COMPOSITIONS FOR CRYOPRESERVATION AND METHODS OF USE THEREOF

CROSS-REFERENCE TO REUATED APPUICATION

[1] This application claims priority to United States Provisional Patent Application 62/612,552, filed December 31, 2017, which is incorporated by reference herein in its entirety.

FIEUD OF INTEREST

[2] Compositions and methods thereof for cryopreservation of mammalian cells, tissues, and organs are described herein.

BACKGROUND

[3] Excessive cell death is a major barrier to the clinical use of cells, tissues, and organs obtained from a live donor, or cells grown in culture. This problem is particularly acute when there is a protracted time period between procuring cells and using them, requiring that the cells be stabilized during prolonged storage. Generally, the process of isolation of donor cells, tissues or organs damages cells in and of itself, as it involves enzymatic or mechanical removal, enzymatic digestion of extracellular proteins, cell transferring between vessels, centrifugation, resuspension in solutions, and filtration steps. Cells can also suffer significant damage during seeding into growth chambers, a process termed trituration. Routine cell manipulation in vitro is likewise a major source of cell damage. Cells in culture are routinely separated from the matrices on which they grow to allow for further amplification of cell numbers and subsequent growth, for procedural manipulation to introduce experimental treatments such as DNA introduction, and routine measurements downstream, which require cells to first be removed from their environment. These procedures often disrupt the integrity of the outer membrane of many cells, causing punctures and ruptures. Such physical damage to the cells causes metabolites to leak from their cytoplasm and into the surrounding extracellular space. On the physiological level, damage to plasma membrane integrity results in excess calcium release within cells, decreased ATP concentrations, decreased NAD concentrations, and activation of pathways that lead to necrosis and necroptosis.

[4] In addition, the isolated cells, tissues, and organs suffer physiological damage due to interruption of circulation which leaves the cells within removed tissues and organs at significant risk of lack of oxygenation, free radical generation, and ischemia. These insults rapidly lead to necrosis and necroptosis within cells in the tissue, resulting in excessive cell death, especially for primary cells derived from animal or human tissue, ultimately leaving insufficient material for subsequent clinical use. It has also been long appreciated that these necrotic cells spill their contents onto neighboring cells and cause them to signal death from the outside. The spilling of these cellular components, characterized as damage-associated molecular patterns (DAMPS), activates an immunogenic response whereby these molecules themselves can activate pattern-recognition receptors and cause neighboring cells to activate necroptosis as a response to perceived inflammation. Furthermore, the cells that do survive may undergo de-differentiation, a process of reversion of committed or differentiated cells with less capacity into ones with greater differentiative capacity.

[5] Physiological damage can be prevented in part through slowing down or halting of cell metabolism during times of stress. One approach is to hold cells and tissues, as well as organs for transplant, at hypothermic temperatures during the period between isolation and later use. Another option for halting cell metabolism and any resultant oxidative damage, which is available in the case of cell cultures and individual cells, is preserving cells at subfreezing temperatures, a process called cryopreservation. However, cryopreservation itself and freeze-thaw cycles associated with cryopreservation, cause damage to cells through ice crystal formation in the immediate vicinity of the cells and, sometimes, within cells, again causing punctures and ruptures of external plasma membrane and of internal organelles (so-called to freeze-thaw damage). The combination of isolation and cryopreservation-associated cell damage can also result in the trans-differentiation of cells from one differentiated state into another cell type, or dedifferentiation of cells, thereby further complicating subsequent use of any isolated cells.

[6] Current methods do not address the ensuing cell damage and death due to these insults, and leave cells and tissues and organs with excessive damage. Additionally, no solutions have been implemented to stop the ensuing necrosis. Likewise, few advances, if any, have led to improvement in cryopreservation techniques, and it is not understood what molecular pathways exactly lead to cell death upon freeze-thaw processes.

[7] Thus, there is an ongoing need for a method of isolating and or storing tissues, cells, and organs that avoids necrosis, necroptosis and de-differentiation stemming from physical and physiological damage the cells suffer in the process of isolation. Further, there is an ongoing need for a method of cryopreserving cells that avoids necrosis, necroptosis and de-differentiation

stemming from physical and physiological damage the cells suffer in the process of cryopreservation. Finally, there is an ongoing need for a reducing cellular plasticity, necroptosis, or necrosis that results from physical and physiological damage cells incur in the course of cell manipulations in vitro , during liberation from tissue.

[8] The composition and methods thereof disclosed herein addresses these needs, wherein use of the composition may reduce the incidence of necroptosis or necrosis during cryopreservation, and may treat, prevent, inhibit, or reduce the incidence of cellular plasticity in a plurality of cells.

SUMMARY

[9] In one aspect, disclosed herein is a composition comprising a necroptosis inhibitor and a Bax channel inhibitor. In another aspect, the necroptosis inhibitor comprises a 5-((7-Chloro-lH-indol-3-yl)methyl)-3-methyl-2,4-imidazolidinedione compound or an analog, derivative, isomer, or pharmaceutically acceptable salt thereof, and the Bax channel inhibitor comprises a 3,6-Dibromo-a-(l-piperazinylmethyl)-9H- carbazole-9-ethanol dihydrochloride compound, or an analog, derivative, isomer, or pharmaceutically acceptable salt thereof.

[10] In a related aspect, a necroptosis inhibitor comprises a 3-p-Methoxyphenyl-5,6-tetramethylenothieno[2,3-d]pyrimidin-4-one-2-mercaptoethylcyanide compound, a (Z)-5-((3-(4-Fluorophenyl)-lH-pyrazol-4-yl)methylene)-2-imino-3-(thiazol-2-yl)thiazolidin-4-one compound, a 5-(Indol-3-ylmethyl)-(2-thio-3-methyl)hydantoin compound, l-([3S,3aS]-3-[3-fluoro-4-[trifluoromethoxy]phenyl]-8-methoxy-3,3a,4,5-tetrahydro-2H-benzo[g]indazol-2-yl)-2-hydroxyethanone compound, (S)-N-(l-[2-chloro-6-fluorophenyl]ethyl)-5-cyano-l-methyl-lH-pyrrole-2-carboxamide compound, methyl-thiohydantoin-tryptophan compound, (E)-N-(4-(N-(3-Methoxypyrazin-2-yl)sulfamoyl)phenyl)-3-(5-nitrothiophene-2-yl)acrylamide compound, l-(4-(4-Aminofuro[2,3-d]pyrimidin-5-yl)phenyl)-3-(2-fluoro-5-(trifluoromethyl)phenyl)urea compound, or 2,2-dimethyl- l-(5(S)-phenyl-4,5-dihydro-pyrazol-l-yl)-propan-l-one compound, or an analog, derivative, isomer, or pharmaceutically acceptable salt of said necroptosis inhibitor compound.

[11] In another related aspect, the concentration of the 5-((7-Chloro-lH-indol-3-yl)methyl)-3-methyl-2,4-imidazolidinedione compound or said analog, derivative, isomer, or pharmaceutically acceptable salt thereof is about 0.2nM - 2mM. In another related aspect, the concentration of said 3,6-Dibromo-a-(l-piperazinylmethyl)-9H-carbazole-9-ethanol dihydrochloride compound, or

analog, derivative, isomer, or pharmaceutically acceptable salt thereof is about 0.5hM-50mM.

[12] In another aspect, a composition disclosed herein further comprises a nicotinamide adenine dinucleotide (NAD), an adenosine triphosphate (ATP), a cyclosporin A, or a superoxide dismutase, or any combination thereof. In a related aspect, the superoxide dismutase comprises a manganese superoxide dismutase or a zinc superoxide dismutase. In another related aspect, the concentration of said NAD is about 5nM - 500mM. In another related aspect, the concentration of said ATP is about lOnM - lmM. In another related aspect, the concentration of said cyclosporin A is about lnM - lmM. In another related aspect, the concentration of said superoxide dismutase is about 0.001- 100 Kunitz Units (KU).

[13] In another aspect, a composition disclosed herein comprises about 20 nM 5-((7-Chloro-lH-indol-3-yl)methyl)-3-methyl-2,4-imidazolidinedione), about 5nM 3,6-Dibromo-a-(l-piperazinylmethyl)-9H- carbazole-9-ethanol dihydrochloride, about 0.05 mM nicotinamide adenine dinucleotide (NAD), about 0.01 mM adenosine triphosphate (ATP), about O.OImM cyclosporin A, and about 0.1 Kunitz Unit manganese superoxide dismutase or zinc superoxide dismutase.

[14] In a related aspect, a composition described herein further comprises a cryoprotective agent. In another related aspect, the cryoprotective agent comprises DMSO, or serum, or any combination thereof.

[15] In a related aspect, a composition described herein further comprises a pharmaceutically acceptable excipients or carriers.

[16] In one aspect, disclosed herein is a method of cryopreservation, the method comprising the steps of: bringing a plurality of cells in contact with a composition comprising a necroptosis inhibitor and a Bax channel inhibitor; and cooling the composition comprising the plurality of cells of step (a), wherein the necroptosis inhibitor comprises a 5-((7-chloro-lH-indol-3-yl)methyl)-3-methyl-2,4-imidazolidinedione compound or an analog, derivative, isomer, or pharmaceutically acceptable salt thereof, and wherein the Bax channel inhibitor comprises 3,6-dibromo-a-(l-pipcrazinylmcthyl)-9/7-carbazolc-9-cthanol dihydrochloride or an analog, derivative, isomer, or pharmaceutically acceptable salt thereof.

[17] In one aspect, disclosed herein is a method of treating, preventing, inhibiting, or reducing the incidence of cellular plasticity, or necroptosis or necrosis in a plurality of cells, said method comprising the step of: bringing said plurality of cells in contact with a composition comprising a necroptosis inhibitor and a Bax channel inhibitor, wherein the necroptosis inhibitor comprises a 5-((7-chloro-lH-indol-3-yl)methyl)-3-methyl-2,4-imidazolidinedione compound or an analog, derivative, isomer, or pharmaceutically acceptable salt thereof, and wherein the Bax channel inhibitor comprises 3,6-dibromo-a-( 1 -pipcrazinylmcthyl)-9//-carbazolc-9-cthanol dihydrochloride or an analog, derivative, isomer, or pharmaceutically acceptable salt thereof.

[18] In a related aspect, the necroptosis or necrosis is associated with aging or disease. In another related aspect, the disease is myocardial infarction, diabetes secondary to beta-cell necroptosis, cholestatic liver disease, stroke, organ ischemia, ischemia-reperfusion injury, liver disease, necrosis from cancer chemotherapy or radiation therapy, traumatic brain injury, necrotizing pancreatitis, pathogen-induced necroptosis, inflammation, or neurodegenerative disease.

[19] In a related aspect, the treating, preventing, inhibiting, or reducing the incidence of cellular plasticity, or necroptosis or necrosis, comprises in vitro or in vivo treating, preventing, inhibiting, or reducing the incidence of cellular plasticity, necroptosis, or necrosis. In another related aspect, the plurality of cells comprises tissue culture cells, primary cells, egg cells, a tissue, or an organ or a portion thereof, or any combination thereof. In another related aspect, the tissue culture cells or primary cells comprise stem cells, adult cells, transdifferentiated cells, dedifferentiated cells, or differentiated cells, or any combination thereof. In another related aspect, the plurality of cells comprises human cells or animal cells. In another related aspect, the bringing in contact is in vivo , ex vivo , or in vitro. In another related aspect, the bringing in contact in vivo or ex vivo comprises perfusion of an animal or a portion thereof, an organ or a portion thereof, or a tissue. In another related aspect, the perfusion is cardiac perfusion. In another related aspect, the bringing in contact in vitro comprises immersing said plurality of cells in said composition, supplementing a growth media of said plurality of cells with said composition, perfusing said plurality of cells with said composition via non-thermal reversible electroporation, or perfusing said plurality of cells with said composition in a bioreactor.

[20] In a related aspect, methods disclosed herein further comprise a step of physical, chemical, or thermodynamic manipulation of said plurality of cells. In a related aspect, the thermodynamic manipulation comprises heating or cooling said plurality of cells. In a related aspect, the cooling comprises cryopreservation or a freeze-thaw cycle, or a combination thereof.

[21] In a related aspect, methods disclosed herein prevent, inhibit, or reduce necrosis or

necroptic death of said plurality of cells during said cryopreservation or a freeze-thaw cycle thereof, compared with an uncontacted plurality of cells.

[22] In a related aspect, the methods disclo sed herein protect said plurality of cells from physical damage during said cryopreservation or freeze-thaw cycles thereof. In another related aspect, the methods enhance growth potential of said plurality of cells during cryopreservation compared with uncontacted cells. In another related aspect, the methods prevent oxidative damage to said plurality of cells during cryopreservation or freeze-thaw cycles thereof. In another related aspect, the methods prevent ischemia of said cells during cryopreservation or freeze-thaw cycles thereof. In another related aspect, the methods inhibit necrosis pathway signaling. In another related aspect, the methods prevent, inhibit, or reduce changes in the differentiation state of said cells, thereby stabilizing the identity of said cells compared with uncontacted cells. In another related aspect, the methods induce a state of metabolic suspension in said cells. In another related aspect, the metabolic suspension comprises reversible cessation of oxygen metabolism. In another related aspect, the methods improve viability or latent viability of said cells compared with an uncontacted plurality of cells.

