Cold storage solution for organs and tissues including whole eyes and methods of use thereof

Abstract

Described herein is a cold storage solution for organs and tissues including a colloid, an impermeant, an ionic component, a nutrient, an antioxidant, a histone deacetylase inhibitor (HDAC inhibitor), a potassium channel blocker, an energy substrate for neurons, and a steroid, wherein the osmolality is 255 to 420 mOsm / kg. A method of preserving an organ or tissue includes cooling the cold storage solution to a temperature of 2 to 6°C and exposing the organ or tissue to the cold storage solution.

Description

P240239US01 107668.286 COLD STORAGE SOLUTION FOR ORGANS AND TISSUES INCLUDING WHOLE EYES AND METHODS OF USE THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application 63 / 566,646 filed on March 18, 2024, which is incorporated herein by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH & DEVELOPMENT This invention was made with government support under W81XWH-16-1-0775 awarded by the ARMY / MRDC. The government has certain rights in the invention. FIELD OF THE DISCLOSURE

[0001] The present disclosure is related to a cold storage solution for organ and tissue preservation, particularly for whole eyes and nervous tissue. BACKGROUND

[0002] Irreversible blindness has a significant socioeconomic impact worldwide. Causes include glaucoma, age-related macular degeneration, diabetic retinopathy, and trauma including battlefield trauma. It has been speculated that whole eye transplant (WET) could be a viable option to restore vision in some individuals. Impediments to the practicality of WET include the ability to adequately perfuse the transplanted tissue, immunogenic rejection, and the refractory nature of the retina including retinal ganglion cells (RGCs) and the regenerative capacity of their axons in the optic nerve. The critical issue of RGC survivability and regeneration remains a significant hurdle.

[0003] What is needed are cold storage solutions that can preserve RGC and potentially enable WET. BRIEF SUMMARY

[0004] In an aspect, a cold storage solution for organs and tissues comprises a colloid, an impermeant, an ionic component, a nutrient, an antioxidant, a histone deacetylase inhibitor (HDAC inhibitor), a potassium channel blocker, an energy substrate for neurons, and a steroid, wherein the osmolality is 255 to 420 mOsm / kg.P240239US01 107668.286

[0005] In another aspect, a method of preserving an organ or tissue comprises cooling the cold storage solution to a temperature of 2 to 6°C, and exposing the organ or tissue to the cold storage solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Figures 1A-L are images of whole eyes stored in modified UW solution (mUW solution) for 24 hours at 4°C, stained with Hematoxylin and Eosin (H&E). These images are representative of the images that were used for evaluation in the scoring system listed in Table 4. The Optimal mUW Solution had the best overall performance, and is the only solution, aside from the baseline, that did not demonstrate retinal detachment after cold storage. Size bar = 1 mm.

[0007] Figures 2A-M show corneal thickness was measured using ImageJ software and compared against the baseline. 2A-L representative images of the corneas from all mUW solutions tested. Size bar = 300 µm. 2M: A graph showing the average corneal thickness from 3 samples each of all solutions. Data from eyes stored in phosphate buffered saline (PBS) is also included (Grey bar). All groups exhibited retinal swelling in the extracellular regions of the stroma (***ANOVA, p<0.0001). Optimal mUW demonstrated the least swelling of all groups tested but was still significantly greater than baseline corneas (t-test, ***p<0.0001).

[0008] Figures 3A-L show immunostaining of retinas for RBPMS (a retinal ganglion cell specific marker) in freshly isolated eyes (3A) or eyes kept in cold storage for 24 h after injection of UW solution (3B) or modified UW solutions containing the supplement(s) indicated (see Table 2) (3C-L). Optimal mUW solution contained valproic acid (VPA), tetraethylammonium (TEA), hydrocortisone, and L-lactate. The ganglion cell layer (GCL) and robust RBPMS staining appeared relatively normal in all conditions tested. Size bar = 50 µm. ONL, outer nuclear layer. INL, inner nuclear layer. 4’,6’-diamidino-2-phenylindole (DAPI) counterstain.

[0009] Figures 4A-L show immunostaining of retinas for RHO (a rod photoreceptor cell specific marker) in freshly isolated eyes (4A) or eyes kept in cold storage for 24 h after injection of UW solution (4B) or modified UW solutions containing the supplement(s) indicated (see Table 2) (4C-L). Optimal mUW solution contained VPA, TEA, hydrocortisone, and L-lactate. RHO staining is most prominent in the outer segments (OS) of the rod photoreceptors, a pattern that was relatively preserved in each of the test conditions, although reduced intensity was noted in both hydrocortisone and L-Lactate conditions. TheP240239US01 107668.286 intensity was robust, however, in optimal mUW which contained both supplements. Size bar = 50 µm. IS, inner segments, ONL, outer nuclear layer. INL, inner nuclear layer. DAPI counterstain.

[0010] Figures 5A-L show immunostaining of retinas for PKC^ (a rod bipolar cell specific marker) in freshly isolated eyes (5A) or eyes kept in cold storage for 24 h after injection of UW solution (5B) or modified UW solutions containing the supplement(s) indicated (see Table 2) (5C-L). Optimal mUW solution contained VPA, TEA, hydrocortisone, and L-lactate. Rod bipolar cell nuclei reside in the outermost region of the inner nuclear layer (INL) and send axons to the dendritic arbors of the retinal ganglion cells in the ganglion cell layer (GCL). Compared to baseline retinas, these cells appeared disorganized and abnormal in mUW solutions containing sunitinib (5C), SB203580 (5D) and TEA (5G). Retinas from eyes stored in Optimal mUW solution, which contains TEA, exhibited cells virtually identical to baseline retinas (5L). Scale bar = 25 µm. ONL, outer nuclear layer. DAPI counterstain, which is shown as a separate channel on the left of each image so that bipolar cell morphology can be clearly shown.

