Cryoprotectant shock-loading, interleaved equilibration, and hyperoncotic washout for cryopreservation by vitrification

Abstract

The present invention relates to cryopreservation of biological samples such as cells, cellular systems, tissues, organoids, organs, embryos, or organisms. Herein, methods and reagents for cryopreservation of difficult to preserve samples are disclosed. The methods disclosed herein are characterized by low complexity and allow for rapid cryopreservation of difficult to preserve biological samples. Furthermore, the methods disclosed herein preserve sample and / or tissue integrity, viability and preserve complex physiological functionalities, including neuronal and myocardial activity.

Description

[0001] New PCT-Patent Application

[0002] Friedrich-Alexander-Universitat Erlangen-Nurnberg

[0003] Vossius Ref.: AK1615 PCT S3

[0004] Cryoprotectant shock-loading, interleaved equilibration, and hyperoncotic washout for cryopreservation by vitrification

[0005] The present invention relates to cryopreservation of biological samples such as cells, cellular systems, tissues, organoids, organs, embryos, or organisms. Herein, methods and reagents for cryopreservation of difficult to preserve samples are disclosed. The methods disclosed herein are characterized by low complexity and allow for rapid cryopreservation of difficult to preserve biological samples. Furthermore, the methods disclosed herein preserve sample and / or tissue integrity, viability and preserve complex physiological functionalities, including neuronal and myocardial activity.

[0006] Field of invention

[0007] The present invention relates to cryopreservation of biological samples including cells, cellular systems, tissues, organoids, organs, embryos, or organisms. Herein, methods and reagents for cryopreservation of difficult to cryopreserve samples, organs and organisms are disclosed. Samples cryopreserved using the disclosed methods maintain their physiological structure and functionality.

[0008] Background of the invention

[0009] Cryopreservation of biological cells, cellular systems, tissues, tissue-engineered constructs, organoids, organs or organisms (herein referred to as biological samples) has many existing and potential applications for their storage and distribution. This includes, but is not limited to, cryopreservation of the following:

[0010] oocytes, sperm, embryos, ovary and testicular tissue in reproductive medicine and animal breeding.

[0011] cells, cellular systems, tissues, tissue-engineered constructs, and organoids in research, personalized and regenerative medicine, including, but not limited to, organotypicslices, as well as replacements and transplants for cartilage, skin, pancreatic islets, vascular grafts, nerves, bladder augmentation, and corneas.

[0012] mammalian organs for transplantation medicine and research.

[0013] the central nervous system, including the mammalian and human brain, for structural brain preservation, cryonics and research [1],

[0014] organisms, including the mammalian and human organism, for deep space exploration, cryonics and research [1],

[0015] Vitrification is a method employed for ice-free cryopreservation, avoiding disruption of sensitive biological samples. During cryopreservation by vitrification, the aqueous phase of biological tissue solidifies into a non-crystalline amorphous glass [2,3], This can be achieved by replacing high proportions of the tissue water with polar solvents. Provided sufficient cooling and rewarming rates, these solvents act as cryoprotectants, inhibiting crystallization due to water-water interactions [4], Ice-free cryopreservation to avoid mechanical disruption of the inter- and intracellular space in complex biological materials has been proposed long ago [5, 6], and has been realized by vitrification [2,7], In larger biological samples which exceed approximately 100 micrometers, successful cryopreservation by vitrification requires the following:

[0016] the addition (herein referred to as loading) of cryoprotectants to lower the probability of ice formation.

[0017] storage below the glass transition temperature of approximately -130°C.

[0018] cooling and warming rates appropriate for the utilized cryoprotectant applied in the metastable temperature region between the glass transition temperature and the melting temperature to avoid crystallization.

[0019] - Thermal profiles which avoid cracking from thermomechanical stress.

[0020] Typical cryoprotectants include, but are not limited to, cell-permeating solvents like dimethylsulfoxide, acetamide, ethylene glycol, propylene glycol, glycerol, formamide, N-methylformamide, dimethylformamide, 3-methoxy-l,2-propanediol, and their combinations. Cryoprotectant solutions can also include non-cell-permeating sugars including trehalose and sucrose, as well as macromolecules including, but not limited to, polyvinylpyrrolidone, X-1000, Z-1000, antifreeze proteins and their combinations.Generally, loading and removal (herein referred to as washout) of cryoprotectants is performed at hypothermic temperatures to reduce toxicity, and using gradual or multistep loading and washout concentrations to avoid excessive osmotic cell shrinking and swelling. E.g., it has been possible to perform successful vitrification of rat kidneys with 54% w / v VMP (16.8% w / v ethylene glycol, 22.3% DMSO, 12.9% formamide, 1% X-1000, 1% Z-1000), US8679735B2 using a gradual loading protocol over 150 minutes [8],

[0021] However, cryopreservation of certain tissues and organs has been a major challenge in the field. For example, in the case of adult mammalian brain tissue and neurons differentiated in culture, full electrophysiological recovery after cryopreservation has thus far not achieved [9-11], Using certain freezing techniques, some recovery of activity was reported in the adult feline brain after several years of storage at -20 °C [12, 13], and in rodent ganglia after up to 24 hours storage at -76 °C

[0014] , both using 15% glycerol. However, freezing of neural tissue results in overt loss of synaptic connections. Achieving full electrophysiological recovery therefore likely necessitates ice-free cryopreservation, i.e., vitrification.

