Compositions and methods for preservation and storage of biological materials

Abstract

Provided are compositions comprising (i) a hydrogen sulfide donor compound, a chalcogenide, or a sulfide salt and (ii) a dual inhibitor of mammalian target of rapamycin (mTOR) and phosphoinositide 3-kinase (PI3K) or a combination of an mTOR inhibitor and a PI3K inhibitor. Also provided are methods for inducing a state of stasis in cells, tissues, or organs with applications to the storage and preservation of biological materials as well as transplantation of biological materials.

Description

COMPOSITIONS AND METHODS FOR PRESERVATION AND STORAGE OFBIOLOGICAL MATERIALSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. provisional application No. 63 / 566,658 filed March 18, 2024, which is incorporated herein by reference in its entirety.FIELD

[0002] The disclosure provides compositions and methods for the storage and preservation of biological materials such as cells, tissues, and organs.BACKGROUND

[0003] Organ transplantation is the sole curative treatment for end-stage organ failure, yet its impact is limited by a critical shortage of viable organs, with over 100,000 patients in the United States alone awaiting transplants and daily fatalities exceeding twenty due to organ unavailability. For example, thousands of donated organs are damaged and discarded annually during transplantation procedures. Current methods of preventing tissue damage, including reduced temperature and perfusion protocols, are too often inadequate to prevent ischemia-reperfusion injury (IRI) which remains an important factor influencing transplant outcome. IRI results from the loss and subsequent gain of blood flow during the transplantation procedure. IRI is associated with delayed graft function, acute tissue injury, acute graft rejection and early graft loss.

[0004] Current methods for reducing the effects of IRI involve donor organ preservation prior to transplantation under hypothermic conditions which lower metabolic activity and allow for improved survival under hypoxic conditions. The most common method includes static cold storage (CS). In accordance with this method, an organ is placed in a preservation solution and then stored on ice. The University of Wisconsin (UW) preservation solution is the current standard for static cold storage. However, IRI still occurs under cold storage conditions. Cooling to —0 - 4°C on ice slows metabolic processes and minimizes damage, extending the viability window. However, while effective at reducing metabolic activity, CS alone cannot fully prevent organ damage, particularly during reperfusion when oxygen and blood supply are reintroduced. This method, while slowing cellular damage, is limited by storage times (<6 hours for hearts, <8 hours for lungs, and < 12 hours for livers), leading to frequent organ loss.

[0005] To overcome the limitations of static CS, normothermic machine perfusion (NMP) has emerged as an alternative that preserves organs at near physiological temperatures while providing continuous nutrient and oxygen support. NMP reduces IRI damage and extends preservation time, but its widespread application is constrained by high costs and logistical challenges.

[0006] H2S gas was shown to induce a suspended animation-like state, significantly reducing metabolism and oxygen consumption in mice under ambient temperature. However, its utility in transplantation has been limited due to key challenges. First, the original study was conducted under reduced oxygen conditions (17.5% 02), making it less applicable to clinical settings. Furthermore, subsequent studies showed that the observed hypometabolism was largely driven by reduced O2 rather than H2S itself, limiting its translational potential. Studies in other research animals showed H2S-induced reductions in oxygen uptake were minimal, highlighting species-specific differences in response and further complicating its application in clinical organ preservation.

[0007] Diapause, another naturally occurring phenomenon, has recently been chemically induced in mouse embryos. However, similar to H2S, this diapause state in mouse embryo was triggered under hypoxic condition (5% O2), limiting its broader practical applications.

[0008] Thus, there remains a need for methods and compositions for storing and preserving cells, tissues and organs.BRIEF SUMMARY

[0009] The present invention provides improved compositions and methods for the storage and preservation of cells, tissues, and organs, including under normothermic (37 °C) or subnormothermic conditions (10-35 °C) as well as near freezing conditions (0-4 °C).

[0010] Provided are compositions and methods for the storage and preservation of living cells, tissues, embryos, and organs. The methods described here include several advantages over prior art methods such as the ability to preserve the viability and functionality of cells, tissues, and organs under normothermic (37 °C) or subnormothermic conditions (10-35 °C) as well as near freezing conditions (0-4 °C). In addition, the methods induce a metabolically paused state in the cells, tissues, or organs under normoxic conditions, not restricted to about 18-22% O2, or about 20-21 % O2. Accordingly, the compositions and methods described here provide a practical, reproducible method for inducing metabolic pausing without relying on hypothermiaor hypoxia, thereby marking a significant advancement in metabolic manipulation and preservation strategies.

[0011] In one aspect, provided is a cell, tissue, embryo or organ preservation composition including (i) a hydrogen sulfide donor compound, a chalcogenide, or a sulfide salt and (ii) a dual inhibitor of mammalian target of rapamycin (mTOR) and phophoinositide 3-kinase (PI3K) or a combination of an mTOR inhibitor and a PI3K inhibitor. Alternatively, the preservation composition may comprise a dual inhibitor of mammalian target of rapamycin (mTOR) and phophoinositide 3-kinase (PI3K), or a combination of an mTOR inhibitor and a PI3K inhibitor, but the preservation composition does not comprise a hydrogen sulfide donor compound, a chalcogenide, or a sulfide salt. The composition may also include where the hydrogen sulfide donor compound is AP39, ATB-346, GIC-1001, SG-1002, GYY4137, or a derivative of any one of the foregoing. The composition may also include where the hydrogen sulfide donor compound is allicin (diallyl thiosulfinate), dially sulfide (DAS), diallyl disulfide (DADS), diallyl triulfidc (DATS), ammonium tctrathiomolybdatc (ATTM or (NID MoS^, anethole dithiolethione (ADT), or 5-(p-hydroxyphenyl)-3H-l,2-dithiole-3-thione (ADT-OH). The composition may also include where the chalcogenide is H2S, H2Se, FETe, or H2P0. The composition may also include where the sulfide salt is sodium hydrosulfide, sodium sulfide, or calcium sulfide. The composition may also include a combination of an mTOR inhibitor and a PI3K inhibitor. The composition may also include a dual mT0R / PI3K inhibitor. The composition may also include GSK2126458 (Omipalisib) or NVP-BEZ235 (Dactolisib). The composition may also include where the mTOR inhibitor is rapamycin, ridaforolimus, or temsirolimus. The composition may also include where the PI3K inhibitor is LY294002, BYL- 719 (Alpelisib), GDC-0941, GSK2636771, TGX-221, or Wortmannin. The composition may also include where the mTOR / PI3K / mTOR+PI3K inhibitor is AZD-8055, GSK2126458 (Omipalisib), GSK-1059615, INK128, NVP-BEZ235 (Dactolisib), or Rapalink. The composition may also include INK128, AZD-8055 or GSK-1059615. In aspects, the composition may be in the form of a tablet, capsule, powder, or solution. In aspects, the cell, tissue, or organ preservation composition may be formulated as a preservation solution or as an additive for a preexisting preservation solution. In aspects, the cell, tissue, embryo or organ preservation composition may be formulated as a preservation medium or as an additive for a preexisting cell culture medium, preferably in the form of a powder or aqueous solution. In aspects, the composition formulated as a preservation medium or as an additive for apreexisting cell culture medium further comprises a suitable buffering system, for example a HEPEs buffer, and optionally an antioxidant, optionally vitamin C, for example 1-500 pg / mL vitamin C, or about 10 pg / mL, 25 pg / mL, 50 pg / mL, 75 pg / mL, 100 pg / mL, 200 pg / mL, 300 pg / mL, 400 pg / mL, or 500 pg / mL vitamin C. Other technical features may be readily apparent to one skilled in the art from the following figures, descriptions, and claims.

[0012] Also provided is a method for inducing a state of metabolic stasis in cells, tissues, or organs where the method includes contacting the cells, tissues or organs with a preservation composition as described herein. A method for preserving cells, tissues, or organs includes contacting the cells, tissues or organs with a preservation composition as described herein. The method may also include where the contacting occurs under normoxic conditions of about 18- 22% O2, optionally about 20% O2, and a normothermic temperature of about 37 °C or a subnormothermic temperature in the range of about about 0-35 °C, optionally about 10-35 °C or about 10-25 °C. The method may also include where the contacting occurs under normoxic conditions of about 18-22% O2, optionally about 20% O2, and a subnormothermic temperature in the range of about 10-35 °C or about 10-18 °C or about 20-26 °C, or about 18-35 °C, or about 25 °C. The method may also include where the contacting occurs under conditions of about 18-22% O2, optionally about 20% O2, and a temperature near 0 °C, for example in the range of 0-4 °C or 0-10 °C or 0-15 °C. The method may also include where the tissue is bone, bone marrow, blood, skin, smooth muscle, skeletal muscle, nerve tissue, vasculature tissue such as vein or artery, or a cornea. The method may also include where the organ is heart, kidney, liver, lung, pancreas, small intestine, or large intestine. The method may also include where the organ is a heart, kidney, liver, or lung. The method may also include where the organ is brain (e.g., for preserving brain specimens). The method may also include where the tissue is an embryo, optionally a blastocyst. The method may also include where the cell is a pluripotent stem cell, optionally an embryonic stem cell (ESC), optionally a human or mouse ESC, or a hematopoietic stem cell.