[23] In another aspect, in methods disclosed herein the necroptosis inhibitor compoundcomprises a 3-p-Methoxyphenyl-5,6-tetramethylenothieno[2,3-d]pyrimidin-4-one-2-mercaptoethylcyanide compound, a (Z)-5-((3-(4-Fluorophenyl)-lH-pyrazol-4-yl)methylene)-2-imino-3-(thiazol-2-yl)thiazolidin-4-one, a 5-(Indol-3-ylmethyl)-(2-thio-3-methyl)hydantoin, a methyl-thiohydantoin-tryptophan compound, a l-([3S,3aS]-3-[3-fluoro-4- [trifluoromethoxy]phenyl]-8-methoxy-3,3a,4,5-tetrahydro-2H-benzo[g]indazol-2-yl)-2-hydroxyethanone, a (S)-N-(l-[2-chloro-6-fluorophenyl]ethyl)-5-cyano-l-methyl-lH-pyrrole-2-carboxamide, a (E)-N-(4-(N-(3-Methoxypyrazin-2-yl)sulfamoyl)phenyl)-3-(5-nitrothiophene-2-yl)acrylamide, a l-(4-(4- Amino furo[2,3-d]pyrimidin-5-yl)phenyl)-3-(2-fluoro-5- (trifluoromethyl)phenyl)urea, or a 2,2-dimethyl- l-(5(S)-phenyl-4,5-dihydro-pyrazol- l-yl)-propan-l-one, or a derivative, isomer, or pharmaceutically acceptable salt of any one of said necroptosis inhibitor compounds.

[24] In a related aspect, in the methods disclosed herein the concentration of the 5-((7-Chloro-lH-indol-3-yl)methyl)-3-methyl-2,4-imidazolidinedione compound or said analog, derivative, isomer, or pharmaceutically acceptable salt thereof is about 0.2nM - 2mM, and wherein the concentration of the 3,6-Dibromo-a-(l-piperazinylmethyl)-9H- carbazole-9-ethanol

dihydrochloride compound, or analog, derivative, isomer, or pharmaceutically acceptable salt thereof is about 0.5nM - 50mM.

[25] In another related aspect, in the methods disclosed herein the composition further comprises, a nicotinamide adenine dinucleotide (NAD), an adenosine triphosphate (ATP), a cyclosporine A, a manganese-superoxide dismutase, or a zinc- superoxide dismutase, or any combination thereof. In another related aspect, in the methods disclosed herein, the concentration of said NAD is about 5nM - 500mM, the concentration of said ATP is about lOnM - lmM, the concentration of said cyclosporin A is about lnM - lmM, and the concentration of said manganese-superoxide dismutase or said zinc- superoxide is about 0.001KU-100.0KU.

[26] In another related aspect, in the methods disclosed herein, the concentration of necroptosis inhibitor 5-((7-chloro-lH-indol-3-yl)methyl)-3-methyl-2,4-imidazolidinedione compound, or the analog, a derivative, a isomer, or a pharmaceutically acceptable salt thereof is about 20nM, the concentration of Bax channel inhibitor 3,6-Dibromo-a-(l-piperazinylmethyl)-9H- carbazole-9-ethanol dihydrochloride compound, or the analog, the derivative, the isomer, or the pharmaceutically acceptable salt thereof is about 5nM, the concentration of said NAD is about 0.05 nM, the concentration of said ATP is about O.OImM, the concentration of said cyclosporin A is about O.OImM, and the concentration of said manganese-superoxide dismutase or the zinc-superoxide is about 0.1KU.

[27] In a related aspect, in the methods disclosed herein, the composition further comprises a cryoprotective agent. In another related aspect, the cryoprotective agent comprises DMSO, or serum, or any combination thereof.

[28] In a related aspect, in the methods disclosed herein, the composition further comprises a pharmaceutically acceptable carriers or excipients.

BRIEF DESCRIPTION OF THE DRAWINGS

[29] The following drawings form part of the present specification and are included to further demonstrate certain embodiments of the present disclosure, the methods described herein may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[30] Figures 1A-C. Tissue dissection and cell manipulation produces reactive oxygen species (ROS) (detection of Noxl), apoptosis (detection of CASP3), necrosis (detection of staining with PI and/or Annexin V) in several cell types. Figure 1A shows mouse cortical neuron tissue dissection and cell manipulation produced ROS (detection of Noxl). Whereas no dissection yielded little ROS (mock), the freshly dissected tissue (fresh) yielded several-fold increase in ROS generation, while inclusion of the cryopreservation solution (Soln) diminished the ROS levels. Figure IB shows that apoptosis was enhanced after dissection (fresh) compared to unmanipulated tissue (mock), although to a lesser extent than ROS generation (Figure 1A) or necrosis (Figure 1C), while inclusion of the cryopreservation solution (+Soln) decreased this back to baseline levels. Figure 1C shows that necrosis was enhanced after tissue dissection and cell manipulation (fresh) compared to no tissue manipulation (mock), an effect that was almost completely removed by inclusion of the cryopreservation solution (+Soln). (For Figures 1A-1C Soln: 20 nM 5-((7-Chloro-lH-indol-3-yl)methyl)-3-methyl-2,4-imidazolidinedione), 5nM 3,6-Dibromo-a-(l-piperazinylmethyl)-9H- carbazole-9-ethanol dihydrochloride, 0.05 mM nicotinamide adenine dinucleotide (NAD), 0.01 mM adenosine triphosphate (ATP), O.OlpM cyclosporin A, and 0.1 Kuntz Unit manganese superoxide dismutase.)

[31] Figures 2A-2C. Cellular freeze / thaw generates significant increases in ROS, apoptosis, and necrosis markers in a number of cell types over time. Figure 2A shows freeze/thaw of neurons produced ROS (detection of Noxl) (freeze) compared to tissue before freezing (mock), an effect which was almost completely abolished by inclusion of the cryopreservation solution (+Soln). Figure 2B shows apoptosis was slightly enhanced after freeze/thaw (freeze) compared to tissue before freezing (mock), an effect that was decreased by inclusion of the cryopreservation solution during the freezing process (+Soln). Figure 2C shows necrosis was greatly enhanced after cell freeze/thaw (freeze) compared to tissue before freezing (mock), an effect that was almost eliminated by inclusion of the cryopreservation solution (+ Soln). (For Figures 2A-2C Soln: 20 nM 5-((7-Chloro-lH-indol-3-yl)methyl)-3-methyl-2,4-imidazolidinedione), 5nM 3,6-Dibromo-a-(l-piperazinylmethyl)-9H- carbazole-9-ethanol dihydrochloride, 0.05 mM nicotinamide adenine dinucleotide (NAD), 0.01 pM adenosine triphosphate (ATP), O.OlpM cyclosporin A, and 0.1 Kuntz Unit manganese superoxide dismutase.) CypD - Cyclophilin D

[32] Figure 3. Cellular freeze/thaw cycle causes stem cells to lose their identity, as measured by the level of CHAT1 in motor neurons. Cryoprotectants in solution used to freeze the cells, maintained motor neuron identity, as measure by the level of CHAT1. Figure 3 shows that stem cells (motor neuron precursors) cultured and expressing CHAT1 (fresh) and then frozen in

solutions comprising O.OImM cyspA (cyclosporine A), or 20nM Neel, or 5nM iMAC2 maintained CHAT1 as compared with stem cells frozen in the absence of these compounds, which showed very low levels of CHAT1+ cells after freezing (frozen). As shown here, each of cyspA, Neel, and IMAC2 provided cryoprotection to stem cells upon freezing, maintaining cellular characteristics.

[33] Figures 4A-4B. Fresh versus frozen cell viability was analyzed by maintenance of dissected cells (hippocampal neurons) in one embodiment of a cryopreservation solution. Figure 4A shows that the cryopreservation solutions disclosed herein, by itself was able to increase the percent viability of freshly dissected cells as well as cells that had been frozen in an embodiment of a cryopreservation solution, and then thawed. (Soln: 20 nM 5-((7-Chloro-lH-indol-3-yl)methyl)-3-methyl-2,4-imidazolidinedione), 5nM 3,6-Dibromo-a-(l-piperazinylmethyl)-9H-carbazole-9-ethanol dihydrochloride, 0.05 mM nicotinamide adenine dinucleotide (NAD), 0.01 mM adenosine triphosphate (ATP), O.OImM cyclosporin A, and 0.1 Kuntz Unit manganese superoxide dismutase.) Key Figure 4A and throughout the figures: Fresh represents Freshly dissected cells; Fresh+Soln represents Freshly dissected cells in composition solution; Freeze represents Frozen / Thawed cells; and Freeze+Soln represents Frozen / Thawed cells in composition solution. Figure 4B shows metabolic suspension of cells (hippocampal neuron) enhanced cell viability, wherein the percent viability provided by different components of cryopreservation solutions is shown. The cell viability for neurons was measure after 7 days in vitro. Vehicle is no cryopreservation solution addition, while necrostatinl (Neel), cyclosporine A (CspA), iMAC2 (iMAC2), or the combination of those 3 reagents (combine), were introduced to cells before the freeze/thaw process. In one embodiment (combine + Perfuse), the 3-reagent cryopreservation solution was perfused into a living pregnant mouse briefly just minutes before the pups were isolated from the dam, and then the hippocampal neurons isolated from the E18 (embryonic day 18) pups. The concentrations of the different components are those concentrations used in Figure 4A.

[34] Figure 5. Freezing process used herein, avoids supercooling effects that damages cells. Figure 5 shows a comparison of the change in temperature over time during freezing in either an embodiment of a cryopreservation solution, disclosed herein (EVERGREEN™ Media Soln: 20 nM 5-((7-Chloro-lH-indol-3-yl)methyl)-3-methyl-2,4-imidazolidinedione), 5nM 3,6-Dibromo-a-(l-piperazinylmethyl)-9H- carbazole-9-ethanol dihydrochloride, 0.05 mM nicotinamide adenine

dinucleotide (NAD), 0.01 mM adenosine triphosphate (ATP), O.OImM cyclosporin A, and 0.1 Kuntz Unit manganese superoxide dismutase; also known as EVERLAST™) versus a traditional media. Notice the absence of the early downward spike in temperature that occurs in traditional media.

[35] Figures 6A-D. Viability and Growth rates: Fresh vs. Frozen Cell Viability Time Course: neurons and fat (adipose tissue) stem cells. Figure 6A shows cortical neuron cell viability was maintained when freshly dissected cells were suspended in an embodiment of a cryopreservation solution (fresh+Soln) disclosed herein, or dissection in and then frozen in an embodiment of a cryopreservation solution disclosed herein (freeze+Soln). Figure 6B shows the time course of percent neuron viability over time (days) after plating cells subjected to these freezing and non-freezing conditions. The inclusion of cryopreservation solution was compared to the same cells exposed to 10% DMSO before freezing (freeze) or to cells that were freshly dissected without any freezing cryoprotectant (fresh). Figure 6C shows fat (adipose tissue-derived) stem cell viability was maintained when freshly dissected cells were suspended in an embodiment of a cryopreservation solution (fresh+Soln) disclosed herein, or dissection in and then frozen in an embodiment of a cryopreservation solution disclosed herein (freeze+Soln). Figure 6D shows the time course of percent fat stem cell viability over time (days) after plating cells subjected to these freezing and non-freezing conditions. The inclusion of cryopreservation solution was compared to the same cells exposed to 10% DMSO before freezing (freeze) or to cells that were freshly dissected without any freezing cryoprotectant (fresh). (For Figures 6A-6D, Soln: 20 nM 5-((7-Chloro-lH-indol-3-yl)methyl)-3-methyl-2,4-imidazolidinedione), 5nM 3,6-Dibromo-a-(l-piperazinylmethyl)-9H- carbazole-9-ethanol dihydrochloride, 0.05 mM nicotinamide adenine dinucleotide (NAD), 0.01 mM adenosine triphosphate (ATP), O.OImM cyclosporin A, and 0.1 Kuntz Unit manganese superoxide dismutase.)

[36] Figures 7A-B. Apoptosis after dissection and freeze/thaw with cryopreservation solution. Figure 7A shows a comparison of the effects of cryoprotectant on early apoptotic cells (primary liver hepatocytes) from fresh and frozen cells suspended in an embodiment of a cryopreservation solution disclosed herein, compared with cell suspended in an absence of such a solution (mock). For cells frozen that were not treated with cryopreservation solution (mock), they were suspended in 10% DMSO before freezing. Caspase3 (green) labels cells in an early apoptotic state in culture. Figure 7B shows the number of apoptotic cells, as defined by annexin V labeling, in fresh or

frozen and thawed cells suspended in an embodiment of a cryopreservation solution disclosed herein or in the absence of such a solution (mock). (For Figures 7A-7B, Soln: 20 nM 5-((7-Chloro-lH-indol-3-yl)methyl)-3-methyl-2,4-imidazolidinedione), 5nM 3,6-Dibromo-a-(l-piperazinylmethyl)-9H- carbazole-9-ethanol dihydrochloride, 0.05 mM nicotinamide adenine dinucleotide (NAD), 0.01 mM adenosine triphosphate (ATP), O.OlpM cyclosporin A, and 0.1 Kuntz Unit manganese superoxide dismutase.)

