Prevention of toxicity from high concentrations of cryoprotective agents

Abstract

Cryoprotectant toxicity is a long-standing and limiting factor for the cryopreservation of many living systems. We now disclose methods by which this toxicity can be reduced or even eliminated by pre-exposing living cells, tissues, organs, and simple organisms to hydrogen peroxide or the equivalent and / or to non-toxic concentrations of cryoprotectants.

Description

[0001] PREVENTION OF TOXICITY FROM HIGH CONCENTRATIONS OF CRYOPROTECTIVE AGENTS

[0002] FIELD OF THE INVENTION

[0003] This invention relates to methods for preventing cryoprotectant toxicity by inducing biochemical defenses against cryoprotectant toxicity.

[0004] BACKGROUND OF THE INVENTION

[0005] The toxicity of cryoprotective agents (CPAs, or cryoprotectants) has been suggested to be the most significant obstacle to the fully successful cryopreservation of most living systems ([5, 6]), and a general solution to this problem would accordingly have broad potential impact. Cryoprotectant toxicity (CT) is a particularly daunting barrier to solving one of the most difficult problems of cryopreservation, the banking of large solid organs, whose revolutionary potential advantages for human organ transplantation have been widely acknowledged (

[0076] ). The importance of CT in this context has been highlighted recently by a report describing the survival of 5 of 5 vitrified rat kidneys after transplantation ([7S]). Although these kidneys supported life, they were severely damaged and only survived because CT was minimized by using cryoprotectant concentrations that may be too low for use with larger organs ([72, 75]). Nevertheless, most of the injury observed after vitrification and transplantation in these experiments likely arose from exposure to the cryoprotectant ([7S]).

[0006] The M22 vitrification solution (

[0075] ) was designed to allow even large organs to escape freezing damage during cooling and warming ([77, 72]). It enabled the survival of a larger vitrified organ, the rabbit kidney, after transplantation (

[0075] ), but toxicity limitations prevented the elimination of devitrification injury during rewarming. A more recent experiment reproduced this survival without apparent ice damage, but creatinine was still elevated for 18 days after transplantation (Fahy et al., unpublished observations). In this context, the ability to limit M22 toxicity could have considerable practical significance in the near term.

[0007] Although useful work has been done on modelling and thereby mitigating factors that affect CT ([7, 25]), there have been very few studies of biochemical interventions that might block or oppose this toxicity. Earlier relevant general studies were reviewed in 1987 ([9]), 1990 (

[0079] ), 1995 ([5]), and 2004 (

[0074] ). More recently, in 2013, Guan et al. reported a useful transcriptomic study on rat liver slices exposed to related solutions (

[0077] ). This study implicated pathways that may be good targets for intervention, and that were consistent with the possibility (

[0072] ) that protein denaturation might be a significant factor; however, they did not demonstrate new means of mitigating CT. Another microarray study investigated mechanisms of ethylene glycol toxicity in endothelial cells ([2]). The results indicated that the cellular responses to ethylene glycol differ from those elicited by M22 exposure, but failed to identify specific mechanisms and again did not provide any potential or actual interventions. Cypser et al. presented an innovative forwardgenetics approach that identified several specific genes that exacerbate M22 and dimethyl sulfoxide toxicity in mouse embryonic stem cells ([3]). This led to the identification of a drug that inhibits the function of one of these genes and reduces M22 toxicity with high statistical significance, at a drug concentration of 100 nM ((

[0072] ); Tom Johnson, personal communication to GMF). However, protection was weak, as might be expected when inhibiting just one protein of a multi-protein response to CPAs, and protection did not immediately translate to rabbit renal cortical slices (Fahy et al., unpublished results), whose epigenetic response to M22 likely differs from the response of mouse ESCs.

[0008] By contrast, the present invention discloses novel interventions that are strongly protective across species as diverse as nematodes and mammals. Although these interventions are general, they are illustrated herein by examples in which toxicity is prevented after exposure to a particularly interesting vitrification solution, M22. Exposing living systems to cryoprotective agents can be damaging due to osmotic effects or due to biochemical effects that are not dependent on osmotic damage. Osmotic injury can generally be made negligible by limiting the speed with which CPAs are introduced and / or removed, and as such they are not as challenging to overcome as biochemical disturbances. The risk of detrimental biochemical effects of CPAs generally increases as the risk of osmotic damage decreases in part because reducing the speed of addition and removal of CPAs to limit osmotic damage prolongs exposure of the molecular machinery of the cell to perturbation by the unnatural presence of intracellular and even extracellular CPAs. Further, osmotic damage can be reduced by increasing the temperature of exposure to cryoprotectants because osmotic damage is caused by the fact that permeating CPAs (pCPAs) do not cross the cell membrane as rapidly as water, but cells are more permeable to CPAs at higher temperatures, so the difference between their permeability and the permeability of water becomes less important at higher temperatures. By the same token, however, biochemical processes are generally accelerated by temperature elevation, which makes imbalances caused by perturbations of enzymatic reaction rates by CPAs more pronounced. Thus, it is the purpose of the present invention to preferentially minimize injury to living cells caused by biochemical effects (“toxicity”) rather than to minimize injury caused by osmotic effects (“osmotic stress” or “osmotic injury”). However, in some embodiments, the disclosed methods of reducing toxicity may also reduce osmotic injury.

[0009] BRIEF DESCRIPTION OF THE INVENTION

[0010] The invention comprises contacting a biological system (specifically, a cell, collection of cells, tissue, vascularized tissue, organ, or simple organism) with a solution of hydrogen peroxide or its equivalent and / or a first cryoprotectant solution in a manner that does not irreversibly damage said biological system and that induces protection of said biological system from toxicity otherwise normally induced by subsequent exposure of the biological system to a second cryoprotectant solution.