[0011] Figures 6A-L show immunostaining of retinas for GFAP (a cell specific marker for astrocytes and Müller cells, the latter being in a reactive state) in freshly isolated eyes (6A) or eyes kept in cold storage for 24 h after injection of UW solution (6B) or modified UW solutions containing the supplement(s) indicated (see Table 2) (6C-L). Optimal mUW solution contained VPA, TEA, hydrocortisone, and L-Lactate. Astrocytes reside on the surface of the retina near the ganglion cell layer (GCL). Overall, cold storage in UW solution and most of the supplemented solutions resulted in a decrease or absence of GFAP staining except for (6J) hydrocortisone, (6K) L-lactate, and (6L) Optimal mUW solutions. The combination of VPA and BaCl2 (6I) appeared to induce increased expression in cells restricted to the astrocyte layer, while L-Lactate also contained sporadic examples of Müller cell end feet processes containing GFAP (arrows in 6K). Size bar = 50 µm. ONL, outer nuclear layer. INL, inner nuclear layer. DAPI counterstain.

[0012] Figure 7 shows an assessment of cell-type specific mRNA abundance in retinas, 24 h after cold storage. Heat map showing the relative change in transcript abundance compared to freshly isolated and prepared eyes. The heat scale bar on the right reflects the ratio of mean mRNA levels of experimental eyes divided by the same transcript level measured in the baseline eyes. Genes included in the mini-array are detailed in Table 4. qPCR results demonstrate a general decrease in mRNA abundance of all marker genes in allP240239US01 107668.286 mUW groups, with the exception of VPA. The effect of VPA, when in combination with other supplements (including Optimal mUW) was not retained, however. RGCs, retinal ganglion cells. PRs, photoreceptors.

[0013] The above-described and other features will be appreciated and understood by those skilled in the art from the following detailed description, drawings, and appended claims. DETAILED DESCRIPTION

[0014] Described herein are cold storage solutions for tissues and organs, particularly cold storage solutions that can stabilize the retina, and in particular the RGC population in donor eyes.

[0015] RGCs are long projection neurons of the central nervous system. In mammals, damage to the RGC axons in the optic nerve initiates the activation of the intrinsic apoptotic program in these cells making the process of removing a donor eye seemingly incompatible with a WET strategy. Additionally, mammalian RGCs have a reduced regenerative capacity relative to cold-blooded vertebrates. Advances in the understanding of the signaling pathways associated with axonal damage may bring some promise to overcoming these critical problems. Axonal damage causes a variety of molecular changes in RGCs, including early apoptotic signaling of the Dual Leucine Zipper Kinase (DLK) – Jun-N-Terminal Kinase (JNK) axis, activation of histone deacetylases (HDACs) which contribute to down-regulation of normal RGC gene expression, and efflux of potassium ions which lead to cell shrinkage and atrophy. Additionally, optic nerve damage leads to changes in other cell types of the retina, particularly micro- and macroglia, which may lose their homeostatic support functions of retinal neurons and contribute to the overall pathology of the retina. Studies have shown some levels of therapeutic benefit by targeting each of these early events in animal models of acute optic nerve damage.

[0016] Described herein is the development of a cold storage solution that could be applied to the donor eye which would stabilize the retina, particularly the RGC population, and preserve it so that a secondary treatment could be applied to induce RGCs to begin regenerating their axons after transplantation. While the experiments herein were performed as augmentations of a standard cold storage solution that is commonly used for donor organ preservation sold as UW® Cold Storage Solution (previously sold under the trade name Viaspan, hereinafter “UW solution”), solutions with properties similar to UW Solution can be used in its place as described herein. Advantageously, UW Solution and its equivalentsP240239US01 107668.286 maintain the functional capacity of individual organs for several days prior to transplantation by keeping cells in metabolic homeostasis while providing a controlled osmotic environment with physiological levels of potassium and sodium ions. While UW Solution has been used successfully to preserve peripheral nerves in allografts and in hand and limb transplantation, UW Solution has not been tested as a cold storage preservative for tissues of the central nervous system.

[0017] The examples show the efficacy of UW Solution alone, or with supplements designed to neuroprotect RGCs, in the architectural preservation of retinas in enucleated eyes to develop a cold storage preservative solution for donor eyes used in WET procedures. Based on a combination of histopathology and individual cell type immunostaining and RNA analysis, a modified UW solution was developed containing supplements directed at reducing K+efflux and HDAC activity, combined with components that are designed to help augment astrocyte function, which appears to be critically suppressed during cold storage.

[0018] In an aspect, a prior art cold storage solution comprises a colloid, an impermeant, an ionic component, a nutrient, and an antioxidant, wherein the cold storage solution has an osmolarity of 255 to 420 mOsm / kg, specifically 320 mOsm / kg.

[0019] Cold storage solutions include a colloid such as a polyethylene glycol, a dextran (dextran 40), and / or a hydroxyethyl starch, to minimize interstitial edema during storage. A preferred colloid is hydroxyethyl starch having a weight average molecular weight of from about 150,000 to about 350,000 daltons, and a degree of substitution of from about 0.4 to about 0.7. A more preferred colloid is hydroxyethyl starch having a weight average molecular weight of from about 200,000 to about 300,000 daltons. Pentafraction, an exemplary hydroxyethyl starch, is a solution of biodegradable hydroxyethyl starch macromolecules with molecular weights of 10 to 100 X 104Da.

[0020] Cold storage solutions include one or more impermeants. Hypothermic- induced cell swelling is due to the accumulation of water. This tendency to swell can be counteracted by adding 110 to 140 mmol (110 to 140 mOsm / kg osmotic force) of substances that are impermeable to the cell (impermeants). The solution also includes one or more impermeants which reduce cell swelling in the solution. Exemplary impermeants include lactobionate, raffinose, trehalose, glucose, mannitol, and combinations thereof, specifically lactobionate and raffinose.

[0021] Cold storage solutions contain ionic components such as ions, buffers, and / or pH adjusting agents to mimic intracellular / extracellular fluid, buffer, and / or balance osmolality. Phosphate and magnesium are ions that are important for many cellularP240239US01 107668.286 functions. Phosphate ions can be provided in the form of sodium or potassium salts, preferably monobasic and / or dibasic potassium phosphate. Magnesium can be provided in the form of magnesium sulfate heptahydrate. Additional ions can include calcium, chloride, sulfate, carbonate, citrate and the like, and combinations thereof. pH adjusting agents include sodium hydroxide and / or hydrochloric acid.