[0013] Also provided is a method for preserving a tissue or organ where the method includes storing or reperfusing the tissue or organ in a preservation composition as described herein. Also provided is a method of decreasing delayed graft function during organ transplantation, where the method includes contacting the organ to be transplanted with a preservation composition as described herein. The method may also include where the storing, reperfusing or contacting occurs under normoxic conditions of about 18-22% O2, optionally about 20% O2,and a normothermic temperature of about 37 °C or a subnormothermic temperature in the range of about 10-35 °C or about 10-18 °C or about 20-26 °C, or about 18-35 °C, or about 25 °C. The method may also include where the contacting occurs under normoxic conditions of about 18- 22% O2, optionally about 20% O2, and a subnormothermic temperature in the range of about 0- 35 °C or a temperature near 0 °C, for example in the range of about 0-4 °C or about 0-10 °C or about 0-15 °C.[00141 Also provided is a method for preserving a donor organ where the method includes perfusing the donor organ in situ with a solution comprising a preservation composition as described herein, removing the donor organ, placing the donor organ in the solution and maintaining the solution at a subnormothermic or normothermic temperature until transplantation to a recipient subject, thereby preserving the donor organ. Also provided is a method for preserving a donor organ, where the method includes perfusing the donor organ in situ with a solution comprising a preservation composition as described herein, removing the donor organ, placing the donor organ in the solution and maintaining the solution at a subnormothermic or normothermic temperature until transplantation to a recipient subject, and administering a hydrogen sulfide donor compound, a chalcogenide, or a sulfide salt to the organ prior to the transplantation procedure, optionally within about 1-2 hours of the procedure.

[0015] Methods provided herein may comprise administration of a composition comprising a dual inhibitor of mammalian target of rapamycin (mTOR) and phophoinositide 3-kinase (PI3K), or a combination of an mTOR inhibitor and a PI3K inhibitor. Methods provided herein performed at room temperature may comprise administration of a composition comprising a dual inhibitor of mammalian target of rapamycin (mTOR) and phophoinositide 3-kinase (PI3K), or a combination of an mTOR inhibitor and a PI3K inhibitor.

[0016] Other technical features may be readily apparent to one skilled in the art from the following figures, descriptions, and claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG. 1 is a schematic diagram illustrating a small molecule screen for inhibitors inducing a dormant state mimicking diapause in pluripotent cells.

[0018] FIG. 2A-B illustrates an aspect of the results of the small molecule screen. A. mESCs treated with GSK2126458 at 100 nM and 200 nM exhibited reversible growth suppression upon inhibitor removal. Higher concentrations (500 nM and 1 pM) maintained suppression. B.Unlike GSK2126458, the mTOR inhibitor INK-128 did not allow for reversible pausing, maintaining growth suppression even after inhibitor withdrawal at 100 nM. C. Dose-response curve demonstrating that Rapalink irreversibly paused mESC proliferation even at the lowest concentration tested (1 nM). D & E. Dose-response curve indicating that NVP-BEZ 235 and AZD8055 induced reversible pausing at 100 nM and near irreversible suppression at 200 nM respectively. F. Dose-response curve showing that DMSO had no effect on mESC proliferation, with cells growing linearly. G. Table summarizing the reversibility from the screen and dosage titration experiment.

[0019] FIG. 3 is a heatmap illustrating results of the small molecule screen.

[0020] FIG. 4 is a schematic illustrating a proposed scientific rationale for the methods of inducing a diapause / hibernation-like state as described herein to preserve tissues and organs, e.g., for transplantation.

[0021] FIG. 5 illustrates results showing that a hydrogen sulfide donor molecule (AP39) protects against liver damage at room temperature.

[0022] FIG. 6A is a line graph showing mean fluorescent intensity (MFI) over time as a marker of proliferation in mESC cells that were either untreated (Ctrl) or treated with increasing doses of GSK2126458 (GSK212) in a range of from 1-1000 nM for the first 5 days, followed by wash-out of the inhibitor and continued culture for an additional 5 days.

[0023] FIG. 6B is a line graph showing fluorescent intensity over time as a marker of proliferation for mESC cells that were either untreated (Ctrl) or treated with increasing doses of INK-128 in a range of from 1-1000 nM for the first 5 days, followed by wash-out of the inhibitor and continued culture for an additional 5 days.

[0024] FIG. 7A shows representative images of blastocysts stored at 37°C and 20% Ch ovcr an approximately four-day period either in untreated medium or medium containing 200 nM of either GSK212 or INK 128 at 9 hours (top panel) and at 108 hours (bottom panel).

[0025] FIG. 7B shows representative images of blastocysts stored at room temperature (~24°C) for 5 days either in untreated (Control) medium or medium containing 200 nM GSK212.

[0026] FIG. 8 A is a schematic illustrating the heterotopic heart transplantation process.

[0027] FIG. 8B is a bar graph showing blood troponin levels (pg / ml) of animals receiving untreated (Ctrl) or treated (GSK+AP) hearts 6 hours after the heterotopic heart transplantation process.

[0028] FIG. 8C is a bar graph showing CK-MB levels (U / L) in tissue of untreated (Ctrl) or treated (GSK+AP) hearts 6 hours after the heterotopic heart transplantation process.

[0029] FIG. 8D is a bar graph showing heartbeats (beats / min) of untreated (Ctrl) or treated (GSK+AP) hearts 6 hours after the heterotopic heart transplantation process.

[0030] FIG. 9A is a schematic illustrating the orthotopic liver transplant procedure.

[0031] FIG. 9B is a bar graph showing blood AST levels in untreated (Control) and treated (GSK+AP) livers.

[0032] FIG. 9C is a bar graph showing blood ALT levels in untreated (Control) and treated (GSK+AP) livers.

[0033] FIG. 9D is a bar graph showing blood ALP levels in untreated (Control) and treated (GSK+AP) livers.

[0034] FIG. 10A-D shows reversible pausing of mouse embryos under normoxia. A. Dosage titration and time course showing potent pausing of mouse embryos under normoxia (20% 02) in GSK212 in a dosage dependent manner. B. Kaplan-Meier curve showing quantification of live embryos from 4A. C. Morulae derived using IVF were used. Over 70% embryos treated with GSK212 at 200nM remained viable after 204h (Embryonic day (El l) or Day Post Coitum (DPC) equivalent) in culture. In contrast, 88% embryos treated with INK- 128 at 200 nM died within 72h in culture (E5.5 DPC equivalent) and compared to 22% dead embryos in the untreated controls when cultured under normoxia at 37°C. D. Kaplan-Meier curve showing quantification of live embryos from 10C.

[0035] FIG. 11A-B shows reversible pausing of mESCs and embryos at room temperature. A. Ie5 mESCs stored at room temperature (23 +2°C) with GSK212 at 200nM show higher viability and cells number after 3 days compared to control. B. 66% of embryos remained alive and paused at room temperature for 5 days with GSK212 at 200nM compared to 0% for the control group.

[0036] FIG. 12A-K shows metabolic pausing enables damage free orthotopic liver transplantation. A. Transplantation schematic illustrating the orthotopic liver transplant procedure in rats following preservation with the indicated small molecules. B-C. Liverspreserved for 18hours at 4°C with the IX dosage (GSK212 2pM + AP39 3pM during preservation, 600nM AP before unclamping) of the small molecule combination exhibited significantly lower AST and ALT levels compared to controls and 3X dosage (GSK212 6pM + AP39 9pM during preservation, 1.8uM AP before unclamping) two hours post reperfusion. D. Representative images of naive (untransplanted) and preserved livers for 18 hours at 4°C just after transplantation into recipients. E-F. Livers preserved for 18 hours at 12°C Liver with IX dosage of the small molecules showed reduced ALT and AST levels compared to the control, 0.3X dosage (GSK212 0.667pM + AP39 IpM during preservation, 200nM AP before unclamping) and 3X dosage. G. Histopathological analysis revealed lower Suzuki scores in the IX dosage treated livers compared to controls. H-I. After 6 hours at 5 hours RT IX dosage of small molecule treated livers maintained lower AST and ALT Levels compared to controls two hours post reperfusion. J. Histopathological analysis and Suzuki Injury Scores at RT shows reduced damage at IX dosage treated livers compared to controls. K. Kaplan-Meier survival curve after 5 hours RT preservation shows significant benefits for livers treated GSK +AP compared to AP (AP39 3pM during preservation) or Control. GSK (GSK212 2pM during preservation) preserved livers show some benefit over the AP39 or control, but not as potent as GSK+AP condition.DETAILED DESCRIPTION

[0037] The present invention provides compositions and methods for inducing in cells, tissues, embryos, and organs a reversible state of stasis effective to protect the cells, tissues, and organs from damage and deterioration ex vivo including during storage and / or transit.