[0011] The invention further comprises contacting a biological system (specifically, a cell, collection of cells, tissue, vascularized tissue, organ, or simple organism) with a first concentration of one or more cryoprotective agents in a manner that does not irreversibly damage said biological system and that induces protection of said biological system from toxicity otherwise normally induced by subsequent exposure of the biological system to a second concentration of one or more cryoprotective agents, wherein said first concentration is lower than said second concentration.

[0012] The invention further comprises treating a biological system (specifically, a cell, collection of cells, tissue, vascularized tissue, organ, or simple organism) with both hydrogen peroxide and a first non-toxic concentration of one or more cryoprotective agents that induces protection of said biological system from toxicity otherwise induced by subsequent exposure of the biological system to a second concentration of one or more cryoprotective agents, wherein said second concentration is higher than said first concentration.

[0013] BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 shows reduction of M22 toxicity in whole adult C. elegans by different pretreatments and a genetic modification. Survival after pretreatment followed by 5-min exposure to 75% M22 at 23°C was assessed 2 hr later and normalized to untreated controls. Numbers above bars indicate p values from 2-tailed heteroscedastic t tests comparing the indicated pretreatments to controls exposed to M22 after mock pre-treatment.

[0015] Figure 2 shows nematode survival over time following exposure to and removal from 50% or 75% M22 at 23°C for 5 min with or without pretreatment with 1-mM hydrogen peroxide. Red circles and lines denote treatment with 50% M22; green triangles and lines indicate exposure to 75% M22. Open symbols signify M22 immersion without H2O2 pretreatment; filled symbols designate M22 exposure after 1-mM H2O2 pretreatment.

[0016] Figure 3 shows the applicability of protection from hydrogen peroxide exposure to mouse kidney tissue. Significances of inter-group differences, indicated above bars, are based on 2- tailed heteroscedastic t tests.

[0017] Figure 4 shows protection against exposure to high concentrations of cryoprotectant by pre-exposure of whole rabbit kidneys to hydrogen peroxide, as demonstrated by permanent transplantation of said kidneys. In A, n values for each group are given in parentheses.

[0018] DETAILED DESCRIPTION OF THE INVENTION

[0019] The invention comprises contacting (treating) a biological system (specifically, for example, a cell, collection of cells, tissue, organoid, vascularized tissue, organ, or simple organism) with effective concentrations of hydrogen peroxide (H2O2) or its equivalent that does not irreversibly damage said biological system and that induces protection of said biological system from toxicity otherwise induced by subsequent exposure of the biological system to one or more cryoprotective agents. Effective concentrations of H2O2 include concentrations from 0.1 to 20 micromolar (pM) in the case of mammalian cells, tissues, vascularized tissues, or organs and 0.1 to 20 mM in the case of relatively impermeable living systems such as adult or immature invertebrates such as nematode worms or aquatic species such as amphibian or fish eggs, zygotes, or embryos (“simple organisms”). Treatment methods that are effective in the invention include immersion for simple organisms or exposed cells or tissues or incorporation into perfusates, superfusion solutions, flushing solutions, and / or bathing solutions in the case of whole organs, vascularized tissues, or organoids for which perfusion or superfusion is required. Treatment methods also include exposure of mammalian cells, tissues, organoids, vascularized tissues, or organs to one or more hydrogen peroxide solutions at temperatures typically from -6 to + 37°C for 1 to 20 min, and most preferably exposure at 0-26°C for 5-10 min, unless it is desired to simultaneously induce heat shock proteins. Simple organisms can be exposed to H2O2 concentrations over the same temperature ranges but are most preferably treated, within this range, with an H2O2 solution at temperatures consistent with those to which the organism is most typically exposed in nature. Simple organisms can be effectively treated with H2O2 solutions for 2-60 min. Non-mammalian cells that are relatively permeable to water and solutes, such as bacteria and yeast, can be exposed at temperatures up to 43°C and should be treated with H2O2 at 0.1-20 pM. It is not the purpose of the present invention to induce heat shock by exposure of living cells to higher temperatures than they normally experience, but the method of the present invention is not excluded for use at said higher temperatures, or when heat shock is also present. Generally, exposure at higher temperatures should be coupled to exposure to lower concentrations or shorter exposure times than are effective at lower temperatures.

[0020] Since hydrogen peroxide is effective in the invention due to its decomposition into hydroxyl free radicals, the invention can also be practiced with other hydroxyl free radical generating agents or systems that produce the same hydroxyl radical concentration ranges as do the stated concentrations of hydrogen peroxide under the stated conditions of hydrogen peroxide use herein.

[0021] The invention further comprises treating a biological system (specifically, for example, a cell, collection of cells, tissue, organoid, vascularized tissue, organ, or simple organism) with a concentration of one or more cryoprotective agents that does not irreversibly damage said biological system and that induces protection of said biological system from toxicity otherwise induced by subsequent exposure of said biological system to a higher concentration of said one or more cryoprotective agents. In one embodiment of this method, a concentration of 1-10% w / v CPA is added to the biological system of interest at -2 to 37°C for 2-180 min and then removed before said biological system is further treated with higher concentrations of CPA prior to cry opreservation by freezing or by vitrification.

[0022] In another embodiment, a concentration of 1-10% w / v, or 0.1M to 1.5M CPA, is added to the biological system of interest at -2 to 37°C by flushing and is then left in said system during a period in which said system is not perfused, during which period said system is prepared for perfusion or superfusion with higher CPA concentrations needed for protection against freezing or for vitrification. This embodiment is explicitly distinguished from a standard step procedure for introducing CPA in a series of concentration steps intended to reduce osmotic stress. For example, it may not be acceptable to expose a living system to 15% w / v CPA in one step due to osmotic damage, but the osmotic damage induced by one-step addition of 15% w / v CPA may hypothetically be prevented by first exposing said living system to 7.5% w / v CPA, whose osmotic effects are acceptable, and then, after a period of equilibration with that concentration, exposing the system to 15% w / v CPA, because equilibration with the first concentration prevents lethal osmotic cell volume reduction during the later step. This is qualitatively different than introducing 3% w / v CPA prior to a gradual gradient loading method culminating in, for example, exposure to a 65% w / v CPA whose damaging effects arise from toxicity rather than from osmotic damage. Any version of CPA pretreatment in which a pretreatment CPA concentration is not removed prior to introducing a higher concentration and in which the protection derived is mostly osmotic in nature is excluded from the metes and bounds of the present invention.