[0022] Cold storage solutions also include nutrients such as tryptophan, adenosine, adenine, glutamic acid, histidine, ketoglutarate, and the like, and combinations thereof. Adenosine triphosphate (ATP) rapidly degrades during hypothermic storage, and this degradation results in the formation of end products (adenosine, inosine, and hypoxanthine) to which the plasma membrane is freely permeable. Organ reperfusion necessitates the rapid regeneration of Na-pump activity, which requires ATP. The availability of ATP precursors, therefore, may be important for successful organ preservation. An exemplary nutrient is adenosine.

[0023] Cold storage solutions also include antioxidants to protect from reactive species such as reactive oxygen and nitrogen species. Exemplary antioxidants include glutathione, allopurinol, vitamin E, tryptophan, and thioredoxin.

[0024] UW® solution, for example, has the composition: Table 1: Composition of UW® solution UW® solution Component Concentration

[0025] The modified sold storage solution described herein has the composition shown in Table 2:P240239US01 107668.286 Table 2: Exemplary Modified Cold Storage Solution General component Specific components Concentration Colloid Pentafraction 25-100 g / L, specifically 50 7 M M .5P240239US01 107668.286

[0026] In the modified storage solution, HDAC inhibitors were included because HDAC activity is selectively up-regulated in damaged neurons including several retinal cell types such as RGCs. HDAC inhibitors include trichostatin-A, valproic acid, trapoxin, RGFP966 (CAS NO.1357389-11-7; (2E)-N-(2-Amino-4-fluorophenyl)-3-[1-(3-phenyl-2- propen-1-yl)-1H-pyrazol-4-yl]-2-propenamide), entinostat (MS-275; (pyridin-3-yl)methyl 4- (2-aminophenylcarbamoyl)benzylcarbamate), and combinations thereof.

[0027] Potassium channel blockers were included because one of the earliest pathological events associated with neuronal death is an efflux of cellular K+ions that leads to a phenomenon known as the apoptotic volume decrease. Studies using agents that block the K+efflux show that they provide resiliency to neurons in the face of an apoptotic challenge. Potassium channel blockers include tetraethylammonium, barium chloride, dalfampridine, dofetilide, amiodarone, and combinations thereof.

[0028] An energy source for neurons was added to provide the transplanted cells with immediate access to an energy substrate and provide a temporal buffer to allow macroglia to regain their normal homeostatic function. This augments the addition of adenosine in UW solution, which was added in an effort to provide preserved cells a necessary building block to begin de novo ATP synthesis once they were removed from the stasis condition of cold storage. Exemplary energy substrates for neurons include lactate, pyruvate, and acetylCoA.

[0029] A steroid was added to help retain GFAP expression, and possibly increase the regenerative potential of the RGCs in the explant. Exemplary steroids include hydrocortisone, cortisone, prednisone, dexamethasone, and combinations thereof.

[0030] The modified cold storage solution described herein may include additional components. A trophic factor may be added to the cold storage solutions to extend the lifespan of RGCs after acute optic nerve damage. Exemplary trophic factors include brain derived neurotrophic factor (BDNF), glial cell-line derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), and combinations thereof.

[0031] A tetracycline derivative can be added to attenuate microglial reactivity. Exemplary tetracyclines include minocycline, doxycycline, demeclocycline, methacycline, and combinations thereof.

[0032] The cold storage solutions described herein can be used for preservation of harvested organs and tissues prior to transplantation. In an aspect, the cold storage solutions are used for static storage of harvested tissues and organs. Preferably, the cold storage solution can maintain tissue and organ viability for 24 hours or even longer. In an aspect, a method of preserving an organ or tissue comprises exposing the organ or tissue to the coldP240239US01 107668.286 storage solution described herein. Exposing can include perfusing the organ or tissue with the solution, followed by cold storage of the flushed organ or tissue. The perfusate may optionally include 2-5 units / mL of heparin. Preferably, the cold storage solution is cooled to a temperature of 2 to 6°C prior to perfusing the organ or tissue, and then the organ or tissue is stored in the cold storage solution at similar temperatures. Exemplary organs for preservation with the solution described herein include organs with extensive nervous tissue such as the brain, spinal cord, peripheral nerves, and the eye. In addition to the whole eye, the cold storage solution can be used to preserve surrounding tissues such as in a hemifacial replacement including the eye. The cold storage solution can also be used to preserve tissues of the central nervous system such as nerves.

[0033] The invention is further illustrated by the following non-limiting examples. Examples Methods

[0034] Animals: Mice were used for these experiments. All mice were handled in accordance with the Association for Research in Vision and Ophthalmology statement for the use of animals for research. Experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Wisconsin and the Animal Care and Use Review office of the Department of Defense. A random mixture of male and female CB6 / F1 mice, between the ages of 2-4 months, were used for experiments. N=3 mice per group. For studies examining the activation response of NF^B, the cis-NF^BEGFPreporter line on a C57BL / 6 was used (a gift from Dr. Christian Jobin at the University of North Carolina).

[0035] Intravitreal Injection of mUW Solution and Storage of Enucleated Whole Eyes: Mice were euthanized by pentobarbital overdose. Prior to enucleation, the head of the mouse was bathed in 7.5% Povidone-iodine (Betadine®, Purdue Products, LLP, Stamford, CT). Eyes were enucleated and the surrounding tissues were cut away with a micro-scissors. For intravitreal injections, a 30 gauge needle was used to puncture the globe at the limbus, to gain access to the vitreous chamber. A NanofilTMSyringe with a 35 gauge NanofilTMneedle (World Precision Instruments, Sarasota, FL) was then used to inject 6 µL of either UW solution (Bridge to Life Ltd., Northbrook IL), phosphate buffered saline (PBS, 50 mM phosphate, 150 mM NaCl, pH 7.4) or UW Solution containing supplements (modified UW solution – mUW) into the vitreous chamber. The needle was held in place for 15 seconds toP240239US01 107668.286 minimize backflow of the solution through the needle hole. Eyes were then fully submerged in ice-cold UW solution and stored at 4°C for 6- 24 hours. Freshly enucleated eyes were used for Baseline control samples. These were frozen or fixed for further analyses immediately following enucleation and puncture with a 30g needle into the vitreous chamber.