[0038] The state of stasis induced by the compositions and methods described herein may be characterized as a diapausc / hibcrnation-likc state. Diapause is a state of developmental pausing that can occur naturally during early mammalian development, generally at the blastocyst stage.During diapause, the embryo may be maintained in a dormant state for a period of from weeks to months. Studies in mice have demonstrated that embryonic cells in diapause retain characteristics of naive pluripotency. Accordingly, the state of reversible stasis may also be referred to herein as a metabolic pause or metabolic stasis.

[0039] Accordingly, in aspects, the disclosure provides methods for inducing a state of stasis in cells, tissues, or organs, the methods comprising contacting the cells, tissues, or organs with a composition comprising (i) a hydrogen sulfide donor compound, a chalcogenide, or a sulfide salt and (ii) a dual inhibitor of mammalian target of rapamycin (mTOR) and phophoinositide 3-kinase (PI3K) or a combination of an mTOR inhibitor and a PI3K inhibitor, or contacting the cells, tissues or organs with a composition comprising a dual inhibitor of mammalian target of rapamycin (mTOR) and phosphoinositide 3-kinase (PI3K), or a combination of an mTOR inhibitor and a PI3K inhibitor, wherein the composition does not comprise a hydrogen sulfide donor compound, a chalcogenide, or a sulfide salt.

[0040] Also provided are methods of preserving cells, tissues, or organs, the methods comprising contacting the cells, tissues, or organs with a composition comprising (i) a hydrogen sulfide donor compound, a chalcogenide, or a sulfide salt and (ii) a dual inhibitor of mammalian target of rapamycin (mTOR) and phophoinositide 3-kinase (PI3K) or a combination of an mTOR inhibitor and a PI3K inhibitor, or contacting the cells, tissues or organs with a composition comprising a dual inhibitor of mammalian target of rapamycin (mTOR) and phosphoinositide 3-kinase (PI3K), or a combination of an mTOR inhibitor and a PT3K inhibitor, wherein the composition does not comprise a hydrogen sulfide donor compound, a chalcogenide, or a sulfide salt. Also provided arc methods of preserving cells, tissues, or organs, the methods comprising contacting the cells, tissues, or organs with a composition comprising a dual inhibitor of mammalian target of rapamycin (mTOR) and phophoinositide 3-kinase (PI3K) or a combination of an mTOR inhibitor and a PI3K inhibitor, or contacting the cells, tissues or organs with a composition comprising a dual inhibitor of mammalian target of rapamycin (mTOR) and phosphoinositide 3-kinase (PI3K), or a combination of an mTOR inhibitor and a PI3K inhibitor, wherein the composition does not comprise a hydrogen sulfide donor compound, a chalcogenide, or a sulfide salt. Also provided are methods of preserving cells, tissues, or organs at room temperature, the methods comprising contacting the cells, tissues, or organs with a composition comprising a dual inhibitor of mammalian target of rapamycin (mTOR) and phophoinositide 3-kinase (PI3K) or a combination of an mTOR inhibitor and a PI3K inhibitor, or contacting the cells, tissues or organs with a composition comprising a dual inhibitor of mammalian target of rapamycin (mTOR) and phosphoinositide 3-kinase (PI3K), or a combination of an mTOR inhibitor and a PI3K inhibitor, wherein the composition does not comprise a hydrogen sulfide donor compound, a chalcogenide, or a sulfide salt.

[0041] Also provided are cell, tissue, or organ preservation compositions comprising (i) a hydrogen sulfide donor compound, a chalcogenide, or a sulfide salt and (ii) a dual inhibitor of mammalian target of rapamycin (mTOR) and phophoinositide 3-kinase (PI3K) or acombination of an mTOR inhibitor and a PI3K inhibitor, or contacting the cells, tissues or organs with a composition comprising a dual inhibitor of mammalian target of rapamycin (mTOR) and phosphoinositide 3-kinase (PI3K), or a combination of an mTOR inhibitor and a PI3K inhibitor, wherein the composition does not comprise a hydrogen sulfide donor compound, a chalcogenide, or a sulfide salt. In aspects, the composition may be in the form of a dry powder or in the form of a solution.[00421 In aspects, the cell, tissue, or organ preservation composition may be formulated as a preservation solution or as an additive for a preexisting preservation solution. In aspects, the preservation solution may contain one or more of colloids such as hydroxyethyl starch (HES), polyethylene glycol (PEG), or dextran; impermeants such as glucose, lactobionate, mannitol, and raffinose; electrolytes such as sodium, potassium, magnesium, calcium, chloride, sulfate, phosphate, carbonate, and citrate ions; antioxidants such as allopurinol and glutathione; and nutrients such as tryptophan, adenine, adenosine, glutamic acid, and histidine. In aspects, the preservation solution may contain histidine, tryptophan, and kctoglutaratc. In aspects, the preservation solution may contain an additive in the form of a gas selected from oxygen (O2), hydrogen (H2), carbon monoxide (CO), nitric oxide (NO), and argon (Ar). Common preservation solutions to which the cell, tissue, or organ preservation composition may be added include but are not limited to the University of Wisconsin Solution marketed as Belzer UW® Cold Storage Solutions, Celsior solution, extracellular-type lung preservation (EP-TU) solution, Custodiol HTK solution, phosphate buffered sucrose (PBS) 140, HP16, HBS, B2, Lifor, Ecosol, Biolasol, renal preservation solution 2 (RPS-2), F-M, AQIXRS-I, WMO-II, and CZ-1 solutions.

[0043] In aspects, the cell, tissue, or organ preservation composition may be formulated as a preservation medium or as an additive for a preexisting cell culture medium, preferably in the form of a powder or aqueous solution. In aspects, the composition formulated as a preservation medium or as an additive for a preexisting cell culture medium further comprises a suitable buffering system, for example a buffering system suitable for storage of cells or embryos at room temperature, such as a HEPEs buffer. In aspects, the compositions formulated as a preservation medium or as an additive for a preexisting cell culture medium comprises an antioxidant, optionally vitamin C, for example 1-500 pg / mL vitamin C, or about 10 pg / mL, 25 pg / mL, 50 pg / mL, 75 pg / mL, 100 pg / mL, 200 pg / mL, 300 pg / mL, 400 pg / mL, or 500 pg / mL vitamin C.

[0044] The state of stasis induced by the compositions and methods described here is a diapause / hibernation-like state defined by reduced metabolic activity and so may be referred to as a metabolically paused state, or a paused state. In some aspects, a cell, tissue, or organ in a state of stasis as defined herein may be characterized by a reduction in the rate or amount of carbon dioxide production and / or the rate or amount of oxygen consumption. Generally the reduction in the rate or amount is at least about 2-fold compared to the cell, tissue, or organ in its normal state. In another aspect, a cell in a state of stasis may be characterized by its lack of proliferation or a markedly reduced rate of proliferation compared to the cell in its normal state, for example at least a 50% reduction in cell proliferation, preferably less than 5% cell proliferation or about 0% cell proliferation compared to its normal state. In the context of an organ or tissue that exhibits movement, the state of stasis may be characterized by at least a 10% or 20% reduction in movement or motility.

[0045] In accordance with the compositions and methods described here, the state of stasis is reversible. In this context, reversibility may be assessed, for example, by comparing the function of the cell, tissue, or organ before undergoing stasis to its function after the conditions of stasis are removed. In one aspect, the function of an organ pre-stasis is compared to the function of the post-stasis transplanted organ. In aspects, stasis is reversed by removing the hydrogen sulfide donor compound and the inhibitors from contact with the cell, tissue, or organ, and optionally washing or reperfusing the cell, tissue, or organ with a medium or solution free of the hydrogen sulfide donor compound and the inhibitors.

[0046] In aspects, the methods may include the storage and preservation of cells, tissues, embryos, or organs under controlled conditions including atmospheric oxygen levels of about 18-22% or about 20% O2 and normothermic (37 °C) or subnormothermic temperature (18-35 °C), including ambient or “room temperature” which is understood to be about 25 °C. As is understood in the art, room temperature is not a single temperature. In illustrative experiments described herein room temperature varied at about 23 ±2°C. In aspects, the methods may include the storage and preservation of cells, tissues, embryos, or organs under controlled conditions including atmospheric oxygen levels of about 18-22% or about 20% O2 and near freezing (about 0 °C) or subnormothermic temperatures in the range of about 5-20 °C or 10-15 °C.

[0047] In aspects, a hydrogen sulfide donor compound is a compound whose reaction with exogenous molecules, degradation or metabolism in vitro or ex vivo produces molecularhydrogen sulfide. In aspects, the hydrogen sulfide donor compound is AP39, ATB-346 (Antibe Therapeutics), GIC-1001 (Glcare Pharma Inc.), SG-1002 (SulfaGENIX), or GYY4137 (Morpholin-4-ium-4-methoxyphenyl-(morpholino)-phosphinodithioate), or a derivative of any one of the foregoing. In aspects, the hydrogen sulfide donor compound is allicin (diallyl thiosulfinate), dially sulfide (DAS), diallyl disulfide (DADS), diallyl triulfide (DATS), ammonium tetrathiomolybdate ("ATTM" or (NID MoS^, anethole dithiolethione (ADT), or 5- (p-hydroxyphenyl)-3H-l,2-dithiole-3-thione (ADT-OH).