[0023] The invention further comprises treating a biological system (specifically, a cell, collection of cells, tissue, vascularized tissue, organ, or simple organism) with both hydrogen peroxide or its equivalent and a concentration of one or more cryoprotective agents without causing irreversible damage to said biological system and wherein said biological system is protected from toxicity otherwise induced by subsequent exposure of the biological system to one or more cryoprotective agents, wherein the conditions of exposure to the hydrogen peroxide or its equivalent and to the said one or more cryoprotective agents are as described above and wherein said exposure to the hydrogen peroxide or its equivalent and to the said one or more cryoprotective agents may be carried out simultaneously or sequentially.

[0024] The invention further comprises exposing a biological system (specifically, a cell, collection of cells, tissue, vascularized tissue, organ, or simple organism) to a first cryoprotectant solution that does not irreversibly damage said biological system but that induces protection of said biological system from toxicity otherwise induced by subsequent exposure of the biological system to a second cryoprotectant solution, whereas said second cryoprotectant solution does not contain exactly the same individual cryoprotectant or cryoprotectants as said first cryoprotectant solution.

[0025] The invention is further described by means of the Examples below.

[0026] DEFINITIONS

[0027] “Cryoprotective agents”, also known as “CPAs” or “cryoprotectants”, are molecules that protect living cells from damage otherwise caused by freezing ([79, 21, 24}). These include molecules that enable living cells to vitrify upon cooling (

[0072] ) rather than being exposed to extra- or intracellular ice. The said protected cells may be individual cells, such as oocytes or zygotes, or they may be cells that are frozen or vitrified in the presence of other cells, for example, in a cell suspension, monolayer, 3-dimensional culture, organoid, natural tissue, engineered or otherwise synthetic tissue (such as tissue produced from induced pluripotent stem cells or genetically or epigenetically modified cells), organ, or organism. The term “cryoprotectant” or “CPA” can refer to one or to more than one CPA present in a solution, such as in the phrase “cryoprotectant addition and washout” when said addition and washout pertains to a mixture of cryoprotective agents, or such as in the phrase “a 10% w / v CPA concentration” when more than one CPA are present and their % w / v concentrations sum to 10% w / v. CPAs may also be cell-permeating (pCPAs) or nonpermeating (npCPAs). (Most pCPAs are under 100 daltons and most npCPAs are over 100 daltons). A nominally permeating CPA may be effectively non-permeating if it is applied or subtracted for an insufficient time for permeation to become significant.

[0028] Phrases such as “75% M22” or “3% v / v M22” refer to a solution composition prepared by diluting the components of the previously described (

[0075] ) M22 solution to the stated percentages of their original concentrations per unit solution volume.

[0029] “Cryoprotectant toxicity” is understood here to be a detrimental change in a living system induced by exposure of that system to one or more cryoprotective agents and that cannot be eliminated by adding and / or removing the CPA more slowly or in smaller steps or by adding and / or removing the CPA at higher temperatures. A detrimental change in a living system that can be eliminated by adding and / or removing the CPA more slowly or in smaller steps or by adding and / or removing the CPA at higher temperatures (or both) is considered to be “osmotic injury.” The present invention is limited to preventing or reducing “cryoprotectant toxicity” rather than preventing or reducing “osmotic injury” unless the disclosed pretreatments also happen to increase membrane permeability to cryoprotectant. A “non-toxic cryoprotectant solution” is one that, after being put in contact with a living system at a particular concentration and then being removed from said system, does not cause detrimental change in said living system.

[0030] A “vitrifiable solution” is here understood to mean a cryoprotectant solution that will vitrify when cooled at the cooling rate employed to cryopreserve the biological system of interest. The present invention, while broadly useful for ameliorating cryoprotectant toxicity, is particularly useful for ameliorating the toxicity of vitrifiable solutions. “Cryopreservation” is understood here to mean the preservation of the viability of a living system when it is either frozen or vitrified and then returned to normal conditions for active life.

[0031] An “H2O2 equivalent” is a hydroxyl free radical generating agent or system that produces the same hydroxyl radical concentration ranges as do the hydrogen peroxide concentrations provided herein under the conditions of hydrogen peroxide use provided herein. But because the cellular response to free radical damage is similar regardless of the specific free radical that induces cellular damage, it is believed that all chemicals that cause free radical or oxidative damage to a cell will induce defensive pathways that will protect against subsequent CPA exposure and are, therefore, also “H2O2 equivalents” provided they do not induce irreversible cellular damage.

[0032] “Irreversible damage” comprises phenotypically important changes that do not spontaneously and adequately resolve within a desired period of time. This term does not include damage that may not be fully repaired but that is not relevant to the continued health of the biological system being treated, including silent or benign mutations. A “non-toxic cryoprotectant solution” does not result in irreversible damage after addition and subtraction from a living system.

[0033] A “simple organism” is one that can be effectively treated for the purposes of the invention with a solution of hydrogen peroxide or its equivalent and / or with a cryoprotectant solution by immersion in or superfusion with said solution or solutions. A “simple organism” does not include, for instance, whole mammals or human beings, but it includes particularly relatively impermeable living systems such as adult or immature invertebrates such as nematode worms or aquatic species such as amphibian or fish eggs, zygotes, or embryos.