[37] Figures 8A-8B. Necrosis after dissection and freeze/thaw of cortical neuron cells suspended in an embodiment of a cryopreservation solution disclosed herein. Figure 8A shows a comparison of the presence of necrotic cells from solutions of fresh and frozen cells suspended in an embodiment of a cryopreservation solution disclosed herein, compared with cell suspended in an absence of such a solution. Anti-cyclophilin antibody labels cells in an early apoptotic state in culture. Figure 8B shows the percent of necrotic cells after dissection performed in an embodiment of a cryopreservation solution, or the absence of such a solution. (For Figures 8A-8B, Soln: 20 nM 5-((7-Chloro-lH-indol-3-yl)methyl)-3-methyl-2,4-imidazolidinedione), 5nM 3,6-Dibromo-a-(l-piperazinylmethyl)-9H- carbazole-9-ethanol dihydrochloride, 0.05 mM nicotinamide adenine dinucleotide (NAD), 0.01 pM adenosine triphosphate (ATP), O.OlpM cyclosporin A, and 0.1 Kuntz Unit manganese superoxide dismutase.)

[38] Figures 9A-9B. Reactive Oxygen Species (ROS) (Noxl as marker) generation after cortical neuron tissue dissection and freeze/thaw. Figure 9A shows Noxl staining in fresh (dissected in saline only, and not frozen) neurons, fresh neurons dissected in cryopreservation solution (Fresh +Soln), neurons frozen while suspended in an embodiment of a cryopreservation solution disclosed herein, then thawed (Freeze + Soln) or in the presence of a conventional cryoprotectant (10% DMSO, dimethyl sulfoxide) (Freeze). Fresh or thawed neurons were plated and allowed to adhere before fixation and labeling for 4 hours. NADPH Oxidase (Noxl) labels ROS in cells. NeuN stains for cortical neurons (not glia). Figure 9B shows ROS generation (Noxl intensity) in freshly dissected cortical neuron tissue alone (fresh, blue bar), fresh cortical neuronal tissue dissected in the suspension of an embodiment of a cryopreservation solution disclosed herein (fresh+ Soln, red bar), frozen cells suspended in an embodiment of a cryopreservation solution disclosed herein (freeze + soln, yellow bar) then thawed, or cells frozen and then thawed in the presence of 10% DMSO (green bar). (For Figures 9A-9B, Soln: 20 nM 5-((7-Chloro-lH-indol-3-yl)methyl)-3-methyl-2,4-imidazolidinedione), 5nM 3,6-Dibromo-a-(l-piperazinylmethyl)-9H-

carbazole-9-ethanol dihydrochloride, 0.05 mM nicotinamide adenine dinucleotide (NAD), 0.01 mM adenosine triphosphate (ATP), O.OImM cyclosporin A, and 0.1 Kuntz Unit manganese superoxide dismutase.)

[39] Figures 10A-B. Neuronal lineage of motor neuron markers are preserved significantly above that using conventional cryoprotectants (10% DMSO) compared to the composition solution. Figure 10A shows the micrographs of neurons before or after freezing, in the presence or absence (10% DMSO only) of a cryopreservation solution disclosed herein. Figure 10B shows motor neuron cell identity (as measure by the presence of CHAT 1 -positive, S lOO-positive immunofluorescence signal) in cells before and after freezing in conventional cryopreservation solutions (10% DMSO), and an embodiment of a cryopreservation solution disclosed herein (“after freeze composition”). (For Figures 10A-10B, Soln: 20 nM 5-((7-Chloro-lH-indol-3-yl)methyl)-3-methyl-2,4-imidazolidinedione), 5nM 3,6-Dibromo-a-(l-piperazinylmethyl)-9H- carbazole-9-ethanol dihydrochloride, 0.05 mM nicotinamide adenine dinucleotide (NAD), 0.01 mM adenosine triphosphate (ATP), O.OImM cyclosporin A, and 0.1 Kuntz Unit manganese superoxide dismutase.)

[40] Figures 11A -11B. Primary mouse liver cells (hepatocytes) show similar physiological characteristics after freeze/thaw in the presence of composition solution as they do compared to freshly dissected tissue. Figure 11A shows a comparison of fresh and frozen liver cells (blue stain), incubated in the presence or absence (fresh, not frozen) of a cryopreservation solution disclosed herein, and either not (-galactosamine) or stimulated with galactosamine briefly before fixation and staining for antibody-conjugated uridine (red stain), as a general measure of galactosamine-induced transcription. Figure 11B shows a comparison of liver cell transcriptional activity in fresh and frozen cells suspended in an embodiment of a cryopreservation solution disclosed herein, wherein the liver cell activity is similar after freeze/thaw when cells are maintained in the presence of a cryopreservation solution disclosed herein. Bars measure anti-body-conjugated uridine staining (arbitrary units). (For Figures 11A-11B, Soln: 20 nM 5-((7-Chloro-lH-indol-3-yl)methyl)-3-methyl-2,4-imidazolidinedione), 5nM 3,6-Dibromo-a-(l-piperazinylmethyl)-9H-carbazole-9-ethanol dihydrochloride, 0.05 mM nicotinamide adenine dinucleotide (NAD), 0.01 mM adenosine triphosphate (ATP), O.OlpM cyclosporin A, and 0.1 Kuntz Unit manganese superoxide dismutase.)

[41] Figure 12. Several analogs of necrostatins work similarly in their cryoprotective effect after freeze/thaw. Figure 12 shows the effects of cryopreservation solutions comprising different necrostatins, wherein the presence of necrostatins (Neel (red bar), Nec2 (yellow bar), Nec3 (green bar) significantly enhanced percent viability of primary mouse cortical neuron cells compared to control cells (vehicle, blue bar) cryopreserved with conventional cryoprotectant (10% DMSO). The concentration of the different necrostatins is 20 nM, and the other components of the“soln” remain the same, that is: 5nM 3,6-Dibromo-a-(l-piperazinylmethyl)-9H- carbazole-9-ethanol dihydrochloride, 0.05 mM nicotinamide adenine dinucleotide (NAD), 0.01 mM adenosine triphosphate (ATP), O.OlpM cyclosporin A, and 0.1 Kuntz Unit manganese superoxide dismutase.

[42] Figures 13A-13B. Necrostatic pathways are halted by the composition solution. Figure 13A shows images of primary mouse liver cells (hepatocytes) before and after freezing in conventional cryopreservation solution (10% DMSO) and in an embodiment of a cryopreservation solution disclosed herein (“composition freeze”). The cells here are staining with an antibody that recognizes HMBG1 (green) as a marker for necroptosis. Figure 13B shows that necroptotic markers were significantly inhibited in a cell suspension in an embodiment of a cryopreservation solution disclosed herein, compared with 10% DMSO (“DMSO freeze”) (cryopreservation solution used herein was: 20 nM 5-((7-Chloro-lH-indol-3-yl)methyl)-3-methyl-2,4-imidazolidinedione), 5nM 3,6-Dibromo-a-(l-piperazinylmethyl)-9H- carbazole-9-ethanol dihydrochloride, 0.05 mM nicotinamide adenine dinucleotide (NAD), 0.01 pM adenosine triphosphate (ATP), O.OlpM cyclosporin A, and 0.1 Kuntz Unit manganese superoxide dismutase.)

[43] Figure 14. TNF-alpha production in necroptosis-induced THP-l cells was significantly diminished with a cryopreservation solution, EVERGREEN™ Media Solution (EVERGREEN™ Media Soln: 20 nM 5-((7-Chloro-lH-indol-3-yl)methyl)-3-methyl-2,4-imidazolidinedione), 5nM 3,6-Dibromo-a-(l-piperazinylmethyl)-9H- carbazole-9-ethanol dihydrochloride, 0.05 mM nicotinamide adenine dinucleotide (NAD), 0.01 pM adenosine triphosphate (ATP), O.OlpM cyclosporin A, and 0.1 Kuntz Unit manganese superoxide dismutase; also known as EVERLAST™). ELISA assay results for TNF-alpha after THP-l cells were cultured and induced by necrotic cell lysates combined with lipopoly saccharide (LPS) to produce pro-inflammatory cytokines. TNF-alpha levels (pg/mL) after mock stimulation (1-3), LPS stimulation alone (4-6), LPS stimulation in the presence of 10% the active level of EVERGREEN™ Media Solution used in the cryopreservation protocol (7-9), LPS stimulation in the presence of 100% active level of EVERGREEN™ Media Solution used in the cryopreservation protocol (10-12). (*) Significant difference (p<0.0l) between mock (1-3) and LPS alone (4-6), and between LPS alone (4-6) and

LPS + 10% EVERGREEN™ Media Solution (7-9). (**) Significant difference (p O.OOl) between LPS alone (4-6) and LPS + 100% EVERGREEN™ Media Solution (10-12).

[44] Figure 15. IL-6 production in necroptosis-induced THP-l cells was significantly diminished with a cryopreservation solution (EVERGREEN™ Media Soln: 20 nM 5-((7-Chloro-lH-indol-3-yl)methyl)-3-methyl-2,4-imidazolidinedione), 5nM 3,6-Dibromo-a-(l-piperazinylmethyl)-9H- carbazole-9-ethanol dihydrochloride, 0.05 mM nicotinamide adenine dinucleotide (NAD), 0.01 mM adenosine triphosphate (ATP), O.OlpM cyclosporin A, and 0.1 Kuntz Unit manganese superoxide dismutase; also known as EVERLAST™). ELISA assay results for IL-6 after THP-l cells were cultured and induced by necrotic cell lysates combined with lipopolysaccharide (LPS) to produce pro-inflammatory cytokines. IL-6 levels (pg/mL) measured after (1-3) mock stimulation, (4-6) LPS stimulation alone, (7-9) LPS stimulation in the presence of 10% the active level of EVERGREEN™ Media Solution used in the cryopreservation protocol, (10-12) LPS stimulation in the presence of 100% active level of EVERGREEN™ Media Solution used in the cryopreservation protocol. (*) Significant difference (p<0.0l) between mock (1-3) and LPS alone (4-6), and between LPS alone (4-6) and LPS + 10% EVERGREEN™ Media Solution (7-9). (**) Significant difference (p<0.00l) between LPS alone (4-6) and LPS + 100% EVERGREEN™ Media Solution (10-12).

[45] Figure 16. A cryopreservation solution (EVERGREEN™ Media Soln: 20 nM 5-((7-Chloro-lH-indol-3-yl)methyl)-3-methyl-2,4-imidazolidinedione), 5nM 3,6-Dibromo-a-(l-piperazinylmethyl)-9H- carbazole-9-ethanol dihydrochloride, 0.05 mM nicotinamide adenine dinucleotide (NAD), 0.01 mM adenosine triphosphate (ATP), O.OlpM cyclosporin A, and 0.1 Kuntz Unit manganese superoxide dismutase; also known as EVERLAST™) produced significant consistency in cytokine release measures. Percent variability was calculated by averaging the percent errors of each (triplicate) experiment (n=3), and expressing each group’s (stimulated, 10% EVERGREEN™, 100% EVERGREEN™) percent variability as a ratio to the mock (control) stimulated group variability. From this, there is -100% increase in variability without the use of EVERGREEN™ Media Solution (both at 10% and 100% levels).

[46] Figure 17. A cryopreservation solution (EVERGREEN™ Media Soln: 20 nM 5-((7-Chloro-lH-indol-3-yl)methyl)-3-methyl-2,4-imidazolidinedione), 5nM 3,6-Dibromo-a-(l-piperazinylmethyl)-9H- carbazole-9-ethanol dihydrochloride, 0.05 mM nicotinamide adenine dinucleotide (NAD), 0.01 pM adenosine triphosphate (ATP), O.OlpM cyclosporin A, and 0.1

Kuntz Unit manganese superoxide dismutase; also known as EVERLAST™) conferred significantly consistent neuronal viability post freeze / thaw compared to DMSO. EVERGREEN™ Media Solution was used to freeze primary mouse hippocampal neurons compared to dimethyl sulfoxide (DMSO) and viability post-thaw was measured and plotted. Five independent experiments were measured.

[47] Figure 18. A cryopreservation solution (EVERGREEN™ Media Soln: 20 nM 5-((7-Chloro-lH-indol-3-yl)methyl)-3-methyl-2,4-imidazolidinedione), 5nM 3,6-Dibromo-a-(l-piperazinylmethyl)-9H- carbazole-9-ethanol dihydrochloride, 0.05 mM nicotinamide adenine dinucleotide (NAD), 0.01 mM adenosine triphosphate (ATP), O.OlpM cyclosporin A, and 0.1 Kuntz Unit manganese superoxide dismutase; also known as EVERLAST™) induced significant consistency in neuronal cell viability post freeze/thaw. Percent variability was calculated by averaging the means of each experiment (n=5) and expressing each group’s individual measurement as a ratio of the individual value to the mean value. From this, the percent deviation was calculated and expressed as a ratio to the mean. From this, there is an -300% increase in variability without the use of EVERGREEN™ Media Solution.

