Compositions and methods for preserving cell viability in low temperature environments

Abstract

Disclosed herein are compositions for treating cells, tissues, or organs to withstand exposure and / or preserve viability when exposed to a low temperature environment, and methods of using such compositions. The cells, tissues or organs are preferably corneas or corneal cells.

Description

[0001] Government Support Statement

[0002] This invention was made with government support under Grant No. 1ZIAEY000488 from the National Eye Institute (NEI). The government has certain rights in this invention.

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of Chinese application No. 202011500962.5 filed on December 17, 2020, the entire contents of which are incorporated herein by reference. Background Art

[0005] Corneal transplantation is used to replace the damaged or diseased cornea of ​​the recipient individual with the donated cornea. Once the corneal tissue is removed from the donor, it is maintained under the conditions of being designed to maximize the cell viability before the tissue is transplanted into the recipient. The successful outcome of corneal transplantation depends on the presence of viable corneal endothelium. The corneal endothelium is a single layer of cells lining the inner surface of the cornea, located at the border between the anterior chamber filled with aqueous humor and the transparent stroma at the rear surface. The corneal endothelium acts as a barrier for fluid movement into the cornea and maintains corneal transparency by regulating the regulation of stroma hydration. These cells are extremely fragile and will not split under normal circumstances, so the function of the corneal endothelium depends on maintaining a high cell population for appropriate repair mechanisms.

[0006] The preservation and storage of corneas containing living cells can be accomplished by cryopreservation (e.g., 2-8°C), cryopreservation, and organ culture. Refrigeration at 2-8°C reduces the metabolic energy requirements of cells and can generally be used to store corneas for 1 to 2 weeks (7-14 days).

[0007] Storage media or corneal preservation solutions have been developed to effectively preserve the cornea. In 1947, McCarey found that corneas stored in MK solution at 4°C for 4 days were as intact as fresh corneas. Chondroitin sulfate and other chemicals were then added to MK solution to develop further preservation solutions such as K-Solution, CSM, Dexsol, etc. In 1990, Optisol-GS was formed by combining K-solution with Dexsol and adding streptomycin. TM, which is currently the most widely used corneal preservation solution. Experimental data show that corneal activity can be maintained for 7-10 days (according to the NEI guidelines for functional preservation of the cornea). Other improved preservation solutions (such as Chen's preservation solution) have also appeared, but the preservation effect is not comparable to Optisol-GS TM compared to.

[0008] However, Optisol-GS TM The traditional organ preservation solution represented by has only a short-term effect on preserving the cornea, and after 12 days, the cornea is usually edematous and blurred and the endothelial cells are killed and shed on a large scale. This cold-preserved cornea often leads to the failure of corneal transplantation. The 12-day shelf life cannot meet the requirements of larger eye banks, and better corneal preservation strategies are urgently needed to meet the requirements.

[0009] Cryopreservation is the process of preserving biological samples (such as cells or whole tissues) by cooling to low temperatures below zero. At such low temperatures, any biological activity, including biochemical reactions that normally lead to cell death, is effectively stopped. Cryopreservation allows the preservation and storage of cells or tissues while preserving the potential biological activity of the cells or tissues. Organ culture involves storing the cornea in a culture medium, such as Eagle's minimum essential medium (MEM) containing 2% fetal bovine serum (FBS), at a temperature of 28-37°C. Organ culture allows storage for up to 4-7 weeks.

[0010] Cold storage at a temperature of 2-8°C is the most common method of corneal storage prior to transplantation. However, during the cold storage of the cornea prior to transplantation, the corneal endothelium is susceptible to damage. If the number of corneal endothelial cells is too low, the repair mechanism may not be sufficient to restore the endothelium and maintain the cornea in an appropriate functional state, that is, able to act as an appropriate permeability barrier and maintain the cornea in its transparent, non-swollen state. In addition, corneal endothelial cells have a very limited ability to proliferate in the human body. Abnormal corneal endothelial function is the root cause of most corneal transplant failures. Therefore, the cornea often needs to be discarded due to the loss of endothelial cells, as evidenced when the cornea becomes swollen or loses transparency. Therefore, preserving the corneal endothelium during storage prior to transplantation is a key issue to ensure the success of corneal transplantation.

[0011] There remains a need for improved compositions and methods for preserving the viability of cells, tissues and organs during long-term storage at temperatures between 2-8°C. Summary of the invention

[0012] Disclosed herein is a method for treating cells, tissues or organs to withstand exposure to a cryogenic environment, the method comprising:

[0013] Providing cells, tissues or organs to be exposed to a cryogenic environment; and

[0014] The cell, tissue or organ is contacted with a composition comprising a compound that activates the FOXO1 protein in the cell, tissue or organ, so that the cell, tissue or organ can withstand exposure to the low temperature.

[0015] Also disclosed herein is a method for preserving the viability of cells, tissues or organs during exposure to a cryogenic environment, the method comprising:

[0016] contacting the cell, tissue or organ with a composition comprising a compound that activates FOX01 protein in the cell, tissue or organ to effectively preserve the viability of the cell, tissue or organ; and

[0017] The contacted cells, tissues or organs are exposed to the hypothermic environment.

[0018] In an embodiment, the cell, tissue or organ is the cornea or a corneal cell.

[0019] In an embodiment, the compound that activates FOXO1 protein is a cell-permeable ceramide analog or apigenin.

[0020] Also disclosed herein is the use of a composition comprising a compound that activates FOXO1 protein in a cell, tissue or organ for treating or preserving the vitality of the cell, tissue or organ to withstand exposure to a low temperature environment.

[0021] Also disclosed herein is the use of a composition comprising a compound that activates FOXO1 protein for preparing a medicament for treating or preserving the vitality of cells, tissues or organs to withstand exposure to a low temperature environment.

[0022] The present invention also discloses a storage solution, which contains the following components in 1 L:

[0023] Amino Acids:

[0024] 1.5-2.2mg of ALA

[0025] 75-93mg arginine

[0026] 14-17 mg of asparagine

[0027] 0.9-1.1 mg of cysteine

[0028] 27-33mg of glycine

[0029] 180-220 mg of glutamine

[0030] 28-34mg histidine

[0031] 94-120mg isoleucine

[0032] 94-120mg of leucine

[0033] 100-130mg lysine

[0034] 27-33 mg of methionine

[0035] 59-73 mg of phenylalanine

[0036] 7-8.5 mg of proline

[0037] 36-46mg of serine

[0038] 85-105mg of threonine

[0039] 14-18 mg of tryptophan

[0040] 60-80 mg of tyrosine

[0041] 84-105 mg of valine

[0042] Vitamins:

[0043] 4-5.2 mg of choline

[0044] 3-4.5 mg of D-calcium pantothenate

[0045] 0.9-1.1 mg pantothenic acid (vitamin B5)

[0046] 4-5 mg of folic acid

[0047] 4-5 mg of niacinamide

[0048] 4-5 mg of pyridoxal (vitamin B6)

[0049] 0.09-0.11 mg of riboflavin (vitamin B2)

[0050] 5-6.5 mg of Thiamine (Vitamin B1)

[0051] 1-1.5 mg of vitamin B12

[0052] Inorganic salts:

[0053] 170-220 mg of CaCl2

[0054] 50-70 mg Fe(NO3)3

[0055] 360-440 mg KCl

[0056] 66-85 mg of NaHCO3

[0057] 4300-5300 mg of NaCl

[0058] 120-160 mg of NaH2PO4

[0059] 69-85 mg MgCl2

[0060] 97-120 mg ZnSO4

[0061] Other components:

[0062] 1.3-1.7 mg of adenosine

[0063] 22,000-28,000 mg of chondroitin sulfate

[0064] 4000-5000 mg of glucose

[0065] 9000-11000mg of beta-glucan

[0066] 90-110 mg of gentamicin

[0067] 9-12 mg of inosine

[0068] 10-14mg of inositol

[0069] 330-420 mg of phenol red

[0070] 0.9-1.1 mg of pyridoxal hydrochloride

[0071] 220-280 mg of MES monohydrate

[0072] 9000-11000 μM MOPS buffer

[0073] 1800-2200 μM HEPES buffer

[0074] 0.9-1.2 mg of sodium pyruvate

[0075] 3.4-4.3 mg of 2-mercaptoethanol,

[0076] The storage solution has a pH of 7.3±2 and an osmotic pressure of 300±50 mOsm.

[0077] In an embodiment, the preservation solution is a corneal preservation solution.

[0078] In an embodiment, the preservation solution further comprises at least one selected from the group consisting of: a compound that activates FOXO1 protein in cells, tissues or organs, and a protease inhibitor cocktail (Pi).

[0079] In an embodiment, the compound that activates FOXO1 protein is a cell-permeable ceramide analog or apigenin.

[0080] Also disclosed herein is a method for treating cells, tissues or organs to withstand exposure to a cryogenic environment, the method comprising:

[0081] Providing cells, tissues or organs to be exposed to a cryogenic environment; and

[0082] The cells, tissues or organs are contacted with a preservation solution as discussed herein to enable the cells, tissues or organs to withstand exposure to the hypothermia.

[0083] Also disclosed herein is a method for preserving the viability of cells, tissues or organs during exposure to a cryogenic environment, the method comprising:

[0084] contacting the cell, tissue or organ with a preservation solution as discussed herein to preserve the viability of the cell, tissue or organ; and

[0085] The contacted cells, tissues or organs are exposed to the hypothermic environment.

[0086] Also disclosed herein is the use of a preservation solution as discussed herein in cells, tissues or organs for treating or preserving the viability of cells, tissues or organs to withstand exposure to a cryogenic environment.

[0087] Also discussed herein is the use of a preservation solution according to any one of claims 40-54 for the preparation of a medicament for treating or preserving the viability of cells, tissues or organs to withstand exposure to a cryogenic environment.

[0088] The above described and other features are exemplified by the following figures and detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] The following figures are of exemplary embodiments, wherein like elements are similarly numbered.

[0090] Figure 1 The corneal staining results of 4-week-old rat corneas and 6-month-old rat corneas are shown. DAPI stains cell nuclei (blue), and PI stains dead cells (red).

[0091] Figure 2The results of FOXO1 staining of 4-week-old rat cornea and 6-month-old rat cornea are shown. DAPI stains the nucleus (blue), phalloidin stains the endothelial cell membrane / F-actin (pink) and stains FOXO1 protein (green).