[0034] A “collection of cells” refers to a small multicellular system such as an embryo, an organoid, or one or more pancreatic islets. A ’’tissue” as used herein refers to a biological tissue sample such as a cornea or skin sample or an organ slice or a biopsy or diced tissue sample that cannot be perfused. A “vascularized tissue” as used herein refers to a biological tissue that can be perfused, such as a section of omentum, bladder wall, coronary artery or other blood vessel or vascularized structure other than a complete organ. Should a method be devised to perfuse a tissue that cannot be perfused by ordinary means, then that tissue becomes a vascularized tissue for purposes of the present invention.

[0035] LM5 and M22 are solutions previously described in the art (

[0075] ).

[0036] A “flushing solution” or “flush solution” is a solution normally used to quickly displace a previously present solution or blood from the vascular bed of a vascularized tissue or organ or the circuit of an in vitro superfusion system and / or to cool said vascularized tissue or organ or system before subsequent perfusion or subsequent storage without perfusion. “Flushing” comprises “perfusion” for typically 1-10 min or, more typically, 1-5 min. “Perfusion” consists of propelling a solution through a vascular system or a perfusion or superfusion system and typically refers to a more prolonged process intended to maintain the perfused system in a stable state or to equilibrate the system in whole or in part with the components of the perfusate, or both.

[0037] A “perfusate” is a solution that is continuously propelled through the vascular bed of a vascularized system or the circuit of an in vitro perfusion or superfusion system. “Superfusion” is the process of propelling a solution through a perfusion system that is not a vascular bed, wherein the perfusate flows over or adjacent to the living system of interest rather than through a vasculature. A perfusate may be propelled through a vascular system or a superfusion circuit by means of a pump or a pressure differential otherwise provided between the arterial and venous sides of said system or circuit.

[0038] EXAMPLES

[0039] Example 1: Protection of Nematode Worms by Free Radical Exposure (Hydrogen Peroxide Pretreatment), CPA Pretreatment, and Both Hydrogen Peroxide and CPA Pretreatment For all nematode experiments described in all examples, wild-type C. elegans, strain

[0040] Bristol-N2 (DRM stock), were maintained in Nematode Growth Medium (NGM) at 20°C and tested at day 4.5 post-hatch (“adult day 2”) for M22 survival. Each group comprised 100 - 300 worms. Worms were suspended in 50% or 75% (v / v) M22 for 5 min at 20°C and then washed three times with S buffer (129 ml of 0.05-MK2HP04, 871 ml 0.05-MKH2P04, and 5.85 gNaCl / L) to remove M22. Nematodes were then placed on agar-NGM plates with a central lawn of E. coli strain OP50 and allowed to recover for 1 - 24 hr at 20°C (typically 2 hr unless otherwise indicated) before monitoring survival by manual assessment of nematode movement in response to gentle prodding with a platinum probe.

[0041] Two worm pre-treatments consisted of pre-exposure to 1 or 10 mM H2O2for 20 min in NGM at 20°C and pre-exposure to 3% of full-strength M22 (i.e., to a total CPA concentration of 1.94% w / v or 284 mM) for 20 min or for 2.5 hr in NGM at 20°C. After pretreatment, worms were allowed to recover on agar-NGM plates with a central lawn of E. coli strain OP50 for 30-60 min before immersion of the worms in M22.

[0042] Figure 1 shows reduction of C. elegans mortality otherwise caused by 5-min incubation in 75% of full-strength M22 at 20°C. Twenty minute pre-exposure to 1 and to 10 mM H2O2at 20°C (data are shown only for 1-mM peroxide) and pretreatment with 3% of full M22 at 20°C for 2.5 hours were both spectacularly protective, with high statistical significance reproduced in multiple experiments. Nematode survival 2 hr after immersion in and washout of 75% v / v M22 increased from 7.5% without pretreatment to 88.5% after pretreatment with 1-mM H2O2and to 86% after brief pretreatment in 3% M22. Powerful protection was also observed for nematodes with the age- l(mg44) mutation, but it is a significant advantage of the present invention that similar protection can be obtained without inducing mutations. Combining peroxide (hydroxyl free radical) and M22 hormesis was also effective, although protection from each pre-treatment individually was so strong that statistical proof of synergy was not demonstrable (data not shown). Other tested treatments, including heat shock, produced modest and at best suggestive improvements. Bars show means ± SEM. In each group, 2-10 independent assays were run (indicated by numbers superimposed on bars).

[0043] Although these results were obtained using the specific cryoprotectant mixture and free radical generator described, any other cryoprotectant solution and free radical generator that induces similar biological defenses are expected to produce similar protection from later cryoprotectant exposure. It is hydroxyl free radical (OH» ) that is the actual mediator of protection from H2O2, and any other hydroxy radical generator should produce similar protection. Further, exposure to any free radical in concentrations that do not produce frank damage to the system being preserved (not just simple organisms) should produce similar benefits. Additionally, it is expected that time limits of 60 and 180 min for H2O2 and cryoprotectant pretreatment, respectively, will be effective for simple organisms in the invention.

[0044] Figure 2 describes the time course of survival after M22 exposure and washout following pre-treatment with 1-mM H2O2 at 23°C. C. elegans survival was 17% after 75% M22 and >90% after 50% M22 without pretreatment, both of which improved, to >90% and to >95%, respectively, after peroxide pretreatment. Worm survival declined only slightly with time, from 1 to 24 hr after the end of M22 treatment. Thus, acute survival measures (usually at 2 hours after M22 removal) are reliable indicators of long-term survival. Robust protection at 1 mM predicts effectiveness at a concentration as low as 0.1 mM.