[48] Figures 19A-19H. Hippocampal neurons show physiological characteristics that are indistinguishable from before (freshly dissected) and after freeze/thaw using a cryopreservation solution (EVERGREEN™ Media Soln: 20 nM 5-((7-Chloro-lH-indol-3-yl)methyl)-3-methyl-2,4-imidazolidinedione), 5nM 3,6-Dibromo-a-(l-piperazinylmethyl)-9H- carbazole-9-ethanol dihydrochloride, 0.05 mM nicotinamide adenine dinucleotide (NAD), 0.01 pM adenosine triphosphate (ATP), O.OlpM cyclosporin A, and 0.1 Kuntz Unit manganese superoxide dismutase; also known as EVERLAST™). Patch clamp of embryonic day 17-19 neurons shows current and voltage profiles from both spontaneous and evoked neuronal activities. Figure 19A shows hippocampal neurons from freshly dissected mouse brain showing whole cell current clamp (at resting membrane potential) recording demonstrates network activity in culture. Vertical bar represents 20 millivolts (mV) magnitude and horizontal bar 2 seconds (sec) of recording. Figure 19B shows hippocampal neuron from freshly dissected mouse brain after freeze/thaw using EVERGREEN™ Media Solution showing whole cell current clamp (at resting membrane potential) recording demonstrates network activity in culture. Vertical bar represents 20 millivolts (mV) magnitude and horizontal bar 2 seconds (sec) of recording. Figure 19C shows Cumulative frequency over 5 minutes recording interval for current clamp data showing average frequency of action potential discharges. Values are mean+SE (standard error) of at least 4 independent experiments, which show no statistically significant differences (p>0.7, Student’s t-test) either before or after freeze/thaw using EVERGREEN™ Media Solution. Figure 19D shows hippocampal neuron from freshly dissected mouse brain showing whole cell voltage clamp (at -60mV) recording demonstrates network activity in culture. Vertical bar represents 200 picoAmps (pA) magnitude and horizontal bar 2 seconds (sec) of recording. Figure 19E shows hippocampal neuron from freshly dissected mouse brain after freeze/thaw using EVERGREEN™ Media Solution showing whole cell voltage clamp (at -60mV) recording demonstrates network activity in culture. Vertical bar represents 200 picoAmps (pA) magnitude and horizontal bar 2 seconds (sec) of recording. Figure 19F shows cumulative frequency over 5 minutes recording interval for voltage clamp data showing average frequency of action potential discharges. Values are mean+SE (standard error) of at least 4 independent experiments, which show no statistically significant difference (p>0.8) either before or after freeze/thaw using EVERGREEN™ Media Solution. Figure 19G shows evoked pre-synaptic action potential shown (lower traces) in current clamp mode at resting potential and the post-synaptic excitatory synaptic response (upper traces) (shown on schematic at left as black circle at end of axon from lower presynaptic neuron) from two adjacent coupled hippocampal neurons, both before (left traces, freshly dissected) and after (right traces) freeze / thaw using EVERGREEN™ Media Solution. Here, the excitatory post synaptic currents (EPSCs) are not statistically different between treatments (actual values 71+/- 23pA before versus 76+/-28pA after). Representative traces from n=l9-22 neurons, at least 3 independent experiments for each treatment (p>0.7, Student’s t-test). Vertical bar represents 20picoAmps (pA) magnitude for current clamped neurons (pre-synaptic, lower traces) and 20milliVolts (mV) for the excitatory synaptic responses (post- synaptic, upper traces); horizontal bar represents 20milliSeconds (ms) of recording. Figure 19H shows evoked action potential amplitude ratios for presynaptic over post-synaptic (pre/post) responses for 19 hippocampal neurons (as measured in part G) both before (closed circles) and after (open circles) freeze/thaw using EVERGREEN™ Media Solution. Average ratios are not statistically different for neurons before and after freeze/thaw using EVERGREEN™ Media Solution (p>0.7, Student’s t-test) (average values 71+/- 23pA for before versus 76+/-28pA for after freeze/thaw). X values correspond to neuron numbers for each treatment.

DETAILED DESCRIPTION

[49] In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the compositions and methods presented herein. In other instances, well-known methods, procedures, and components have not been described in detail so as not to obscure the compositions and methods and resultant inhibition of necrosis and necroptosis, suppression of ischemia and oxidative damage, improvement in cell viability, enhancement of cells’ growth potential, and stabilization of cell identity, as disclosed herein.

[50] In one embodiment, disclosed herein is a composition for cryopreservation of mammalian cells, tissues, organs and portions thereof, comprising a necroptosis inhibitor and a Bax channel inhibitor. In another embodiment, disclosed herein is a method for cryopreservation of mammalian cells, tissues, and organs, comprising the steps of (a) bringing said cells, tissues, or organs or portions thereof in contact with a composition comprising a necroptosis inhibitor and a Bax channel inhibitor, and (b) cooling the composition comprising said cells, tissues, or organs or portions thereof. In a further embodiment, disclosed herein is method for suppressing, preventing or avoiding cell necrosis that results from physical and physiological damage cells incur in the course of cell manipulations in vitro , during liberation from tissue, and during cryopreservation and freeze-thaw cycles thereof.

Compositions

[51] In one embodiment, disclosed herein is a composition comprising a necroptosis inhibitor and a Bax channel inhibitor. A skilled artisan would appreciate that necroptosis may encompass any form of active necrosis (death of living cells or tissues). In some embodiments, necrosis comprises cell membrane and organelle disruption, cell swelling and mitochondrial impairment, followed by cell lysis and accompanied by inflammatory response. In some embodiments, necroptosis comprises response to inflammatory signals and stress in particular from tissues. In some embodiments, necroptosis comprises regulated necrosis.

[52] In one embodiment, necroptosis comprises an active or programed form of necrosis (cell death). In another embodiment, necroptosis comprises a necrotic cell death dependent on receptor interacting protein kinase- 1 (RIPK1). In another embodiment, necroptosis comprises a necrotic cell death dependent on receptor-interacting protein kinase-3 (RIPK3). In another embodiment, necroptosis comprises a regulated caspase-independent cell death mechanism that results in morphological features resembling necrosis. In some embodiments, activation of TNF-receptor leads

to RIPK1 activation and subsequent recruitment of RIPK3 forming the necrosome. In another embodiment, necroptosis comprises a non-apoptotic cell death pathway.

[53] In one embodiment, a necroptosis inhibitor comprises a compound that inhibits any form of active necrosis. In another embodiment, a necroptosis inhibitor comprises a compound that inhibits any form of necrosis. In another embodiment, a necroptosis inhibitor comprises a compound that inhibits any form of necroptosis. In another embodiment, a necroptosis inhibitor comprises an inhibitor of necrotic cell death dependent on receptor- interacting protein kinase- 1 (RIPK1). In another embodiment, a necroptosis inhibitor comprises an inhibitor of necrotic cell death dependent on receptor-interacting protein kinase-3 (RIPK3). In another embodiment, a necroptosis inhibitor comprises an inhibitor of regulated caspase-independent cell death mechanism that results in morphological features resembling necrosis.

[54] While not wishing to be bound by theory, the activity of receptor interacting protein kinases (RIPK) has been shown to be important for cells to undergo necroptosis. Furthermore, RIP kinases’ activity is also known to promote the release of inflammatory mediators such as TNF alpha from cells which can induce inflammation and also promote further necroptosis. In one embodiment, a necroptosis inhibitor comprises a RIPK inhibitor. In another embodiment, a necroptosis inhibitor comprises a RIP1 inhibitor. In another embodiment, a necroptosis inhibitor comprises a RIP3 inhibitor.

[55] In one embodiment, the necroptosis inhibitor comprises a 5-((7-Chloro-lH-indol-3-yl)methyl)-3-methyl-2,4-imidazolidinedione (necrostatin-ls) compound, or an analog, a derivative, an isomer, or a pharmaceutically acceptable salt thereof. In another embodiment, the structure of

necrostatin-ls is represented in Formula
Formula I.

[56] As used herein, the terms “5-((7-Chloro-lH-indol-3-yl)methyl)-3-methyl-2,4-imidazolidinedione”, “5-((7-Chloro-lH-indol-3-yl)methyl)-3-methylimidazolidine-2,4-dione”, ‘7-Cl-O-MH-Trp”, “necrostatin-ls”, “Nec-ls”, “Nec-l stable”, “RIPK1 Inhibitor II”, “Receptor-Interacting Protein 1 Inhibitor II”,“Necrosis Inhibitor IV”, and“7N-1” may be used interchangeably having all the same meanings and qualities.

[57] One of ordinary skill in the art would appreciate that term“analog” may encompass any compound having a structure similar to that of another one, but differing from it in respect of a certain component such as a functional group or a substructure. The term“analog” is used interchangeably with“analog compound”.

[58] One of ordinary skill in the art would appreciate that term“necro statin- ls analog” may encompass a compound having a structure similar to that of necrostatin-ls, but differing from it in respect of a certain component such as replacement of one atom or a group of atoms or a functional group or a substructure with another atom or group of atoms or functional group or a substructure.

In another embodiment, the necroptosis inhibitior is any necrostatin-ls analog known in the art.

[59] In another embodiment, the necroptosis inhibitor comprises a 5-((7-Chloro-lH-indol-3-yl)methyl)-3-methyl-2,4-imidazolidinedione (necrostatin-ls) compound or an analog, a derivative, an isomer, or a pharmaceutically acceptable salt thereof. In another embodiment, a necroptosis inhibitor compound comprises a 3-p-Methoxyphenyl-5,6-tetramethylenothieno[2,3-d]pyrimidin-4-one-2-mercaptoethylcyanide (Nec-5) compound or an analog, a derivative, an isomer, or a pharmaceutically acceptable salt thereof. In another embodiment, a necroptosis inhibitor compound comprises a (Z)-5-((3-(4-Fluorophenyl)-lH-pyrazol-4-yl)methylene)-2-imino-3-(thiazol-2-yl)thiazolidin-4-one compound (Nec-7) or an analog, a derivative, an isomer, or a pharmaceutically acceptable salt thereof. In another embodiment, a necroptosis inhibitor compound comprises a 5-(Indol-3-ylmethyl)-(2-thio-3-methyl)hydantoin compound (necrostatin-l; Nec-l), or an analog, a derivative, an isomer, or a pharmaceutically acceptable salt thereof. In one embodiment, Nec-l is represented by a structure of Formula II

Formula II.

[60] In another embodiment, a necroptosis inhibitor compound comprises a methyl-thiohydantoin-tryptophan compound, or an analog, a derivative, an isomer, or a pharmaceutically acceptable salt thereof. In another embodiment, a necroptosis inhibitor compound comprises a 1-([3S,3aS]-3-[3-fluoro-4-[trifluoromethoxy]phenyl]-8-methoxy-3,3a,4,5-tetrahydro-2H-benzo[g]indazol-2-yl)-2- hydroxyethanone compound (Nec-3a), or an analog, a derivative, an

isomer, or a pharmaceutically acceptable salt thereof. In another embodiment, a necroptosis inhibitor compound comprises a (S)-N-(l-[2-chloro-6-fluorophenyl]ethyl)-5-cyano-l-methyl-lH-pyrrole-2-carboxamide compound (Nec-4), or an analog, a derivative, an isomer, or a pharmaceutically acceptable salt thereof. In another embodiment, a necroptosis inhibitor compound comprises a (£)-N-(4-(N-(3-Methoxypyrazin-2-yl)sulfamoyl)phenyl)-3-(5-nitrothiophene-2-yl)acrylamide compound (necrosulfonamide), or an analog, a derivative, an isomer, or a pharmaceutically acceptable salt thereof. In another embodiment, a necroptosis inhibitor compound comprises a l-(4-(4-Aminofuro[2,3-d]pyrimidin-5-yl)phenyl)-3-(2-fluoro-5-(trifluoromethyl) phenyl)urea compound (RIP1 Inhibitor III), or an analog, a derivative, an isomer, or a pharmaceutically acceptable salt thereof.

[61] In another embodiment, a necroptosis inhibitor compound comprises a 3-p-Methoxyphenyl-5,6-tetramethylenothieno[2,3-d]pyrimidin-4-one-2-mercaptoethylcyanide (Nec-5) compound, a (Z)-5-((3-(4-Fluorophenyl)- lH-pyrazol-4-yl)methylene)-2-imino-3-(thiazol-2-yl)thiazolidin-4-one compound (Nec-7), a 5-(Indol-3-ylmethyl)-(2-thio-3-methyl)hydantoin compound (Nec-l), a methyl-thiohydantoin-tryptophan compound, a l-([3S,3aS]-3-[3-fluoro-4-[trifluoromethoxy]phenyl]-8-methoxy-3,3a,4,5-tetrahydro-2H-benzo[g]indazol-2-yl)-2-hydroxyethanone compound (Nec-3a), a (S)-N-(l-[2-chloro-6-fluorophenyl]ethyl)-5-cyano-l-methyl-lH-pyrrole-2-carboxamide compound (Nec-4), a (£’)-N-(4-(N-(3-Methoxypyrazin-2-yl)sulfamoyl)phenyl)-3-(5-nitrothiophene-2-yl)acrylamide compound (necrosulfonamide), a l-(4-(4-Aminofuro[2,3-d]pyrimidin-5-yl)phenyl)-3-(2-fluoro-5-(trifluoromethyl)phenyl)urea compound (RIP1 Inhibitor III), 2,2-dimethyl- l-(5(S)-phenyl-4,5-dihydro-pyrazol-l-yl)-propan-l-one (GSK’963), or an analog, a derivative, an isomer, or a pharmaceutically acceptable salt of any one of said necroptosis inhibitor compounds.