[0048] In aspects, the hydrogen sulfide donor compound is a phosphonamidothioate, a dithiolthione, a A-benzoylthiobenzamide, an acyl perthiol, a dithioperoxyanhydride, a polysulfide, an arylthioamide, or an S-aroylthiooxime.

[0049] In aspects, the chalcogenide is H2S, thSe, PETe, or H2P0.

[0050] In aspects, the sulfide salt is sodium hydrosulfide, sodium sulfide, or calcium sulfide. In general, the sulfide salt may be any compound that disassociates in aqueous media into hydrogensulfide anions (HS‘), sulfide anions (S2‘), and molecular hydrogen sulfide (H2S).

[0051] In aspects, the mTOR inhibitor is rapamycin, ridaforolimus, or temsirolimus.

[0052] In aspects, the phophoinositide 3-kinase (PI3K) inhibitor is LY294002, BYL-719 (Alpelisib), GDC-0941, GSK2636771, TGX-221, or Wortmannin.

[0053] In aspects, the dual inhibitor of mTOR and PI3K includes AZD-8055, GSK2126458 (Omipalisib), GSK-1059615, INK128, NVP-BEZ235 (Dactolisib), or Rapalink.

[0054] In aspects, provided is a composition comprising (i) a hydrogen sulfide donor compound selected from AP39, ATB-346 (Antibe Therapeutics), GIC-1001 (Glcare Pharma Inc.), SG-1002 (SulfaGENIX), or GYY4137 (Morpholin-4-ium-4-mcthoxyphcnyl- (morpholino)-phosphinodithioate), or a derivative of any one of the foregoing; and (ii) a dual inhibitor of mTOR and PI3K selected from AZD-8055, GSK2126458 (Omipalisib), GSK- 1059615, INK128, NVP-BEZ235 (Dactolisib), or Rapalink.

[0055] In aspects, provided is a composition comprising (i) a hydrogen sulfide donor compound selected from AP39, ATB-346 (Antibe Therapeutics), GIC-1001 (Glcare Pharma Inc.), SG-1002 (SulfaGENIX), or GYY4137 (Morpholin-4-ium-4-methoxyphenyl- (morpholino)-phosphinodithioate), or a derivative of any one of the foregoing; and (ii) GSK2126458.

[0056] In aspects, provided is a composition comprising AP39 and GSK2126458. In aspects the composition may also include one or more of a HEPES buffer and an antioxidant, optionally vitamin C.

[0057] In accordance with the methods described here, the cells, tissue, or organ can be any type of cell, tissue, or organ. For example, the cells, tissues, or organs may be an embryo, heart, lung, kidney, liver, bone marrow, pancreas, skin, bone, vein, artery, cornea, blood, small intestine, large intestine, larynx, brain, spinal cord, smooth muscle, nerves, skeletal muscle, ovary, testis, uterus, and umbilical cord cells, tissues, or organs.

[0058] In some aspects of the methods described here, the cell, tissue, or organ is transferred back into the donor organism from which it was derived. In such cases the cell, tissue, or organ may be referred to as an “autologous” cell, tissue, or organ. In other aspects, the cell, tissue, or organ is transferred into a recipient different from the donor. In such cases, the cell, tissue or organ may be referred to as “heterologous”. Thus, the terms autologous and heterologous relate to the origin of the donor cell, tissue, or organ relative to the recipient subject.

[0059] In accordance with an aspect of the methods described here, the hydrogen sulfide donor compound, chalcogenide, or sulfide salt is administered just prior to transplantation to mitigate ischemia-reperfusion injury (IRI) damages. In one aspect, the hydrogen sulfide donor compound is AP39, ATB-346 (Antibe Therapeutics), GIC-1001 (Glcare Pharma Inc.), SG-1002 (SulfaGENIX), or GYY4137 (Morpholin-4-ium-4-methoxyphenyl-(morpholino)- phosphinodi thioate), or a derivative of any one of the foregoing. In aspects, the hydrogen sulfide donor compound is allicin (diallyl thiosulfinate), dially sulfide (DAS), diallyl disulfide (DADS), diallyl triulfide (DATS), ammonium tetrathiomolybdate ("ATTM" or (NFE MoS^, anethole dithiolcthionc (ADT), or 5-(p-hydroxyphcnyl)-3H-l,2-dithiolc-3-thionc (ADT-OH). In one aspect, the hydrogen sulfide donor compound is AP39. In accordance with this method, the hydrogen sulfide donor compound, chalcogenide, or sulfide salt is utilized in combination with metabolic pausing induced by a dual inhibitor of mammalian target of rapamycin (mTOR) and phophoinositide 3-kinase (PI3K) or a combination of an mTOR inhibitor and a PI3K inhibitor. Without begin bound by any specific theory, it is believed that this combination approach reduces ischemia / reperfusion injury (IRI) in the graft.

[0060] An “embodiment” may refer to an illustrative representation of a method or article in which a disclosed concept or feature may be provided or embodied, or a representation of a manner in which a concept or feature may be provided or embodied. Such illustratedembodiments are to be understood as examples (unless otherwise stated), and other manners of embodying the described concepts or features, such as may be understood by one of ordinary skill in the art upon learning the concepts or features from the present disclosure, are within the scope of the disclosure. Accordingly, disclosed embodiments are to be considered in all respects as illustrative and not restrictive, the scope of the claimed subject matter being indicated by the appended claims, and not limited to the foregoing description or particular embodiments or arrangements described or illustrated herein. It is intended that the present subject matter covers such modifications and variations as come within the scope of the appended claims and their equivalents.

[0061] Unless defined otherwise, technical terms used herein are to be understood as commonly understood by one of ordinary skill in the art to which the disclosure belongs.

[0062] The phrases “at least one”, “one or more”, and “and / or”, as used herein, are open- ended expressions that are both conjunctive and disjunctive in operation. The terms “a”, “an”, “the”, “first”, “second”, etc., do not preclude a plurality. For example, the term “a” or “an” entity, as used herein, refers to one or more of that entity. As such, the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably herein.

[0063] The term “about” when used before a numerical designation, e.g., temperature, time, amount, concentration, and such other, including a range, indicates approximations which may vary by ( + ) or ( - ) 10%, 5%, 1%, or any subrange or subvalue there between. Preferably, the term “about” means that the value may vary by + / - 10%.

[0064] The term “comprises / comprising” does not exclude the presence of other elements, components, features, regions, integers, steps, operations, etc. Additionally, although individual features may be included in different claims, these may possibly advantageously be combined, and the inclusion in different claims does not imply that a combination of features is not feasible and / or advantageous. By contrast, the transitional phrase “consisting of’ excludes any element, step, or ingredient not specified in the claim. The transitional phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention.

[0065] “Subject” refers to a living organism and includes birds, mammals, non-human primates and humans. The term “patient” refers to a human subject. In aspects, the subject is a mammal. Non-limiting examples of mammals include rodents (e.g., mice and rats), non-human primates (e.g., monkeys, apes), rabbits, dogs, horses, cats, and livestock including pigs,bovines, donkeys, mules, bison, goats, camels, and sheep. In some aspects, the subject may be a bird including e.g., chickens and turkeys.

[0066] GSK2126458 is sometimes abbreviated as GSK212 herein.

[0067] As used herein, “may,” “may comprise,” “may be,” “can,” “can comprise,” “can be” and “is contemplated herein” all indicate something envisaged by the inventors that is functional and available as part of the subject matter provided.

[0068] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure.

[0069] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials for the purpose for which the publications are cited. If any material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.Examples

[0070] Small molecule screenings were conducted in mouse embryonic stem cells (mESCs) and embryos to identify compounds capable of inducing reversible metabolic pausing without reduced oxygen levels. A dual mT0R / PI3K inhibitor, GSK2126458, was identified which overcomes lUS’s metabolic limitations under normoxia. Combining GSK2126458 with AP39, a mitochondrial-targeted H2S donor, metabolic pausing was achieved, a novel preservation process that is scalable, cost-effective, and efficient. In orthotopic liver transplantation models, metabolic pausing significantly extended preservation viability at 18 hours at 4 and 12°C and up to six hours at room temperature (23 ±2°C) for graft survival while reducing IRE Metabolic pausing is contemplated herein to revolutionize organ preservation by expanding the transplant pool and overcoming geographical limitations, thereby addressing critical organ shortages and improving patient outcomes.Example 1Metabolic Pausing of Embryonic Stem Cells (ESC)

[0071] FIG. 1 is a schematic illustrating the screening assay. A fluorescently labeled singlecell cloned mESC line was utilized to ensure consistent mean fluorescent intensity (MFI). After tagging with a PiggyBac vector carrying tdTomato, a clone exhibiting ideal fluorescence anddevelopmental potential, verified via mouse-mouse chimera assays (data not shown), was selected.