[0022] Recently, it has been demonstrated that cryopreservation of brain tissue can be achieved using 59% w / v V3 (16.8% w / v ethylene glycol, 22.3% DMSO, 12.9% formamide, 7% PVP K12) in a 7-step loading protocol over 32 minutes

[0015] , After re-warming, cryoprotectants are washed out by reversing the loading steps, with the exception that solutions may be supplemented with mannitol to avoid excessive cell or tissue swelling. Gradual and multi-step protocols have thus far failed for whole mammalian brains, due to lethal shrinking of the organ during cryoprotectant loading [1],

[0023] The use of gradual or multistep loading concentration profiles has been motivated by the empirical observation that cells shrinkage to approximately 70% of their isotonic volume during cryopreservation - even when exposed to comparatively low concentrations of cryoprotectant, such as 10% w / v = 1.28 mol / l dimethylsulfoxide

[0016] or 11,4% w / v = 1.5 mol / l propylene glycol

[0017] , This shrinking can be explained by higher cell permeability for water compared to cell permeability for cryoprotectants, resulting in a rapid efflux of intracellular water until the extracellular osmolarity is balanced by increased intracellular concentration of solutes. During slow influx of cryoprotectant, the cells reswell. This shrink-reswell dynamics can be described by a two-parameter formalism modeling water as a solvent and cryoprotectant as a solute, see equations below and Figure 1. As shown in Figure 1, modellingsingle-step cryopreservation protocols (such as shock-loading) based on these known formalisms suggests that such protocols should not be used for cryopreservation. Thus, the state of the art clearly teaches away from using single-step cryopreservation protocol. Surprisingly, and contrary to these modelling results, we here report a single-step cryopreservation protocol, which enables rapid cryopreservation, maintains tissue integrity and physiological function upon sample recovery.

[0024] dVw, ..

[0025] = - LpA RT(M‘ - M )(1)dN„ . ..

[0026] = PSA(M* - Mj)(2)

[0027]

[0028] Vwis the intracellular volume, Lpis the water permeability, A is the cell surface, R is the universal gas constant, T is the absolute temperature, Meand Mlare the total extracellular and intracellular osmotic concentrations, Nsis the intracellular amount of the cryoprotectant, Psis the cryoprotectant permeability,

[0029]

[0030] and Mslare the extracellular and intracellular osmotic concentrations of the cryoprotectant.

[0031] It must be noted that the above two-parameter formalism of solute-solvent flux is neglecting the volume effects of cryoprotectants, which can also act as solvents. Cryoprotectants constitute the dominating solvents in known stable vitrification solutions with >50% w / v cryoprotectant, i.e., the solute-solvent relationship with water becomes inverted. It must also be noted that the above two-parameter formalism is neglecting saturation effects at aquaporins, interfacial energy barriers, intermolecular forces between cryoprotectants and water that are affecting their respective chemical potentials, as well as their diffusion characteristics and membrane permeability. Theoretical models which capture these relevant non-idealities are lacking

[0018] , To our knowledge, empirical measurements of the shrink-reswell dynamics during exposure to vitrification solutions with relevant non-ideality are lacking as well

[0019] ,

[0032] Technical problem

[0033] In view of the prior art, taking into account these beneficial and surprising effects, the technical problem underlying the present invention is the provision of improved methods forcryopreservation of a biological sample. The technical problem is solved by provision of the embodiments characterized in the claims and the embodiments described hereinbelow. The methods provided herein achieve, inter alia, rapid cryopreservation of biological samples, suitable to preserve tissue integrity and biological / physiological functionality upon sample recovery.

[0034] The present invention solves the above identified technical problem since, as documented herein below and in the appended examples, it was surprisingly found that cryopreservation fulfilling these criteria may be achieved in a one-step process using a single cryopreservation solution - hereinafter referred to as "shock loading".

[0035] Methods disclosed in the prior art have relied on the use of carrier solutions which are gradually or incrementally supplemented with increasing quantities of the desired cryoprotectant and required concomitant adaptation of sample temperatures to ensure tissue integrity and prevent toxicity of the employed cryoprotectants.

[0036] In contrast, the method of the present invention provides an easy and rapid cryopreservation protocol. Importantly, the disclosed method is compatible with hard-to-conserve tissues or whole organs, including mammalian brain tissues and / or whole mammalian brains. The disclosed method is short enough to enable - for the first time - cryopreservation of complex and transient physiological properties, such as preservation of physiological brain functionality.

[0037] The present invention further provides methods for recovery of cryopreserved samples with improved viability, tissue integrity and / or physiological functionality.

[0038] Surprisingly, and contrary to the teachings of the prior art, the invention disclosed herein provides superior and simplified new methods for cryoprotectant loading and washout.

[0039] Summary of the invention

[0040] The present invention provides novel methods for cryopreservation by vitrification of biological samples including cells, cellular systems, tissues, organoids, organs, or organisms. Specifically, the invention entails three techniques: shock-loading, interleaved equilibration, and hyperoncotic washout which are described in more detail further below.The present invention is, inter alia, characterized by the following items 1 to 27.

[0041] 1. A method for cryopreservation of a biological sample comprising the step:

[0042] AA. contacting the biological sample with a cryoprotectant solution comprising at least about 45 % cryoprotectant (shock loading), and

[0043] lowering the temperature of the biological sample; or

[0044] AB. contacting the biological sample sequentially with a cryoprotectant solution comprising at least about 45 % cryoprotectant and a second solution (interleaved equilibration), and

[0045] lowering the temperature of the biological sample.

[0046] 2. The method of item 1, wherein the cryoprotectant solution comprising at least about 45 % cryoprotectant in step (AA) or (AB) is the sole cryoprotectant solution with which the biological sample is contacted.

[0047] 3. The method of item 1, wherein step (AA) alternatively comprises contacting the biological sample with a cryoprotectant solution comprising at least about 50 % and does not comprise contacting the biological sample with a cryoprotectant solution comprising less than about 50 % cryoprotectant

[0048] 4. The method of any one of items 1 to 3, further comprising a step B of storing the biological sample.

[0049] 5. The method of any one of items 1 to 4, further comprising a step C of removing the cryoprotectant solution from the biological sample and, optionally, recovering the biological sample.