[62] In another embodiment, the necroptosis inhibitor comprises a 5-(lH-Indol-3-ylmethyl)-3-methyl-2-thioxo-4-Imidazolidinone (necro statin- 1) compound, or an analog, a derivative, an isomer, or a pharmaceutically acceptable salt thereof. As used herein, the term“5-(lH-Indol-3-ylmethyl)-3-methyl-2-thioxo-4-Imidazolidinone”, may be used interchangeably with necro statin- 1,“5-(Indol-3-ylmethyl)-3-methyl-2-thio-Hydantoin”, “Methylthiohydantoin-DL-tryptophan”, or “MTH-DL-Tryptophan”, “MTH-Trp”, “RIP1 Inhibitor I”, “Necrosome Inhibitor I”, “Receptor-Interacting Protein 1 Inhibitor I”,“Necrosis Inhibitor II” or“Nec-l”, having all the same qualities and meanings.

[63] One of ordinary skill in the art would appreciate that term“necro statin- 1 analog” may

encompass a compound having a structure similar to that of necro statin- 1, but differing from it in respect of a certain component such as replacement of one atom or a group of atoms or a functional group or a substructure with another atom or group of atoms or functional group or a substructure. Multiple necrostatin-l analogs have been described in the art and are contemplated herein (see e.g. U.S. Patent 8,324,262, columns 1-31 and 51-52; U.S. Patent 8,658,689 columns 2-31; U.S. Patent 9,108,955 columns 2-31; and 9,499,521 columns 2-5, 12-20 and 49-57; U.S. Patent Application Publications Ser. No: 2005/0119260, paragraphs [0008]-[0l03]; 2013/0158024, paragraphs [0009]-[0166]; 2010/0190836, paragraphs [0009] -[0168]; 2012/0122889, paragraphs [0008] -[0266]; and 2014/0024657, paragraphs [0008]-[0423] and [447]; PCT Patent Application Publications Ser. No. WO 2014152182, pages 2-4, 6-9, 11-14; and WO 2016094846 , pages 2-4, 13-19 and 39-50; and EPO Patent Application Publication Ser. No. EP 3017825 paragraphs [0009], [0021], and [0025], all of which are hereby incorporated by reference in their entirety).

[64] In another embodiment, the necroptosis inhibitior is any necrostatin-l analog or necrostatin-ls inhibitor known in the art.

[65] In another embodiment, a necroptosis inhibitor comprises a 5-((7-Chloro-lH-indol-3-yl)methyl)-3-methyl-2,4-imidazolidinedione (Nec-ls), a 3-p-Methoxyphenyl-5,6-tetramethylenothieno[2,3-d]pyrimidin-4-one-2-mercaptoethylcyanide (Nec-5) compound, a (Z)-5-((3-(4-Fluorophenyl)-lH-pyrazol-4-yl)methylene)-2-imino-3-(thiazol-2-yl)thiazolidin-4-one compound (Nec-7), a 5-(Indol-3-ylmethyl)-(2-thio-3-methyl)hydantoin compound (Nec- 1), a methyl-thiohydantoin-tryptophan compound, a l-([3S,3aS]-3-[3-fluoro-4-[trifluoromethoxy]phenyl]-8-methoxy-3,3a,4,5-tetrahydro-2H-benzo[g]indazol-2-yl)-2- hydroxyethanone compound (Nec-3a), a (S)-N-(l-[2-chloro-6-fluorophenyl]ethyl)-5-cyano- l-methyl- lH-pyrrole-2-carboxamide compound (Nec-4), a (£’)-N-(4-(N-(3-Methoxypyrazin-2-yl)sulfamoyl)phenyl)-3-(5-nitrothiophene-2-yl)acrylamide compound (necrosulfonamide), a l-(4-(4-Aminofuro[2,3-d]pyrimidin-5-yl)phenyl)-3-(2-fluoro-5-(trifluoromethyl)phenyl)urea compound (RIP1 Inhibitor III), or 2,2-dimethyl- l-(5(S)-phenyl-4,5-dihydro-pyrazol-l-yl)-propan-l-one compound (GSK’963), or an analog, a derivative, an isomer, or a pharmaceutically acceptable salt of any one of said necroptosis inhibitors.

[66] While not wishing to be bound by theory, it is believed that mitochondria are the central orchestrators of both apoptosis and regulated necrosis. In some embodiments, during regulated necrosis the mitochondria become dysfunctional in a process that is, termed "maladaptive" where mitochondria swell, lose their ability to generate ATP, and subsequently rupture. In some

embodiments, the maladaptive process is driven in part by the opening of the mitochondrial permeability transition pores (MPTP), a process that is in turn activates Bax and Bak protein-mediated signaling. In some embodiments, Bax (Bcl-2-associated X protein) is a protein that primarily found in cytosol. In some embodiments, upon initiation of necrosis, Bax undergoes a conformational shift and becomes associated with mitochondrial membrane, where it forms a multimeric pore within the mitochondrial membrane.

[67] Multiple Bax channel inhibitors have been described in the art and are contemplated herein ( see e.g. PCT Patent Application Publications Ser. No. WO 2014110476, pages 18-40; and EPO Patent Application Publication Ser. No. EP 1094063, pages 5- 14, all of which are hereby incorporated by reference in their entirety). In another embodiment, the Bax channel inhibitor is any Bax channel inhibitor known in the art. In another embodiment, the Bax channel inhibitor comprises an analog, a derivative, an isomer, or a pharmaceutically acceptable salt of a Bax channel inhibitor.

[68] In one embodiment, the Bax channel inhibitor comprises a 3,6-Dibromo-a-(l-piperazinylmethyl)-9H- carbazole-9-ethanol dihydrochloride compound, or an analog, a derivative, an isomer, or a pharmaceutically acceptable salt thereof.

[69] In one embodiment, a composition disclosed herein comprises a necroptosis inhibitor comprising a 5-((7-Chloro-lH-indol-3-yl)methyl)-3-methyl-2,4-imidazolidinedione compound or an analog, derivative, isomer, or pharmaceutically acceptable salt thereof, and a Bax channel inhibitor comprising a 3,6-Dibromo-a-(l-piperazinylmethyl)-9H- carbazole-9-ethanol dihydrochloride compound, or an analog, derivative, isomer, or pharmaceutically acceptable salt thereof.

[70] In one embodiment, the composition comprises a necroptosis inhibitor selected from the group comprising a 5-((7-Chloro-lH-indol-3-yl)methyl)-3-methyl-2,4-imidazolidinedione , a 3-p-Methoxyphenyl-5,6-tetramethylenothieno[2,3-d]pyrimidin-4-one-2-mercaptoethylcyanide compound, a (Z)-5-((3-(4-Fluorophenyl)- lH-pyrazol-4-yl)methylene)-2-imino-3-(thiazol-2-yl)thiazolidin-4-one compound, a 5-(Indol-3-ylmethyl)-(2-thio-3-methyl)hydantoin compound, 1-([3S,3aS]-3-[3-fluoro-4-[trifluoromethoxy]phenyl]-8-methoxy-3,3a,4,5-tetrahydro-2H-benzo[g]indazol-2-yl)-2- hydroxyethanone compound, (S)-N-(l-[2-chloro-6-fluorophenyl]ethyl)-5-cyano- 1 -methyl- lH-pyrrole-2-carboxamide compound, methyl-thiohydanto in-tryptophan compound, (E)-N-(4-(N-(3-Methoxypyrazin-2-yl)sulfamoyl)phenyl)-3-(5-nitrothiophene-2-yl)acrylamide compound, or l-(4-(4-Aminofuro[2,3-d]pyrimidin-5-yl)phenyl)-3-(2-fluoro-5-(trifluoromethyl) phenyl)urea compound, or 2,2-dimethyl- l-(5(S)-phenyl-4,5-dihydro-pyrazol-l-yl)- propan- 1 -one compound, or an analog, derivative, isomer, or pharmaceutically acceptable salt of said necroptosis inhibitor compound, and a Bax channel inhibitor or an analog, derivative, isomer, or pharmaceutically acceptable salt thereof.

[71] In another embodiment, the composition comprises a necroptosis inhibitor selected from the group comprising a 5-((7-Chloro-lH-indol-3-yl)methyl)-3-methyl-2,4-imidazolidinedione, a 3-p-Methoxyphenyl-5,6-tetramethylenothieno[2,3-d]pyrimidin-4-one-2-mercaptoethylcyanide compound, a (Z)-5-((3-(4-Fluorophenyl)- lH-pyrazol-4-yl)methylene)-2-imino-3-(thiazol-2-yl)thiazolidin-4-one compound, a 5-(Indol-3-ylmethyl)-(2-thio-3-methyl)hydantoin compound, 1-([3S,3aS]-3-[3-fluoro-4-[trifluoromethoxy]phenyl]-8-methoxy-3,3a,4,5-tetrahydro-2H-benzo[g]indazol-2-yl)-2- hydroxyethanone compound, (S)-N-(l-[2-chloro-6-fluorophenyl]ethyl)-5-cyano- 1 -methyl- lH-pyrrole-2-carboxamide compound, methyl-thiohydanto in-tryptophan compound, (E)-N-(4-(N-(3-Methoxypyrazin-2-yl)sulfamoyl)phenyl)-3-(5-nitrothiophene-2-yl) acrylamide compound, l-(4-(4- Amino furo[2,3-d]pyrimidin-5-yl)phenyl)-3-(2-fluoro-5- (trifluoromethyl)phenyl)urea compound, or 2,2-dimethyl- l-(5(S)-phenyl-4,5-dihydro-pyrazol- l-yl)-propan-l-one compound, or an analog, derivative, isomer, or pharmaceutically acceptable salt of said necroptosis inhibitor compound, and a Bax channel inhibitor comprising a 3,6-Dibromo-a-(l-piperazinylmethyl)-9H- carbazole-9-ethanol dihydrochloride compound, or an analog, derivative, isomer, or pharmaceutically acceptable salt thereof.

[72] One of ordinary skill in the art would appreciate that term“isomer” may encompass an optical isomers and analogs, structural isomers and analogs, conformational isomers and analogs, and the like. Optical isomers, are also known as enantiomers and may in one embodiment comprise one of two stereoisomers that are mirror images of each other that are non- superpo sable (not identical),

[73] In one embodiment, an isomer comprises optical isomers of the necroptosis inhibitor compound. It will be appreciated by those skilled in the art that the necroptosis inhibitor compound disclosed herein may contain at least one chiral center. Accordingly, the necroptosis inhibitor compound used in the compositions and methods disclosed herein may exist in optically- active or racemic forms. Some compounds may also exhibit polymorphism. It is to be understood that in one embodiment, the necroptosis inhibitor compounds may encompass any racemic, optically-active, polymorphic, or stereo-isomeric form, or mixtures thereof, which form possesses properties useful in methods of cryopreservation herein. In another embodiment, any racemic, optically-active, polymorphic, or stereo-isomeric form, or mixtures thereof, of a necroptosis inhibitor compound

described herein, may possess properties useful in method of treating, preventing, inhibiting, or reducing the incidence of cellular plasticity disclosed herein.

[74] One of ordinary skill in the art would appreciate that term "enantiomer", may encompass compound having a center of chirality and being one of two stereoisomers that are non-superposable complete mirror images of each other. As known in the art, enantiomers differ from each other in their ability to rotate plane-polarized light and may be classified according to the CIP (Cahn-Ingold-Prelog)-convention as S- or R-enantiomer. The S- and R- configurations represent the three-dimensional orientation of the four substituents about the chiral center carbon atom.

[75] In one embodiment, a necroptosis inhibitor compound is the pure (A)- isomer. In another embodiment, a necroptosis inhibitor compound is the pure (S)-isomer. In another embodiment, a necroptosis inhibitor compound comprises a mixture of the ( R ) and the (S) isomers. In another embodiment, a necroptosis inhibitor compound is a racemic mixture comprising an equal amount of the (R) and the (S) isomers. It is well known in the art how to prepare optically- active forms (for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically- active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase).

[76] In one embodiment, a Bax channel inhibitor compound is the pure (R)-isomer. In another embodiment, a Bax channel inhibitor compound is the pure (S)-isomer. In another embodiment, a Bax channel inhibitor compound comprises a mixture of the (R) and the (S) isomers. In another embodiment, a Bax channel inhibitor compound is a racemic mixture comprising an equal amount of the ( R ) and the (S) isomers. It is well known in the art how to prepare optically- active forms (for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically- active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase).