[0092] Figure 3 The results of FOXO1 staining before and after C6 injection into the anterior chamber of the cornea of ​​6-month-old rats are shown, and the effect of C6 on the nuclear translocation of FOXO1 in rat corneal endothelial cells is shown. DAPI: cell nucleus (blue), phalloidin: F-actin (pink) and FOXO1 protein (green).

[0093] Figure 4 The results of FOXO1 staining before and after apigenin was injected into the anterior chamber of the cornea of ​​6-month-old rats and after rewarming are shown, and the effect of apigenin on the nuclear entry of FOXO1 in rat corneal endothelial cells is shown; DAPI: cell nucleus (blue), phalloidin: F-actin (pink), FOXO1 protein (green).

[0094] Figure 5A and Figure 5B The results of FOXO1 staining are shown after C6 and goat serum were injected into the anterior chamber of the cornea of ​​6-month-old rats, the corneas were removed, and then the corneas were cold stored in different corneal preservation solutions for 3 weeks. The data show that C6 and Pi can be added to Optisol-GS TM and / or novel corneal preservation solution (MCM) and enhances the survival of rat corneal endothelial cells. DAPI: cell nuclei (blue), PI: dead cells (red), phalloidin: F-actin (pink), FOXO1 protein (green).

[0095] Figure 6 A graph showing the corneal endothelial cell mortality rate after injection of C6 and goat serum into the anterior chamber of the cornea of ​​6-month-old rats and subsequent cold storage of the cornea in different corneal preservation solutions for 3 weeks.

[0096] Figure 7 Shown are the results of corneal permeability in 6-month-old rats after injection of C6 and goat serum into the anterior chamber, removal of the cornea, and then cryopreservation of the cornea in different corneal preservation solutions for 3 weeks.

[0097] Fig. 8A and Figure 8BThe results of FOXO1 staining after C6 and goat serum were injected into the anterior chamber of the cornea of ​​6-month-old rats and the corneas were subsequently refrigerated in different corneal preservation solutions for 4 weeks are shown. The staining result images show DAPI: cell nuclei (blue), PI: dead cells (red), phalloidin: F-actin (pink), FOXO1 protein (green).

[0098] Fig. 9 Shown are the results of corneal permeability in 6 month old rats after intracameral injection of C6 and goat serum and subsequent refrigeration of the corneas in different corneal preservation solutions for 4 weeks.

[0099] Fig.10 Shown is the corneal endothelial cell mortality rate after injection of C6 and goat serum into the anterior chamber of the corneas of 6-month-old rats and subsequent cold storage of the corneas in different corneal preservation solutions for 4 weeks.

[0100] Fig.11 Shown are the results of FOXO1 staining after apigenin and goat serum were injected into the anterior chamber of the cornea of ​​6-month-old rats and the corneas were subsequently refrigerated in different corneal preservation solutions for 3 weeks. DAPI: cell nuclei (blue), PI: dead cells (red), phalloidin: F-actin (pink), FOXO1 protein (green).

[0101] Fig.12 The results of FOXO1 staining after apigenin and goat serum were injected into the anterior chamber of the cornea of ​​6-month-old rats and then the corneas were refrigerated in different corneal preservation solutions for 4 weeks. The data show that when apigenin is used, the rat cornea can be stored at 4°C for 28 days, and the endothelium is basically intact. DAPI: cell nucleus (blue), PI: dead cells (red), phalloidin: F-actin (pink), FOXO1 protein (green).

[0102] Fig.13 Shown is corneal endothelial mortality following injection of C6 and goat serum into the anterior chamber of the corneas of 6 month old rats and subsequent cold storage of the corneas in various corneal preservation solutions for 3 and 4 weeks.

[0103] Fig.14 Images of rat eyes 3 weeks after corneal transplantation are shown. C6 and goat serum were injected into fresh corneas and anterior chambers of corneas of 6-month-old rats, and the corneas were then cold-stored in various corneal preservation solutions for 4 weeks.

[0104] Fig.15 Transmission electron microscopy micrographs of corneal endothelial subcellular structures 6 months after corneal transplantation are shown. Except for the 'fresh' cornea control, the other 3 groups were from cryopreserved corneas 6 months after transplantation surgery.

[0105] Fig.16A Nuclear accumulation of FOXO1 in the retina of slow ground squirrel (TLGS) and active TLGS after cryopreservation is shown. Fig. 16B Shown is the nuclear accumulation of FOXO1 in TLGS iPSC neurons cultured under the indicated conditions.

[0106] Fig.17 Panels A to D show FOXO1 nuclear accumulation in young and adult human iPSC neurons cultured under the indicated conditions.

[0107] Fig.18 It was shown that cold survival of human H1 embryonic stem cells is dependent on nuclear import of FOXO1 and is aggravated by 'aging' factors.

[0108] Fig.19A and Fig.19B Shown are FOXO1 in mouse heart sections under the indicated conditions.

[0109] Fig. 20A and 20B Shown is FOXO1 in zebrafish larvae from the indicated conditions.

[0110] Fig.21 Panels A to C show that cold-induced nuclear import of FOXO1 is enabled by SUMO E3 ligase RANBP2 and transporter importin-7, and inhibited by transporter exportin-1. Functional deterioration of RANBP2 and importin-7 may be the reason why FOXO1 nuclear import is inhibited after cold stress in older H1 cells.

[0111] Fig. 22 Panels A and B show that cold-induced nuclear import of FOXO1 is also determined by the key SUMO-interacting motif (SIM) on the N-terminus of the FOXO1 protein; if the SIM motif is mutated, the FOXO1 protein will accumulate in the cell nucleus under normal conditions.

[0112] Fig.23 Various images using mouse pancreatic islets as a model are shown in panels A to E. Key components in the FOXO1-dependent survival pathway are not well maintained in the current gold standard organ preservation solution (UW, University of Wisconsin solution). Fig.24C) When added to UW solution, islet cold survival was improved; if basal preservation solution (HS) was used, islet cold survival was even better; 3) Even in HS solution, if SUMOylation inhibitor 2-D08 or ginkgolic acid (GA, Fig.24 B and Fig.24 D), cold-induced nuclear translocation of FOXO1 was inhibited and islet cell death became severe.

[0113] Fig.24 Images of sections of mouse islets and mouse kidneys with transplanted islets are shown in panels A to C. N-EM and protease inhibitors were added to HS to make HS+. Mouse islets can be stored in HS+ at 4°C for up to 14 days, transplanted into streptozotocin (STZ)-induced type I diabetic mice, and successfully reduced blood glucose levels in recipient mice.

[0114] Fig.25 Graphs of insulin production / secretion due to storage in UW or HS+ storage solution are shown in panels A to C. As can be seen, HS+ storage solution can significantly improve the quality of human islets after prolonged refrigeration. DETAILED DESCRIPTION

[0115] Disclosed herein are compositions and methods of using the compositions to treat cells, tissues or organs to withstand exposure, preserve viability and / or reduce damage when exposed to a cryogenic environment. In embodiments, the cells, tissues or organs may be corneas or corneal cells or pancreatic islets or cells, preferably corneas or corneal cells.

[0116] Forkhead box O1 (FOXO1) protein is a transcription factor belonging to the family of transcription factors with a forkhead box or motif, and is a DNA binding domain with about 80 to 100 amino acids and composed of three helices and two characteristic macroloops. The FOXO1 protein is involved in the regulation of metabolic homeostasis in response to oxidative stress and shuttles between the nucleus and cytoplasm of cells. Phosphorylation of FOXO1 results in inactivation of the protein and in the exclusion of the phosphorylated FOXO1 from the nucleus, where it is ubiquitinated and degraded. In contrast, non-phosphorylated and activated FOXO1 is located in the nucleus, where it induces the expression of various genes involved in apoptosis, glucose metabolism, cell cycle progression, and differentiation.

[0117] It has been unexpectedly found that exposure to cold temperatures induces nuclear accumulation and activation of FOXO1 in human corneal cells derived from young donors, and that FOXO1 expression is associated with long-term survival of human corneal cells in vitro. Fig.18A to Fig.18 B. FIG. 19A to FIG. 1 9D、Figure 20、 Fig.21 A to Fig.21 B and Fig. 22 A to Fig. 22 B. It has also been unexpectedly discovered that treatment of corneas with a composition comprising a compound that causes FOXO1 activation in corneal endothelial cells results in increased viability of corneal endothelial cells during long-term storage of the cornea under cryogenic conditions. In embodiments, corneas treated with a composition comprising a compound that causes FOXO1 activation can be refrigerated for at least 4 weeks or 5 weeks with endothelial cell survival greater than 95%. The compound that causes FOXO1 activation can be a cell-permeable ceramide analog or apigenin. In contrast, corneas refrigerated only in standard corneal storage media result in endothelial cell mortality rates as high as 90-100% (survival rates 0-10%) over a similar period of time.

[0118] As used herein, "treatment" or "treating" includes providing as an active agent an amount of a compound disclosed herein effective to measurably obtain a desired effect (eg, maintaining vitality and / or reducing damage).

[0119] An "effective amount" of an active ingredient, or a composition comprising the active ingredient, is an amount effective to provide a therapeutic benefit when administered.

[0120] A significant change refers to any detectable change that is statistically significant in a standard parametric test of statistical significance (eg, Student's T-test) with p < 0.05.

[0121] As used herein, "cold storage" or "refrigerated storage" are used interchangeably and refer to temperatures of 2-8°C unless otherwise specified.

[0122] A "viable" cell or the "viability" of a cell refers to the ability of a cell to perform certain natural functions (e.g., metabolism, growth, motility, proliferation, certain forms of responsiveness and adaptation). "Cell survival" has a similar meaning.

[0123] As used herein, "preserve viability" or "preserving viability" means that the number of viable cells present in a cell population or a tissue comprising the cell population is substantially the same after cold storage as the number of viable cells present before cold storage. For example, preserving the viability of a cell population or a tissue comprising the cell population means that at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% of the cells are viable after a defined cold storage period. Preserving viability also means that 20% or less, 10% or less, 8% or less, 5% or less, 3% or less, or 1% or less of the cells are not viable (not viable) when the number of viable cells present after cold storage is compared to the number of viable cells present before cold storage begins.