[0072] Briefly, for ESC derivation, naturally mated CD-I female mice (3-4 weeks) with confirmed vaginal plugs at E0.5 were allowed to develop until embryonic day 3.5 (E3.5). Blastocyst-stage embryos were directly flushed out and rinsed in M2 medium. Single embryos were transferred into 12-well plates (Falcon) onto feeder layers using N2B27-2iL medium. The mESC line SUN107, or its derivatives was employed for all ESC experiments. Specifically, the SUN107.4 line, a fluorescently labeled single-cell cloned mESC line derived from SUN107, was utilized to ensure consistent mean fluorescent intensity (MFI). This line was tagged with a Piggy Bac vector carrying tdTomato, and a clone exhibiting optimal fluorescence and developmental potential was selected following verification through mouse-mouse chimera assays (data not shown). SGE2 mESCs were maintained in DMEM high glucose media supplemented with lx glutamax, sodium pyruvate, non-essetial amino acids, beta-mercaptoethanol (all Thermofisher scientific) with 10% FBS (Sigma F2442) and 2iL. For routine culture, all mESCs were cultured and maintained on feeder layers in their respective medium, passaged at a density of 7,000-10,000 cells / cm2every 3-4 days to ensure optimal growth and maintenance of pluripotency.

[0073] All small molecules used in the study were purchased from SelleckChem and predissolved at a concentration of 10 mM in dimethyl sulfoxide (DMSO). For the screening assays, each inhibitor was utilized at a final concentration of 1 pM, prepared by diluting the stock solution in N2B27 medium only.

[0074] For dosage titration experiments, small molecules were sourced from MedChemExpress or Cayman Chemical. All lyophilized powders were dissolved in anhydrous DMSO (Thermo Fisher Scientific) and stored as single-use aliquots at concentrations exceeding 10,000X stock in -80°C until further use.

[0075] In SUN107.4 mESCs experimental setups, cells were seeded at a density of 10,000 cells / cm2in N2B27-2iL medium supplemented with the indicated concentrations of small molecules to induce pausing, ensuring the final DMSO concentration did not exceed 0.1%.

[0076] Cells were imaged two hours post-seeding and subsequently every 24 hours for the next 3-5 days. After the initial imaging period, cells were washed three times with phosphate- buffered saline (PBS) to remove any residual inhibitors before being returned to fresh N2B27- 2iL medium. Imaging continued at 24-hour intervals for an additional 3-5 days. All imagingdata were processed and analyzed using FIJI with custom scripts developed in-house to quantify cellular responses to the small molecule treatments.

[0077] Initially, mESCs received 1 uM of each inhibitor.

[0078] The effects of the inhibitors on metabolic pausing were evaluated by fluorescent imaging to evaluate cell proliferation. In this assay, mean fluorescent intensity (MFI) correlates directly with cell growth (higher MFI means more cells). Imaging commenced 12h posttreatment, repeating every 24h for five days. To reverse the metabolic pause, cells were thoroughly washed with PBS on the fifth day post- treatment, then returned to standard culture media without inhibitors. Imaging post-wash was immediate, followed by further observationevery 24h for four days. MFI was analyzed using ImageJ, with higher MFI indicating increased cell numbers, thus reflecting reversal of paused state.

[0079] mESCs treated with GSK2126458 at 100 nM and 200 nM exhibited reversible growth suppression upon inhibitor removal. Higher concentrations (500 nM and 1 pM) maintained suppression. Unlike GSK2126458, the mTOR inhibitor INK-128 did not allow for reversible pausing, maintaining growth suppression even after inhibitor withdrawal at 100 nM. Rapalink irreversibly paused mESC proliferation even at the lowest concentration tested (1 nM). NVP- BEZ 235 and AZD8O55 induced reversible pausing at 100 nM and near irreversible suppression at 200 nM respectively. DMSO had no effect on mESC proliferation, with cells growing linearly. (FIG. 2A-F)

[0080] FIG. 2G shows a select list of inhibitors and their impacts on in vitro proliferation of the fluorescently labelled homogeneous single cell-cloned mESC line. Several small molecule inhibitors targeting nutrient sensing and metabolic regulators within the mTOR / PI3K / AKT pathway were screened. The screen included mTORCl- specific inhibitors (e.g., Rapamycin, Temsirolimus), mT0RC2 inhibitors via PI3K inhibition, and dual inhibitors of both mTOR complexes (mTORCl and mT0RC2). Additionally, pan-PI3K inhibitors and inhibitors of PI3K-like kinases (PIKK) were tested, alongside the calcineurin inhibitor Tacrolimus as a control.

[0081] FIG. 3 is a heatmap summarizing the impact of various small molecule inhibitors on the proliferation of the fluorescently labelled homogeneous single cell-cloned mESC line. As discussed above, in this assay mean fluorescent intensity (MFI) correlates directly with cell growth such that a higher MFI equates to more cells. An MFI value of 100 signifies full confluence with no inhibition, while a value of 1 represents maximal inhibition. As illustrated in line two of the figure, GSK2126458 (GSK212) significantly reduces cell confluence, showcasing its potent inhibitory action. GSK212 targets both mTOR complexes and PI3K pathways. Pan-PI3K inhibitors such as GDC-0941 and LY294002 target all PI3K isoforms, whereas BYL-719 specifically inhibits the PI3Ka isoform, and TGX-221 targets PI3Kp.

[0082] Inhibitors targeting both mTORCl and mT0RC2 (via Pi3K), particularly GSK212, were the most effective at inducing metabolic pausing, achieving over 90% suppression of cell proliferation at IpM concentration by day 4. Pan-PI3K inhibitors like GDC-0941 or NVP- BEZ235 also demonstrated inhibitory effects but were less potent than GSK212.

[0083] Single-target inhibitors, such as mTORCl inhibitors (Rapamycin and Ridaforolimus) or PI3K inhibitors, showed insufficient suppression of mESC proliferation. Similarly, activators of PI3K (GSK621) had minimal effects on cell growth, underscoring the importance of dual inhibition for achieving metabolic pausing. Interestingly, Tacrolimus exhibited little to no effect on mESC proliferation, confirming that direct inhibition of mTOR kinase activity is essential for inducing a metabolic pause.Example 2Room-Temperature Preservation of Rat Liver

[0084] FIG. 4 is a schematic illustrating the advantages of the methods described here for organ preservation.

[0085] As shown in FIG. 5, room-temperature preservation of rat liver with the AP39 (H2S donor) small molecule effectively prevented any pathologically observable damage for 6 hours. For this experiment, donor rats were perfused with pre-warmed UW solution at 37°C with or without AP39. After the initial flush, the perfusion solution was allowed to cool down to room temperature (~24°C) during and after the perfusion. Following perfusion, organs were subjected to a simulated moderate warm ischemia for 5 minutes, before storing the perfused livers in the UW solution with or without AP39 for 6 hours at room temperature. Shown is histological section of the stored liver samples after H&E staining.Example 3Reversible Pausing of Rodent Embryos

[0086] In order to test the reversibility of the inhibitor-induced metabolic pausing, mESC were cultured cither without treatment or treated with increasing doses of an inhibitor (cither GSK2126458 or INK- 128) for 5 days, followed by removal of the inhibitor and culture for an additional 5 days. Mean fluorescent intensity (MFI) was used as a proxy for cell number. mESCs were seeded on day 0 with an inhibitor dose of either 1 nM, 10 nM, 100 nM, 200 nM, 500 nM, or 1000 nM to induce pausing. Then on day 5 cells were washed three to four times with PBS and given fresh media without inhibitor.

[0087] As shown in FIG. 6A, doses of 100 nM GSK212 and above effectively paused the mESCs, as evidenced by the lack of cell proliferation between days 0 and 5 at doses of 100 nM, 200 nM, 500 nM, and 1000 nM. When the inhibitors were removed, the cells that had been exposed to GSK212 at either 100 or 200 nM resumed proliferation, showing that at these dosesthe metabolic pause induced by GSK212 was reversible. At higher doses (500 nM and 1000 nM) the cells did not resume proliferation.

[0088] Unlike what was seen for GSK212, the inhibition of proliferation by 100 or 200 nM INK-128 was not reversible, as illustrated in FIG. 6B, where at doses of 100 nM INK-128 and above the cells did not proliferate either in the presence of the inhibitor during days 0-5, or after removal of the inhibitor during days 5-10.Example 4 Maintaining Viable Blastocysts at Room Temperature

[0089] Following the promising results with mESC, experiments were undertaken to test whether the pause time could be extended with an optimized combination of inhibitors. Murine blastocyst stage embryos were at stored at 37°C and 20% Ch over an approximately four day period either in untreated medium or medium containing 200 nM of either GSK212 or INK128. FIG. 7A shows representative images of the blastocysts at 9 hours (top panel) and at 108 hours (bottom panel). Remarkably, INK128 not only failed to preserve the blastocysts under these conditions but seemed to adversely impact their viability relative to untreated controls (compare first two rows in bottom panel which shows marked loss of viability in INK128 treated blastocysts). In contrast, most of the blastocysts treated with GSK212 remained viable out to 108 hours under these conditions.