[0050] 6. The method of any one of items 1 to 5, wherein said cryoprotectant solution comprises at least about 45% w / v cryoprotectant selected from the group consisting of dimethyl sulfoxide, ethylene glycol, propylene glycol, glycerol, formamide, N- methylformamide, dimethylformamide, 3-methoxy-l,2-propanediol, and any combination thereof, optionally supplemented with non-cell-permeating sugars or macromolecules.The method of any one of items 1 to 6, wherein said cryoprotectant solution comprises preferably at least about 50 % cryoprotectant, for example, 61% w / v ethylene glycol or 59% w / v V3.

[0051] The method of any one of items 5 to 7, wherein the cryoprotectants are removed from said biological sample by contacting the sample with hyperoncotic solutions (hyperoncotic washout), preferably with colloidal solutions of oncotic pressure greater than physiological levels.

[0052] The method of item 8, wherein said colloidal solution comprises about 10% to 30% w / v dextran.

[0053] The method of any one of items 1 to 9, wherein the biological sample is / are or comprises cells, cellular systems, tissues, tissue-engineered constructs, organoids, organs, or organisms.

[0054] The method of any one of items 1 to 10 wherein the biological sample is a sample from a vertebrate, preferably a mammal.

[0055] The method of any one of items 1 to 10, wherein the biological sample is a brain, preferably the whole brain, and / or wherein said contacting with said cryoprotectant solution and / or removing of said cryoprotectant solution do not require deliberate opening of the blood-brain barrier.

[0056] The method of any one of items 1 to 12, wherein contacting with the cryoprotectant solutions and / or removing of the cryoprotectant solutions are performed via perfusion, superperfusion, immersion, or combinations thereof.

[0057] The method of any one of items 1 to 12, wherein the method does not comprise contacting the biological sample with a cryoprotectant solution comprising less than about 45 % cryoprotectant, preferably less than about 50 % cryoprotectant.The method of any one of items 1 to 12, wherein the method further comprises a pre-cooling step prior to step (AA) or (AB).

[0058] The method of any one of items 1 to 12 and 15, wherein the method further comprises vitrifying said biological sample after prior to step (AA) or (AB).

[0059] The method of item 16, wherein the method further comprises storing said biological sample subsequent to vitrification.

[0060] The method of item 16 or 17, wherein the method further comprises removing said cryoprotectant from said biological sample subsequent to vitrification and / or storage.

[0061] The method of item 18, wherein the sample maintains one or more physiological functionalities as compared to a a biological sample that has not been cryopreserved.

[0062] The method of items 18 or 19, wherein removal of said cryoprotectant comprises contacting the sample with a hyperoncotic or hyperosmotic solution.

[0063] The method of item 20, wherein said solution is a hyperoncotic solution comprising between 10 and 30% w / v dextran or PVP, such as PVP K12.

[0064] The method of item 20, wherein said solution is a hyperosmotic solution comprising between 10 and 30% w / v glucose.

[0065] The method of any one of items 12 to 14, wherein when the biological sample is the whole mammalian brain, the method comprises the following steps:

[0066] A. performing blood washout and connecting said brain to a perfusion system; AA. perfusing the brain with vitrification solution containing at least 45% w / v cryoprotectant at approximately half of physiological baseline flow rates; orAB. perfusing the brain sequentially with a vitrification solution containing at least 50% w / v cryoprotectant and a carrier solution by intermittently alternating perfusion between said vitrification solution and carrier solution

[0067] B. vitrifying and storing said brain below the glass transition temperature;

[0068] BB. rewarming said brain above the melting temperature without causing crystallization; and

[0069] C. perfusing colloidal solutions at reduced flow rates adjusted to solution viscosity.

[0070] The method of any one of items 1 to 22, comprising the steps of:

[0071] AA. immersing said biological sample in vitrification solution containing at least 45% w / v cryoprotectant until said samples sink or equilibrate;

[0072] B. vitrifying and storing said biological samples below the glass transition temperature;

[0073] BB. rewarming said biological samples above the melting temperature without crystallization;

[0074] C. immersing said biological samples in a colloidal solution; and

[0075] D. subsequent immersion in isotonic carrier solutions.

[0076] The method of any one of items 1 to 24, wherein said biological samples are viable after removal of the cyroprotectant solution and, optionally, are assessed after recovery, by methods such as, but not limited to, electrophysiological, structural, biochemical, immunohistochemical, functional, or molecular assessment postcryopreservation.

[0077] The method of any one of items 1 to 25, wherein said biological samples include human neurospheres, organotypic brain slices, acute brain slices, mammalian including human organs and organisms, natural and engineered cells, stem cells, oocytes, sperm, embryos, ovarian and testicular tissue, pancreatic islets, and tissue-engineered constructs such as skin, cartilage, vascular grafts, nerves, bladder augmentation, and corneas.

[0078] A biological sample cryopreserved by the method of any one of items 1 to 26.Shock-loading involves rapid, direct exposure of the biological sample to highly concentrated vitrification solutions to harness non-idealities of solute-solvent flux. Surprisingly and contrary to prior expectations, this approach reduces shrinkage and toxicity, and allows for shorter and less complex cryopreservation protocols, even in challenging systems such as whole mammalian brains. Importantly, this method does not require deliberate opening of the blood-brain barrier prior to cryopreservation.

[0079] Interleaved equilibration employs intermittent, alternating exposure of samples to cryoprotectant solutions and isotonic carrier solutions or lower-concentration cryoprotectant solutions. This alternation reduces overall osmotic excursion and concomitant tissue shrinkage.

[0080] Hyperoncotic washout employs high-concentration colloidal solutions; i.e. a solution comprising one or more macromolecular oncotic agents. This technique significantly reduces swelling upon cryoprotectant removal compared to conventional osmotic buffers (e.g. conventional buffers comprising 300mM mannitol). Furthermore, reducing the number of washout steps also shortens protocol times and complexity.