[0036] Supplements Tested in Modified UW solution: Table 3 shows a summary of the individual supplements tested and the rationale and concentration. Supplements included the kinase inhibitors sunitinib and SB203580, the HDAC inhibitors trichostatin A (TSA) and valproic acid (VPA), the K+channel blockers tetraethylammonium (TEA) and BaCl2, the steroid hydrocortisone, and the energy substrate L-lactate. The final concentrations of each supplement were chosen based on effective doses published for in vivo experiments in the literature. Stock solutions were stored at -20°C, -80°C (hydrocortisone) or 4°C (L-lactate). A stock solution was not created for BaCl2, as it precipitated out of solution at higher concentrations. The correct amount of crystalline BaCl2was added directly to one milliliter of UW Solution at the time of use. Osmolality of mUW solutions were measured in a VAPRO® vapor pressure osmometer 5520 (Wescor, Logan, UT) according to the manufacturer. The pH of mUW solutions were measured using an UltraBASIC UB-10 pH meter (Denver Instrument Co., Arvada, CO). Table 3: Individual supplements tested Test compound category Example Concentration Pan-kinase inhibitor Sunitinib 1 µM

[0037] Quantitative Reverse-Transcription Polymerase Chain Reaction (qPCR): After cold storage, whole eyes, except for the optic nerve and surrounding tissue that were removed previously, were frozen in dry ice and stored at -80˚C until processed. RNA isolation was completed using the ‘RNA / DNA / Protein Extraction Kit’ with RNase-Free DNase-1 Set (IBI Scientific, Dubuque, IA). Frozen whole globes were submersed in 400 µLP240239US01 107668.286 of lysis buffer (DR Buffer, IBI Scientific) and 4 µL of 2-mercaptoethanol (Bio-Rad Laboratories, Hercules, CA) was added to each sample and incubated on ice for 5 minutes. The samples were then sonicated 3x10 pulses, with a 2-3 minute cooling period on ice in between each 10 pulse set. The samples were then incubated at room temperature for 5 minutes. Total RNA was extracted according to kit protocol. RNA from each treatment group was pooled, and RNA concentration and quality was determined using a Thermo NanodropTMOne-C spectrophotometer (Thermo Fisher Scientific, Waltham, MA). RNA was aliquoted and stored at -80°C until cDNA synthesis was performed.

[0038] For qPCR, 2 µg of total RNA from each treatment group was used combined with MMLV- reverse transcriptase, 10 mM dNTPs, and Oligo(dT) (Promega, Madison, WI) to create cDNA. The cDNA was then diluted 10-fold, and 55 µL of cDNA from each group was mixed 1:1 with TaqMan™ Gene Expression Master Mix (Thermo Fisher Scientific) before being loaded into one of eight wells on a customized TaqMan™ Custom qPCR Array Card (Thermo Fisher Scientific). Each sample was run in triplicate. Target genes on this card are listed in Table 4. Quantitative PCR analyses were then performed using the QuantStudioTM7 Flex system (Thermo Fisher Scientific) and a qPCR procedure of 40 cycles of 15 seconds at 95°C, and 60 seconds at 60°C. Changes in the quantity of mRNA of each gene of interest between the samples and baseline were quantified using the ΔΔCt method, using the 18S rRNA abundance for each sample to normalize the total amount of cDNA and then calculating fold change between the experimental and baseline control retinas for each group. Table 4: Gene mRNA targets interrogated using quantitative RT-PCR (qPCR) microfluidic cards. Quantitative RT-PCR Targets Gene Name Gene of Interest FunctionP240239US01 107668.286 Glial Fibrillary Acidic Macroglial (astrocytes and Protein Müller cells). Upregulated in Gfa stressed retinas s. gBetaIII Tubulin expression is not exclusive to RGCs, but is most abundant in these cells and therefore is commonly used as a marker for RGCs. ***Red and green opsins have nearly identical sequences so it is likely that both cell types are interrogated by qPCR of Opn1mw, which is the specific name for the green cone opsin gene. **** Allograft Inflammatory Factor 1 is also named Ionized Calcium Binding Adaptor Molecule 1 (IBA1).

[0039] Paraffin sectioning, Frozen sectioning, H&E and Immunofluorescence staining: In preparation for paraffin sectioning, whole eye samples were fixed in 2% glutaraldehyde and 4% paraformaldehyde in PBS at 4°C overnight and then embedded in paraffin. Samples were then sectioned and stained with hematoxylin and eosin (H&E). The H&E-stained samples were then imaged with a Zeiss Axioimager Z2 upright microscope (Carl Zeiss, Oberkochen, Germany) and processed with Zen Blue image analysis software (v2.3, Carl Zeiss). Digital images were then evaluated using a four-point scoring system evaluating the integrity of multiple regions of the eye. Images were evaluated for: (i) Protein Leakage in Vitreous and Anterior Chambers: The amount of protein leakage in the vitreal and anterior chambers was ranked to quantify the level of damage to the eye (by staining intensity), with no protein accumulation having a score of one (baseline control) and the most severe accumulation within the data sets ranked as a four; (ii) Lens Distress: Lens structural quality and integrity was ranked on a scale of one to four, with one being equal / similar to the integrity of the lens of the baseline control (most intact), and a 4 being the most severe lens disintegration / condition within our data set (least intact); (iii) Retinal Edema: Healthy retinas exhibit very low levels of edema. Retinal edema was scored based on the number of swollen cells in the retinal nuclear and plexiform layers and the nerve fiber layer, with a score of one correlating to that of the retinas most similar to the baseline, and four representing retinas with the most swollen cells / edema within our data set. Since there was not a noticeableP240239US01 107668.286 difference in the amount of edema present between the individual layers of each retina, retinal edema was consolidated to a single category; (iv) Retinal Detachment: Retinal detachment occurs when the outer segment of the retina is detached from the retinal pigmented epithelium (RPE). This would be a significant obstacle to WET, as it would prevent proper visual function. Retinal detachment was scored using a binary scale consisting of either zero, which represented a retina, without detachment of the outer segment from the RPE, or one, which indicated detachment of the outer segment from the RPE.