[0090] A further experiment was conducted to test whether the blastocysts could be maintained at room temperature (~24°C) in the presence of GSK212. FIG. 7B illustrates the results of this experiment which showed 0% viability for untreated blastocysts after 5 days at room temperature, compared with 66% viability for GSK212 treated blastocysts. To our knowledge, this is the first demonstration of a method for maintaining viable blastocysts at room temperature for up to 5 days.

[0091] For this experiment, blastocysts were maintained in a base media of potassium simplex optimization medium with amino acids (KSOMaa) without albumin or phenol red, available from commercial sources including Sigma or Cytospring. The base media was buffered with 22 mM HEPES to support buffering capacity under room temperature and atmospheric oxygen conditions. Untreated (control) blastocysts were stored in the base media. Treatment groups received 100 pg / mL vitamin C and 200 nM GSK212. Both groups were kept in cryovials at room temperature, with the caps sealed using parafilm to prevent gas exchange.Example 5Successful Transplantation of Metabolically Paused Rat Hearts

[0092] Next, the ability of treatment with combination inhibitors to preserve hearts stored on ice was evaluated using a rat model system. FIG. 8A shows a schematic of the heterotopic heart transplantation process. Male Wistar “donor” rats were subjected to perfusion through the heart using an ice cold commercially available solution (Belzer UW® Cold Storage Solution), either without treatment (Control or Ctrl) or treated with a combination of GSK2126458 (2 uM) and AP39 (3 uM). Donor hearts were stored on ice for 18 hours. Prior to transplantation, AP39 (600 nM) was directly administered directly into the left ventricle via apical injection. Donor heart was harvested 6 hours post-transplantation for analysis. Recipient blood was also harvested at the same time point.

[0093] As shown in FIG. 8B, troponin levels were significantly reduced in the blood of animals receiving treated hearts, indicating minimal cardiac cell death compared to untreated controls.

[0094] As shown in FIG. 8C, lower levels of CK-MB, with values nearing normal ranges, were also observed for hearts receiving treatment, indicating reduced myocardial damage in these hearts.

[0095] Finally, treated hearts maintained near normal electrical activity at 6 hours posttransplantation, as evidenced by heart rates. As shown in FIG. 8D, treated hearts maintained about 225 beats per minute (bpm) as compared to 250 bpm measured at donor harvest. In contrast, untreated hearts showed a significantly reduced heart rate of about 120 bpm.

[0096] Together, the data in this experiment highlights the efficacy of the claimed methods in preserving cardiac function following prolonged cold storage.Example 6Metabolic Pausing in Orthotopic Liver Transplantation

[0097] FIG. 9A shows a schematic of the orthotopic liver transplant procedure. Following heparinization, the donor rat's descending thoracic aorta is clamped, and the supra-hepatic inferior vena cava severed. Blood is then flushed with pre-warmed saline, succeeded by University of Wisconsin (UW) solution through the abdominal aorta, simulating warm ischemia for 5-10 minutes before liver harvest and storage at 12°C in UW solution with or without treatment where treatment was with a combination of GSK2126458 (2uM) and AP39 (3uM) for 18 hours. Prior to transplantation, AP39 (600nM) was directly administered intravenously into the portal vein. Blood samples were collected 2 hours post-transplantation for biochemicalanalysis of alkaline phosphatase (ALP), alanine transaminase (ALT), and aspartate aminotransferase (AST).

[0098] FIG. 9B shows blood AST levels in untreated (Control) and treated (GSK+AP) livers.

[0099] FIG. 9C shows blood ALT levels in untreated (Control) and treated (GSK+AP) livers.

[0100] FIG. 9D shows blood ALP levels in untreated (Control) and treated (GSK+AP) livers.

[0101] In summary, treated livers showed significantly lower levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), indicating reduced cellular damage. Moreover, elevated levels of alkaline phosphatase (ALP) were observed, which, in the context of liver transplantation, may reflect improved bile duct integrity or function. These biochemical markers collectively signify a healthier state of the transplanted liver, showcasing the efficacy of treatment with a preservation solution as described herein in enhancing organ viability post-transplant.Example 7Metabolic Pausing Achieves Embryo Preservation under Room Temperature Storage Conditions

[0102] Given the superior reversibility of GSK2126458 and its dual inhibition of mTOR and PI3K, investigations were extended to mouse embryos. Mouse embryo derivation experiments (including derivation of ESCs above) were reviewed and approved by the Stanford University Administrative Panel on Laboratory Animal Care under protocol 29042.

[0103] Briefly, mouse embryos were collected from super ovulated CD- I females (3-4 weeks) that were paired with CD-I stud males. Successful mating was confirmed by checking for the presence of a vaginal plug on embryonic day 0.5 (E0.5). Embryos were collected at the 2-cell stage (E1.5) by flushing out the oviducts and subsequently rinsed three times with M2 medium to remove any residual fluid.

[0104] For imaging, the 2-cell stage embryos were then transferred to fresh KSOM-AA medium without phenol red (Cytospring, Cosmobio, or EMD Millipore) and incubated at 37 °C with atmospheric oxygen levels and 5% CO2 for two hours before imaging. Embryos were cultured in non-tissue culture-treated 96-well U-bottom plates (Falcon), with one embryo per well and the outer wells filled with Milli-Q (MQ) water to prevent edge effects. The incubation was carried out in an Operetta imaging system (PerkinElmer) maintained at 37°C withatmospheric oxygen levels and 5% CO2. Images were captured every hour for a duration of up to 7 days to monitor embryonic development.

[0105] For inhibition experiments, the naturally fertilized 2-cell mouse embryos were harvested and cultured for 6 days at 37°C and 20% O2 in either untreated medium or medium containing 0.2, 2, 20 or 200 nM of GSK2126458 or INK- 128.

[0106] Remarkably, embryos treated only with INK- 128 exhibited adverse toxicity particularly at 200 nM, with visible toxicity by day 3 (DPC 4.5 equivalent). Even at the lower dosage of 20nM, INK- 128 treated embryos showed impaired growth. In contrast, embryos treated with GSK212 across concentrations remained viable, indicating that GSK2126458 could preserve embryonic viability under these conditions. (FIG. 10A and B)

[0107] Next, the pausing time was extended to El l DPC equivalent by mouse moruale stage embryos derived using IVF and imaged for 8.5 days. For imaging, IVF derived morula stage embryos were transferred to fresh KSOM-AA medium without phenol red (Cytospring, Cosmobio, or EMD Millipore) and incubated at 37°C with atmospheric oxygen levels and 5% CO2 for two hours before imaging. Embryos were cultured in non-tissue culture-treated 96-well U-bottom plates (Falcon), with one embryo per well and the outer wells filled with Milli-Q (MQ) water to prevent edge effects. The incubation was carried out in an Operetta imaging system (PerkinElmer) maintained at 37°C with atmospheric oxygen levels and 5% CO2. Images were captured every hour for a duration of up to 8.5 days to monitor embryonic development.

[0108] As shown in FIG. 10C and D, the pausing of mouse embryos was successfully prolonged when treated with 200nM GSK2126458 at 37°C for over 8 days. In comparison, 88% of embryos treated with 200nM INK-128 died within 72h of culture (DPC 5.5 equivalent).Interestingly, treatment with INK128 at 200nM at 20% oxygen appears to be more significantly more damaging than the untreated controls (DMSO only), where only 22% of died within 72h of culture.

[0109] Following the successful results at normoxic O2 (20%) with GSK2126458 in maintaining viable paused mESCs and embryos at 37°C for several days, this success was achieved at room temperature (23 ±2°C).

[0110] For the room temperature (23 ±2°C) mESC storage experiment, SGE2 mESCs were used. Briefly, cells were counted using NucleoCounter NC-3000 (ChemoMetec) and seeded at 100,000 cells / ml in the culture medium supplemented with the indicated concentrations of small molecules, ensuring the final DMSO concentration did not exceed 0.1% in cryotube. Tubes were stored at roomtemperature for 3 days away from direct light. On day 3, cells were homogeneously mixed by pipetting before being counted.

[0111] As seen in FIG. 11A, mESCs paused at room temperature with GSK2126458 at 200nM showed higher viability and live cells compared to controls. Next, by titrating pH and adding reactive oxygen species scavengers in combination with our small molecule inhibitor GSK2126458 at 200nM, mouse embryos were maintained at room temperature for five days. As illustrated in FIG. 1 IB, untreated blastocysts exhibited 0% viability after five days at room temperature, whereas GSK2126458-treated blastocysts maintained 66% viability. These conditions facilitated approximately 66% of blastocyst stage embryos to remain paused at room temperature for 5 days, an accomplishment previously unattainable in the field.Example 8Metabolic Pausing Achieves Superior Liver Preservation Across Sub-Normothermic and Room Temperature Storage Conditions

[0112] Following the identification of GSK2126458 as a key component in inducing a metabolic pause above, the combined effect of GSK2126458 and the mitochondrial-targeting H2S donor, AP39, was tested for the ability to induce a paused state for an entire organ and assessing the ability of this combination during organ preservation by protecting them against IRE

[0113] Liver preservation was evaluated first, given the liver's high susceptibility to IRI. Livers were preserved under sub-normothermic (4 and 12°C) and room temperature (23 ± 2°C) conditions to test whether our small molecule combination could mitigate IRI, improve liver function markers, and ultimately support long-term post-transplant survival using an orthotopic transplantation model.