[0081] Accordingly, it is understood that the washout solution according to the present invention is preferably a washout solution which reduces swelling upon cryoprotectant removal compared to conventional osmotic buffers. Such reduced swelling may be affected by using hyperoncotic and / or hyperosmotic washout solutions; i.e. solutions with higher oncotic and / or osmotic pressure than conventional osmotic / oncotic buffers. Accordingly, the present invention encompasses hyperoncotic washout and, in separate embodiments, hyperosmotic washout. Unless otherwise indicated, 'hyperoncotic washout' refers to washout in which the principal anti-swelling effect is mediated by elevated oncotic pressure, whereas 'hyperosmotic washout' refers to washout in which the principal anti-swelling effect is mediated by elevated osmotic pressure.

[0082] Together, these methods accomplish accelerated, simplified, and significantly improved vitrification protocols for a diverse spectrum of sensitive biological samples, including whole mammalian brains, brain slices, and neurospheres.Gradual and multi-step cryoprotectant loading protocols expose the sample to hypothermia and cryoprotectants for prolonged periods of time, (see, e.g., US8679735B2 and [8]). These prolonged time intervals may have detrimental effects on tissue integrity and physiological function. By comparison, shock-loading minimizes exposure time to hypothermia and cryoprotectants, delivering vitrifiable amounts of cryoprotectant to the brain within 6 minutes, and achieving sufficient loading in the liver and kidney within 20 minutes. It also simplifies procedural complexity. Given the time-dependent toxicity of cryoprotectants, the superiority of shock-loading compared to the state of the art in cryoprotectant loading of peripheral organs is assumed. Hyperoncotic washout accelerates and simplifies the state of the art with respect to gradual and multi-step washout protocols as well.

[0083] Shock-loading: Reduced cell shrinking and equilibration times are achieved by directly exposing the biological sample to highly concentrated vitrification solutions which exhibit relevant non-idealities with respect to the two-parameter formalism of solute-solvent flux, including, but not limited to, aquaporin saturation effects, interfacial energy barriers, solutesolvent inversion, dominating adhesive and cohesive intermolecular forces and altered diffusion characteristics and membrane permeability (referred to herein as shock-loading). Vitrification solutions which possess relevant non-ideal modeling characteristics for shockloading, include, but are not limited to, solutions containing >50% w / v cryoprotectant, e.g., 61% w / v ethylene glycol, or 59% w / v V3. Contrary to previous expectations, shock-loading allows rapid equilibration of biological samples with vitrifiable concentrations of cryoprotectants, with reduced protocol duration, complexity and toxicity compared to gradual or multi-step loading. For the first time, shock-loading enables equilibration of the whole mammalian brain with vitrifiable concentrations of cryoprotectant without the need for opening the blood-brain barrier

[0020] , see Figures 2 and 3.

[0084] Interleaved equilibration: Alternating exposure to cell-permeating cryoprotectants and carrier solution, or concentrations of the cryoprotectants that are lowerthan in the biological sample (herein referred to as interleaved equilibration) can reduce the maximum amount of cellshrinking and swelling, already under the idealized assumptions of the two-parameter formalism of solute-solvent flux, see Figure 1. This approach extends the methods known in the prior art of gradual and multi-step loading and washout, and brief overshoot

[0016] ,Interleaved equilibration only requires that cryoprotectant and water permeabilities are different in the biological sample. In typical biological samples, permeability for water is significantly higher than for cryoprotectants and loading by interleaved equilibration results in repeated reswelling and rehydration of the biological sample, see Figure 1. Interleaved equilibration can also be effectively applied to cryoprotectant washout. Interleaved equilibration is of particular use for repeated shock-loading, further emphasizing the nonidealities of the diffusion dynamics of highly concentrated vitrification solutions with respect to the two-parameter formalism. In case of the mammalian brain, interleaved cryoprotectant equilibration results in significantly reduced shrinking, outperforming the lethal shrinking observed using gradual loading by far, see Figures 2 and 3.

[0085] It will be understood that a wide range of cycle times are appropriate for emphasizing the favorable diffusion dynamics of shock-loading, limited by very short cycle times which lead to mixing of the two solutions before they reach the capillary bed, which can be as short as 5 seconds in the mouse brain, and very long cycle times which lead to harmful shrink-reswell excursion of above 20%, which can be as long as 4 minutes for the mouse brain.

[0086] Hyperoncotic washout:

[0087] In one example, reduced swelling and improved survival are achieved by removing cryoprotectant from biological samples by exposing them to colloidal solutions with oncotic pressure greater than physiological levels (referred to herein as hyperoncotic washout). This is including, but not limited to, previously unexplored solutions like 30-10% w / v dextran, or 10-3x of its isooncotic concentration, or 30-10% w / v PVP K12.

[0088] As used herein, the term 'hyperoncotic washout solution' refers to a washout solution comprising one or more macromolecular oncotic agents that remain predominantly extracellular during the washout interval and that generate an oncotic pressure above physiological levels, thereby opposing tissue swelling during cryoprotectant removal.