[0040] Corneal thickness was determined with the ImageJ software measurement tool. Five separate measurements from 1 image each of 3 different eyes of the same treatment group. The average of the 15 measurements from each group was plotted on a graph and an unpaired t-test was performed to determine significance between 2 individual groups while a one-way Analysis of Variance (ANOVA) was used to compare multiple groups.

[0041] For immunofluorescence, whole eye samples (n=3 eyes / group) were fixed in 4% paraformaldehyde in PBS for 1 h at room temperature, and then infiltrated with 30% sucrose in PBS overnight at 4°C, before embedding in Optimal Cutting Temperature media (Scigen, Paramount, CA) and frozen on dry ice. Eight µm sections were taken through the equator of the globe to visualize all ocular components. Sections were incubated in PBS for 10 minutes at room temperature and blocked with 2% goat serum in 0.3% TritonTM-X- 100 / PBS overnight at 4°C in a humidified chamber. Slides were then treated with a primary antibody in blocking solution at a concentration of 1:1000. Primary antibodies were anti- RNA-binding protein with multiple splicing (cat# PA5-31231, RBPMS, Invitrogen Corporation, Rockford, IL), anti-glial fibrillary acidic protein (cat# PA1-10019, GFAP, Invitrogen Corporation, Rockford, IL), anti-rhodopsin (cat# MAB5356, RHO, Millipore Sigma, St. Louis, MO) or anti-protein kinase C^ (cat# sc-208, PKC^, Santa Cruz Biotechnology Inc., Dallas, TX). Slides were incubated either overnight (anti-RBPMS, anti- rhodopsin, and anti-PKC) or for 3 days (anti-GFAP) at 4°C in a humidified chamber. Slides were washed three times in PBS before applying the appropriate Alexa Fluor® 488 (Jackson Immuno-Research, West Grove, PA) conjugated goat anti-rabbit (anti-RBPMS, anti-GFAP) or goat anti-mouse (anti-PKC^, anti-rhodopsin) IgG. Slides were then incubated overnight at 4°C in a humidified chamber. Slides were washed in PBS three times at room temperature and mounted with Vectashield® containing 4′6-diamidino-2-phenylindole (DAPI) and Fisherbrand®Microscope cover glass (Thermo Fisher Scientific) before imaging with a ZeissP240239US01 107668.286 Axioimager Z2 upright microscope and processed with Zen Blue image analysis software (v2.3).

[0042] DNA fragmentation assay using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL): TUNEL staining was performed using the DeadEndTMFluorometric TUNEL System (Promega). Fixed eyes were embedded and frozen sections were obtained as described above. As a control, some sections were treated with 10 units / ml RQ1 DNase I (Promega) for 20 minutes at room temperature. Slides were then stained according to the manufacturer’s protocol. After washing, slides were mounted with VectashieldTMand imaged as described above. Example 1: Initial studies with UW® Solution

[0043] Initial experiments compared the effects of cold storage of mouse eyes emersed in either PBS or unmodified UW® Solution. Straight emersion of globes into either solution resulted in varying levels of retinal edema (assessed by histopathology), within 6 hrs for PBS and 24 hrs for UW Solution. This effect was mitigated for both solutions if they were directly injected into the vitreous cavity immediately after enucleation (data not shown) although edematous regions of the retina, optic nerve, and cornea persisted in eyes injected with PBS and incubated for 24 hrs (data not shown). Active cell death in the retina was examined using TUNEL staining and no evidence of nuclear DNA degradation was found in either PBS or UW treated retinas at any time point up to 24 hrs (data not shown). Finally, eyes from cisNF^BeGFPtransgenic reporter mice were used to assess regions exhibiting cellular ischemic responses. Only eyes treated with PBS exhibited any evidence of NF^B activation and this was restricted to the sclera of the limbus near Schlemm’s canal (data not shown). Example 2: Characteristics of modified UW (mUW) solutions

[0044] After addition of all test reagents, the osmolality and pH of the mUW solution was measured to determine if addition of different compounds affected the physiological parameters of the starting UW solution. Table 5 shows that most test mUW solutions moderately increased the osmolality of the starting solution with the most dramatic effect observed for 15 µM TSA. All solutions retained a pH of 7.4. The addition of BaCl2to UW Solution caused a marked increase in turbidity, possibly as a consequence of the chelatingP240239US01 107668.286 characteristic of lactobionate, which is known to chelate both iron and calcium ions. Nevertheless, this solution, and the combination of VPA and BaCl2was also tested. Table 5: Osmolality of mUW Test Solutions at 20˚C. All solutions, including UW solution and PBS were at pH 7.4 Treatment: Osmolality (mOsmol / kg)Example 3: Histopathology of eyes after cold storage

[0045] H&E staining was performed for histopathological analyses of all treatment groups. Longitudinal sections of whole eyes were used to evaluate eyes from each treatment group in several categories, including the relative amount of protein accumulation in the vitreous and anterior chambers, the condition of the lens, the relative amount of edema in the cornea, the relative amount of edema in the retina, and retinal detachment from the RPE (Figure 1A-L). Higher magnification images of the retinas, shows that each test solution maintained retinal architecture that was substantially unchanged from the baseline (data not shown). In each category, the eyes were scored on a 4-point scale with 1 showing no abnormal appearance and 4 showing extensive pathology, with the exception of retinal detachment which was scored as either completely attached (“0”) or detached in a majority of the section (“1”). The individual scores for each test reagent are shown in Table 6. A final score was calculated from the sum of all other scores in order to determine the reagents withP240239US01 107668.286 the best overall performance. The baseline control received a final score of “4”. The top five performing solutions were hydrocortisone, VPA, TEA, L-lactate, and BaCl2. All five outperformed UW solution alone. Eyes injected with hydrocortisone, TEA, or L-lactate did not exhibit protein leakage into the vitreous chamber. Hydrocortisone, TEA, VPA, and BaCl2showed mild leakage into the anterior chamber, while L-lactate had moderate protein leakage into this chamber. Retinal edema was moderate in eyes injected with TEA and mild in eyes injected with VPA, L-lactate, and hydrocortisone. Eyes treated with BaCl2had levels of retinal edema similar to that of the baseline control. The remaining groups, VPA / BaCl2, sunitinib, TSA, and SB203580 performed worse than plain UW solution and exhibited severe pathology in at least one of the 5 areas analyzed. These groups were not considered as candidates for the optimal solution. Table 6: Pathology scores of H&E stained sections of globes incubated in cold storage for 24 hours Group Protein in Protein in Lens Retinal Retinal Final Vitreous Anterior Distress Edema Detachment Score