[0124] "Alkyl" means a straight-chain or branched saturated monovalent hydrocarbon group (eg, methyl or hexyl).

[0125] As used herein, "keratocytes" are cells contained within the intact cornea, and are not intended to refer to cells that have been isolated from the cornea and cultured independently.

[0126] Disclosed herein is a composition for preserving the viability of cells, tissues or organs and reducing damage to the cells, tissues or organs during exposure to a cryogenic environment. In an embodiment, the composition comprises a compound that causes FOXO1 activation in the cells, tissues or organs.

[0127] In an embodiment, the cell, tissue or organ is a mammalian cell, tissue or organ, including a neuronal cell, a retinal cell, a cornea, a retina, a skin, a heart, a lung, a pancreas, a kidney, a liver, an intestine, a stem cell, a blood, or any other cell, tissue or organ suitable for transplantation. In an embodiment, the cell, tissue or organ may be a cornea or a corneal cell or a pancreatic islet or a pancreatic cell, preferably a cornea or a corneal cell, even more preferably a corneal endothelial cell.

[0128] In an embodiment, the compound may be any compound that drives FOXO1 translocation to the nucleus or prevents FOXO1 from being expelled from the nucleus. In an embodiment, the compound may be a cell-permeable ceramide analog, apigenin, N-EM (deSUMOylation inhibitor), resveratrol or Leptomycin B (LMB). N-EM (deSUMOylation inhibitor) and resveratrol induce FOXO1 translocation to the nucleus. Leptomycin B (LMB) is a nuclear export inhibitor of exportin-1 / CRM1, which causes FOXO1 to accumulate in the nucleus. In an embodiment, the compound is a cell-permeable ceramide analog or apigenin.

[0129] In an embodiment, the compound that causes FOXO1 activation is a cell-permeable ceramide analog, wherein the cell-permeable ceramide analog comprises a compound represented by Formula 1,

[0130]

[0131] wherein R is a C1 to C10 alkyl group.

[0132] In an embodiment, the cell permeable analog comprises a compound represented by Formula 2, also known as C6 ceramide.

[0133]

[0134] In an embodiment, the compound that causes activation of FOXO1 is apigenin.

[0135] In an embodiment, the compound that causes FOXO1 activation can be dissolved in any physiological solution considered suitable for the cell, tissue or organ before contacting the cell, tissue or organ. The compound can also be combined with a novel medium that is commercially available or designed for low-temperature cell, tissue or organ storage (preservation solution) as discussed herein. In an embodiment, the composition comprises a compound that causes FOXO1 activation and a preservation solution. Any suitable physiological solution or preservation solution that does not interfere with the function of the compound that causes FOXO1 activation can be used, whether commercially available or novel as discussed herein. In an embodiment, the cell, tissue or organ can be the cornea or corneal cell or pancreatic islet or pancreatic cell, preferably the cornea or corneal cell, and even more preferably the corneal endothelial cell.

[0136] In embodiments, the preservation solution is a corneal preservation solution. In embodiments, the preservation solution is known. In embodiments, the preservation solution is a novel preservation solution as discussed herein.

[0137] Examples of known corneal preservation solutions developed for clinical use include K-Sol (Cilco, Huntington, West Virginia), chondroitin sulfate storage medium (CSM), Dexsol, Optisol-GS (Chiron Ophthalmics Inc. Irvine, California), Chen's medium (Chen Laboratories, Phoenix, MD, https: / / jamanetwork.com / journals / jamaophthalmology / fullarticle / 272206), and Likorol (OpsiaPharma, France). Other examples of corneal preservation solutions include Life4°C (Numedis, US), Cornea Cold TM , Kerasave (AL.CHI.MI.ASrl). Other examples of preservation solutions include University of Wisconsin (UW) solution and similar agents for liver and kidney cold storage. Optisol-GS is a widely used commercially available medium for corneal cold storage and contains dextran, chondroitin sulfate, vitamins, and precursors of adenine triphosphate (adenosine, inosine, and adenine). In embodiments, the corneal preservation solution may contain dextran, chondroitin sulfate, or a combination thereof. In embodiments, the corneal preservation solution contains chondroitin sulfate.

[0138] The compound that causes FOXO1 activation can be mixed with a physiological solution and / or a preservation solution before contacting the cell, tissue or organ. The preservation solution can be commercially available, or can be a novel cryopreservation solution as discussed herein. The composition comprising the compound that causes FOXO1 activation can be prepared by dissolving the compound that causes FOXO1 activation directly into a physiological solution and / or a preservation solution to a concentration optimized for the cell, tissue or organ. In an embodiment, the cell, tissue or organ can be the cornea or corneal cell or pancreatic islet or pancreatic cell, preferably the cornea or corneal cell, even more preferably the corneal endothelial cell.

[0139] The concentration of the compound that causes FOXO1 activation in the composition is a physiologically effective amount that, when administered to a cell, tissue or organ, maintains the viability of the cell, tissue or organ during refrigeration. In an embodiment, the concentration of the compound that causes FOXO1 analog activation in the composition is a physiologically effective amount that, when administered to a cell, tissue or organ, causes activation of the FOXO1 protein. In an embodiment, the cell, tissue or organ may be the cornea or corneal cell or pancreatic islet or cell, preferably the cornea or corneal cell. In an embodiment, the corneal cell is a corneal endothelial cell.

[0140] In embodiments, the concentration of the cell permeable ceramide analog in the composition can be 0.1 micromolar (μM) to 100 μM, or 1 μM to 50 μM, or 1 μM to 25 μM, or 2 μM to 20 μM, or 5 μM to 15 μM.

[0141] In embodiments, the concentration of apigenin in the composition may be 0.1 μM to 30 μM, or 0.1 μM to 20 μM, or 0.1 μM to 10 μM, or 0.1 μM to 5 μM.

[0142] The composition may further comprise a pharmaceutically acceptable additive or excipient. As used herein, a pharmaceutically acceptable excipient refers to an inactive pharmaceutical ingredient (activepharmaceutical ingredient, "API") substance used to formulate a pharmaceutical composition or product, such as a disintegrant, a binder, a filler, and a lubricant. Each of these substances is generally safe for administration to humans according to established government standards, including standards promulgated by the U.S. Food and Drug Administration ("FDA").

[0143] Examples of disintegrants include agar, algin, calcium carbonate, carboxymethyl cellulose, cellulose, clay, colloidal silicon dioxide, cross-linked carboxymethyl cellulose sodium, crospovidone, gum, magnesium aluminum silicate, methyl cellulose, polacrilin potassium, sodium alginate, low-substituted hydroxypropyl cellulose and cross-linked polyvinyl pyrrolidone hydroxypropyl cellulose, sodium starch glycolate and starch, or combinations thereof, but are not limited thereto.

[0144] Examples of the binder include microcrystalline cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, or a combination thereof, but are not limited thereto.

[0145] Examples of fillers include, but are not limited to, calcium carbonate, calcium phosphate, dibasic calcium phosphate, tribasic calcium sulfate, carboxymethylcellulose calcium, cellulose, dextrin derivatives, dextrin, dextrose, fructose, lactitol, lactose, magnesium carbonate, magnesium oxide, maltitol, maltodextrin, maltose, sorbitol, starch, sucrose, sugar, xylitol, or combinations thereof.

[0146] Examples of lubricants include agar, calcium stearate, ethyl oleate, ethyl laurate, glycerin, glyceryl palmitostearate, hydrogenated vegetable oil, magnesium oxide, magnesium stearate, mannitol, poloxamer, glycols, sodium benzoate, sodium lauryl sulfate, sodium stearyl, sorbitol, stearic acid, talc, zinc stearate, or a combination thereof, but are not limited thereto.

[0147] The compound in the composition that causes FOXO1 activation is used in a method for improving the viability of cells, tissues or organs during extended cold storage periods. The composition can be used to pretreat cells, organs or tissues before exposure to a low temperature environment. The composition can also be used to maintain the viability of cells, organs or tissues during storage in a low temperature environment. In an embodiment, the cell, tissue or organ can be the cornea or corneal cells or pancreatic islets or pancreatic cells, preferably the cornea or corneal cells, and even more preferably corneal endothelial cells.

[0148] Also disclosed herein is a method for treating a cell, tissue or organ to withstand exposure to a low temperature environment, the method comprising providing a cell, tissue or organ to be exposed to a low temperature environment; and contacting the cell, tissue or organ with a composition comprising an amount of a compound that causes FOXO1 activation, which effectively enables the cell, tissue or organ to withstand exposure to the low temperature.

[0149] Also disclosed herein is a method for preserving the viability of cells, tissues or organs during exposure to a low temperature environment, the method comprising contacting the cells, tissues or organs with a composition comprising a certain amount of a compound that causes FOXO1 activation to effectively preserve the viability of the cells, tissues or organs; and exposing the cells, tissues or organs to the low temperature environment.

[0150] In embodiments, the methods disclosed herein also reduce damage to cells, tissues, or organs when exposed to hypothermic environments.

[0151] In embodiments, the cell, tissue or organ may be a cornea or corneal cell or islet or cell, preferably a cornea or corneal cell. In embodiments, the corneal cell is an endothelial cell, an epithelial cell, a corneal stromal cell or a combination thereof. The corneal cell is contained within a complete cornea. In embodiments, the corneal cell is an endothelial cell and is contained within the corneal endothelium of the cornea.

[0152] In an embodiment, a method of treating a cell, tissue or organ to withstand exposure to a low temperature environment comprises providing a cell, tissue or organ to be exposed to a low temperature environment; and contacting the cell, tissue or organ with a composition comprising a certain amount of a compound that causes FOXO1 activation, which effectively enables the cell, tissue or organ to withstand exposure to the low temperature. In an embodiment, a method of preserving the viability of a cell, tissue or organ during exposure to a low temperature environment is also disclosed herein, the method comprising contacting the cell, tissue or organ with a composition comprising a certain amount of a compound that causes FOXO1 activation to effectively preserve the viability of the cell, tissue or organ; and exposing the cell, tissue or organ to the low temperature environment.