[0089] As used herein, the term 'hyperosmotic washout solution' refers to a washout solution having a total osmotic concentration above that of the relevant physiological carrier solution and / or extracellular fluid, thereby reducing water influx and swelling during cryoprotectant removal. In some embodiments, the washout solution may provide both hyperoncotic and hyperosmotic effects.During gradual cryoprotectant washout as known in the prior art, 300 mmol / l mannitol or other non-permeating osmolytes are added as osmotic buffer to reduce the extracellular chemical potential of water and to counteract osmotic swelling US8679735B2, [8,15], Previously used osmotic buffers have not effectively prevented brain swelling during gradual or single-step cryoprotectant washout procedures. For the first time, good recovery and avoidance of brain swelling could successfully be achieved with hyperoncotic washout, see Figures 2 and 3. Perfusion flow must be reduced according to the viscosity of the hyperoncotic washout solution. Hyperoncotic washout is also accelerating and simplifying multi-step washout protocols by immersion known in the prior art

[0015] , Hyperoncotic solutions were empirically found to exert little to no toxicity on human fibroblasts during the timescales required for cryoprotectant washout at hypothermic conditions. Their superior effectiveness compared to small osmolytes is not fully understood but might be explained by reduced permeability, increased viscosity, steric hindrance, excluded volume, and macromolecular crowding effects.

[0090] As used herein, the term "cryoprotectant solution" refers to solutions comprising one or more cryoprotectants. Such solutions are capable of reducing, preventing, or mitigating physicochemical damage to biological or synthetic materials during exposure to sub-zero temperatures. In particular, cryoprotectant solution may modulate ice nucleation and growth, control osmotic flux, stabilize biomolecular structures, alter thermal or glass-forming properties, or otherwise maintain sample integrity throughout cooling, storage, and rewarming.

[0091] A cryoprotectant solution, as defined in the context of the present invention, includes any solution that performs at least one (preferably all) of the following actions: suppress formation or enlargement of intracellular or extracellular ice crystals; regulate water transport across cellular or polymeric boundaries to prevent excessive shrinkage or swelling; increase viscosity and glass-forming capacity to support vitrification; inhibit recrystallization during storage or warming; or enhance thermal buffering to provide uniform cooling conditions.A cryoprotectant solution according to the present invention may comprise one or more cryoprotectant agents (also referred to as cryoprotectants). A solution qualifies as a cryoprotectant solution when it has one or more (preferably all) of the following characteristics: it measurably reduces post-thaw damage, shifts the freezing or nucleation temperature, improves recovery of biological or functional activity, or facilitates formation of an amorphous glassy state under defined cooling conditions. Unless otherwise indicated, cryoprotectant solutions may be aqueous or non-aqueous.

[0092] As used herein, the term 'cell-permeable cryoprotectant' refers to a cryoprotective agent that, under the loading conditions used herein, crosses the plasma membrane on the relevant experimental timescale and thereby contributes to suppression of intracellular ice formation and / or intracellular vitrification. Non-limiting examples include dimethyl sulfoxide (DMSO), ethylene glycol (EG), propylene glycol, glycerol, formamide, N-methylformamide, dimethylformamide, and 3-methoxy-l,2-propanediol.

[0093] As used herein, the term 'non-cell-permeating cryoprotectant' refers to a cryoprotective additive that remains predominantly extracellular on the relevant experimental timescale and primarily modulates extracellular osmolality, viscosity, and / or glass-forming behavior. Nonlimiting examples include sucrose, trehalose, dextran, and polyvinylpyrrolidone (PVP, e.g. PVP K12) .

[0094] Cell permeability depends on the physicochemical properties of the agent, the membrane, temperature, and exposure time, and is not determined solely by molecular mass.

[0095] In preferred embodiments of shock-loading and interleaved equilibration for viable tissues and organs, the cryoprotectant solution comprises at least one cell-permeable cryoprotectant and may optionally further comprise one or more non-cell-permeating cryoprotectants or macromolecular additives

[0096] The cryoprotectant solution to be used herein preferably is an aqueous solution.

[0097] The present invention relates to:

[0098] A method for cryopreservation of a biological sample comprising the step:AA. contacting the biological sample with a cryoprotectant solution comprising at least about 45 % cryoprotectant (shock loading), and

[0099] lowering the temperature of the biological sample; or

[0100] AB. contacting the biological sample sequentially with a cryoprotectant solution comprising at least about 45 % cryoprotectant and a second solution (interleaved equilibration), and

[0101] lowering the temperature of the biological sample.

[0102] The term "cryopreservation" is known in the art and commonly refers to a process where biological material (cells, tissues, or organs or organisms) are frozen to preserve the material for an extended period of time. At low temperatures (typically -80 °C, -130 or -196 °C using liquid nitrogen) cell metabolism which might cause damage to the biological material in question is effectively stopped. Cryopreservation is known as an effective way to transport biological samples over long distances, store samples for prolonged periods of time, and create a bank of samples for users.

[0103] The biological sample which can be subjected to the cryopreservation in accordance with the method of the present invention is not particularly limited to a specific kind of sample. Any possible cell, cellular system, tissue, tissue-engineered construct, organoid, organ or organism can be subjected to the cryopreservation in accordance with the present invention.

[0104] Non-limiting examples of the biological sample are outlined in more detail further below. In a preferred embodiment, the biological sample is or can be derived from a "patient", a "subject" and an "individuum", terms, which are used interchangeably. The patient / subject / individuum is in a preferred embodiment a mammal, such as a dog, cat, pig, cow, sheep, horse, rodent, e.g., rat, mouse, and guinea pig, or a primate, e.g., gorilla, chimpanzee and a human. In a most preferable embodiment, the subject is a human. The term "can be derived" can mean in this context an organ or a tissue sample from the patient / subject / individuum, and may include an oocyte and / or a sperm cell and / or an embryo. The term "embryo" as used herein may specifically refer to a blastocyst, e.g. a stage of embryo development that occurs about 2 to 6 days, e.g. five days, after fertilization, for example when the embryo is ready for implantation (e.g. for in vitro fertilization).In one example, the biological sample is not a whole organism, such as a whole animal or human, but is instead an organ or a tissue sample, preferably an organ or tissue sample derived from a mammal, such as a rodent and / or a human. As used herein, an embryo may be considered a whole organism.