[0046] The reagents in the Optimal mUW Solution (Table 7) were chosen based on their ranking relative to the Baseline. TEA, a potent pan-HDAC inhibitor, was substituted for BaCl2to eliminate the increase in turbidity associated with BaCl2. The combination of reagents in the optimal solution yielded histopathology scores that were similar to the baseline in all areas, with the exception of mild protein leakage into the anterior chamber.P240239US01 107668.286 Remarkably, the retinas of all tested eyes treated with the Optimal mUW Solution remained attached to the RPE layer. This characteristic appeared to be a function of L-lactate, since eyes treated with Optimal mUW solution lacking L-lactate again exhibited retinal detachment (data not shown). Overall, the combination of test reagents in the Optimal mUW Solution performed better than any of the individual components alone. Table 7: Optimal mUW Solution Optimal mUW Solution Valproic acid 2 mMthickness (Figure 2A-M) (P<0.0001, 1-way ANOVA, for all test reagents relative to baseline and P<0.0001 for Optimal mUW Solution relative to baseline, t-test). Importantly, the increase in thickness was not attributable to the swelling of epithelial, stromal, or endothelial cells that were commonly detected in PBS treated eyes (data not shown). Instead, swelling appeared to be restricted to extracellular regions of the stroma. Example 4: Immunostaining for retinal cell type-specific markers in eyes after cold storage

[0048] Frozen retinal sections were stained for RPBMS (RGCs), PKC^ (rod bipolar cells), GFAP (astrocytes and Müller cells), and RHO (rod photoreceptors) to assess the effect of cold storage solutions on presence of markers of different retinal cell types. RBPMS (Figure 3A-L) and RHO (Figure 4A-L) staining was relatively uniform across all the different test solutions and not markedly changed from the baseline. Similarly, staining for PKC^ was present in all test reagents, although cell morphology was considered sub-optimal in the sunitinib, SB203580, and TEA supplemented reagents, which were marked by fewer cells being stained and disorganization of the dendritic arbor regions of cells that were positive (Figure 5A-L). This effect was not carried over in the Optimal mUW Solution, which contained TEA. The most prominent difference in staining was for GFAP, which was generally reduced or absent across most of the test reagents with the exception VPA+BaCl2and L-lactate (Figure 6A-L). Importantly, there was sporadic evidence of GFAP upregulation in Müller cells in the L-lactate condition, suggesting that a minority of these cells were undergoing reactive gliotic changes in response to cold storage. This effect was not observedP240239US01 107668.286 in Optimized mUW Solution, which exhibited GFAP staining in astrocytes similar to the baseline condition. Example 5: Assessment of cell-type specific mRNA abundance in retinas after cold storage

[0049] In addition to immunostaining, the change in mRNA abundance was monitored for several retina cell-types using qPCR. In most test reagents, there was a decrease in mRNA levels for all target genes, with the exception of mUW solution that was supplemented with only VPA (Figure 7). This effect of VPA was not carried over to either UW solution modified with both VPA and BaCl2, or to the final Optimized mUW Solution. Notably, the decrease in transcript abundance was not appreciably reflected in a loss of immunostaining for many of the same gene products, suggesting that most immunostaining was of latent protein that had not been turned over and that mRNA levels may be a more sensitive quantitative measurement of cells that had entered a period of stasis while in cold storage. Discussion

[0050] Described herein are modified cold storage solutions exemplified by modifications of UW Solution that were both designed to mitigate apoptotic signaling within the RGCs so that they survive and to preserve the associated cells in the retina so that they could function normally once the transplant was complete. Additional components were evaluated for overall retinal architecture by both classical histopathology and by selectively immuno-staining for cell type specific antigens. Transcript abundance of a variety of cell- type specific markers was also evaluated for either dramatic increases or catastrophic decreases in abundance as an indicator of overall retinal health.

[0051] Overall, eyes stored in any of the test solutions, including PBS, showed no indication of active cell death (as assessed by TUNEL) or a retinal inflammatory response. Cells in the limbal region of the globe did exhibit upregulation / activation of NF^B activity in eyes stored in PBS, but this was mitigated by using UW Solution or mUW solutions. Other signs of histopathology, notably the formation of edematous pockets throughout the tissues of the globe, were principally evident only in solutions such as PBS, that did not have cell impermeant agents that prevent cell swelling.

[0052] Several of the components were specifically tested based on their known effects in animals that have had acute optic nerve damage, which were reasoned to mostP240239US01 107668.286 closely mimicked the condition of a transplanted globe with normal perfusion. HDAC inhibitors were selected because HDAC activity is selectively upregulated in damaged neurons including several retinal cell types such as RGCs. Importantly, inhibition of HDAC activity is strongly protective in a diverse range of neurodegenerative conditions including RGCs in models of acute optic nerve damage. Of the two pan-HDAC inhibitors tested (VPA and TSA), VPA was selected because it yielded minimal pathological effects such as the generation of extracellular fluid when assessed by histology, helped preserve GFAP expression in astrocytes, and performed extremely well in maintaining mRNA abundance levels in retinas of cold stored eyes. None of these elements was provided by TSA. Notably, however, the effect of VPA on mRNA levels was not maintained in the Optimal mUW solution.