[77] One of ordinary skill in the art would appreciate that term "tautomer" may encompass compounds produced by the phenomenon wherein a proton of one atom of a molecule shifts to another atom, thereby forming a structural isomer of the original compound. See, Jerry March, Advanced Organic Chemistry: Reactions, Mechanisms and Structures, Fourth Edition, John Wiley & Sons, pages 69-74 (1992). The tautomers also refer to one of two or more structural isomers that exist in equilibrium and are readily converted from one isomeric form to another. Examples of include keto-

enol tautomers, such as acetone/propen- 2-ol and the like, ring-chain tautomers, such as glucose/2, 3, 4, 5, 6-pentahydroxy-hexanal, aromatic tautomers and the like. The compounds described herein may have one or more tautomers and therefore include various isomers. All such isomeric forms of these compounds are expressly included in the compositions disclosed herein.

[78] One of ordinary skill in the art would appreciate that term“derivative”, may encompass any pharmaceutically acceptable derivative or non-pharmaceutically acceptable derivative which is suitable for use in the process disclosed herein. The skilled person will appreciate that non-pharmaceutically acceptable derivatives may be used to prepare compounds and derivatives suitable for pharmaceutical use. In one embodiment, the derivatives used or prepared in the compositions disclosed herein are pharmaceutically acceptable derivatives.

[79] One of ordinary skill in the art would appreciate that term“pharmaceutically acceptable salt” may encompass salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response and the like and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For a review on suitable salts see Berge et al., J. Pharm. Sci., 1977, 66, 1-19.

[80] Typically, a pharmaceutically acceptable salt may be readily prepared by using a desired acid or base as appropriate. The salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent. The salt can also be prepared in situ during the final isolation and purification of the compounds of a composition disclosed herein or separately by reacting the free base group with a suitable organic acid.

[81] Salts my comprise acid addition salts resulting from reaction of an acid with a basic nitrogen atom. Salts encompassed within the term“pharmaceutically acceptable salts” refer to non-toxic salts of the compounds disclosed herein. Suitable addition salts are formed from acids which form non toxic salts and examples are acetate, p-aminobenzoate, ascorbate, aspartate, benzenesulfonate, benzoate, bicarbonate, bismethylenesalicylate, bisulfate, bitartrate, borate, bromide, calcium edetate, camsylate, carbonate, chloride, clavulanate, citrate, cyclohexylsulfamate, dihydrochloride, edetate, edisylate, estolate, esylate, ethanedisulfonate, ethanesulfonate, formate, fumarate, gluceptate, gluconate, glutamate, glutarate, glycollate, glycollylarsanilate, hemisulfate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydrogen phosphate, hydroiodide, hydroxynaphthoate, iodide, isethionate, itaconate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylbromide, methyinitrate, methylsulfate, monopotassium maleate, mucate, napsylate, nitrate, N-methylglucamine, oxalate, oxaloacetate, pamoate (embonate), palmate, palmitate, pantothenate, phosphate/diphosphate, piruvate, polygalacturonate, propionate, saccharate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, teoclate, tosylate, triethiodide, trifluoroacetate and valerate. In some embodiments, salts prepared in the compositions disclosed herein include the succinate, glutarate and hemisulfate salts.

[82] In one embodiment, the concentration of a necroptosis inhibitor comprised in a composition disclosed herein, comprises a range of about 0.2 nM to 2mM. In another embodiment, the concentration of the necroptosis inhibitor comprises a range of about 0.2 nM to about 0.5 nM. In another embodiment, the concentration of the necroptosis inhibitor comprises a range of about 0.5 nM to about 5 nM. In another embodiment, the concentration of a necroptosis inhibitor comprises a range of about 5 nM to about 50 nM. In another embodiment, the concentration of a necroptosis inhibitor comprises a range of about 50 nM to about 500 nM. In another embodiment, the concentration of a necroptosis inhibitor comprises a range of about 50 nM to about 100 nM. In another embodiment, the concentration of a necroptosis inhibitor comprises a range of about 100 nM to about 200 nM. In another embodiment, the concentration of a necroptosis inhibitor comprises a range of about 200 nM to about 300 nM. In another embodiment, the concentration of a necroptosis inhibitor comprises a range of about 300 nM to about 400 nM. In another embodiment, the concentration of a necroptosis inhibitor comprises a range of about 400 nM to about 500 nM. In another embodiment, the concentration of a necroptosis inhibitor comprises a range of about 500 nM to about 1 mM. In another embodiment, the concentration of a necroptosis inhibitor comprises a range of about 500 nM to about 600 nM. In another embodiment, the concentration of a necroptosis inhibitor comprises a range of about 600 nM to about 700 nM. In another embodiment, the concentration of a necroptosis inhibitor comprises a range of about 700 nM to about 800 nM. In another embodiment, the concentration of a necroptosis inhibitor comprises a range of about 800 nM to about 900 nM. In another embodiment, the concentration of a necroptosis inhibitor comprises a range of about 900 nM to about 1 mM. In another embodiment, the concentration of a necroptosis inhibitor comprises a range of about lpM to about 2 pM. In another embodiment, the concentration of a necroptosis inhibitor comprises a range of about lpM to about 1.1 pM. In another embodiment, the concentration of a necroptosis inhibitor comprises a range of about lpM to about 1.2 pM. In another embodiment, the concentration of a necroptosis inhibitor comprises a range of about 1.2 pM to about 1.3 pM. In another embodiment, the concentration of a necroptosis inhibitor comprises a range of about 1.3 mM to about 1.4 mM. In another embodiment, the concentration of a necroptosis inhibitor comprises a range of about 1.4 pM to about 1.5 pM. In another embodiment, the concentration of a necroptosis inhibitor comprises a range of about 1.5 pM to about 1.6 pM. In another embodiment, the concentration of a necroptosis inhibitor comprises a range of about 1.6 pM to about 1.7 pM. In another embodiment, the concentration of a necroptosis inhibitor comprises a range of about 1.7 pM to about 1.8 pM. In another embodiment, the concentration of a necroptosis inhibitor comprises a range of about 1.8 pM to about 1.9 pM. In another embodiment, the concentration of a necroptosis inhibitor comprises a range of about 1.9 pM to about 2.0 pM. In another embodiment, the concentration of a necroptosis inhibitor comprises a range of about 2 pM to about 200 pM. In another embodiment, the concentration of a necroptosis inhibitor comprises a range of about200 pM to about 500 pM.

[83] In one embodiment, the final concentration of the necroptosis inhibitor comprised in a composition disclosed herein is about 0.2nM. In another embodiment, the final concentration of the necroptosis inhibitor is about 2.0 nM. In another embodiment, the final concentration of the necroptosis inhibitor is about 20 nM. In another embodiment, the final concentration of the necroptosis inhibitor is about 200 nM. In another embodiment, the final concentration of the necroptosis inhibitor is about 2 pM.

[84] In one embodiment, the concentration of a necrostatin-ls compound comprised in a composition disclosed herein, comprises a range of about 0.2 nM to 2pM. In another embodiment, the concentration of the necrostatin- 1 s compound comprises a range of about 0.2 nM to about 0.5 nM. In another embodiment, the concentration of the necrostatin- 1 s compound comprises a range of about 0.5 nM to about 5 nM. In another embodiment, the concentration of a necrostatin-ls compound comprises a range of about 5 nM to about 50 nM. In another embodiment, the concentration of a necrostatin- 1 s compound comprises a range of about 50 nM to about 500 nM. In another embodiment, the concentration of a necrostatin-ls compound comprises a range of about 50 nM to about 100 nM. In another embodiment, the concentration of a necrostatin-ls compound comprises a range of about 100 nM to about 200 nM. In another embodiment, the concentration of a necrostatin-ls compound comprises a range of about 200 nM to about 300 nM. In another embodiment, the concentration of a necrostatin-ls compound comprises a range of about 300 nM to about 400 nM. In another embodiment, the concentration of a necrostatin-ls compound comprises a range of about 400 nM to about 500 nM. In another embodiment, the concentration of a necrostatin-ls compound comprises

a range of about 500 nM to about 1 mM. In another embodiment, the concentration of a necrostatin- ls compound comprises a range of about 500 nM to about 600 nM. In another embodiment, the concentration of a necro statin- ls compound comprises a range of about 600 nM to about 700 nM. In another embodiment, the concentration of a necrostatin-ls compound comprises a range of about 700 nM to about 800 nM. In another embodiment, the concentration of a necrostatin-ls compound comprises a range of about 800 nM to about 900 nM. In another embodiment, the concentration of a necrostatin- 1 s compound comprises a range of about 900 nM to about 1 mM. In another embodiment, the concentration of a necrostatin-ls compound comprises a range of about ImM to about 2 mM. In another embodiment, the concentration of a necrostatin-ls compound comprises a range of about ImM to about 1.1 mM. In another embodiment, the concentration of a necrostatin-ls compound comprises a range of about ImM to about 1.2 mM. In another embodiment, the concentration of a necrostatin-ls compound comprises a range of about 1.2 mM to about 1.3 pM. In another embodiment, the concentration of a necrostatin-ls compound comprises a range of about 1.3 pM to about 1.4 mM. In another embodiment, the concentration of a necrostatin-ls compound comprises a range of about 1.4 mM to about 1.5 pM. In another embodiment, the concentration of a necrostatin- ls compound comprises a range of about 1.5 pM to about 1.6 pM. In another embodiment, the concentration of a necrostatin-ls compound comprises a range of about 1.6 pM to about 1.7 mM. In another embodiment, the concentration of a necrostatin-ls compound comprises a range of about 1.7 mM to about 1.8 pM. In another embodiment, the concentration of a necrostatin-ls compound comprises a range of about 1.8 pM to about 1.9 pM. In another embodiment, the concentration of a necrostatin-ls compound comprises a range of about 1.9 pM to about 2.0 pM. In another embodiment, the concentration of a necrostatin-ls compound comprises a range of about 2 mM to about 200 pM. In another embodiment, the concentration of a necrostatin-ls compound comprises a range of about200 pM to about 500 pM.

[85] In one embodiment, the final concentration of necrostatin-ls compound comprised in a composition disclosed herein, or an analog, derivative, isomer, or pharmaceutically acceptable salt of the necrostatin-ls, is about 0.2nM. In another embodiment, the final concentration of necrostatin-ls compound comprised in a composition disclosed herein, or an analog, derivative, isomer, or pharmaceutically acceptable salt thereof, is about 2.0 nM. In another embodiment, the final concentration of necrostatin-ls compound comprised in a composition disclosed herein, or an analog, derivative, isomer, or pharmaceutically acceptable salt thereof, is about 20 nM. In another

embodiment, the final concentration of necrostatin- 1 s compound comprised in a composition disclosed herein, or an analog, derivative, isomer, or pharmaceutically acceptable salt thereof, is about 200 nM. In another embodiment, the final concentration of necrostatin- ls compound comprised in a composition disclosed herein, or an analog, derivative, isomer, or pharmaceutically acceptable salt thereof, is about 2 mM.

[86] In one embodiment, the concentration of Bax channel inhibitor comprised in a composition disclosed herein comprises a range of about 0.5 nM to about 50 mM. In another embodiment, the concentration of Bax channel inhibitor comprises a range of about 0.5 nM to about 5nM. In another embodiment, the concentration of Bax channel inhibitor comprises a range of about 5 nM to about 50 nM. In another embodiment, the concentration of Bax channel inhibitor comprises a range of about 50 nM to about 500 nM. In another embodiment, the concentration of Bax channel inhibitor comprises a range of about 500 nM to about 5 pM. In another embodiment, the concentration of Bax channel inhibitor comprises a range of about 5 pM to about 50 pM.

[87] In one embodiment, the concentration of a 3,6-Dibromo-a-(l-piperazinylmethyl)-9H- carbazole-9-ethanol dihydrochloride compound comprised in a composition disclosed herein, or an analog, derivative, isomer, or pharmaceutically acceptable salt thereof comprises a range of about 0.5 nM to about 50 pM. In another embodiment, the concentration of 3,6-Dibromo-a-(l- piperazinylmethyl)-9H-carbazole-9-ethanol dihydrochloride compound comprised in a composition disclosed herein, or an analog, derivative, isomer, or pharmaceutically acceptable salt thereof comprises a range of about 0.5 nM to about 5nM. In another embodiment, the concentration of 3,6- Dibromo-a-(l-piperazinylmethyl)-9H-carbazole-9-ethanol dihydrochloride compound comprised in a composition disclosed herein, or an analog, derivative, isomer, or pharmaceutically acceptable salt thereof comprises a range of about 5 nM to about 50 nM. In another embodiment, the concentration of 3,6-Dibromo-a-(l-piperazinylmethyl)-9H-carbazole-9-ethanol dihydrochloride compound comprised in a composition disclosed herein, or an analog, derivative, isomer, or pharmaceutically acceptable salt thereof comprises a range of about 50 nM to about 500 nM. In another embodiment, the concentration of 3,6-Dibromo-a-(l-piperazinylmethyl)-9H-carbazole-9-ethanol dihydrochloride compound comprised in a composition disclosed herein, or an analog, derivative, isomer, or pharmaceutically acceptable salt thereof comprises a range of about 500 nM to about 5 pM. In another embodiment, the concentration of 3,6-Dibromo-a-(l-piperazinylmethyl)-9H-carbazole-9-ethanol dihydrochloride compound comprised in a composition disclosed herein, or an analog, derivative,

isomer, or pharmaceutically acceptable salt thereof comprises a range of about 5 mM to about 50 pM.