[0105] In one example, the biological sample is an organ, an organoid or a tissue sample derived from an organ. As shown in the appended non-limiting example, the cryopreservation protocol of the present invention is particularly useful in the preservation of organs such as brain, kidney, liver, heart or skin, neurological organoids or neurospheres, or other neurological tissue samples (e.g. brain slices or nerve preparation).

[0106] The cryopreservation protocol according to the present invention allows cryopreservation and recovery of a sample comprising cells that maintain their physiological functionality. For example, where the sample is a neurological tissue sample, a whole brain, a whole heart or a neurological organoid, the cryopreservation protocol according to the present invention allows cryopreservation and recovery of tissues comprising cells that maintain their physiological functionality.

[0107] As used herein, "physiological functionality" refers to the ability of a biological cell, tissue, or system to perform its inherent, native, and biologically normal functions. The term encompasses the maintenance of structural integrity, biochemical activity, metabolic processes, and responsiveness to intrinsic or extrinsic stimuli as characteristic of the corresponding cell type in vivo. In one example, the physiological functionality may be a physiological functionality of cells of the nervous system.

[0108] Non-limiting examples for CNS-resident cell type— including, without limitation, neurons, astrocytes, oligodendrocytes, microglia, ependymal cells, and neural stem or progenitor cells

[0109] Non-limiting examples for such physiological functionalities include the execution of cell-type-specific functional activities, such as synaptic signalling in neurons, neurotransmitter uptake and metabolic support in astrocytes, myelination and axonal insulation by oligodendrocytes, immune surveillance and phagocytic activity by microglia, cerebrospinalfluid regulation by ependymal cells, and proliferative or differentiative capacity in neural stem / progenitor cells.

[0110] In another example, the physiological functionality may be a physiological functionality of cells of the heart. Non-limiting examples for such cells comprise cardiomyocytes, pacemaker cells, conducting system cells, and cardiac fibroblasts. Non-limiting examples for such physiological functionalities include muscle contraction, excitation-contraction coupling, and signal transduction.

[0111] Furthermore, and as shown in the appended non-limiting examples, the cryopreservation protocol of the present invention does not require permeabilization of the blood brain barrier when cryopreserving whole brains and prevents lethal shrinkage of neurological tissues. Thus, the present invention provides a protocol that allows the cryopreservation and recovery of intact neurological tissues capable of eliciting their physiological functionalities, as discussed herein above.

[0112] As used herein, 'deliberate opening' or 'permeabilization' of the blood-brain barrier (BBB) refers to an intentional intervention to increase BBB permeability beyond its native state prior to or during cryoprotectant delivery. The skilled person is aware of means and methods to permeabilize the BBB. Non-limiting examples comprise osmotic disruption with hyperosmotic agents such as mannitol, focused ultrasound with microbubbles, chemical mediators such as bradykinin analogues, detergents or solvents, inflammatory mediators, or mechanical or invasive disruption. The main role of the BBB is to restrict the access of foreign solutes to the brain. For this reason, BBB permeabilization has readily been employed in the prior art to allow uniform contacting of the brain with CPA solutions. In previous studies, cryopreservation of whole brains and recovery of whole brains capable of eliciting one or more physiological functions without BBB permeabilization has been unsuccessful. While BBB permeabilization has been required for cryopreservation in the prior art, this procedure is generally undesirable because it may cause endothelial injury, edema, haemorrhage, inflammation, and non-physiological exposure of neural tissue; i.e. BBB permeabilization prevents cryopreservation of whole brains in a state of physiological functionality.In contrast, the present inventors have surprisingly found that BBB permeabilization is not necessary when using the means and methods discloser herein for cryopreservation of whole brains. Thus, it is herein preferred that cryoprotectant delivery is achieved without deliberate opening of the BBB.

[0113] As mentioned above, the method for cryopreservation of a biological sample comprises at least two major steps, a contacting step and a step of lowering the temperature.

[0114] In the method of the present invention, (a) further step(s) may be performed before, between an / or after these two major steps. As outlined further below, in a preferred embodiment, there may be a step prior to step A (i.e., prior to the alternative step (AA) or prior to the alternative step (AB)) wherein the biological sample is contacted with a „cooldown solution". This initial step prior to step A may be termed "cooldown step". Without being bound by theory, the biological sample may be cooled down to below 4°C, before contacting it with cryoprotectants to reduce toxicity.

[0115] Yet, in preferred embodiments, no further step is performed between these two major steps. In other words, in preferred embodiments, the step of lowering the temperature follows immediately the step of contacting the biological sample without any further intermediate step(s) and, preferably, without any delay in time.

[0116] Regarding the first major step of the present invention, i.e., the contacting step, the method of the present invention provides two alternative ways, i.e.,

[0117] AA. contacting the biological sample with a cryoprotectant solution comprising at least about 45 % cryoprotectant (shock loading); or

[0118] AB. contacting the biological sample sequentially with a cryoprotectant solution (first solution) comprising at least about 45 % cryoprotectant and a second solution (interleaved equilibration).

[0119] Both steps have in common that subsequent to the contacting step, there is a step of "lowering the temperature of the biological sample". This step of "lowering the temperature of the biological sample" is described in more detail further below.

[0120] In preferred embodiments, said "lowering the temperature of the biological sample"-step is immediately following the contacting step without any further steps in between.The "contacting" of the contacting step of the method for cryopreservation of a biological sample of the present invention can be performed in different ways and is not particularly limited as long as the biological sample is thereby brought into contact with / is exposed to the (cryoprotectant) solution(s).

[0121] This "contacting" can, in its simplest form, e.g., be carried out in any kind of container that is suitable to hold the biological material as well as (cryoprotectant solution and, accordingly, is not limited. Without being bound by theory, the contacting step of the method according to the present invention can, e.g., be carried out in a vessel, a test tube, a (tissue) culture dish, or in a Petri dish. For example, Fig. 4 shows a thermally controlled container that may be used in the herein disclosed method. Using such containers "contacting" may be performed by immersion / immersing the sample in the (cryoprotectant) solution(s).