[0153] In an embodiment, the cell, tissue or organ is a mammalian cell, tissue or organ, including a neuronal cell, a retinal cell, a cornea, a retina, a skin, a heart, a lung, a pancreas, a kidney, a liver, an intestine, a stem cell, a blood, or any other cell, tissue or organ suitable for transplantation. In an embodiment, the cell, tissue or organ may be a cornea or a corneal cell or a pancreatic islet or a pancreatic cell, preferably a cornea or a corneal cell, even more preferably a corneal endothelial cell. The cornea is a transplantable tissue that is removed from the eye of a donor and stored at low temperatures for a period of time prior to transplantation.

[0154] In the disclosed methods, contacting the cell, tissue or organ with the composition can occur before exposing the cell, tissue or organ to a cryogenic environment, during exposing the cell, tissue or organ to a cryogenic environment, or a combination thereof. In an embodiment, contacting the cell, tissue or organ with the composition occurs before exposing the cell, tissue or organ to a cryogenic environment.

[0155] In embodiments, contacting the cell, tissue or organ with the composition (pretreatment) occurs before the cell, tissue or organ is removed from the donor, after the cell, tissue or organ is removed from the donor, or a combination thereof. In embodiments, the cell, tissue or organ is cornea or corneal cells, and contacting the cornea or corneal cells with the composition may occur before the cornea is removed from the donor's eye, after the cornea is removed from the donor's eye, or a combination thereof. In each case, the contacting occurs before cold storage begins.

[0156] In an embodiment, the cell, tissue or organ is a cornea, which is contacted with the composition prior to removal from the donor's eye (pretreatment). The composition comprising a compound that causes FOXO1 activation is injected into the anterior chamber of the eye, which is the aqueous humor-filled space in the front of the eye between the cornea and the iris. In the anterior chamber, the composition contacts endothelial cells within the corneal endothelium at the posterior surface of the cornea. After a defined period of time, the cornea is removed from the donor eye.

[0157] In embodiments, the cornea is contacted with the composition after it has been removed from the eye of the donor (pretreatment).The cornea is removed from the eye of the donor and placed in a container containing the composition for a defined period of time.

[0158] In embodiments, the defined time period for pre-treating cells, tissues or organs with the composition is 5 minutes to 240 minutes, 10 minutes to 150 minutes, 20 minutes to 120 minutes, 20 minutes to 90 minutes, or 45 minutes to 90 minutes, or 45 minutes to 60 minutes. In embodiments, the contact time is 20 minutes to 90 minutes. The cells, tissues or organs can be pre-treated with a composition comprising a compound that causes FOXO1 activation at a temperature of about 22°C to 37°C (e.g., 25°C to 37°C).

[0159] After pre-treating cells, tissues or organs with the composition, the cells, tissues or organs are placed in a medium suitable for maintaining (storing) the cells, tissues or organs in a low temperature environment, and then placed in a low temperature environment. In an embodiment, the medium is a preservation solution. In an embodiment, the preservation solution may contain a compound that has been pre-added to cause FOXO1 activation.

[0160] In embodiments, the cell, tissue or organ is a cornea or corneal cell, and the cornea can be stored in a corneal preservation solution (commercially known or discussed herein), optionally pre-added with a compound that causes FOXO1 activation. In embodiments, the cornea or corneal cell is maintained in a cryogenic environment in the presence of a composition comprising a compound that causes FOXO1 activation.

[0161] The cell, tissue or organ can be maintained in a cryogenic environment for a period of 1 day (24 hours) to 56 days (8 weeks) in the presence of a preservation solution. In an embodiment, the cell, tissue or organ is a cornea or corneal cell, and the cornea can be maintained in a cryogenic environment for a period of 1 day (24 hours) to 42 days (6 weeks), 1 day to 35 days, 5 days to 35 days, 10 days to 35 days, 14 days to 35 days, 10 days to 28 days, or 14 days to 28 days in the presence of a preservation solution. In an embodiment, exposing cells, tissues or organs to a cryogenic environment includes storing at least one week at a temperature of 2°C to 8°C, preferably at least 1 week to 5 weeks, more preferably at least 1 week to 4 weeks. The cell, tissue or organ can be maintained in a cryogenic environment for a period of at least 2 weeks, at least 3 weeks, at least 4 weeks, or at least 5 weeks in the presence of a preservation solution, and the viability of the cell, tissue or organ is not significantly lost.

[0162] In embodiments, the cell, tissue or organ is cornea or corneal cell. In embodiments, corneal cell is endothelial cell present in intact corneal endothelium. After storage at a temperature of 0°C to 8°C for a period of 4 weeks, the activity of corneal endothelium is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. In embodiments, maintaining the cornea at a temperature of 0°C to 8°C for a period of 4-5 weeks results in preservation of 80% to 100%, or 85% to 100%, or 90% to 100%, or 95% to 100% activity of endothelial cells in corneal endothelium.

[0163] Also disclosed is the use of a composition comprising a compound that activates a FOXO1 protein as discussed herein in a cell, tissue or organ for treating or preserving the viability of the cell, tissue or organ to withstand exposure to a low temperature environment.

[0164] Also disclosed is the use of a composition comprising a compound that activates a FOXO1 protein as discussed herein for the preparation of a medicament for treating or preserving the viability of cells, tissues or organs to withstand exposure to a hypothermic environment.

[0165] In embodiments, the uses disclosed herein also reduce damage to cells, tissues or organs when exposed to hypothermic environments.

[0166] The present invention also discloses a novel preservation solution for preserving cells, tissues or organs, the novel preservation solution comprising a combination of amino acids, vitamin preparations, inorganic salts and other components. In an embodiment, the novel preservation solution is used for corneal preservation. In an embodiment, the preservation solution comprises the following components per 1L:

[0167] Amino Acids:

[0168] 1.5-2.2mg of ALA

[0169] 75-93mg arginine

[0170] 14-17 mg of asparagine

[0171] 0.9-1.1 mg of cysteine

[0172] 27-33mg of glycine

[0173] 180-220 mg of glutamine

[0174] 28-34mg histidine

[0175] 94-120mg isoleucine

[0176] 94-120mg of leucine

[0177] 100-130mg lysine

[0178] 27-33 mg of methionine

[0179] 59-73 mg of phenylalanine

[0180] 7-8.5 mg of proline

[0181] 36-46mg of serine

[0182] 85-105mg of threonine

[0183] 14-18 mg of tryptophan

[0184] 60-80 mg of tyrosine

[0185] 84-105 mg of valine

[0186] Vitamins:

[0187] 4-5.2 mg of choline

[0188] 3-4.5 mg of D-calcium pantothenate

[0189] 0.9-1.1 mg pantothenic acid (vitamin B5)

[0190] 4-5 mg of folic acid

[0191] 4-5 mg of niacinamide

[0192] 4-5 mg of pyridoxal (vitamin B6)

[0193] 0.09-0.11 mg of riboflavin (vitamin B2)

[0194] 5-6.5 mg of Thiamine (Vitamin B1)

[0195] 1-1.5 mg of vitamin B12

[0196] Inorganic salts:

[0197] 170-220 mg of CaCl2

[0198] 50-70 mg Fe(NO3)3

[0199] 360-440 mg KCl

[0200] 66-85 mg of NaHCO3

[0201] 4300-5300 mg of NaCl

[0202] 120-160 mg of NaH2PO4

[0203] 69-85 mg MgCl2

[0204] 97-120 mg ZnSO4

[0205] Other components:

[0206] 1.3-1.7 mg of adenosine

[0207] 22,000-28,000 mg of chondroitin sulfate

[0208] 4000-5000 mg of glucose

[0209] 9000-11000mg of beta-glucan

[0210] 90-110 mg of gentamicin

[0211] 9-12 mg of inosine

[0212] 10-14mg of inositol

[0213] 330-420 mg of phenol red

[0214] 0.9-1.1 mg of pyridoxal hydrochloride

[0215] 220-280 mg of MES monohydrate

[0216] 9000-11000 μM MOPS buffer

[0217] 1800-2200 μM HEPES buffer

[0218] 0.9-1.2 mg of sodium pyruvate

[0219] 3.4-4.3 mg of 2-mercaptoethanol.

[0220] In an embodiment, the storage solution has a pH of 7.3±2 and an osmotic pressure of 300±50 mOsm.

[0221] In embodiments, the storage solutions discussed herein can extend the storage life of cells, tissues or organs from about 12 days to about 28-35 days. In embodiments, the cells, tissues or organs are mammalian cells, tissues or organs, including neuronal cells, retinal cells, corneas, retina, skin, heart, lungs, pancreas, kidneys, liver, intestines, stem cells, blood, or any other cells, tissues or organs suitable for transplantation. In embodiments, the cells, tissues or organs can be corneas or corneal cells or islets or pancreatic cells, preferably corneas or corneal cells, and even more preferably corneal endothelial cells. It has been observed that corneas after 28 days of cold storage can still be successfully transplanted into rat recipients.

[0222] In embodiments, the disclosed storage solution further comprises a protease inhibitor cocktail (Pi) (which is described in WO2019 / 040359A1) and / or a compound that causes FOXO1 activation as discussed herein.

[0223] In embodiments, the compound that causes FOXO1 activation is a cell-permeable ceramide analog, apigenin, N-EM (deSUMOylation inhibitor), resveratrol, or leptomycin B (LMB), as discussed herein. N-EM (deSUMOylation inhibitor) and resveratrol induce translocation of FOXO1 to the nucleus. Leptomycin B (LMB) is a nuclear export inhibitor of exportin-1 / CRM1, which causes FOXO1 to accumulate in the nucleus. In embodiments, the compound can be a cell-permeable ceramide analog or apigenin, as discussed herein.

[0224] In an embodiment, the compound that causes FOXO1 activation is a cell-permeable ceramide analog, wherein the cell-permeable ceramide analog comprises a compound represented by Formula 1,

[0225]

[0226] wherein R is a C1 to C10 alkyl group.

[0227] In an embodiment, the cell permeable analog comprises a compound represented by Formula 2, also known as C6 ceramide.

[0228]

[0229] In embodiments, the concentration of the cell-permeable ceramide analog in the storage solution may be 0.1 μM / μL to 100 μM / μL, or 1 μM / μL to 50 μM / μL, or 1 μM / μL to 25 μM / μL, or 5 μM / μL to 20 μM / μL, or 5 μM / μL to 15 μM / μL.

[0230] In an embodiment, the compound that causes FOXO1 activation is apigenin. In an embodiment, the concentration of apigenin in the storage solution can be 0.1 μM / μL to 30 μM / μL, or 0.1 μM / μL to 20 μM / μL, or 0.1 μM / μL to 10 μM / μL, or 0.1 μM / μL to 5 μM / μL.

