[0106] Fluorescent Immunohistochemistry

[0107] Corneas were paraffin-embedded and sectioned as for histology. Samples were deparaffinised in xylene and rehydrated in a series of graded ethanol solutions. Antigen retrieval was performed in a pH 6.0 sodium citrate buffer (Vector) at 95° C. for 60 min. Sections were permeabilised in 0.1% (v/v) Triton-X100 for 10 minutes and subsequently washed three times for 5 minutes in PBS. Non-specific protein binding was blocked using a solution of PBS with 1% bovine serum albumin (BSA), 0.3 M glycine and 3% (v/v) donkey serum, for 1 hour at RT. Primary antibodies were diluted in PBS containing 1% BSA and 0.3 M glycine as follows: polyclonal mouse anti-Collagen-I (Sigma-Aldrich, dilution 1:200) and polyclonal rabbit anti-laminin (Millipore, dilution 1:100) Sections were incubated with the primary antibodies for 1 hour at room temperature before washing three times in PBS. Either donkey anti-mouse Alexa-Fluor 594 or donkey anti-rabbit Alexa Fluor 488 secondary antibodies (Life Technologies, dilution 1:300) were applied to the samples at room temperature for 1 hour. Samples were rinsed in PBS three times and counterstained with 4′,6-diamidino-2-phenylindole (DAPI; 1:200,000, Life Technologies). Samples were mounted in fluorescent mounting medium (Dako, UK) and imaged using a Leica DMIL LED inverted microscope with a Leica DFC camera.

[0108] Rehydration

[0109] Rehydration was performed by immersing corneas in 5 mL of various rehydration solutions: Optisol (Bausch+Lomb), 0.2% sodium hyaluronate (HYLO-FORTE Eyedrops, Scope, UK), contact lens saline (Tesco, UK), distilled water (dH.sub.2O), dH.sub.2O with 5% (w/v) dextran, dH2O with 10% (v/v) glycerol, 0.9% NaCl, 0.9% NaCl with 5% (w/v) dextran, Hank's Balanced Salt Solution (HBSS, Gibco, ThermoFisher, UK), and HBSS with 5% (w/v) dextran.

[0110] Transmission Electron Microscopy

[0111] Samples were fixed in 3% glutaraldehyde in sodium cacodylate buffer solution (0.2 M, pH 7.2) for 24 h at 4° C. Corneas were post-fixed with 1% osmium tetroxide in sodium cacodylate buffer solution for 2 h at room temperature, followed by washing in sodium cacodylate buffer (0.1 M, pH 7.2), serial dehydrations in ethanol, and washing in propylene oxide (TAAB Laboratories Equipment Ltd). The corneas were embedded in araldite resin (TAAB Laboratories Equipment Ltd) and sectioned (Leica EM UC6; Leica Biosystems). Ultrathin sections of 90 nm were contrasted with uranyl acetate and lead citrate and observed on a transmission electron microscope (TEM, FEI Tecnai Biotwin T12), operating at 100 kV. Images were taken using a SIS Megaview digital camera (Olympus).

[0112] Recellularisation

[0113] Preparation of Dried Corneas for Recellularisation

[0114] Dried whole corneas were rehydrated in sterile NaCl with 5% (w/v) dextran for one hour. A sterile trephine was used to cut out a central corneal button of 8.5 mm diameter. Prior to CSC injection seeding, corneas were swollen in dH2O for 1 hour.

[0115] Immortalised Human Corneal Epithelial Cells (ihCEC)

[0116] SV40-immortalised human corneal epithelial cells (ihCEC) [Araki-Sasaki, K., et al., Invest Ophthalmol Vis Sci, 1995. 36(3): p. 614-21] were cultured in supplemented basal epithelial cell medium EpiLife® containing 5 mL human keratinocyte growth supplement and 1% antibiotic-antimycotic (AbAm, Sigma-Aldrich). ihCEC were seeded on the basement membrane side at 1×10.sup.6 cells per corneal button, and cultured in 3 mL of supplemented EpiLife containing 5% (w/v) dextran.

[0117] Corneal Stromal Cells (CSCs)

[0118] Corneal stromal cells were extracted from human corneoscleral rims by enzymatic means and cultured as described previously in Stem Cell Medium (SCM) consisting of DMEM/F12 with Glutamax supplemented with 20% (v/v) knock-out serum replacement (KSR), 1% (v/v) non-essential amino acids, 4 ng/mL bFGF, 5 ng/mL hLIF (New England Biolabs, Hertfordshire, UK) and 1% AbAm [Sidney, L. E., et al. Cytotherapy, 2015. 17(12): p. 1706-1722]. For recellularisation, CSC were re-suspended in 250 μL SCM containing 5% (w/v) dextran, and injected through a 20 gauge hypodermic needle into the stroma of swollen corneas through the endothelial side. CSC injected into corneas were then cultured in SCM containing 5% (w/v) dextran.