[0122] In a preferred embodiment, the contacting can be performed by "immersion". "Immersion" is a passive way of contacting a sample with a cryopreservant, in contrast to, e.g., "perfusion" as described in the following.

[0123] In a preferred embodiment, the "contacting" can be performed by perfusion. The term "perfusion" is known in the art and refers, in its general terms, to the passage of some kind of fluid through the circulatory system or lymphatic system to an organ or a tissue. Perfusion may, e.g., be assisted by a pump device or pumping system that provides the biological sample (e.g., an organ or a tissue) with a certain solution at a certain flow rate. The skilled person knows how to operate corresponding device and to adjust the flow rate to allow a desired perfusion. A corresponding pump device may, e.g., be a peristaltic pump. Fig. 2 illustrates a pumping system that may be used in the herein disclosed method.

[0124] In another preferred embodiment, "contacting" can be performed by simultaneous and / or sequential "immersion" and "perfusion", as described above. In the context of the present invention, "Immersion" and "perfusion" are considered representative methods achieve "loading" of a biological sample with a cryoprotectant (solution).

[0125] Where the biological sample is a whole organ, such as a brain, kidney, liver or heart, contacting preferably comprises perfusion.In one example, where the contacting step is (AB), i.e., the contacting of the biological sample sequentially with a cryoprotectant solution comprising at least about 45 % cryoprotectant and a second solution (interleaved equilibration), said contacting may be carried out by perfusion.

[0126] The temperature at which the contacting step is to be performed is not particularly limited. Yet, it is preferred that the contacting step of the method of the present invention is effected at a temperature from above 0°C to about 20°C. Preferably, temperatures above 42°C should be avoided, if it is desired to avoid denaturation processes to occur. Thus, in more preferred embodiments, the temperature is in the range of about 1°C to about 20°C. Thus, the temperature may be about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41°C, or about 42°C. In a more preferred embodiment, the temperature may be about 4°C; i.e. the temperature of the contacting step is preferably 4°C.

[0127] In certain embodiments the sample may be pre-cooled prior to the contacting step in order to preserve the sample's physiological and / or biological function. In a preferred embodiment, the sample is pre-cooled to the range between about 1°C and 10 °C. Thus, the temperature may be about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, or about 10°C. In a more preferred embodiment, the temperature may be about 10 °C.

[0128] While it is preferred to perform the "contacting" of the contacting step of the method for cryopreservation of a biological sample of the present invention only once (single step of "contacting"), the contacting of the biological sample with the cryoprotectant solution can be repeated several times. The cryoprotectant solution may be a vitrification solution. The term "cryoprotectant solution" is meant to refer to a solution suitable for cryopreserving a biological sample. The term vitrification solution is meant to refer to a solution suitable forvitrifying a biological sample and / or for cryopreserving a biological sample by, inter alia, vitrification.

[0129] The period of time of the contacting step of the method for cryopreservation during which the biological sample is to be contacted with is not particularly limited. In preferred embodiments, the biological sample is contacted with the cryoprotectant solution for a sufficient time to allow the entire immersion of the biological sample with the cryoprotectant solution. Sufficient or successful immersion can be monitored by the skilled person by methods in the art. Without being bound by theory, for large samples loaded by immersion, e.g., organoids having about 4 mm in diameter, loading time can be up to 4 hours. Sufficient equilibration is usually indicated by sinking of the sample in the vitrification solution. Minimum loading time of a murine brain by perfusion is typically 6 minutes to permit vitrification. Full loading of brain, liver and kidney is achieved after 30 minutes of perfusion.

[0130] As regards the contacting step (AA), i.e., the contacting of the biological sample with a cryoprotectant solution comprising at least about 45 % cryoprotectant (shock loading), it is understood that the contacting of the biological sample with a cryoprotectant solution comprising at least about 45 % cryoprotectant is typically performed in a single step only. Thus, the herein disclosed method, and particularly the herein disclosed contacting step (AA), does typically not comprise contacting the biological sample in two or more subsequent steps wherein, e.g., the biological sample is contacted with different cryoprotectant solutions (having, e.g., different concentrations of the cryoprotectant) and / or wherein the biological sample is contacted with a gradient of cryoprotectant solutions. A gradient may be a continuous increase of the concentration of the cryoprotectant over time or a linear ramp of cryoprotectant concentration overtime. In a preferred aspect, the contacting step (AA) or (AB) is the sole contacting step with a cryoprotectant solution, i.e. the herein disclosed method does not comprise any other contacting step with a cryoprotectant solution, e.g. either preceding step (AA) or (AB) or subsequent to step (AA) or (AB).

[0131] In one example, the contacting step (AA) or (AB) is the sole contacting step with a cryoprotectant solution before lowering the temperature; i.e. the biological sample is not being contacted with another cryoprotectant solution comprising at least 45% w / v CPA,preferably at least about 55% w / v CPA such as 55% w / v V3 in LM5, most preferably 59% w / v CPA, such as 59% w / v V3 in LM5, before lowering the temperature of the biological sample.

[0132] Thus, in preferred embodiments, the contacting of the biological sample "solely" with a cryoprotectant solution does not include (a) step(s) of changing the cryoprotectant solution (e.g., by using one or more cryoprotectant solutions having a different concentration of the cryoprotectant than a solution comprising at least about 45 % cryoprotectant). In other words, the biological sample is to be contacted with only one cryoprotectant solution comprising at least about 45 % cryoprotectant, i.e. "solely" with the cryoprotectant solution. Thus, the cryoprotectant solution comprising at least about 45 % cryoprotectant is preferably the only cryoprotectant solution with which the biological sample is to be contacted. The herein disclosed method, and particularly the herein disclosed contacting step (AA), does typically not comprise contacting the biological sample with a cryoprotectant solution comprising less than about 45 % cryoprotectant, preferably less than about 50 % cryoprotectant.