[0231] In an embodiment, the components and concentrations of the protease inhibitor cocktail (Pi) of the novel storage solution are as follows:

[0232] Aspartic protease inhibitors (e.g., pepstatin, pepstatin A): 1-20 μM;

[0233] Serine and cysteine ​​protease inhibitors (eg, leupeptin): 2-40 μM.

[0234] In an embodiment, the protease inhibitor cocktail Pi further comprises at least one of the following components and concentrations for the novel storage solution:

[0235] Metalloproteinase inhibitors (e.g., Betadine, Bestatin): 5-50 μM;

[0236] Cysteine ​​protease inhibitors (e.g., E-64): 1.5-30 μM;

[0237] Serine protease inhibitors (e.g., aprotinin): 0.08-2 μM;

[0238] AEBSF (4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride): 10-100 μM.

[0239] Preferably, the protease inhibitor cocktail (Pi) comprises the following components and concentrations for use in the novel storage solution:

[0240] 1 μM pepstatin;

[0241] 2 μM leupeptin;

[0242] 5 μM bestatin;

[0243] 1.5 μM E-64;

[0244] 0.08 μM aprotinin;

[0245] 100 μM AEBSF.

[0246] The disclosed preservation solutions are used in methods for improving the viability of cells, organs or tissues during extended cold storage periods. The disclosed preservation solutions can be used to pretreat the cells, organs or tissues prior to exposure to a cryogenic environment. The novel preservation solutions can also be used to maintain the viability of cells, tissues or organs during storage in a cryogenic environment.

[0247] Disclosed herein is a method for treating cells, organs or tissues to withstand exposure to a low temperature environment, the method comprising providing the cells, tissues or organs to be exposed to the low temperature environment; and contacting the cells, organs or tissues with the disclosed preservation solution discussed herein so that the cells, organs or tissues can withstand exposure to the low temperature.

[0248] Disclosed herein is a method for preserving the viability of cells, organs or tissues during exposure to a cryogenic environment, the method comprising contacting the cells, organs or tissues with a preservation solution disclosed as discussed herein to preserve the viability of the cells, organs or tissues; and exposing the cells, organs or tissues to the cryogenic environment.

[0249] In embodiments, the methods disclosed herein also reduce damage to cells, organs, or tissues when exposed to hypothermic environments.

[0250] In an embodiment, the cell, tissue or organ is a mammalian cell, tissue or organ, including neuronal cells, retinal cells, cornea, retina, skin, heart, lung, pancreas, kidney, liver, intestine, stem cell, blood, or any other suitable cell, tissue or organ for transplantation. In an embodiment, the cell, tissue or organ can be cornea or corneal cell or islet or cell, preferably cornea or corneal cell. In an embodiment, corneal cell is endothelial cell, epithelial cell, corneal stromal cell or a combination thereof. Corneal cell is contained in a complete cornea. In an embodiment, corneal cell is endothelial cell and is contained in the corneal endothelium of the cornea.

[0251] In an embodiment, a method for treating a cell, organ or tissue to withstand exposure to a low temperature environment comprises providing the cell, organ or tissue to be exposed to the low temperature environment, and contacting the cell, organ or tissue with a novel preservation solution as discussed herein to effectively enable the cell, organ or tissue to withstand exposure to the low temperature. In an embodiment, a method for preserving the viability of a cell, organ or tissue during exposure to a low temperature environment is also disclosed herein, the method comprising contacting the cell, organ or tissue with a novel preservation solution as discussed herein to effectively preserve the viability of the cell, organ or tissue; and exposing the cell, organ or tissue to the low temperature environment.

[0252] In an embodiment, contacting the cell, tissue or organ with the novel preservation solution and / or the composition comprising a compound that causes FOXO1 activation can occur before the cell, tissue or organ is exposed to a low temperature environment. Contacting the cell, tissue or organ with the novel preservation solution or composition can occur before the cell, tissue or organ is removed from a donor, after the cell, tissue or organ is removed from a donor, or a combination thereof. In each case, the contacting occurs before cold storage begins.

[0253] The cornea is a transplantable tissue that is removed from a donor's eye and stored at low temperatures for a period of time prior to transplantation. In the disclosed methods, contacting the cornea or corneal cells with the novel preservation solution can occur before exposing the cornea to a low temperature environment, during exposing the cornea to a low temperature environment, or a combination thereof.

[0254] In an embodiment, the cornea is contacted with the novel preservation solution and / or composition before being removed from the donor's eye (pretreatment). The novel preservation solution and / or composition comprising a compound that causes FOXO1 activation is injected into the anterior chamber of the eye, which is the aqueous humor-filled space in the front of the eye between the cornea and the iris. In the anterior chamber, the composition contacts endothelial cells within the corneal endothelium at the posterior surface of the cornea. After a defined period of time, the cornea is removed from the donor eye.

[0255] In embodiments, the cornea is contacted with the novel preservation solution and / or composition after it is removed from the donor's eye (pretreatment).The cornea is removed from the donor's eye and placed in a container containing the novel preservation solution and / or composition for a defined period of time.

[0256] In embodiments, the cells, tissues or organs may be pretreated with the novel preservation solution and / or composition for a period of 5 minutes to 240 minutes, 10 minutes to 150 minutes, 20 minutes to 120 minutes, 20 minutes to 90 minutes, or 45 minutes to 90 minutes, or 45 minutes to 60 minutes. In embodiments, the contact time is 20 minutes to 90 minutes. The cells, tissues or organs may be pretreated with the novel preservation solution and / or composition comprising a compound that causes FOXO1 activation at a temperature of 22°C to 37°C (e.g., 25°C to 37°C).

[0257] In an embodiment, the cell, tissue or organ is a cornea or a corneal cell, and after pre-treating the cornea with the novel preservation solution and / or composition, the cornea is placed in a medium suitable for maintaining (storing) the cornea in a low temperature environment and then placed in a low temperature environment. The cornea can be stored in a novel corneal preservation solution discussed herein. In an embodiment, the novel corneal preservation solution may contain a compound and / or Pi that causes FOXO1 activation. In an embodiment, the cornea or corneal cells are maintained in a low temperature environment in the presence of a novel preservation solution containing a compound and / or Pi that causes FOXO1 activation.

[0258] In the presence of a novel preservation solution, the cells, tissues or organs are maintained in a low temperature environment for a period of 1 day (24 hours) to 42 days (6 weeks), 1 day to 35 days, 5 days to 35 days, 10 days to 35 days, 14 days to 35 days, 10 days to 28 days, or 14 days to 28 days. In an embodiment, exposing cells, tissues or organs to a low temperature environment includes storage at a temperature of 2°C to 8°C for at least one week, preferably at least 1 week to 5 weeks, more preferably at least 1 week to 4 weeks. The cells, tissues or organs can be maintained in a low temperature environment in the presence of a novel preservation solution for a period of at least 2 weeks, at least 3 weeks, at least 4 weeks, or at least 5 weeks, without significant loss of viability of the cells, tissues or organs.

[0259] In embodiments, the cells, tissues or organs are corneal cells, preferably endothelial cells present in intact corneal endothelium. After storage at a temperature of 0°C to 8°C for a period of 4 weeks, the viability of the corneal endothelium is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. In embodiments, maintaining the cornea at a temperature of 0°C to 8°C for a period of 4 weeks results in preservation of 80% to 100%, or 85% to 100%, or 90% to 100%, or 95% to 100% of the viability of the endothelial cells in the corneal endothelium.

[0260] Also disclosed is the use of a novel preservation solution as discussed herein in cells, organs or tissues for treating or preserving the vitality of the cells, tissues or organs to withstand exposure to a cryogenic environment. Also disclosed is the use of a novel preservation solution as discussed herein for preparing a medicament for treating or preserving the vitality of cells, tissues or organs to withstand exposure to a cryogenic environment. In embodiments, the uses disclosed herein also reduce damage to cells, tissues or organs when exposed to a cryogenic environment.

[0261] The present disclosure is further illustrated by the following non-limiting examples.

[0262] Example

[0263] Example 1-

[0264] This example shows that activation of FOXO1 provides young corneas with better cold adaptation. TM The corneas were stored in a corneal preservation solution at 4°C for 3 weeks. Figure 1 As shown, it was found that corneal endothelial cells of 4-week-old rats showed increased survival rate / lower mortality compared with 6-month-old rats, indicating that corneal tissue of young individuals has better cold adaptation ability.

[0265] By immunofluorescence staining, it was found that ( Figure 2 ) In 4-week-old fresh corneas, the FOXO1 transcription factor translocated from the cytoplasm ("inactive") to the nucleus ("active"), while in 6-month-old corneas, FOXO1 was mainly present in the cytoplasm. This again suggests that FOXO1 may be associated with the better cold adaptation ability of young rat corneas.

[0266] In tardive ground squirrels (TLGS), when the ambient temperature dropped from 37°C to 4°C, the transcription factor FOXO1 translocated from the cytoplasm ('inactive') to the nucleus ('active') in TLBS iPSC-neurons and young human iPSC-neurons, but not in adult iPSC-neurons ( FIG. 16A to FIG. 16B and Fig.17 A to Fig.17 D) If FOXO1 is inhibited pharmacologically, or naturally in adult cells, the same duration of incubation at 4°C will result in significantly higher cell death.

[0267] Furthermore, the temperature-sensitive characteristics of FOXO1 are retained in various cold-intolerant species. Fig.18 , FIG. 19A to FIG. 19B and FIG. 20A to FIG. 20B . FOXO1 nuclear transport is regulated by RANBP2 / importin-7 / exportin-1; RANBP2 is a SUMO (small ubiquitin protein-like modifier) ​​E3 ligase. Addition of a SUMOylation inhibitor inhibited FOXO1 nuclear import; addition of the deSUMOylation inhibitor N-ethylmaleimide enabled longer-term cryopreservation of human and mouse islets. Fig.21 A to Fig.21 C shows that cold-induced nuclear import of FOXO1 is enabled by SUMO E3 ligase RANBP2 and transporter importin-7, and inhibited by transporter exportin-1. Functional deterioration of RANBP2 and importin-7 may be the reason why FOXO1 nuclear import is inhibited after cold stress in older H1 cells.