[0045] To test the possibility that the improved survival of worms in M22 may be attributed to suppression of oral M22 intake by peroxide pre-treatment, we immersed worms in 1-mM hydrogen peroxide as described, and then fed them fluorescently tagged bacteria, suspended in 75% v / v M22. E. coli strain HT115, commonly used as nematode food in RNAi knockdown experiments, was transformed with a plasmid expressing Green Fluorescent Protein (GFP). A suspension of these bacteria in nematode growth medium (NGM) was mixed with M22 at a volume ratio of 1 :3 (achieving a 75% v / v final concentration of M22) and C. elegans adults were placed in this mixture to assess their food (and hence, M22) intake, with or without prior peroxide exposure. The results showed that the uptake of [M22 + bacteria] was not reduced, and that if anything, it was even possibly increased (but did not differ significantly), for pre-conditioned worms relative to untreated worms. Therefore, protection by hydrogen peroxide was not due to prevention of oral uptake of M22 into the worms.

[0046] While not wishing to be bound by any theory, it is believed that the reduced damage observed by application of both methods is due to a reduction of CPA toxicity more than it is due to a reduction in osmotic injury. It is not clear how exposure to peroxide could block the vast majority of observed damage if that damage were primarily osmotic. Further, there are four osmotic stress resistance (OSR) genes in C. elegans (

[0022] ), but these were apparently not activated by pretreatment with 3% M22 or by hydrogen peroxide. Further, exposure was carried out at elevated temperatures, which augment toxic injury and reduce osmotic injury. Finally, physical damage to the oral area that would be expected from osmotic damage was not observed.

[0047] Example 2: Hydrogen Peroxide and CPA Pretreatment Induce Different Protective Mechanisms

[0048] A combination of hydrogen peroxide and CPA pretreatment will have additive protection under some circumstances because these interventions induce protective genetic responses that do not completely overlap: activating more protective mechanism will produce more total protection than activating fewer such mechanisms. Day-2 adult wild-type Bristol-N2 / DRM worms (day 5 post-hatch) were exposed for 20 min at 20°C to either diluted M22 (3% of full strength) or to 1- mM hydrogen peroxide to induce mechanisms protective against higher M22 concentrations. The major fraction of each group was immediately processed for total RNA isolation, and a smaller population of each was assessed for survival of M22 to ensure that RNAseq differential-expression analysis reflected worms that have acquired resistance to M22 toxicity in response to pretreatments. Using two biological replicates for each pre-treatment and for untreated control nematodes, we confirmed the expected protection from M22 toxicity for each experimental group relative to controls, enabling us to run a genome- wide RNAseq analysis on worms known to share the common phenotype of M22 resistance. Following paired-read RNAseq analysis, we compared the normalized read-counts between each experimental group and untreated controls to assess differential expression. Genes that were up- and down-regulated in experimental vs. control samples were required to meet two criteria: p < 0.05 after adjustment for multiple endpoints, and fold change > 2. With these thresholds, we observed that, of >17,000 transcripts studied for each sample, 672 genes shifted significantly relative to controls by oxidative stress (1-mM H2O2) and 410 genes shifted after pre-exposure to a 3% dilution of M22 but, remarkably, we found only four annotation terms that showed downregulation by both interventions and only one annotation term that showed upregulation by both interventions. This implies that many of the mechanisms of protection by these dissimilar interventions are likely to be independent and will, therefore, be additive when the interventions are correctly combined.

[0049] Example 3: Hydrogen Peroxide Pretreatment Enables Preservation of Renal Cortical Membrane Integrity after M22 Treatment at Room Temperature Given that over 80% of protein-coding genes and pathways in C. elegans are conserved in mammals (

[0020] ), we tested the possibility that H2O2 pretreatment (hydroxyl radical exposure) could protect mouse kidney slices from subsequent exposure to M22. C57BL / 6J mouse kidneys were isolated immediately after sacrifice, decapsulated, and sliced using a Vibratome Series 1500 into 200-pm sections in Krebs-Hensel eit buffer and washed in Dulbecco’s Modified Eagle’s Medium (DMEM), all at 0°C. They were then immersed for 5 min in DMEM or DMEM with (1 or 10 pM) or without hydrogen peroxide at 0°C, although any physiological alternative to DMEM would also be effective in the invention. They were then placed in 75% v / v M22 (three volumes of M22 plus one volume of LM5) at 23 °C for 20 min and then in 100% M22 at 23 °C for another 20 min. The slices were then washed in LM5 and then in DMEM prior to staining. Membrane integrity was assessed by staining for 30 min at 23 °C with calcein-AM (for plasma membrane integrity sufficient to retain cytoplasmic esterase activity, visualizable by the presence of green cells) and ethidium homodimer-I (to detect permeabilization of nuclei by M22, as visualized by orange / red nuclei). Alternatively, cells were stained with Hoechst 33342 (a cell-permeant dye that stains all nuclei) and Sytox Green (which indirectly measures plasma membrane integrity by crossing permeabilized plasma membranes and then staining nuclei). All dyes were used at concentrations recommended by their manufacturers. Fluorescent images were captured using a Keyence laser-scanning confocal fluorescence microscope or a Nikon Eclipse microscope (each with the corresponding proprietary z-stacking and integration software (

[0023] ). Stacked confocal images were combined to reconstruct 3D spatial images, from which we assessed viability of cells in the interior of each slice.