[88] In one embodiment, the final concentration of said Bax channel inhibitor comprised in a composition disclosed herein is about 0.5nM. In another embodiment, the final concentration of said Bax channel inhibitor comprised in a composition disclosed herein is about 5nM. In another embodiment, the final concentration of said Bax channel inhibitor comprised in a composition disclosed herein is about 50 nM. In another embodiment, the final concentration of said Bax channel inhibitor disclosed herein is about 500 nM. In another embodiment, the final concentration of said Bax channel inhibitor comprised in a composition disclosed herein is about 5 pM. In another embodiment, the final concentration of said Bax channel inhibitor comprised in a composition disclosed herein is about 50 pM.

[89] In one embodiment, the final concentration of 3,6-Dibromo-a-(l-piperazinylmethyl)-9H-carbazole-9-ethanol dihydrochloride compound comprised in a composition disclosed herein, or an analog, derivative, isomer, or pharmaceutically acceptable salt thereof is about 0.5nM. In another embodiment, the final concentration of 3,6-Dibromo-a-(l-piperazinylmethyl)-9H-carbazole-9-ethanol dihydrochloride compound comprised in a composition disclosed herein, or an analog, derivative, isomer, or pharmaceutically acceptable salt thereof is about 5nM. In another embodiment, the final concentration of 3,6-Dibromo-a-(l-piperazinylmethyl)-9H-carbazole-9-ethanol dihydrochloride compound comprised in a composition disclosed herein, or an analog, derivative, isomer, or pharmaceutically acceptable salt thereof is about 50 nM. In another embodiment, the final concentration of 3,6-Dibromo-a-(l-piperazinylmethyl)-9H-carbazole-9-ethanol dihydrochloride compound comprised in a composition disclosed herein, or an analog, derivative, isomer, or pharmaceutically acceptable salt thereof is about 500 nM. In another embodiment, the final concentration of 3,6-Dibromo-a-(l-piperazinylmethyl)-9H-carbazole-9-ethanol dihydrochloride compound comprised in a composition disclosed herein, or an analog, derivative, isomer, or pharmaceutically acceptable salt thereof is about 5 pM. In another embodiment, the final concentration of 3,6-Dibromo-a-(l-piperazinylmethyl)-9H-carbazole-9-ethanol dihydrochloride compound comprised in a composition disclosed herein, or an analog, derivative, isomer, or pharmaceutically acceptable salt thereof is about 50 pM.

[90] In some embodiments, the compositions disclosed herein further comprise an Adenosine triphosphate (ATP). While not wishing to be bound by theory, it is believed that in some embodiments the loss of cellular ATP and reactive oxygen species are produced (both as the result of mitochondrial maladaptive process) are harmful to cells and further push toward necrotic signaling. In addition, in some embodiments, depletion of ATP allows Ca2+ uptake by mitochondria, resulting in permeability transition pore (PTP) opening, which leads, in some embodiments, to cytochrome C release, mitochondrial swelling and death. Thus, in some embodiments, said ATP in the composition reverses or mitigates the loss of ATP due to cell membrane disruption caused by cell dissociation from tissue and cryopreservation, thereby relieving the necroptotic pressure. In additional embodiments, ATP in the composition preserves mitochondrial function through minimizing the slowdown in mitochondrial respiration and also aids in replenishment of lost cellular metabolites lost in the process of breaking up cells.

[91] In one embodiment, said ATP is present at a concentration of from about 10 nM to about 1 mM. In another embodiment, said ATP is present at a concentration of from about 10 nM to about 100 nM. In another embodiment, said ATP is present at a concentration of from about 100 nM to about ImM. In another embodiment, said ATP is present at a concentration of from about ImM to about 10 mM. In another embodiment, said ATP is present at a concentration of from about 10 pM to about 100 pM. In another embodiment, said ATP is present at a concentration of from about 100 pM to about 100 mM.

[92] In some embodiments, the final concentration of ATP comprised in a composition disclosed herein is about 10 nM. In another embodiment, the final concentration of ATP comprised in a composition disclosed herein is about 100 nM. In another embodiment, the final concentration of ATP comprised in a composition disclosed herein is about lpM. In another embodiment, the final concentration of ATP comprised in a composition disclosed herein is about 10 pM. In another embodiment, the final concentration of ATP comprised in a composition disclosed herein is about 100 pM. In another embodiment, the final concentration of ATP comprised in a composition disclosed herein is about 1 mM.

[93] In some embodiments, the compositions disclosed herein further comprise a nicotinamide adenine dinucleotide (NAD) compound. While not wishing to be bound by theory, it is believed that, in some embodiments, the loss cellular of NAD additionally contributes to cellular necrosis through various mechanisms, including, in some embodiments, inhibition of glycolysis and decreasing activity of the sirtuin family of proteins. An additional effect of NAD depletion is, in some embodiments, the reduction of NADP/NADPH production. In some embodiments, NADPH provides the reducing equivalents for biosynthetic reactions and in protecting against the necroptotic-inducing signals of ROS (reactive oxygen species). Therefore, in some embodiments low NAD levels are believed to be a trigger for activation of several mediators of necroptotic cell death. Thus, in some embodiments, said NAD in the composition reverses or mitigates the loss of NAD due to cell membrane disruption caused, in some embodiments, by cell dissociation from tissue or cryopreservation. In other embodiments said NAD in the composition suppresses necroptotic signaling.

[94] In some embodiments, replenishment of NAD prevents decrease of NAD levels in hippocampus and subsequent release of cathepsin B from lysosomes after ischemia / reperfusion injury. In some embodiments, high intracellular NAPDH causes CaMKII to phosphorylate and inactivate caspase-2, which has multiple effects including prevention of necrosis by keeping caspase-2 in its pro-caspase inactive enzyme form. In some embodiments, NAD also influences the differentiation state of cells, maintaining their active state to prevent de-differentiation and self renewal programs. Thus, in some embodiments, the addition of NAD to the freezing composition prevents excessive cell death and loss by ensuring the regeneration of chemical reducing pathways. In addition, sufficient cellular NADPH, which derives from NAD, causes caspases to remain at sufficiently low activity to avoid cell death (necrosis) while preventing regression along the differentiation pathways. Thus, in some embodiments NAD in the composition suppresses cell differentiation and supports maintenance of cellular identity in cells experiencing stress due to membrane disruption caused, in some embodiments, by cell dissociation from tissue or cryopreservation.

[95] In one embodiment, said NAD is present at a concentration of from about 5nM to about 500 mM. In another embodiment, said NAD is present at a concentration of from about 5nM to about 50 nM. In another embodiment, said NAD is present at a concentration of from about 5nM to about 50 nM. In another embodiment, said NAD is present at a concentration of from about 50 nM to about 500 nM. In another embodiment, said NAD is present at a concentration of from about 500 nM to about 5 mM. In another embodiment, said NAD is present at a concentration of from about 5 pM to about 50 pM. In another embodiment, said NAD is present at a concentration of from about 50 pM to about 500 pM.

[96] In one embodiment, the final concentration of NAD comprised in a composition disclosed herein is about 5nM. In another embodiment, the final concentration of comprised in a composition disclosed herein is about 50 nM. In another embodiment, the final concentration of NAD comprised in a composition disclosed herein is about 500 nM. In another embodiment, the final concentration of NAD comprised in a composition disclosed herein is about 5 mM. In another embodiment, the final concentration of NAD comprised in a composition disclosed herein is about 50 pM. In another embodiment, the final concentration of NAD comprised in a composition disclosed herein is about 500 pM.

[97] In some embodiments, the compositions disclosed herein further comprise Cyclosporin A. In one embodiment, the compositions disclosed herein further comprise an inhibitor of Cyclophilin D (CypD). In another embodiment, said inhibitor is Cyclosporin A. In some embodiments, Cyclosporin A is an immunosuppressant drug that interferes with the swelling of mitochondria secondary to calcium activation, and, in some embodiments, has been shown to protect against reactive oxygen species-induced necrotic death in many cell types, including liver. In some embodiments, Cyclophilin D (cypD) is a peptidyl-prolyl isomerase that resides in the mitochondrial matrix and controls the MPTP, and, in some embodiments, is central to the early determination of mitochondrial dysfunction leading to necrotic cell death.

[98] While not wishing to be bound by theory, it is believed that, in some embodiments, cyclophilin D modulates regulated necrosis, or necroptosis, through controlling the opening state of the MPTP, and its inhibition by cyclosporin A was shown to be protective in calcium- and oxidative stress induced death of hepatocytes. In some embodiments, CypD inhibition also results in protection from ischemic injury, and, in some embodiments, can rescue several aspects of neurodegenerative diseases including muscular dystrophy and also in liver toxicity models. Thus, necroptosis resulting from ischemia-reperfusion injury is, in some embodiments, sensitive to CypD activity, implicating it directly in this form of injury-mediated cell death.

[99] In one embodiment, said cyclosporin A is present at a concentration of from about 1 nM to about 1 mM. In another embodiment, said cyclosporin A is present at a concentration of from about 1 nM to about 10 nM. In another embodiment, said cyclosporin A is present at a concentration of from about 10 nM to about 100 nM. In another embodiment, said cyclosporin A is present at a concentration of from about 100 nM to about 1 mM. In another embodiment, said cyclosporin A is present at a concentration of from about 1 pM to about 10 pM. In another embodiment, said cyclosporin A is present at a concentration of from about 10 pM to about 100 pM. In another embodiment, said cyclosporin A is present at a concentration of from about 100 pM to about 1 mM.

[100] In one embodiment, the final concentration of cyclosporin A comprised in a composition

disclosed herein is about 1 nM. In another embodiment, the final concentration of cyclosporin A comprised in a composition disclosed herein is about 10 nM. In another embodiment, the final concentration of cyclosporin A comprised in a composition disclosed herein is about 100 nM. In another embodiment, the final concentration of cyclosporin A comprised in a composition disclosed herein is about 1 mM. In another embodiment, the final concentration of cyclosporin A comprised in a composition disclosed herein is about 10 mM. In another embodiment, the final concentration of cyclosporin A comprised in a composition disclosed herein is about 100 pM. In another embodiment, the final concentration of cyclosporin A comprised in a composition disclosed herein is about 1 mM.

CLAIMS

What is claimed is:

1. A composition comprising a necroptosis inhibitor compound and a Bax channel inhibitor compound.

2. The composition of claim 1, wherein said necroptosis inhibitor compound comprises a 5-((7-Chloro-lH-indol-3-yl)methyl)-3-methyl-2,4-imidazolidinedione compound or an analog, derivative, isomer, or pharmaceutically acceptable salt thereof, and said Bax channel inhibitor comprises a 3,6-Dibromo-a-(l-piperazinylmethyl)-9H- carbazole-9-ethanol dihydrochloride compound, or an analog, derivative, isomer, or pharmaceutically acceptable salt thereof.

3. The composition of claim 1, wherein said necroptosis inhibitor compound comprises a 5- ((7-Chloro- lH-indol-3-yl)methyl)-3-methyl-2,4-imidazolidinedione, a 3-p-Methoxyphenyl-5,6-tetramethylenothieno[2,3-d]pyrimidin-4-one-2-mercaptoethylcyanide compound, a (Z)-5-((3-(4-Fluorophenyl)-lH-pyrazol-4-yl)methylene)-2-imino-3-(thiazol-2-yl)thiazolidin-4-one compound, a 5-(Indol-3-ylmethyl)-(2-thio-3-methyl)hydantoin compound, l-([3S,3aS]-3-[3-fluoro-4-[trifluoromethoxy]phenyl]-8-methoxy-3,3a,4,5-tetrahydro-2H-benzo[g]indazol-2-yl)-2-hydroxyethanone compound, (S)-N-(l-[2-chloro-6-fluorophenyl]ethyl)-5-cyano-l-methyl-lH-pyrrole-2-carboxamide compound, methyl-thiohydantoin-tryptophan compound, (E)-N-(4-(N-(3-Methoxypyrazin-2-yl)sulfamoyl)phenyl)-3-(5-nitrothiophene-2-yl)acrylamide compound, l-(4-(4-Aminofuro[2,3-d]pyrimidin-5-yl)phenyl)-3-(2-fluoro-5-(trifluoromethyl) phenyl)urea compound, or 2,2-dimethyl- l-(5(S)-phenyl-4,5-dihydro-pyrazol-l-yl)-propan-l-one compound, or an analog, derivative, isomer, or pharmaceutically acceptable salt of said necroptosis inhibitor compound.

4. The composition of any one of claims 1-3, wherein the concentration of the 5-((7-Chloro-lH-indol-3-yl)methyl)-3-methyl-2,4-imidazolidinedione compound or said analog, derivative, isomer, or pharmaceutically acceptable salt thereof is about 0.2nM - 2mM.