[0268] Fig. 22 A to Fig. 22 B indicates that cold-induced nuclear translocation of FOXO1 is also determined by the key SUMO-interacting motif (SIM) on the N-terminus of the FOXO1 protein; if the SIM motif is mutated, the FOXO1 protein will accumulate in the nucleus under normal conditions. 2) Therefore, it is possible to design drugs or peptides that interact with the FOXO1 SIM before cells are exposed to cold, thereby "activating" the translocation of the FOXO1 protein into the nucleus and enhancing cell cold survival.

[0269] Example 2-

[0270] The donor sources for corneal transplantation are mainly adults and the elderly, and the question is whether adult corneas can acquire the cold adaptation ability of young individuals' corneas by activating FOXO1. The present inventors demonstrated that C6 ceramide (abbreviated as C6) and apigenin can activate FOXO1 in adult rat corneas.

[0271] The inventors injected two drugs, C6 ceramide (C6 at a concentration of 20 μM / μL, dissolved in goat serum solvent) and apigenin (at a concentration of 4 μM / μL, dissolved in goat serum solvent) into the anterior chamber of the eyes of 6-month-old rats, and found that the corneal endothelial cells of the rats activated FOXO1 to enter the nucleus, and after C6 injection, the nuclear entry signal was enhanced after cold stimulation ( Figure 3 ). Injection of apigenin does not cause FOXO1 to enter the nucleus. After cold stimulation, the nuclear import signal activates FOXO1 to enter the nucleus, and the weak nuclear import effect is still maintained after rewarming ( Figure 4 ).

[0272] Therefore, the present inventors found that two drugs, C6 ceramide and apigenin, can be used to activate FOXO1 to enter the nucleus and enhance the cold adaptation ability of adult rat cornea.

[0273] Example 3 - Novel corneal preservation solution formulation

[0274] The present inventors adjusted the components of the preservation solution according to the metabolic characteristics of the dormant period and mainly used polar neutral amino acids, basic amino acids and water-soluble vitamins to meet the normal physiological needs of cells during the cold storage period. The composition of the improved corneal preservation solution is shown in Table 1:

[0275] Table 1: Improved corneal preservation solution (MCM) formulation

[0276]

[0277]

[0278]

[0279] Example 4 - Novel corneal preservation solution formulation

[0280] The composition of the novel cornea preservation solution of this example is shown in Table 2.

[0281] Table 2 Novel corneal preservation solution (MCM) formula

[0282]

[0283]

[0284]

[0285] Example 5 - Novel corneal preservation solution formulation

[0286] The composition of the novel cornea preservation solution of this example is shown in Table 3.

[0287] Table 3 Novel corneal preservation solution (MCM) formula

[0288]

[0289]

[0290]

[0291] Example 6 - Novel Cold Cornea Preservation Strategy

[0292] A novel corneal preservation solution was prepared according to the novel corneal preservation solution formula as discussed herein, and the optimal preservation strategy was adjusted by using apigenin or C6. The specific process is as follows:

[0293] (1)C6

[0294] After 0.5 μL of C6 solution (10 μM / μL in goat serum solvent) was injected into the anterior chamber of the eye through the limbal stroma. After 90 minutes, the cornea was removed and refrigerated in corneal preservation solution. After 3 and 4 weeks of refrigeration, the refrigeration results were evaluated by immunofluorescence staining.

[0295] The corneal preservation solution of this example is the novel corneal preservation solution prepared in Example 3, to which 20 μM C6 and protease inhibitor mixture Pi (as described in WO2019 / 040359A1) are added, and the final concentrations of the components in the protease inhibitor mixture Pi in the cold preservation solution are: 1 μM pepstatin; 2 μM leupeptin; 5 μM bestatin; .5 μM E-641; 0.08 μM aprotinin; 100 μM AEBSF.

[0296] The results are FIG. 5A to FIG. 5B The corneal endothelial cell mortality rate in the group using the novel corneal preservation solution was lower than that in the group using Optisol-GS TM Corneal endothelial cell death rate in cold-preserved groups (with and without C6). The number of dead corneal endothelial cells decreased after the addition of C6 compared to the group without C6. In addition, the corneal endothelial cells in the corneal preservation solution containing the combination of C6 and Pi maintained a better hexagonal structure compared to the corneal preservation solution with only C6 added. Optisol-GS TM The solution plus C6 showed F-actin degradation. Figure 5A It is shown that C6 and Pi can be added to Optisol-GS TM Neutralizes and enhances the survival rate of rat corneal endothelial cells. Figure 5B It was shown that C6 and Pi could be added to a novel corneal preservation solution (MCM) and enhance the survival rate of rat corneal endothelial cells.

[0297] The mortality rate of each group was calculated by counting the number of PI-positive cells in the total number of cells in the field of view, and the results, such as Figure 6 As shown, Optisol-GS was found TM After 3 weeks of storage, the cell death rate was close to 100%, and many endothelial cells were rescued after using C6 and Pi, with death rates of 18.9% and 8.5%, respectively. The death rate of endothelial cells in the novel corneal preservation solution was lower than that in Optisol-GS. TM The endothelial cell mortality rate in the novel corneal preservation solution plus C6 and Pi group was 0.18%.

[0298] To compare corneal permeability, record a picture of the "A" character observed through the cornea. Figure 7 As shown, corneas treated with the novel preservation solution plus C6 and Pi maintained constant permeability after 3 weeks of cold storage, while the novel preservation solution plus C6 alone ranked second. TM Corneal edema of the treated cornea caused blurring in which a clear letter "A" could not be seen.

[0299] To further test the observed effects, the group with the better 3-week cold storage effect was selected and the cold storage time was extended to 4 weeks. Fig. 8A and Figure 8B As shown, rat corneas can be stored at 4°C for 28 days with the endothelium essentially intact. TM Rat corneas stored in 2% MgCl2O3 still had a high mortality rate. TM The corneal endothelial cells still had a high mortality rate when the corneal endothelial cells were treated with the novel corneal preservation solution (containing C6 and Pi) because the corneal endothelial cells were maintained in a better hexagonal structure. F-actin of the C6 group was added to the novel corneal fluid to form a block. The novel corneal preservation solution containing C6 showed a large amount of F-actin.

[0300] Permeability comparison showed that the corneas using the novel corneal preservation solution (containing C6 and Pi) still maintained a certain permeability after 4 weeks, while the corneas using Optisol-GS TM Corneas of the 100% corneal preservation solution (containing C6 and Pi) and the novel corneal preservation solution (containing C6) showed that the edges of the letter "A" were blurred and difficult to distinguish ( Fig. 9 ).

[0301] The ratio of dead endothelial cells was counted, and it was found that the novel corneal preservation solution (containing C4 and Pi) resulted in the best effect after 4 weeks of cold storage, with a corneal endothelial cell death rate of about 14.3% ( Fig.10 ).

[0302] (2) Apigenin

[0303] 0.5 μL of apigenin solution (4 μM / μL, dissolved in goat serum solvent) was injected into the anterior chamber through the limbal stroma, and 20 minutes later, the cornea was removed and stored in the novel preservation solution (containing 4 μM apigenin and previously added Pi) prepared in Example 3. After 3 and 4 weeks of cold storage, the cold storage results were evaluated by immunofluorescence staining.

[0304] Based on the results of C6, the novel corneal preservation solution plus apigenin was compared with the novel corneal preservation solution plus apigenin and Pi. Fig.11 As shown, corneal endothelial cells showed reduced mortality after apigenin was applied. Cells in the novel corneal preservation solution containing apigenin and Pi showed better corneal endothelial hexagonal structure. However, when preserved in the novel corneal preservation solution containing only apigenin, the corneal endothelium showed partial damage.

[0305] The refrigeration time was then extended to 4 weeks. The data showed that rat corneas could be stored at 4°C for 28 days with the endothelium largely intact. Fig.12 It shows that after adding apigenin, the nucleus presents a full / plump shape, and PI positive cells (PI stains dead cells) are significantly reduced. When combined with Pi, corneal endothelial cells retain a better hexagonal structure. However, the nucleus preserved in the novel corneal preservation solution and apigenin presents a slight pyknosis (slightly wrinkled shape).

[0306] According to the data ( Fig.13 ), the novel corneal preservation solution plus apigenin and Pi showed a good preservation effect within 3 weeks. By 4 weeks, the novel corneal preservation solution plus apigenin and Pi showed only 2.5% mortality.

[0307] After 4 weeks of preservation of the corneas of 6-month-old rats using C6 or a combination of apigenin and Pi in a novel corneal preservation solution, corneal transplantation was performed. Fig.14 ) showed that within 1 week after implantation, the transplanted cornea slowly healed, and by the third week, the fresh cornea was completely healed and transparent / translucent. TM The corneas refrigerated for 4 weeks atrophied, blanched, and showed scar healing (showing signs of degeneration and more severe inflammation); the corneas using the novel storage solution containing the combination of C6 and Pi for 4 weeks were slightly cloudy / cloudy, but generally good; the corneas using the novel storage solution containing the combination of apigenin and Pi were slightly cloudy / cloudy after 4 weeks, but still had good clarity. Fig.15 As seen in the transmission electron micrograph, Optisol-GS TMCorneal endothelial subcellular structure was poor in stored corneas; except for the 'fresh' cornea control, the other 3 groups were from cryopreserved corneas 6 months after transplantation surgery.

[0308] In summary, the inventors have developed a novel corneal cold storage strategy. The novel corneal preservation solution plus C6 or apigenin and optionally with Pi have a good cold preservation effect, wherein the novel corneal preservation solution plus apigenin or C6 and Pi group has the best preservation effect in 4 weeks, the corneal endothelial cell mortality rate is only 2.5%, the transplantation operation can be performed, and there is good transparency and brightness after surgery.

[0309] Example 7 - Novel preservation solution shown to be effective in different tissues and species

[0310] The present inventors have found that the novel preservation solution can achieve the effect of improving the cold preservation effect on cells of different tissues and different species.