[0050] Figure 3A shows merged green / red images of kidney slices with and without exposure to M22 as described above and with or without prior brief exposure to hydrogen peroxide. Upper left: no treatment. Orange / red staining in the baseline (untreated) state is believed to be due to surface cells damaged by tissue cutting (a background artifact). Upper right: increased nuclear staining after M22 treatment without protective pretreatment. Lower left: reduced nuclear staining when M22 treatment as described is preceded by pretreatment with 1 pM H2O2. Lower right: further reduction in nuclear staining when M22 treatment as described is preceded by pretreatment with 10 pM H2O2. Figure 3B presents the quantitative results of Experiment 2 [see Table 1; the bars depict the mean percent of “live” (green) cells, ± 1 SEM, per group, for 9 fields per group]. In Experiment 2, 1-pM H2O2 pretreatment raised the fraction of esterase-positive (green) cells from -30% after M22 exposure without pretreatment to -58% (p < 2 x 106), which approximates the

[0051] 62% of green cells observed without M22 treatment; 10-pM H2O2 increased the fraction of green cells to -67% (p - 4 x 107vs. controls without pretreatment), which is not statistically different from untreated slices despite M22 contact with the slices at room temperature. Table 1 shows that similarly robust results were seen in two of three independent experiments, and positive results were seen in the third experiment as well.

[0052] In summary, the results indicate that hydrogen peroxide pre-exposure protects mouse renal tissue against M22 toxicity as it does for C. elegans nematodes, astonishingly maintaining membrane integrity at levels very close to those observed in tissue never exposed to M22 despite the extreme measure of exposing tissue to M22 at room temperature. This experiment was repeated several times, with similar results (Table 1), including one experiment using a different pair of stains: Hoechst 33342 (a cell-permeant dye staining nuclei of all cells, alive or dead) plus SYTOX Green (staining only nuclei of “dead” cells).

[0053] Although these results were obtained with H2O2 and with M22 pretreatment, H2O2 equivalents and other CPA solutions are expected to confer similar protection.

[0054] Example 4: Hydrogen Peroxide Protects Rabbit Renal Cortical Slice Viability

[0055] Because vital staining results reflect only membrane integrity and enzymatic activity, we carried out two additional experiments to determine if hydrogen peroxide pretreatment can improve functional measures of cellular viability. All living cells must accumulate potassium and extrude sodium in order to sustain life, and these active transport processes require not only membrane integrity but also mitochondrial integrity to produce the ATP required to drive ion transport as well as a functional Na,K-ATPase membrane pump. Accordingly, rabbit kidneys were removed under surgical anesthesia, decapsulated, bisected, and sliced into ~0.5 mm thick cortical slices using a Stadie-Riggs microtome at 0-4°C as described elsewhere ([4]) and rinsed in LM5 (

[0075] ). They were then either exposed to hydrogen peroxide at 0°C for 5 min with or without subsequent treatment with CPA, or they were exposed to CPA or to no treatment without hydrogen peroxide exposure. The CPA exposure protocol was ( 1 / 16)X, (1 / 8)X, (1 / 4)X, (1 / 2)X, IX, (1 / 2)X+, (1 / 4)X+, (1 / 8)X+, (1 / 16)X+, 0X+, and OX, where X refers to the full strength concentration being tested, “+” refers to the presence of 300 mM mannitol, and all concentration steps are carried out at 0°C and are 20 min in duration, with occasional swirling of the slices in the solution so as to keep the solutions well-stirred. Viability was tested by incubating slices in Cross-Taggart solution ([V]) for 90 min at room temperature with constant oxygenation and agitation followed by brief rinsing of the slices in isotonic mannitol to reduce the slice content of extracellular cations, after which the slices were transferred to 3% trichloroacetic acid for ion extraction at least overnight. Ion concentrations were measured using an Instrumentation Laboratory Model 943 flame photometer. To compute percent of control recovery, 0.373 was subtracted from all K+ / Na+ratios because the K+ / Na+ratio of the Cross-Taggart solution is 0.373, so only K+ / Na+ratios above this signify levels of slice viability.

[0056] The results of the first experiment are shown in Table 2. Treating slices with either 0.5 or 5 pM H2O2 as described above was not detrimental, and slice pretreatment with both concentrations raised the K+ / Na+ratio of the slices after exposure to a high concentration of a novel cryoprotectant from about 92% (p=0.015 vs controls by one-tailed t-test) to about 96-100% (not significantly different from the control K+ / Na+ratio.) In fact, the improvement in K+ / Na+ratio with the use of 0.5 pM H2O2 was statistically significant (p=0.0076 by one-tailed test, or 0.0152 by two-tailed test), as was the difference between the 50% CPA group and the lumped 50% CPA plus H2O2 groups (0.0176 by two-tailed test).

[0057] The results of the second experiment, which employed the same methodology as the experiment shown in Table 2 except for the chosen concentration of H2O2, are shown in Table 3. They strongly confirm the results of Experiment 1. Together, the experiments described in Table 2 and 3 demonstrate that H2O2, and presumably its equivalents, can prevent toxicity upon subsequent exposure to any otherwise-toxic CPA solution, and not just upon exposure to M22.

[0058] Further, the effectiveness of 0.5 pM H2O2 implies a lower effective concentration limit of 0.1-0.2 pM for mammalian cells, within or outside of an organ.

[0059] Example 5: Hydrogen Peroxide Pre-treatment Protects Whole Organs from Injury Caused by Perfusion with a Verifiable Solution

[0060] It is not clear a priori how hydrogen peroxide pretreatment can be applied to a donor organ. Infusing this agent intravenously or perfusing it at elevated temperatures is likely to be damaging. On the other hand, absent the teachings of the present invention, introducing it into a cold perfusate after blood is removed would be predicted to have no protective effect because low temperatures are expected to inhibit the induction of protective biological mechanisms just as they preserve organs by slowing their metabolism to less than 10% of normal at 0-4°C. Further, although protection would not be expected, damage would be expected, because the chemical reactivity of hydroxyl free radicals and the rate of production of hydroxyl free radicals from decomposition of H2O2 are comparatively uninhibited at the same temperatures. The present invention newly removes the general barrier to low temperature use of H2O2 or its equivalent by showing that 5 min of exposure near 0°C is sufficient to induce profound protection of isolated, non-perfused mammalian tissues, but use for whole organs requires additional knowledge.