5. The composition of any one of claims 1-4, wherein the concentration of said 3,6-Dibromo-a-(l-piperazinylmethyl)-9H-carbazole-9-ethanol dihydrochloride compound, or analog, derivative, isomer, or pharmaceutically acceptable salt thereof is about 0.5hM-50mM.

6. The composition of any one of claims 1-5, further comprising a nicotinamide adenine

dinucleotide (NAD), an adenosine triphosphate (ATP), a cyclosporin A, or a superoxide dismutase, or any combination thereof.

7. The composition of claim 6, wherein said superoxide dismutase comprises a manganese superoxide dismutase or a zinc superoxide dismutase.

8. The composition of any one of claims 6-7, wherein the concentration of said NAD is about 5nM - 500mM.

9. The composition of any one of claims 6-8, wherein the concentration of said ATP is about lOnM - lmM.

10. The composition of any one of claims 6-9, wherein the concentration of said cyclosporin A is about lnM - lmM.

11. The composition of any one of claims 6-10, wherein the concentration of said superoxide dismutase is about 0.001- 100 Kunitz Units (KU).

12. The composition of any one of claims 6-11, comprising about 20 nM 5-((7-Chloro-lH-indol-3-yl)methyl)-3-methyl-2,4-imidazolidinedione), about 5nM 3,6-Dibromo-a-(l-piperazinylmethyl)-9H- carbazole-9-ethanol dihydrochloride, about 0.05 mM nicotinamide adenine dinucleotide (NAD), about 0.01 mM adenosine triphosphate (ATP), about O.OImM cyclosporin A, and about 0.1 Kunitz Unit manganese superoxide dismutase or zinc superoxide dismutase.

13. The composition of any one of claims 1-12, further comprising a cryoprotective agent.

14. The composition of claim 13, wherein said cryoprotective agent comprises DMSO, or serum, or any combination thereof.

15. The composition of any one of claims 1-14, further comprising pharmaceutically acceptable excipients or carriers.

16. A method of cryopreservation, the method comprising the steps of:

(a) bringing a plurality of cells in contact with a composition comprising a necroptosis inhibitor compound and a Bax channel inhibitor compound; and

(b) cooling the composition comprising the plurality of cells of step (a),

wherein the necroptosis inhibitor compound comprises a 5-((7-chloro-lH-indol-3-yl)methyl)-3-methyl-2,4-imidazolidinedione compound or an analog, derivative, isomer, or pharmaceutically acceptable salt thereof, and wherein the Bax channel inhibitor comprises 3,6-dibromo-a-(l-pipcra/inyl met hyl)-9/7-carba/olc-9-ct hand dihydrochloride or an analog, derivative, isomer, or pharmaceutically acceptable salt thereof.

17. A method of treating, preventing, inhibiting, or reducing the incidence of cellular plasticity, or necroptosis or necrosis in a plurality of cells, said method comprising the step of: bringing said plurality of cells in contact with a composition comprising a necroptosis inhibitor compound and a Bax channel inhibitor compound, wherein the necroptosis inhibitor compound comprises a 5-((7-chloro-lH-indol-3-yl)methyl)-3-methyl-2,4-imidazolidinedione compound or an analog, derivative, isomer, or pharmaceutically acceptable salt thereof, and wherein the Bax channel inhibitor comprises 3,6-dibromo-a-(l-piperazinylmethyl)-97/-carbazole-9-ethanol dihydrochloride or an analog, derivative, isomer, or pharmaceutically acceptable salt thereof.

18. The method of claim 17, wherein said necroptosis or necrosis is associated with aging or disease.

19. The method of claim 18, wherein said disease is myocardial infarction, diabetes secondary to beta-cell necroptosis, cholestatic liver disease, stroke, organ ischemia, ischemia-reperfusion injury, liver disease, necrosis from cancer chemotherapy or radiation therapy, traumatic brain injury, necrotizing pancreatitis, pathogen-induced necroptosis, inflammation, or neurodegenerative disease.

20. The method of any one of claims 17-19, wherein said treating, preventing, inhibiting, or reducing the incidence of cellular plasticity, or necroptosis or necrosis, comprises in vitro or in vivo or ex vivo treating, preventing, inhibiting, or reducing the incidence of cellular plasticity, necroptosis, or necrosis.

21. The method of any one of claims 16-20, wherein the plurality of cells comprises tissue culture cells, primary cells, egg cells, a tissue, or an organ or a portion thereof, or any combination thereof.

22. The method claim 2l, wherein said tissue culture cells or primary cells comprise stem cells, adult cells, transdifferentiated cells, dedifferentiated cells, or differentiated cells, or any combination thereof.

23. The method of any one of claims 16-22, wherein the plurality of cells comprises human cells or animal cells.

24. The method of any one of claims 16-23, wherein bringing in contact is in vivo , ex vivo , or in vitro.

25. The method of claim 24, wherein bringing in contact in vivo or ex vivo comprises perfusion of an animal or a portion thereof, an organ or a portion thereof, or a tissue.

26. The method of claim 25, wherein said perfusion is cardiac perfusion.

27. The method of claim 24, wherein bringing in contact in vitro comprises immersing said plurality of cells in said composition, supplementing a growth media of said plurality of cells with said composition, perfusing said plurality of cells with said composition via non-thermal reversible electroporation, or perfusing said plurality of cells with said composition in a bioreactor.

28. The method of any one of claims 16-27, further comprising the step of physical, chemical, or thermodynamic manipulation of said plurality of cells.

29. The method of claim 28, wherein the thermodynamic manipulation comprises heating or cooling said plurality of cells.

30. The method of claims 29, wherein said cooling comprises cryopreservation or a freeze-thaw cycle, or a combination thereof.

31. The method of any one of claims 16 and 21-30, wherein said method prevents, inhibits, or reduces necrosis or necroptic death of said plurality of cells during said cryopreservation or a freeze-thaw cycle thereof, compared with an uncontacted plurality of cells.

32. The method of any one of claims 16 and 21-31, wherein said method protects said plurality of cells from physical damage during said cryopreservation or freeze-thaw cycles thereof.

33. The method of any one of claims 16 and 21-32, wherein said method enhances growth

potential of said plurality of cells during cryopreservation compared with uncontacted cells.

34. The method of any one of claims 16 and 21-33, wherein said method prevents oxidative damage to said plurality of cells during cryopreservation or freeze-thaw cycles thereof.

35. The method of any one of claims 16 and 21-34, wherein said method prevents ischemia of said cells during cryopreservation or freeze-thaw cycles thereof.

36. The method of any one of claims 16-35, wherein said method inhibits necrosis pathway signaling.

37. The method of any one of claims 16-36, wherein said method prevents, inhibits, or reduces changes in the differentiation state of said cells, thereby stabilizing the identity of said cells compared with uncontacted cells.

38. The method of any one of claims 16-37, wherein said method induces a state of metabolic suspension in said cells.

39. The method of claim 38, wherein said metabolic suspension comprises reversible cessation of oxygen metabolism.

40. The method of any one of claims 16-39, wherein said method improves viability or latent viability of said cells compared with an uncontacted plurality of cells.

41. The method of any one of claims 16-40, wherein the necroptosis inhibitor comprises 5-((7-chloro-lH-indol-3-yl)methyl)-3-methyl-2,4-imidazolidinedione compound, a 3-p-Methoxyphenyl-5,6-tetramethylenothieno[2,3-d]pyrimidin-4-one-2-mercaptoethylcyanide compound, a (Z)-5-((3-(4-Fluorophenyl)- lH-pyrazol-4-yl)methylene)-2-imino-3-(thiazol-2-yl)thiazolidin-4-one, a 5-(Indol-3-ylmethyl)-(2-thio-3-methyl)hydantoin, a methyl-thiohydantoin-tryptophan compound, a l-([3S,3aS]-3-[3-fluoro-4-[trifluoromethoxy]phenyl]-8-methoxy-3,3a,4,5-tetrahydro-2H-benzo[g]indazol-2-yl)-2- hydroxyethanone, a (S)-N-(l-[2-chloro-6-fluorophenyl]ethyl)-5-cyano-l-methyl-lH-pyrrole-2-carboxamide, a (E)-N-(4-(N-(3-Methoxypyrazin-2-yl)sulfamoyl)phenyl)-3-(5-nitrothiophene-2-yl)acrylamide, a l-(4-(4-Aminofuro[2,3-d]pyrimidin-5-yl)phenyl)-3-(2-fluoro-5-(trifluoromethyl)phenyl)urea, or 2,2-dimethyl- l-(5(S)-phenyl-4,5-dihydro-pyrazol-l-yl)-propan-l-one, or a derivative, isomer, or pharmaceutically acceptable salt of any one of said necroptosis inhibitors.

42. The method of any one of claims 16-41, wherein the concentration of the 5-((7-Chloro-lH-indol-3-yl)methyl)-3-methyl-2,4-imidazolidinedione compound or said analog, derivative, isomer, or pharmaceutically acceptable salt thereof is about 0.2nM - 2mM, and wherein the concentration of the 3,6-Dibromo-a-(l-piperazinylmethyl)-9H- carbazole-9-ethanol dihydrochloride compound, or analog, derivative, isomer, or pharmaceutically acceptable salt thereof is about 0.5nM - 50mM.

43. The method of any one of the claims 16-42, wherein the composition further comprises, a nicotinamide adenine dinucleotide (NAD), an adenosine triphosphate (ATP), a cyclosporine A, a manganese-superoxide dismutase, or a zinc- superoxide dismutase, or any combination thereof.

44. The method of claim 43, wherein the concentration of said NAD is about 5nM - 500mM, the concentration of said ATP is about lOnM - lmM, the concentration of said cyclosporine A is about lnM - lmM, and the concentration of said manganese-superoxide dismutase or said zinc-superoxide is about 0.001KU-100.0KU.

45. The method of claim 44, wherein the concentration of necroptosis inhibitor compound 5-((7-chloro-lH-indol-3-yl)methyl)-3-methyl-2,4-imidazolidinedione compound, or the analog, a derivative, a isomer, or a pharmaceutically acceptable salt thereof is about 20nM, the concentration of Bax channel inhibitor 3,6-Dibromo-a-(l-piperazinylmethyl)-9H- carbazole-9-ethanol dihydrochloride compound, or the analog, the derivative, the isomer, or the pharmaceutically acceptable salt thereof is about 5nM, the concentration of said NAD is about 0.05 nM, the concentration of said ATP is about O.OImM, the concentration of said cyclosporine A is about O.OImM, and the concentration of said manganese-superoxide dismutase or the zinc-superoxide is about 0.1KU.

46. The method of any one of claims 16-45, wherein the composition further comprises a cryoprotective agent.

47. The method of claim 46, wherein said cryoprotective agent comprises DMSO, or serum, or any combination thereof.

48. The method of any one of claims 16-47, wherein the composition further comprises a pharmaceutically acceptable carriers or excipients.

49. A composition for use in preventing excessive necrosis and necroptotic cell death, the composition comprising at least one inhibitor of necrosis- or necroptosis-inducing damage-associated molecular patterns (DAMPs).

50. The composition of claim 49, wherein said inhibitors comprise antibodies, proteins, peptides, small molecules or any combination thereof.

51. The composition of any one of claims 49-50, wherein said DAMPs comprise, lipoxygenases inhibitors, sphingomyelinases inhibitors, phospholipases inhibitors, ceramidases inhibitors pattern-recognition receptors (PRRs) ligands, Toll-like receptors (TLRs) ligands, retinoic acid-inducible gene I-like receptors (RIG-I-like) receptors (RLRs) ligands, nucleotide binding domain and leucine-rich repeat containing molecules (NLRs) ligands, C-type lectin receptors (CLRs) ligands, or death receptors ligands.

52. The composition of claim 51, wherein said ligands comprise high mobility group box 1 protein (HMGB 1), heat- shock proteins (HSPs), ribonucleoproteins (RNPs), messenger ribonucleic acids (mRNAs), high mobility group nucleosome binding domain 1 (HMGN1), hyaluronan, biglycan, heparin sulfate, Ul small nucleolar ribonucleoprotein (snRNP), mitochondrial DNA, or double- stranded RNA (dsRNA).

53. The composition of claim 51, wherein said death receptors comprise tumor necrosis factor receptor 1 (TNFR1), tumor necrosis factor receptor 2 (TNFR2), CD95/FAS receptor, TNF-related apoptosis-inducing ligand receptor 1 (TRAILR1), and TRAILR2.

54. The composition of claim 51, wherein said death receptor ligands comprise, deubiquitylating enzymes, FAS-associated protein with a death domain (FADD), riboflavin kinase (RFK), tumor necrosis factor, TNFR2, FAS ligand (FASL or CD95L), and TNFR-associated death domain (TRADD).

55. The composition of claim 54 wherein said deubiquitylating enzymes comprise, A20 (TNFAIP3), OTUD7b, cylindromatosis (cylD) and USP21.

56. The composition according to any one of the claims 49-55, further comprising pharmaceutically acceptable excipients or carriers comprising pH stabilizers, sugars, amino acids, or antibiotics, or any combination thereof.