[0311] Using mouse pancreatic islets as a model, Fig.23 A to Fig.23 E It can be seen that 1) key components of the FOXO1-dependent survival pathway are not well maintained in the current gold standard organ preservation solution (UW, University of Wisconsin solution); 2) if the deSUMOylation inhibitor N-ethylmaleimide (N-EM; see Fig.23 C) When added to UW solution, islet cold survival was improved; if the basal preservation solution (HS) developed by us was used, islet cold survival was even better; 3) Even in HS solution, if the SUMOylation inhibitor 2-D08 or ginkgolic acid (GA, see Fig.23 B and Fig.23 D), the cold-induced nuclear entry of FOXO1 was inhibited and islet cell death became severe. N-EM and protease inhibitors were added to HS to make HS+. As can be seen from Figures 26A to 26C, mouse islets can be stored in HS+ at 4°C for up to 14 days, transplanted into streptozotocin (STZ)-induced type I diabetic mice, and successfully reduced the blood glucose levels of the recipient mice. Fig.25 A to Fig.25 B shows that HS+ can significantly improve the quality of human islets after long-term refrigeration.

[0312] In another example, mouse islets were extracted and stored in a refrigerator at 4°C. The commonly used conventional 1640 culture medium was used as a control group. As a result, islet cells died in large numbers in the 1640 culture medium, while islet cell death was less in the novel preservation solution (MM), and the addition of C6 and apigenin to the islets further reduced the number of dead cells.

[0313] In another example, human pancreases were refrigerated and 50 mg of pancreatic tissue was extracted at two time points: 24 and 48 hours, and insulin content was measured by chemiluminescence. Fig.25 C. Compared with the classical UW preservation solution used clinically, the pancreas refrigerated in the novel preservation solution of the present invention had higher pancreatic insulin content at both the 24-hour and 48-hour time points, and the preservation effect of the novel preservation solution was better.

[0314] This suggests that the novel preservation solution has application prospects across different tissues and different species.

[0315] The compositions, methods and articles may alternatively comprise, consist of or consist essentially of any suitable material, step or component disclosed herein. The compositions, methods and articles may additionally or alternatively be formulated to be free of or essentially free of any material (or type), step or component that is otherwise unnecessary for achieving the function or purpose of the compositions, methods and articles.

[0316] Unless otherwise indicated herein, the description of a range of values ​​is merely intended to serve as a shorthand method of referring to each separate value falling within the range, 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 are independently combinable.

[0317] "Combination" includes blends, mixtures, alloys, reaction products, etc. The terms "first", "second", etc. do not represent any order, amount or importance, but are used to distinguish one element from another element. Unless otherwise specified herein or clearly contradictory to the context, the terms "one", "one (kind)" and "said" do not represent quantity restrictions, and should be interpreted as covering both the singular and the plural. Unless otherwise clearly stated, "or" means "and / or". Throughout this specification, references to "some embodiments", "one embodiment", etc. mean that the specific elements described in conjunction with the embodiments are included in at least one embodiment described herein, and may or may not exist in other embodiments. In addition, it should be understood that the elements described can be combined in any suitable manner in various embodiments. "Their combination" is open, and includes any combination of at least one of the listed components or properties and any optional unlisted similar or equivalent components or properties.

[0318] Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs. All cited patents, patent applications and other references are incorporated herein by reference in their entirety. However, if a term in the present application contradicts or conflicts with a term in an incorporated reference, the term in the present application takes precedence over the conflicting term from the incorporated reference.

[0319] Compounds are described using standard nomenclature. For example, any position not substituted by any specified group is understood to have its valency filled by a specified bond or hydrogen atom. A dash ("-") that is not between two letters or symbols is used to indicate the point of attachment of a substituent. For example, -CHO is attached through the carbon of a carbonyl group.

[0320] Unless otherwise specified herein or obviously contradictory to the context, all methods described herein can be performed in a suitable order. Unless otherwise required for protection, the use of any and all examples or illustrative language (e.g., "for example") provided herein is only intended to better illustrate the present invention, rather than to limit the scope of the present invention. Any language in the specification should not be interpreted as indicating that any unprotected element is essential for the practice of the present invention as used herein. Unless otherwise defined, the technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in the field of the present disclosure.

[0321] In addition, the present disclosure encompasses all variations, combinations and permutations of introducing one or more limitations, elements, clauses and descriptive terms from one or more of the listed claims into another claim. For example, any claim that is dependent on another claim can be amended to include one or more limitations found in any other claim that is dependent on the same basic claim. When elements are presented as a list (e.g., in Markush group format), various subgroups of the elements are also disclosed, and any element can be removed from the group.

[0322] Although specific embodiments have been described, the applicant or other persons skilled in the art may conceive of alternatives, modifications, variations, improvements, and substantial equivalents that are not currently foreseen or may not currently be foreseen. Therefore, the appended claims as filed and as they may be amended are intended to cover all such alternatives, modifications, variations, improvements, and substantial equivalents.

Claims

1. A method for treating a cell, tissue or organ to withstand exposure to a cryogenic environment, the method comprising: Providing the cells, tissues or organs to be exposed to the low temperature environment; as well as The cell, tissue or organ is contacted with a composition comprising a compound that activates the FOXO1 protein in the cell, tissue or organ, so that the cell, tissue or organ can withstand exposure to the low temperature.

2. A method for preserving the vitality of cells, tissues or organs during exposure to a cryogenic environment, the method comprising: contacting the cell, tissue or organ with a composition comprising a compound that activates the FOX01 protein in the cell, tissue or organ to effectively preserve the viability of the cell, tissue or organ; as well as The contacted cells, tissues or organs are exposed to the hypothermic environment.

3. The method according to any one of claims 1-2, wherein the cell, tissue or organ is cornea or corneal cells.

4. The method according to any one of claims 1 to 3, wherein the cell, tissue or organ is an endothelial cell of the corneal endothelium. 5 . The method according to any one of claims 1 to 4 , wherein the compound that activates FOX01 protein is a cell-permeable ceramide analog or apigenin. 6 . The method according to any one of claims 1 to 5 , wherein the compound that activates FOX01 protein is a cell-permeable ceramide analog.

7. The method according to claim 6, wherein the cell-permeable ceramide analog is a compound represented by Formula 1 wherein R is a C1 to C9 alkyl group.

8. The method according to claim 6, wherein the cell-permeable ceramide analog is a compound represented by Formula 2 9. The method according to any one of claims 6 to 8, wherein the concentration of the cell-permeable ceramide analog in the composition is 0.1 μM to 100 μM.

10. The method according to any one of claims 6-8, wherein the concentration of the cell-permeable ceramide analog in the composition is 2 μM to 20 μM.

11. The method according to any one of claims 1 to 5, wherein the compound that activates FOX01 protein is apigenin. 12 . The method according to claim 11 , wherein the concentration of the apigenin in the composition is 0.1 μM to 30 μM.

13. The method according to claim 11, wherein the concentration of the apigenin in the composition is 0.1 μM to 10 μM.

14. The method of any one of claims 1-13, wherein the composition further comprises a preservation solution.

15. The method of claim 14, wherein the preservation solution is a corneal preservation solution and comprises dextran, chondroitin sulfate, or a combination thereof.

16. The method according to claim 14, wherein the storage solution comprises the following components in 1 L: Amino Acids: 1.5-2.2mg of ALA 75-93mg arginine 14-17 mg of asparagine 0.9-1.1 mg of cysteine 27-33mg of glycine 180-220 mg of glutamine 28-34mg histidine 94-120mg isoleucine 94-120 mg leucine 100-130 mg lysine 27-33 mg methionine 59-73 mg phenylalanine 7-8.5 mg proline 36-46 mg of serine 85-105 mg of threonine 14-18 mg of tryptophan 60-80 mg of tyrosine 84-105 mg of valine Vitamins: 4-5.2 mg of choline 3-4.5 mg of calcium D-pantothenate 0.9-1.1 mg of pantothenic acid (vitamin B5) 4-5 mg of folic acid 4-5 mg of niacinamide 4-5 mg of pyridoxal (vitamin B6) 0.09-0.11 mg of riboflavin (vitamin B2) 5-6.5 mg of thiamine (vitamin B1) 1-1.5 mg of vitamin B12 Inorganic salts: 170-220 mg of CaCl2 50-70 mg Fe(NO3)3 360-440 mg KCl 66-85 mg of NaHCO3 4300-5300 mg of NaCl 120-160 mg of NaH2PO4 69-85 mg MgCl2 97-120 mg ZnSO4 Other components: 1.3-1.7 mg of adenosine 22,000-28,000 mg of chondroitin sulfate 4000-5000 mg of glucose 9000-11000mg of beta-glucan 90-110 mg of gentamicin 9-12 mg of inosine 10-14mg of inositol 330-420 mg of phenol red 0.9-1.1 mg of pyridoxal hydrochloride 220-280 mg of MES monohydrate 9000-11000 μM MOPS buffer 1800-2200 μM HEPES buffer 0.9-1.2 mg of sodium pyruvate 3.4-4.3 mg of 2-mercaptoethanol, The storage solution has a pH of 7.3±2 and an osmotic pressure of 300±50 mOsm.

17. The method of any one of claims 1-16, wherein the storage solution further comprises a protease inhibitor cocktail (Pi) comprising 1-20 μM aspartic protease inhibitors and 2-40 μM serine and cysteine ​​protease inhibitors, and optionally further comprises at least one of: 5-50 μM metalloproteinase inhibitors, 1.5-30 μM cysteine ​​protease inhibitors, 0.08-2 μM serine protease inhibitors, and 10-100 μM AEBSF (4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride).

18. The method according to claim 17, wherein The aspartic protease inhibitor is pepstatin, The serine and cysteine ​​protease inhibitor is leupeptin, The metalloproteinase inhibitor is bestatin, The cysteine ​​protease inhibitor is E-64, and The serine protease inhibitor is aprotinin.

19. The method according to any one of claims 17-18, wherein the protease inhibitor cocktail (Pi) comprises: 1 μM pepstatin; 2 μM leupeptin; 5 μM bestatin; 1.5 μM E-64; 0.08 μM aprotinin; and 100 μM AEBSF.

20. The method of any one of claims 1-19, wherein contacting the cell, tissue or organ with the composition occurs before and / or during exposure of the cell, tissue or organ to the cryogenic environment.

21. The method of any one of claims 1-20, wherein contacting the cell, tissue or organ with the composition occurs prior to removing the cell, tissue or organ from a donor.

22. The method of claim 21, wherein the cell, tissue or organ is the cornea or corneal cells, and the cornea or corneal cells are contacted with the composition prior to removal of the cornea or corneal cells from the eye.