[0061] Whole organs must be perfused, and perfusion introduces unanswered questions as to whether H2O2 can be distributed adequately to the organ parenchyma without over-exposure of the vascular endothelium, the time required for adequate distribution of H2O2, and the times and temperatures over which exposure to H2O2 is both safe and effective for the purposes of the invention as well as the specific perfusion means that must be employed. The present invention provides critical and novel methodology that enables application of the invention to whole organs as follows.

[0062] Whole rabbit kidneys were washed free of blood in two steps, one step at room temperature (20-26°C) and one step at 0-6°C, with a variation of Renasol-14 (similar to Transcend B described elsewhere ([ / 4]) as the flush solution, but the method will be effective for any chosen flush solution). The syringes used for flushing were connected to a stopcock, so the time gap between the two flushes was less than 30 seconds. Control kidneys were washed free of blood with no H2O2 in the flush solution, and experimental kidneys were similarly flushed with 0.5, 1, or 8.5 pM H2O2 in each flush solution. The time required for flushing was approximately 1-1.25 minutes for each step, or around 2-2.5 min for both, but acceptable times range from 0.5-5 min per flush, or 1- 10 min for both. Other acceptable variations include flushing with only a cold (<15°C) flush solution or flushing with a solution whose temperature continuously varies from 25 °C or above to 10°C or below. After flushing, the kidneys were weighed and transported without perfusion to a perfusion machine, after which the weight was entered into a computer program before the kidneys were perfused. The time required between completing the second flush and the onset of perfusion in the perfusion machine was generally 2-3 minutes but may vary from less than 1 min to 5 min or longer; however, it is desirable to keep this time as short as possible, and most desirably below 5- 10 min, when H2O2 concentrations above 0.5-1 gM are used. The kidneys were then perfused with the same solution minus H2O2 for 10 min, with renal effluent discarded to ensure purging of H2O2 from the kidney and the perfusate. Thus, the total contact time between the kidneys and H2O2 prior to washing out the H2O2 was generally 4-6 min, but this may vary from 3 to 15 min, or more desirably, from 3-10 min. However, when H2O2 concentrations < 0.5gM are perfused, the kidney can, after flushing, be perfused with additional H2O2, for a total H2O2 perfusion time of up to 20 min.

[0063] After the 10-min H2O2 washout period, which may vary from 1 min to 20 min to an hour or more, the kidneys were gradually loaded with a vitrifiable 9.4M M22 variant solution and gradually unloaded largely as described elsewhere ([7, 75]), but the process of the present invention will be effective for any vitrifiable cryoprotectant solution and any appropriate CPA loading and unloading process. After washout of the CPA, the kidneys were transplanted back to their donors for in vivo renal functional observations over the next 14 days.

[0064] As shown in Figure 4, H2O2 pre-treatment dose-dependently lowered mean postoperative peak creatinine and reduced the time to return creatinine to 2.0 mg / dL by about one-third (A, B). Thus, the method of the present invention, i.e., the pretreatment of a living system with hydroxyl free radical (via H2O2) or its equivalent to reduce damage otherwise caused by subsequent exposure to cryoprotectants, is applicable even to mammalian organs suitable for transplantation, and the range of effective H2O2 concentrations is expected to be 0.2-20 pM, but preferably 0.5-10 pM.

[0065] Given that the protection against cryoprotectant toxicity is conserved from worms to mammalian cells to a whole kidney, it is expected that the protection demonstrated for kidneys will also convey to other mammalian organs, including, for example, the heart, liver, lung, pancreas, ovary, uterus, and testicle.

[0066] References

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[0068] 2. Cordeiro, R.M., et al., Insights on cryoprotectant toxicity from gene expression profiling of endothelial cells exposed to ethylene glycol. Cryobiology, 2015. 71: 405-412.

[0069] 3. Cypser, J.R., et aL, Genetic suppression of cryoprotectant toxicity. Cryobiology, 2019. 86: 95-102.

[0070] 4. Fahy, G.M., Cryoprotectant toxicity: biochemical or osmotic? Cryo-Letters, 1984. 5: 79-90.

[0071] 5. Fahy, G.M., The relevance of cryoprotectant "toxicity" to cryobiology. Cryobiology, 1986. 23: 1- 13.

[0072] 6. Fahy, G.M., Cryoprotectant toxicity neutralization. Cryobiology, 2010. 60: S45-S53.

[0073] 7. Fahy, G.M., Elimination of most damage after perfusing rabbit kidneys with M22 solutions.

[0074] Cryobiology, 2016. 73(3): 407.

[0075] 8. Fahy, G.M., et al., Cellular injury associated with organ cryopreservation: chemical toxicity and cooling injury, in Cell Biology of Trauma, JJ. Lemasters and C. Oliver, Editors. 1995, CRC Press: Boca Raton.

[0076] 9. Fahy, G.M., D.l. Levy, and S.E. Ali, Some emerging principles underlying the physical properties, biological actions, and utility of vitrification solutions. Cryobiology, 1987. 24: 196-213.

[0077] 10. Fahy, G.M., et al., Cryoprotectant toxicity and cryoprotectant toxicity reduction: in search of molecular mechanisms. Cryobiology, 1990. 27: 247-268.

[0078] 11. Fahy, G.M., et al., Vitrification as an approach to cryopreservation. Cryobiology, 1984. 21: 407- 426.

[0079] 12. Fahy, G.M. and B. Wowk, Principles of ice-free cryopreservation by vitrification, in

[0080] Cryopreservaiton and freeze-drying protocols (Methods Mol Biol 2180), W.F. Wolkers and H.

[0081] Oldenhof, Editors. 2021, Humana Press: New York. p. l-°n.

[0082] 13. Fahy, G.M., et al., Physical and biological aspects of renal vitrification. Organogenesis, 2009. 5: 167-175.