[0053] K+channel blockers were tested based on the rationale that one of the earliest pathological events associated with neuronal death is an efflux of cellular K+ions that leads to a phenomenon known as the apoptotic volume decrease. Studies using agents that block the K+efflux show that they provide resiliency to neurons in the face of an apoptotic challenge. In rodent models of acute and chronic optic nerve damage, RGCs exhibit nuclear atrophy, which can be attenuated if the eyes are injected with BaCl2to block the K+efflux. Addition of BaCl2to UW solution yielded generally favorable results histologically, but did not provide evidence of maintaining GFAP expression in astrocytes. Additionally, it was noted that the addition of BaCl2created a precipitate that made the mUW solution turbid. As a consequence, BaCl2was replaced with TEA, which has also been used extensively in the literature are a K+channel blocker. TEA essentially mimicked the attributes of BaCl2without creating the formation of a precipitate, although some signs of retinal edema were noted in eyes treated with this supplement.

[0054] The third major category that was targeted with supplements was protein kinase activity using the pan-kinase inhibitor sunitinib or the selective p38MAPK inhibitor SB203580. Sunitinib was chosen because it had previously been shown to affect the axis between Dual Leucine Zipper Kinase (DLK) and JUN-N-terminal Kinases (JNKs). Several lines of experimental evidence indicate that the DLK-JNK axis is the principal mediator of initiating the activation of the intrinsic apoptotic program in RGCs in response to axonal damage. Surprisingly, addition of either of these kinase inhibitors to UW solution yielded poor overall outcomes to retinal architecture after cold storage, with both leading to excessive protein accumulation in the anterior and posterior chambers and regions of focal retinalP240239US01 107668.286 edema. Without being held to theory, it is believed that these inhibitors have a deleterious effect on kinase functions in other ocular cell types.

[0055] Retinal macroglia include astrocytes which are situated in close proximity to the nerve fibers of the RGCs in the inner retina (i.e., adjacent to the vitreous) and Müller glia, which have cell bodies in the inner nuclear layer and extend processes to both the nerve fiber layer and the outer limiting membrane that is defined by the base of the photoreceptor outer segments. These cells play critical roles in neuronal maintenance, including providing energy substrates to the neurons in the form of L-lactate, maintaining homeostasis of ion and small molecule environments, and providing neurotrophic support. To assess the effect of cold storage solutions on the macroglia, both the pattern and level of expression of GFAP were interrogated. This served two purposes. The first was to determine if Gfap expression remained restricted to astrocytes, and therefore normal, and the second was to evaluate the level of glial activation, which is characterized by an increase in Gfap expression in both cell types. Initial studies with mUW solutions showed that rather than increases in GFAP there was a dramatic decrease in expression, suggesting that astrocytes were particularly susceptible to the cold storage process, which was interpreted as being particularly detrimental to neuronal health, particularly in their ability to recover after transplantation. Studies with retinal explants, however, showed that addition of hydrocortisone to the media was able to not only help retain GFAP expression, but actually induced increased expression which was associated with an increase in the regenerative potential of the RGCs in the explant. Based on immunostaining, the addition of hydrocortisone to UW solution was able to help preserve GFAP expression in the astrocyte layer.

[0056] Given that macroglial activity may be partially suppressed during cold storage, and therefore unable to provide critical energy support to adjacent neurons, the addition of L- lactate in UW solution was also tested. The rationale for this was that the cells in the transplanted retina would have immediate access to this energy substrate and provide a buffer to allow macroglia to regain their normal homeostatic function. This augments the addition of adenosine in UW solution, which was added in an effort to provide preserved cells a necessary building block to begin de novo ATP synthesis once they were removed from the stasis condition of cold storage. L-lactate had several effects on the efficacy of mUW solution. By itself, it was able to help preserve GFAP immunostaining in the astrocyte layer, but it also induced moderate upregulation of GFAP protein accumulation in Müller cells. The reasoning for this is not clear, but it is known that L-lactate is also a critical energy substrate for these cells, and that they are actively involved in L-lactate homeostasis inP240239US01 107668.286 healthy retinas. Unexpectedly, L-lactate, in combination with other supplements in Optimal mUW Solution, prevented retinal detachment in eyes maintained in cold storage. L-lactate is also an energy substrate for RPE cells. These cells surround the distal ends of photoreceptor outer segments and provide structural and metabolic support, so it is likely that the L-lactate helps maintain this important role for the RPE by keeping them active.

[0057] In combination, the supplements that comprise the Optimized mUW Solution, provided exceptional preservation of retinal and cellular architecture after 24 hrs in cold storage. Cells, while appearing normal, did exhibit a decrease in mRNA abundance across the retina, suggesting that cold storage was pushing the cells into a period of stasis that was characterized by reduced levels of transcription but not mRNA turnover. Evaluation of mRNA levels may provide an important indicator of overall retinal health and be a major factor in determining how long the donor tissue can be held in storage prior to transplantation.

[0058] A common feature of UW solution, and most of the mUW solutions, was the decrease in abundance of cell-specific mRNAs during the period of cold storage. With the exception of GFAP, this was not reflected in loss of protein levels as a function of immunostaining, although the latter evaluation was not quantitative. Without being held to theory, this finding suggests a decrease in transcriptional activity caused by hypothermia. This did not appear to affect the process of mRNA degradation and may have even contributed to an increase in it. Example 6: Evaluation of trophic factors added to optimized UW solution

[0059] There is extensive literature suggesting that trophic factors extend the lifespan of RGCs after acute optic nerve damage. Brain derived neurotrophic factor (BDNF), glial cell-line derived neurotrophic factor (GDNF), and ciliary neurotrophic factor (CNTF), all of which have protective and regenerative potential in vivo, will be tested initially individually consistent with the pattern of testing of the current supplements, and then in combination. For experiments, validation of preservation, or lack thereof, would include histopathology, immunostaining, and molecular analysis as described herein. It is predicted that these neurotrophic factors will augment the neuroprotective effect of optimized UW solution, principally once they recover from cold storage and prior to reestablishment of an endogenous source of trophic support after transplantation.P240239US01 107668.286 Example 7: Evaluation of supplements that suppress reactive gliosis in vivo

[0060] Preliminary, rewarming experiments showed that marker genes for both macroglia and microglia become strongly activated leading to the hypothesis that these cell types were exhibiting a reactive phenotype. Upon confirmation of the reactive gliosis, this would be followed up by immunostaining with GFAP and IBA1 to evaluate cell type, cell phenotype, and localization of reactive cells in the retina. A full single-cell RNA-Seq screen of isolated cell types (i.e., astrocytes, Müller cells, microglia) will be conducted to assess the full spectrum of gliotic phenotypes. Cytokine ELISA panels will also be run to evaluate the inflammatory environment created by the reactive gliosis with an emphasis on TNF^, C1QA, and IL1-^, which have been identified as microglial-derived factors that stimulate conversion of A2 astrocytes to an A1 phenotype that leads to an increase in RGC susceptibility to optic nerve damage.