[0114] Male Lewis rats (7-8 weeks of age) were sourced from Charles River Laboratories and housed under specific pathogen-free conditions. The housing environment was controlled for temperature and humidity, with a 12-hour light-dark cycle. Rats had unrestricted access to water and standard chow pellets. All animal experiments were performed in accordance with the ethical guidelines and standards set by the Stanford University Administrative Panel on Laboratory Animal Care, which reviewed and approved protocols (#9821) related to animal handling and care.

[0115] The protocol for orthotopic liver transplantation was adapted from established methods. Briefly, after donor rats were heparinized with 1 lU / g (Fresenius Kabi, Fake Zurich,IL), the descending thoracic aorta was clamped, and the supra-hepatic inferior vena cava (SHIVC) was cut. The liver was flushed using a catheter inserted into the abdominal aorta with 50 mL of phosphate-buffered saline (PBS), followed by 20 mL of University of Wisconsin (UW) solution (Bridge to Life Ltd., Northbrook, IL). The liver was then excised and immediately immersed in a basin of UW solution. For the treatment group, 2 pM of GSK2126458 and 3 pM of AP39 were added to the UW solution. A cuff fashioned from a 14- gauge ethylene tetrafluoroethylene (ETFE) catheter was applied to the portal vein (PV) during back-table preparation. The liver was stored in UW solution for either 12 hours at 18°C or for 5-6 hours at room temperature (23 ± 2°C). Immediately before transplantation, the liver was flushed with 5 mL of phosphate-buffered saline (PBS). For the treatment group, PBS was supplemented with 600 nM AP39, a dosage twice that used in previous preservation studies, to mitigate ischemia-reperfusion injury (IRI) based on the cardioprotective effects of slow- releasing hydrogen sulfide (FES) donors observed in prior research.

[0116] The liver was then orthotopically transplanted into the recipient, with the SHIVC anastomosed end-to-end using continuous 7-0 polypropylene sutures. The PV was reconstructed using a cuff technique, and reperfusion was achieved within 16 minutes of clamping. The infra-hepatic IVC (IHIVC) was anastomosed end-to-end with continuous 8-0 polypropylene sutures, and the hepatic artery (HA) was reconstructed by inserting the recipient’s proper HA into the celiac artery of the liver graft. The bile duct was reconstructed end-to-end using a 24-gauge ETFE catheter as a stent.

[0117] At 2 hours post-transplantation, serum and liver samples were collected. Serum levels of AST and ALT were analyzed at Stanford’s Animal Diagnostic Laboratory. Liver tissue samples were fixed in 10% formaldehyde, embedded in paraffin, and sectioned at 5 pm for H&E staining. Histological assessment of ischemia / reperfusion injury, including necrosis, sinusoidal congestion, and centrilobular ballooning, was performed using the modified Suzuki criteria.

[0118] Transitioning from cellular assays to whole-organ preservation, the commonly accepted 10X scaling factor was used to adjust small molecule dosages. Thus, GSK2126458 was tested at 2 pM based on its optimal 200 nM dosage in mESCs, and AP39 was used at 3 pM, consistent with previous studies in cellular contexts. First, an 18-hour static CS duration was tested as it represents one of the longest preservation times previously documented and assessed the ability of the small molecules in reducing IRI, given the liver's high susceptibilityto IRI. Two dosages were assessed, where (A), IX dosage means the liver was perfused and preserved with GSK2126458 2pM and AP39 3pM, and 600nM AP was used before unclamping prior to transplantation as described in FIG. 12A, (B) 3X dosage means the liver was perfused and preserved with GSK2126458 6pM and AP39 9pM, and 1.8pM AP was used before unclamping prior to transplantation as described in FIG. 13A. As seen in FIG. 12B-D, livers preserved for 18hours at 4°C with the IX dosage of the small molecule combination exhibited significantly lower AST and ALT levels compared to controls and 3X dosage two hours post reperfusion.

[0119] The ability of the small molecule combination was assessed in preserving livers at 12°C for 18 hours. As illustrated in FIG. 12E-G, the lx dose treated group exhibited significantly better outcomes compared to the control group. The median aspartate aminotransferase (AST) levels in the control group were 8025 U / L (range: 7926-8123 U / L), whereas the treatment group showed markedly reduced levels at 4240 U / L (range: 4109-5761 U / L). Similarly, alanine aminotransferase (ALT) levels followed this trend, with the treatment group showing 5991 U / L (range: 5739-7398 U / L), compared to 12,026 U / L (range: 10,102- 13,949 U / L) in the control group. Histopathological analysis using the Suzuki score also reflected improved outcomes in the lx dose treated group, with a median score of 7 (range: 5- 7) compared to a median of 8 (range: 8-9) in the control group. This reduction in tissue damage confirms the protective effect of the small molecule combination under sub-normothermic conditions.

[0120] To further challenge the limits of liver preservation, livers were stored for 6 hours at room temperature (23 ±2°C). As depicted in FIG. 12H-J, the lx dose-treated group continued to show improved outcomes, with a median AST level of 2937 U / L (range: 2265-4594 U / L) compared to 5217 U / L (range: 4790-5942 U / L) in the control group. Similarly, ALT levels were reduced to 3496 U / L (range: 2851-6656 U / L) in the treatment group, while the control group had values of 6678 U / L (range: 6129-8325 U / L). Histopathological examination of the livers harvested 2 hours post-transplantation showed a median Suzuki score of 7 (range: 7-7) in the lx dose treatment group, in contrast to 12 (range: 11-12) in the control group, further highlighting GSK2126458’s ability to protect against IRI even in extreme storage conditions.

[0121] Finally, the ability of livers preserved using our small molecules for 5 hours of room temperature to allow long term survival of the recipient animal was assessed. As shown in FIG. 12L, all recipients 4 rats in the control and AP (AP39 3pM during preservation) groups diedwithin 12h post-transplant. As expected, GSK +AP (GSK2126458 2pM + AP39 3pM during preservation) show significantly better survival compared to control and AP. Interesting, GSK2126458 (GSK2126458 2pM during preservation) alone worked better than the Control or AP only group, but at per with the GSK+AP group. This demonstrates the potential of such a small molecule combination to extend liver preservation and improve post-transplant outcomes.Discussion of the Examples

[0122] The use of the dual mT0R / PI3K inhibitor GSK2126458 small molecule and combinations thereof as provided herein represents a significant advancement in cell, embryo and organ preservation. For example, metabolic pausing as described herein offers an accessible and cost-effective alternative that not only reduces IRI but also extends the viability of various organ types during transplantation.

[0123] The results in preclinical models of liver transplantation herein demonstrate translational potential. For liver preservation, the combination of the dual mT0R / PI3K inhibitor GSK2126458 and the mitochondrial-targeting H2S donor AP39 significantly reduced damage in both sub-normothermic (4 and 12°C) and room temperature (23 ±2°C) storage conditions, as evidenced by decreased AST and AFT levels, improved histopathological outcomes. Notably, the small molecule combination extended liver preservation viability to up to six hours at room temperature, a six-fold improvement over current standards, which typically limit warm ischemia time to 30-60 minutes. Furthermore, the room-temperature preserved liver enabled normal survival of the recipient rats for over two weeks, compared to approximately 12 hours for the control graft or AP only group. This breakthrough is contemplated to revolutionize liver preservation, especially in contexts where transportation distances or logistical challenges restrict the effectiveness of traditional cold storage.

[0124] The simplicity and scalability of this small molecule combination further enhances its potential impact. Unlike NMP, which requires expensive equipment and specialized personnel, metabolic pausing as described herein can be seamlessly integrated into existing storage protocols, including CS and potentially machine perfusion, without significant infrastructure modifications. This makes it accessible not only to top-tier transplant centers but also to economically constrained regions, democratizing organ preservation and expanding its global applicability.

[0125] Moreover, metabolic pausing as provided herein is effective for blastocyst-stage embryos under normoxic conditions (20% O2) at both physiological (37°C) and room temperature (~23°C). This capability significantly extends the available timeframe for comprehensive prenatal diagnostics beyond current methods, which are limited by rapid embryo progression past the blastocyst stage or the need for cryop reservation. By maintaining blastocyst viability without freezing, extensive genomic and omics-based diagnostic tests can now be performed, offering deeper embryo characterization without compromising their subsequent use for IVF. It is contemplated herein that this profoundly enhances prenatal diagnostics and embryo selection processes. Methods provided herein for metabolic pausing at near-physiological temperatures also provide significant potential for ex vivo gene therapy of organs. Currently, such procedures require normothermic machine perfusion (NMP), which is expensive, logistically challenging, and not widely accessible. Methods provided herein overcome these limitations by allowing efficient, cost-effective, and simplified ex vivo gene therapies, thus expanding treatment options for patients needing targeted organ- specific gene corrections or enhancements.