[0083] 14. Fahy, G.M., et al., Improved vitrification solutions based on predictability of vitrification solution toxicity. Cryobiology, 2004. 48: 22-35.

[0084] 15. Fahy, G.M., et al., Cryopreservation of organs by vitrification: perspectives and recent advances. Cryobiology, 2004. 48: 157-178.

[0085] 16. Giwa, S., et al., The promise of organ and tissue preservation to transform medicine. Nature Biotechnology, 2017. 35: 530-542. Guan, N., et al., Analysis of gene expression changes to elucidate the mechanism of chilling injury in precision-cut liver slices. Toxicol in Vitro, 2013. 27: 890-899. Han, Z., et al., Vitrification and nanowarming enable long-term organ cryopreservation and lifesustaining kidney transplantation in a rat model. Nature Communications, 2023. 14: 1-12. Karow, A.M., Jr, Cryoprotectants - a new class of drugs. Journal of Pharmacy and Pharmacology, 1969. 21: 209-223. Lai, C.H., et al., Identification of novel human genes evolutionarily conserved in Caenorhabditis elegans by comparative proteomics. Genome Res, 2000. 10(5): 703-13. Lovelock, J.E., The protective action of neutral solutes against haemolysis by freezing and thawing. Biochemical Journal, 1954. 56: 265-270. Solomon, A., et al., Caenorhabditis elegans OSR-1 regulates behavioral and physiological responses to hyperosmotic environments. Genetics, 2004. 167: 161-170. Takahashi, K., et al., Comparison of Histological Sample Volumes among Various Endoscopic Ultrasound-Guided Biopsy Needles. J Clin Med, 2021. 10(16). Takahashi, T., et al., Mechanism of cryoprotection by extracellular polymeric solutes. Biophysical Journal, 1988. 54: 509-518. Warner, R.M., et al., Rapid quantification of multi-cryoprotectant toxicity using an automated liquid handling method. Cryobiology, 2021. 98: 219-232.

Claims

Claims1. A method for reducing injury caused by exposing a biological system comprising a simple organism, a cell, a collection of cells, a tissue, a vascularized tissue, or an organ to a cryoprotectant solution that would normally be toxic to said biological system, comprising contacting said biological system with a solution containing hydrogen peroxide, removing said hydrogen peroxide, and then exposing said biological system to said normally toxic cryoprotectant solution.

2. The method of claim 1, wherein said contacting is accomplished by immersion of, superfusion of, or perfusion of said biological system with said hydrogen peroxide solution.

3. The method of claim 1, wherein said solution containing hydrogen peroxide contains 0.1- 20 mM hydrogen peroxide solution.

4. The method of claim 1, wherein said hydrogen peroxide solution is in contact with said biological system for 1-60 min.

5. The method of claim 1, wherein said cell, collection of cells, tissue, or organ is in contact with a 0.1-20 pM hydrogen peroxide solution for 1-20 min.

6. The method of claim 1, wherein said hydrogen peroxide solution does not irreversibly damage said biological system.

7. The method of claim 1, wherein contact with said hydrogen peroxide solution is done at -6 to 37°C.

8. The method of claim 1, wherein contact with said hydrogen peroxide solution is done for 4-30 min for a simple organism or for 4-10 min for said cell, collection of cells, tissue, or organ.

9. The method of claim 1, wherein contact with said hydrogen peroxide solution is done at a temperature that also induces heat shock proteins.

10. The method of claim 1, wherein said hydrogen peroxide solution comprises 0.2-20 pM hydrogen peroxide and wherein said organ or vascularized tissue is perfused with said hydrogen peroxide solution for 1-20 min and wherein said perfusion with said hydrogen peroxide solution does not irreversibly damage said organ or vascularized tissue.

11. The method of claim 10, wherein said hydrogen peroxide solution comprises 0.5-10 pM H2O2 and is perfused for 4-10 min.

12. The method of claim 10, wherein said perfusion with said hydrogen peroxide solution takes place at 0-37°C.

13. A method for reducing injury caused by exposing a biological system comprising a simple organism, a cell, a collection of cells, a tissue, a vascularized tissue, or an organ to a normally toxic cryoprotectant solution, comprising contacting said biological system with a non-toxic cryoprotectant solution, removing that non-toxic cryoprotectant solution, and then exposing said biological system to said normally toxic cryoprotectant solution.

14. The method of claim 13, wherein said non-toxic cryoprotectant solution has a concentration of 0.1 to 1.5M or 1-10% w / v.

15. The method of claim 13, wherein said non-toxic cryoprotectant solution is put in contact with said biological system at -6 to 42°C.

16. The method of claim 14, wherein said non-toxic cryoprotectant solution comprises a concentration range of 1-4% w / v or 0.1-0.7M and said biological system is put in contact with said solution at 10-37°C.

17. The method of claims 14 and 16, wherein said biological system is put in contact with said non-toxic cryoprotectant solution for 2 to 180 min.

18. The method of claim 1, wherein said hydrogen peroxide solution contains a non-toxic concentration of cryoprotectant.

19. The method of claim 1, further comprising, after contacting said biological system with a solution containing hydrogen peroxide and removing said hydrogen peroxide, and before said step of exposing said biological system to said normally toxic cryoprotectant solution, exposing said biological system to a non-toxic cryoprotectant solution.

20. The method of claim 1, further comprising, before contacting said biological system with a solution containing hydrogen peroxide and removing said hydrogen peroxide, exposing the biological system to a non-toxic cryoprotectant solution.

21. The method of claim 13, wherein the said non-toxic cryoprotectant solution does not have the same composition as the said normally toxic cryoprotectant solution.

22. The method of claim 13, wherein said non-toxic cryoprotectant solution reduces toxicity of the normally toxic cryoprotectant solution but is not removed before introducing said normally toxic cryoprotectant solution. 1

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