[0061] Confirmation of reactive gliosis in rewarmed eyes warrants the testing of supplements that have been shown to suppress reactive gliosis in vivo. For example, the tetracycline derivative minocycline has been shown to attenuate microglial reactivity (and therefore possibly down-stream reactive macrogliosis) in the rodent CNS including providing neuroprotection to retinal ganglion cells after acute and chronic optic nerve damage. Using the assays described herein to assess glial reactivity and the induction of a neuroinflammatory cytokine environment, different levels of minocycline (1-100 µg / mL) added to optimized mUW solution will be evaluated in rewarmed eyes previously kept in cold storage. It is predicted that minocycline will attenuate reactive gliosis and prevent the production of inflammatory cytokines.

[0062] The use of the terms “a” and “an” and “the” and similar referents (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms first, second etc. as used herein are not meant to denote any particular ordering, but simply for convenience to denote a plurality of, for example, layers. The terms “comprising”, “having”, “including”, and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to”) unless otherwise noted. Recitation of ranges of values are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. The endpoints of all ranges are included within the range and independently combinable. All methodsP240239US01 107668.286 described herein can be performed in a suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”), is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention as used herein.

[0063] While the invention has been described with reference to an exemplary embodiment, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims. Any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

P240239US01 107668.286 Claims 1. A cold storage solution for organs and tissues, comprising a colloid, an impermeant, an ionic component, a nutrient, an antioxidant, a histone deacetylase inhibitor (HDAC inhibitor), a potassium channel blocker, an energy substrate for neurons, and a steroid, wherein the osmolality is 255 to 420 mOsm.

2. The cold storage solution of claim 1, comprising 25 g / L to 100 g / L of the colloid, 40 g / L to 80 g / L of the impermeant, 5 g / L to 20 g / L of the ionic component, 0.5 g / L to 5 g / L of the nutrient, 0.5 g / L to 5 g / L of the antioxidant, 5 µM to 10 mM of the HDAC inhibitor, 0.5 mM to 5 mM of the potassium channel blocker, 0.5 mM to 20 mM of the energy substrate for neurons, and 0.1 µM to 1 µM of the steroid.

3. The cold storage solution of claim 1 or 2, wherein the colloid is a hydroxyethyl starch, a polyethylene glycol, a dextran, or a combination thereof; the impermeant is lactobionate, raffinose, trehalose, glucose, mannitol, or a combination thereof; the ionic component comprises potassium, magnesium, and phosphate; the antioxidant comprises glutathione; and the nutrient comprises adenosine.

4. The cold storage solution of claim 1 or 2, wherein the colloid is a hydroxyethyl starch; the impermeant is lactobionate and raffinose; the ionic componentP240239US01 107668.286 comprises potassium hydroxide, magnesium sulfate, and potassium phosphate; the antioxidant comprises glutathione; and the nutrient comprises adenosine.

5. The cold storage solution of any of claims 1-4, wherein the HDAC inhibitor is trichostatin-A, valproic acid, trapoxin, RGFP966, entinostat, or a combination thereof; the potassium channel blocker is tetraethylammonium, barium chloride, dalfampridine, dofetilide, amiodarone, or a combination thereof; the energy substrate for neurons is lactate; and the steroid is hydrocortisone, cortisone, prednisone, dexamethasone, or a combination thereof.

6. The cold storage solution of any of claims 1-4, wherein the HDAC inhibitor is valproic acid; the potassium channel blocker is tetraethylammonium; the energy substrate for neurons is lactate; and the steroid is hydrocortisone.

7. The cold storage solution of any of claims 1-6 wherein the pH is 7 to 8.

8. The cold storage solution of claim 7, wherein the pH is 7.

4.

9. The cold storage solution of any of claims 1-8, wherein the osmolality is 320 mOsm / kg.

10. The cold storage solution of claim 1, having the composition: 50 g / L of hydroxyethyl starch as the colloid, 35.83 g / L lactobionate and 17.83 g / L raffinose as the impermeant, 3.4 g / L potassium phosphate, 1.23 g / L magnesium sulfate, and 5.61 g / L potassium hydroxide as the ionic component, 1.34 g / L adenosine as the nutrient, 0.992 g / L glutathione as the antioxidant, 15 µM trichostatin-A and / or 2 mM valproic acid as the HDAC inhibitor, 5 mM tetraethylammonium as the potassium channel blocker, 10 mM L-lactate as the energy substrate for neurons, and 0.36 µM hydrocortisone as the steroid, wherein the pH is 7.4, and the osmolality is 320 mOsm / kg.P240239US01 107668.286 11. The cold storage solution of any of the foregoing claims, further comprising a trophic factor.

12. The cold storage solution of claim 11, wherein the trophic factor is brain derived neurotrophic factor (BDNF), glial cell-line derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), or a combination thereof.

13. The cold storage solution of any of the foregoing claims, further comprising a tetracycline.

14. The cold storage solution of claim 13, wherein the tetracycline is minocycline, doxycycline, demeclocycline, methacycline, or a combination thereof.

15. A method of preserving an organ or tissue, comprising cooling the cold storage solution of any of claims 1-14 to a temperature of 2 to 6°C, and exposing the organ or tissue to the cold storage solution.

16. The method of claim 15, wherein exposing comprises perfusing the organ or tissue with the cold storage solution, followed by cold storage of the flushed organ or tissue.

17. The method of claim 15 or 16, wherein the organ or tissue comprises brain, spinal cord, a whole eye, central nervous system tissue, or isolated nerves.

18. The method of claim 15 or 16, wherein the organ or tissue comprises a whole eye.

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