[0126] While the invention has been described by means of specific embodiments and applications thereof, modifications and variations could be made thereto by those skilled in the art without departing from the scope set forth in the claims.

[0127] The present invention is set forth in various levels of detail. In certain instances, details not necessary for one of ordinary skill in the art to understand the invention may have been omitted.

[0128] Section headings are for organizational purposes only and are not to be construed as limiting the subject matter described.

Claims

CLAIMSWhat is claimed is:

1. A cell, tissue, embryo or organ preservation composition comprising: i) a hydrogen sulfide donor compound, a chalcogenide, or a sulfide salt and ii) a dual inhibitor of mammalian target of rapamycin (mTOR) and phophoinositide 3- kinase (PI3K), or a combination of an mTOR inhibitor and a PI3K inhibitor.

2. The composition of claim 1, wherein the hydrogen sulfide donor compound is AP39, ATB- 346, GIC-1001, SG-1002, GYY4137, or a derivative of any one of the foregoing.

3. The composition of claim 1, wherein the hydrogen sulfide donor compound is allicin (diallyl thiosulfinate), dially sulfide (DAS), diallyl disulfide (DADS), diallyl triulfide (DATS), ammonium tetrathiomolybdate (ATTM or (NfD MoS^, anethole dithiolethione (ADT), or 5- (p-hydroxyphenyl)-3H- 1 ,2-dithiole-3-thione (ADT-OH).

4. The composition of claim 1, wherein the chalcogenide is H2S, PESe, PhTe, or H2P0.

5. The composition of claim 1, wherein the sulfide salt is sodium hydrosulfide, sodium sulfide, or calcium sulfide.

6. The composition of any one of claims 1 to 5, wherein the composition comprises a combination of an mTOR inhibitor and a PI3K inhibitor.

7. The composition of claim 6, wherein the mTOR inhibitor is rapamycin, ridaforolimus, or temsirolimus.

8. The composition of claim 6 or 7, wherein the PI3K inhibitor is LY294002, BYL-719 (Alpelisib), GDC-0941, GSK2636771, TGX-221, or Wortmannin.

9. The composition of any one of claims 1 to 5, wherein the composition comprises a dual mT0R / PI3K inhibitor.

10. The composition of claim 9, wherein the dual mT0R / PI3K inhibitor is AZD-8055, GSK2126458 (Omipalisib), GSK-1059615, INK128, NVP-BEZ235 (Dactolisib), or Rapalink.

11. The composition of any one of claims 1 to 5, wherein the composition comprises GSK2126458 (Omipalisib) or NVP-BEZ235 (Dactolisib).

12. The composition of claim 11, wherein the composition comprises INK128, AZD-8055 or GSK-1059615.

13. The composition of any one of claims 1 to 9, wherein the composition is in the form of a tablet, capsule, powder, or solution.

14. A method for inducing a state of metabolic stasis in cells, tissues, or organs the method comprising contacting the cells, tissues or organs with the composition of any one of claims 1 to 13, or with a composition comprising iii) a dual inhibitor of mammalian target of rapamycin (mTOR) and phophoinositide 3-kinase (PI3K), or a combination of an mTOR inhibitor and a PI3K inhibitor.

15. A method for preserving cells, tissues, embryos or organs comprising contacting the cells, tissues, embryos or organs with the composition of any one of claims 1 to 13, or with a composition comprising iii) a dual inhibitor of mammalian target of rapamycin (mTOR) and phophoinositide 3-kinase (PI3K), or a combination of an mTOR inhibitor and a PI3K inhibitor.

16. The method of claim 15, wherein the hydrogen sulfide donor compound is AP39, ATB-346, G1C-1001, SG-1002, GYY4137, or a derivative of any one of the foregoing.

17. The method of claim 15, wherein the hydrogen sulfide donor compound is allicin (diallyl thiosulfinate), dially sulfide (DAS), diallyl disulfide (DADS), diallyl triulfide (DATS), ammonium tetrathiomolybdate (ATTM or (NID MoS^, anethole dithiolethione (ADT), or 5- (p-hydroxyphenyl)-3H-l,2-dithiole-3-thione (ADT-OH).

18. The method of claim 15, wherein the chalcogenide is H2S, HzSe, JDTe, or H2P0.

19. The method of any one of claims 15 to 18, wherein the mTOR inhibitor is rapamycin, ridaforolimus, or temsirolimus.

20. The method of any one of claims 15 to 19, wherein the PI3K inhibitor is LY294002, BYL- 719 (Alpelisib), GDC-0941, GSK2636771, TGX-221, or Wortmannin.

21. The method of any one of claims 15 to 18, wherein the dual mT0R / PI3K inhibitor is AZD- 8055, GSK2126458 (Omipalisib), GSK- 1059615, INK128, NVP-BEZ235 (Dactolisib), or Rapalink.

22. The method of claim 15, wherein the composition comprises AP39 as the hydrogen sulfide donor compound.

23. The method of claim 22, wherein the composition comprises GSK2126458 (Omipalisib) as the dual inhibitor of mTOR and PI3K.

24. The method of any one of claims 14 to 23, wherein the contacting occurs under normoxic conditions of about 18-22% O2, optionally about 20% O2, and a normothermic temperature of about 37 °C or a subnormothermic temperature in the range of about 0-35 °C, optionally about 10-35 °C or about 10-25 °C, or about 18-35 °C, or about 25 °C.

25. The method of claim 24, wherein the contacting occurs under normoxic conditions of about 18-22% O2, optionally about 20% O2, and a subnormothermic temperature in the range of about 0-35 °C or about 0-4 °C or about 0-10 °C or about 0-15 °C.

26. The method of any one of claims 15 to 25, wherein the tissue is an embryo, bone, bone marrow, blood, skin, smooth muscle, skeletal muscle, nerve tissue, vasculature tissue such as vein or artery, or a cornea.

27. The method of claim 26, wherein the organ is heart, kidney, liver, lung, pancreas, small intestine, or large intestine.

28. The method of claim 26, wherein the organ is a heart, kidney, liver, or lung.

29. The method of claim 26, wherein the embryo is optionally a blastocyst.

30. The method of any one of claims 15 to 25, wherein the cell is a pluripotent stem cell, optionally an embryonic stem cell (ESC), optionally a human or mouse ESC, or a hematopoietic stem cell.

31. A method for preserving a tissue or organ comprising storing or reperfusing the tissue or organ with the composition of any one of claims 1 to 13, or with a composition comprising iii) a dual inhibitor of mammalian target of rapamycin (mTOR) and phophoinositide 3-kinase (PI3K), or a combination of an mTOR inhibitor and a PI3K inhibitor.

32. A method of decreasing delayed graft function during organ transplantation, the method comprising contacting the organ to be transplanted with the composition of any one of claims 1to 13, or with a composition comprising iii) a dual inhibitor of mammalian target of rapamycin (mTOR) and phophoinositide 3-kinase (PI3K), or a combination of an mTOR inhibitor and a PI3K inhibitor.

33. The method of claim 31 or 32, wherein the storing, reperfusing or contacting occurs under normoxic conditions of about 18-22% O2, optionally about 20% O2, and a normothermic temperature of about 37 °C or a subnormothermic temperature in the range of about 0-35 °C, optionally about 10-35 °C or about 10-25 °C, or about 18-35 °C, or about 25 °C.

34. A method for preserving a donor organ, the method comprising removing the donor organ from a donor subject; placing the donor organ in a buffered solution comprising the composition any one of claims 1 to 13, or with a composition comprising iii) a dual inhibitor of mammalian target of rapamycin (mTOR) and phophoinositide 3-kinase (PI3K), or a combination of an mTOR inhibitor and a PI3K inhibitor, and maintaining the solution at a subnormothermic or normothermic temperature until transplantation to a recipient subject, thereby preserving the donor organ.

35. A method for preserving a donor organ, the method comprising removing the donor organ from a donor subject; placing the donor organ in a buffered solution comprising the composition any one of claims 1 to 13, or with a composition comprising iii) a dual inhibitor of mammalian target of rapamycin (mTOR) and phophoinositide 3-kinase (PI3K), or a combination of an mTOR inhibitor and a PI3K inhibitor, and maintaining the solution at a subnormothermic or normothermic temperature until transplantation to a recipient subject; administering a hydrogen sulfide donor compound, a chalcogenide, or a sulfide salt to the donor organ prior to the transplantation procedure, optionally within about 1-2 hours of the procedure.

36. The method of claim 34 or claim 35, wherein the donor organ is maintained under normoxic conditions.

37. The method of any one of claims 34 to 36, wherein the method comprises perfusing the donor organ in situ with a solution comprising the composition of any one of claims 1 to 13, or with a composition comprising iii) a dual inhibitor of mammalian target of rapamycin (mTOR)and phophoinositide 3-kinase (PI3K), or a combination of an mTOR inhibitor and a PI3K inhibitor, prior to removal from the donor subject.

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