23. The method of any one of claims 1-20, wherein contacting the cell, tissue or organ with the composition occurs after the cell, tissue or organ is removed from a donor.

24. The method of claim 23, wherein the cell, tissue or organ is the cornea or corneal cells, and the cornea or corneal cells are contacted with the composition after the cornea or corneal cells are removed from the eye.

25. The method of any one of claims 1-24, wherein the cell, tissue or organ is contacted with the composition for a period of 5 minutes to 240 minutes.

26. The method of any one of claims 1-25, wherein after contacting the cell, tissue or organ with the composition, the method further comprises maintaining the cell, tissue or organ in the cryogenic environment in the presence of the composition for a period of 1-4 weeks.

27. The method of any one of claims 1-26, wherein the exposure to a low temperature environment comprises storage at a temperature of 2°C to 8°C.

28. The method of any one of claims 1-27, wherein the cell, tissue or organ is a corneal endothelial cell present in intact corneal endothelium and the viability of the corneal endothelium after storage at a temperature of 2°C to 8°C for a period of 4 weeks is at least 90%.

29. The method of any one of claims 1-28, wherein the cell, tissue or organ is a corneal endothelial cell present in intact corneal endothelium and the viability of the corneal endothelium after storage at a temperature of 2°C to 8°C for a period of 4 weeks is at least 95%.

30. Use of a composition comprising a compound that activates FOX01 protein in cells, tissues or organs for treating or preserving the vitality of the cells, tissues or organs to withstand exposure to a low temperature environment.

31. Use of a composition comprising a compound that activates FOX01 protein for preparing a medicament for treating or preserving the vitality of cells, tissues or organs to withstand exposure to a low temperature environment.

32. Use of the composition according to claim 30 or 31, wherein the cell, tissue or organ is cornea or corneal cells.

33. The use of the composition according to any one of claims 30 to 32, wherein the compound that activates FOX01 protein is a cell-permeable ceramide analog or apigenin.

34. The use of the composition according to any one of claims 30 to 33, wherein the compound that activates FOX01 protein is a cell-permeable ceramide analog.

35. The use of the composition according to claim 34, wherein the cell-permeable ceramide analog is a compound represented by Formula 1 wherein R is a C1 to C9 alkyl group.

36. The use of the composition according to claim 35, wherein the cell-permeable ceramide analog is a compound represented by Formula 2 37. Use of a composition according to any one of claims 34 to 36, wherein the concentration of the cell-permeable ceramide in the composition is 2 μM to 20 μM.

38. The use of the composition according to any one of claims 30-33, wherein the compound that activates FOX01 protein is apigenin.

39. Use of the composition according to claim 38, wherein the concentration of the apigenin in the composition is 0.1 μM to 30 μM.

40. A storage solution, calculated as 1 L, comprising the following components: Amino Acids: 1.5-2.2mg of ALA 75-93mg arginine 14-17 mg of asparagine 0.9-1.1 mg of cysteine 27-33mg of glycine 180-220 mg of glutamine 28-34mg histidine 94-120mg isoleucine 94-120mg of leucine 100-130mg lysine 27-33 mg of methionine 59-73 mg of phenylalanine 7-8.5 mg of proline 36-46mg of serine 85-105mg of threonine 14-18 mg of tryptophan 60-80mg of tyrosine 84-105 mg of valine Vitamins: 4-5.2 mg of choline 3-4.5 mg of calcium D-pantothenate 0.9-1.1 mg of pantothenic acid (vitamin B5) 4-5 mg of folic acid 4-5 mg of niacinamide 4-5 mg of pyridoxal (vitamin B6) 0.09-0.11 mg of riboflavin (vitamin B2) 5-6.5 mg of thiamine (vitamin B1) 1-1.5 mg of vitamin B12 Inorganic salts: 170-220 mg of CaCl2 50-70 mg Fe(NO3)3 360-440 mg KCl 66-85 mg of NaHCO3 4300-5300 mg of NaCl 120-160 mg of NaH2PO4 69-85 mg MgCl2 97-120 mg ZnSO4 Other components: 1.3-1.7 mg of adenosine 22000-28000mg chondroitin sulfate 4000-5000mg glucose 9000-11000mg dextran 90-110mg gentamicin 9-12mg inosine 10-14mg of inositol 330-420 mg phenol red 0.9-1.1 mg pyridoxal hydrochloride 220-280 mg MES monohydrate 9000-11000 μM MOPS buffer 1800-2200 μM HEPES buffer 0.9-1.2 mg of sodium pyruvate 3.4-4.3 mg of 2-mercaptoethanol, The storage solution has a pH of 7.3±2 and an osmotic pressure of 300±50 mOsm.

41. The preservation solution of claim 40, wherein the preservation solution is a corneal preservation solution.

42. The preservation solution according to any one of claims 40-41, further comprising at least one selected from the group consisting of: a compound that activates FOX01 protein in the cell, tissue or organ, and a protease inhibitor cocktail (Pi).

43. The preservation solution according to claim 42, wherein the compound that activates FOX01 protein is a cell-permeable ceramide analog or apigenin.

44. The preservation solution of claim 42, wherein the compound that activates FOX01 protein is a cell-permeable ceramide analog.

45. The preservation solution according to claim 44, wherein the cell-permeable ceramide analog is a compound represented by Formula 1 wherein R is a C1 to C9 alkyl group.

46. ​​The preservation solution according to any one of claims 44-45, wherein the cell-permeable ceramide analog is a compound represented by Formula 2 47. The preservation solution of any one of claims 44-46, wherein the concentration of the cell-permeable ceramide in the solution is 0.1 μM / μL to 100 μM / μL.

48. The preservation solution of any one of claims 44-46, wherein the concentration of the cell-permeable ceramide analog in the solution is 2 μM / μL to 20 μM / μL.

49. The preservation solution of claim 42, wherein the compound that activates FOX01 protein is apigenin.

50. The preservation solution of claim 49, wherein the concentration of the apigenin in the solution is 0.1 μM / μL to 30 μM / μL.

51. The preservation solution of claim 49, wherein the concentration of the apigenin in the solution is 0.1 μM / μL to 10 μM / μL.

52. The preservation solution of claim 42, wherein the protease inhibitor cocktail (Pi) comprises 1-20 μM aspartic protease inhibitors and 2-40 μM serine and cysteine ​​protease inhibitors, and optionally further comprises at least one of: 5-50 μM metalloproteinase inhibitors, 1.5-30 μM cysteine ​​protease inhibitors, 0.08-2 μM serine protease inhibitors, and 10-100 μM AEBSF (4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride).

53. The preservation solution of claim 52, wherein The aspartic protease inhibitor is pepstatin, The serine and cysteine ​​protease inhibitor is leupeptin, The metalloproteinase inhibitor is bestatin, The cysteine ​​protease inhibitor is E-64, and The serine protease inhibitor is aprotinin.

54. The corneal preservation solution of claim 52, wherein the protease inhibitor cocktail (Pi) comprises: 1 μM pepstatin; 2 μM leupeptin; 5 μM bestatin; 1.5 μM E-64; 0.08 μM aprotinin; and 100 μM AEBSF.

55. A method of treating a cell, tissue or organ to withstand exposure to a cryogenic environment, the method comprising: Providing the cells, tissues or organs to be exposed to the low temperature environment; as well as The cells, tissues or organs are contacted with a preservation solution according to any one of claims 40-54 to enable the cells, tissues or organs to withstand exposure to the low temperature.

56. A method for preserving the viability of a cell, tissue or organ during exposure to a cryogenic environment, the method comprising: contacting the cell, tissue or organ with a preservation solution according to any one of claims 40 to 54 to preserve the viability of the cell, tissue or organ; as well as The contacted cells, tissues or organs are exposed to the hypothermic environment.

57. The method of any one of claims 55-56, wherein the cells, tissues or organs are maintained in the cryogenic environment in the presence of the preservation solution for a period of 1-4 weeks.

58. The method of any one of claims 55-57, wherein the exposure to a low temperature environment comprises storage at a temperature of 2°C to 8°C.

59. The method of any one of claims 55-58, wherein the cell, tissue or organ is cornea or corneal cells.

60. The method of claim 59, wherein the corneal cells are endothelial cells present in intact corneal endothelium and the viability of the corneal endothelium after storage at a temperature of 2°C to 8°C for a period of 4 weeks is at least 90%.

61. The method of claim 59, wherein the corneal cells are endothelial cells present in intact corneal endothelium and the viability of the corneal endothelium after storage at a temperature of 2°C to 8°C for a period of 4 weeks is at least 95%.

62. Use of the preservation solution according to any one of claims 40 to 54 in cells, tissues or organs for treating or preserving the viability of the cells, tissues or organs to withstand exposure to a cryogenic environment.

63. Use of the preservation solution according to any one of claims 40 to 54 for the preparation of a medicament for treating or preserving the viability of cells, tissues or organs to withstand exposure to a cryogenic environment.

64. Use of a preservation solution according to any one of claims 62-63, wherein the cell, tissue or organ is cornea or corneal cells.

65. A composition comprising a compound that causes activation of FOXO1 in a cell, tissue or organ and a preservation solution for cryogenic storage of the cell, tissue or organ, The composition preserves the viability of the cells, tissues or organs during exposure to a cryogenic environment.

66. The composition of claim 65, wherein the cell, tissue or organ is cornea or corneal cells.

67. The composition of claim 65 or 66, wherein the compound that activates FOX01 protein is a cell-permeable ceramide analog or apigenin.

68. The composition according to any one of claims 65-67, wherein the compound that activates FOX01 protein is a cell-permeable ceramide analog.

69. The composition according to any one of claims 65-68, wherein the cell-permeable ceramide analog is a compound represented by Formula 1 wherein R is a C1 to C9 alkyl group.

70. The composition of any one of claims 65-69, wherein the cell-permeable ceramide analog is a compound represented by Formula 2 71. The composition of any one of claims 65-70, wherein the concentration of the cell-permeable ceramide analog in the composition is 2 μM to 20 μM.

72. The composition of any one of claims 65-67, wherein the compound that activates FOX01 protein is apigenin.

73. The composition of claim 72, wherein the concentration of the apigenin in the composition is 0.1 μM to 30 μM.

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