[0119] B4G12 Corneal Endothelial Cells (Endo Cells)

[0120] The B4G12 corneal endothelial cell line was cultured in Endothelial Serum-Free Growth Medium (Endo-SFM, Gibco, ThermoFisher) supplemented with 10 ng/mL basic fibroblast growth factor (ThermoFisher) and 1% AbAm. Endo cells were seeded on the Descemet's side at 1×10.sup.6 cells per corneal button, and cultured in 3 mL of supplemented Endo SFM containing 5% (w/v) dextran.

Example 1

The Effect of Drying on Corneal Characteristics

[0121] The data presented herein demonstrates that the dried weight of corneas is approximately 20% of initial weight, regardless of the agitation media used.

[0122] Rehydration of corneas dried according the invention was shown to return the corneas back to their original weight within 3 hours (see FIG. 2A). Pre-treatment in had a significant negative effect on transparency of the corneas (see FIG. 2B).

[0123] Non-dried corneas are shown to retain significant metabolic activity in the cells (cell viability), and drying is shown to lead to a loss of this metabolic activity. When pre-treated with PBS or dextran metabolic activity is completely lost (cells are non-viable).

[0124] Measuring the amount of LDH released into the rehydration media crudely measures the number of lysed cells and therefore the level of cell membrane bursting. No difference was observed in dried versus the non-dried corneas, and when corneas were treated with an SDS lysis agent, levels of LDH released were significantly higher than in any other group (see FIG. 2D).

[0125] Corneal buttons look very similar after drying and rehydration to how they look pre-dried (see FIG. 2E).

Example 2

Corneal Structure

[0126] No apparent change in corneal structure was caused by drying and rehydration (see FIG. 3A). No difference in structure was observed when different pre-treatments were used. No loss of collagen or sGAG content was observed by drying and rehydration (see FIGS. 3B and 3C).

Example 3

The Effect of Dextran on Corneal Structure

[0127] Dextran is shown to prevent further absorption of dye after 30 minutes, therefore demonstrating its ability to prevent swelling of the cornea to let in more dye (see FIG. 4A). Without dextran present the cornea was observed to swell to beyond its original weight and thickness (see FIGS. 4B and 4C). The difference in the levels of dye penetrating the corneas can be seen in the images (see FIG. 4D).

[0128] The presence of dextran meant that the temperature of agitation had no effect.

Example 4

The Effect of Different Drying Times on Corneal Structure

[0129] Optimal drying time is shown in the data presented herein to be 4 hours. After 4 hours the weight/thickness of the cornea does not reduce any further. Drying for 24 hours is shown not to change the corneal structure (see FIGS. 5A to 5C, 5E and 5F). Dried corneas are observed to retain the basement/Bowman's membrane and Descemet's membranes (showed by laminin staining) but there are no epithelial or endothelial cells (see FIG. 5D).

Example 5

The Effect of Corneal Rehydration Solutions on Corneal Structure

[0130] Rehydration of a corneal structure with a solution containing NaCl and dextran produced a rehydrated tissue closest to initial weight and thickness and did not affect structure or transparency (see FIG. 6). Water alone was shown to cause significant swelling and loss in transparency on rehydration.

Example 6

The Effect of Storage Prior to Drying

[0131] Storage of corneas in Optisol™ for longer than one month prior to drying resulted in a loss of epithelial cells and endothelial cells, but not a loss of basement membrane or Descemet's (see FIGS. 7A to 7E). Cell viability is also lost over storage time (see FIG. 7F), but metabolic activity is maintained for 3 months. A loss of transparency over storage time was also observed (see FIG. 7G). Macro images show that rehydrated corneas look similar to those before drying. Pink staining of pre-drying is due to Optisol™ storage (see FIG. 7H). TEM images show the microstructure of collagen fibrils are not affected by drying and rehydration (See FIG. 7I). Stromal cells are encapsulated within the collagen fibrils.

Example 7

Analysis of Rehydrated Corneas

[0132] Dried and rehydrated corneas are biocompatible with all three major cellular types of the cornea. Epithelial cells attach to the basement membrane (see FIGS. 8A and 8B). Endothelial cells attached to the Descemet's membrane. CSCs fill pockets within the corneal stroma and proliferate within the cornea. Transparency is not significantly affected by recellularisation (see FIG. 8D).