[0133] In certain aspects, the contacting with the cryoprotectant solution comprising at least about 45 % cryoprotectant may be repeated, e.g. by repeatedly / subsequently immersing the biological in different containers comprising the solution. In a preferred aspect, if there is multiple contacting of the biological sample with the cryoprotectant solution, the identical or same cryoprotectant solution comprising at least about 45 % cryoprotectant is used, or at least a cryoprotectant solution comprising the same concentration of the cryoprotectant. "Same concentration" means for example that the solution(s) each comprise(s) 45 % cryoprotectant, or each comprise 50 % cryoprotectant, or each comprise 59 % cryoprotectant, and so on. "Identical or same cryoprotectant solution" means that the solution has the same / identical components, preferably the same / identical components in the same / identical concentration (e.g. % w / v or % v / v).

[0134] Thus, in preferred embodiments, the herein disclosed method, and particularly the contacting of the biological sample with a cryoprotectant solution does not include / comprise (a) step(s) of using (a) gradient(s) of cryoprotectant solutions, for example, does not comprise contacting (subsequently) the sample with (a) gradient(s) of cryoprotectant solutions, e.g. with at least 2, 3, 4, 5 or more cryoprotectant solutions having different or increasing concentrations of cryoprotectant, for example with cryoprotectant solutions comprising 2%, 4%, 8%, 16% and / or30% cryoprotectant e.g. at least 2, 3, 4, 5 cryoprotectant solutions comprising 2%, 4%, 8%, 16% and / or 30% cryoprotectant.

[0135] In a preferred aspect, the herein disclosed method comprises contacting the biological sample with a cryoprotectant solution comprising at least about 45 % cryoprotectant, and does not comprise contacting the biological sample with a cryoprotectant solution comprising less than the indicated concentration / amount e.g. less than about 45 % cryoprotectant, e.g. 44 %, 43 %, 42 % and so on. The same applies, mutatis mutandis, for any concentration given herein for cryoprotectants in cryoprotectant solution to be used herein. The term "at least" includes the indicated value (e.g. 45%) and any greater value (e.g. 46 %). The term "less than" does not include the indicated value (e.g. 45 %), but is meant to include any lower values, e.g. 44 %, 43 %, 42 % and so on.

[0136] In a preferred aspect, the contacting step (AA) or (AB) is the sole contacting step with a cryoprotectant solution, i.e. the herein disclosed method does not comprise any other contacting step with a cryoprotectant solution, e.g. either preceding step (AA) or (AB) or subsequent to step (AA) or (AB).

[0137] It is understood that the term "X % cryoprotectant" and the like typically refers to "% w / v" of the cryoprotectant and the like in a solution. The solution typically is an aqueous solution. Without being bound by theory, the contacting of the biological sample "solely" with a cryoprotectant solution comprising at least about 45 % cryoprotectant (shock loading) leads, in part, to the above unexpected beneficial effects of the method of the present invention over prior art cryopreservation methods because, in particular, quickly (i.e., within in short period of time and without any further additional contacting steps) and at a high concentration (i.e., at least about 45 % cryoprotectant) said cryoprotectant is contacted with the biological sample. As shown in the appended Examples, this leads, inter alia, to the beneficial effects of a rapid cryopreservation of biological samples that preserves tissue integrity and biological / physiological functionality upon sample recovery.

[0138] The term "shock loading" in terms of the present invention describes the contacting step in a way that illustrates that the contacting step of the biological sample with the cryoprotectantis rapidly and directly performed while the biological sample is exposed to highly concentrated cryoprotectant solutions (e.g. , the vitrification solution as described herein) possessing solution non-idealities with the result of achieving reduced shrinkage, toxicity, and protocol duration compared to gradual or multistep cryoprotectant loading methods.

[0139] Where the contacting according to the present invention is performed by "shock loading" (i.e. loading step (AA)), the contacting may be performed by perfusion and / or immersion.

[0140] The duration of such contacting depends on whether perfusion and / or immersion is being employed and is preferably the duration recited herein below for the duration of a single step.

[0141] In one particular example, where the biological sample is a slide or small tissue sample, the sample may be contacted by immersion. Non-limiting examples for such slides or small tissue samples comprise brain slides, myocardial slices, organoids, neurospheres and / or nerve preparations. Similarly, skin tissue samples may be contacted by immersion.

[0142] As shown in the appended non-limiting examples, the contacting time for tissue slides may be between 5 minutes and 30 minutes, which contacting may be split into three contacting intervals of about 5 minutes, the third of which may be extended until the sample fully submerges.

[0143] In another non-limiting example, the contacting time for small biological samples, such as organoids or spheroids, may be at least 5 minutes, may be between 5 and 10 minutes, preferably about 8 minutes.

[0144] In one particular example, where the biological sample is a whole organ, the sample may be contacted by perfusion. The duration of said perfusion depends on the size of the organ. In a non-limiting example, where the organ is a murine organ, the perfusion time is about 10 minutes. The skilled artisan is aware that the duration and perfusion speed may be adapted when perfusing organs from a different organism. It is within the knowledge of the skilled artisan to adapt the duration and perfusion speed accordingly.

[0145] Where the contacting according to the present invention is performed by "shock loading" (i.e. loading step (AA)), the contacting is preferably carried out using a cryoprotectant solution as described herein below and at the contacting temperatures disclosed herein.In one particular example of shock-loading by perfusion of a whole organ, such as a whole kidney, cryopreservation may be performed as follows: the organ may be perfused ex vivo with a cryopreservation solution comprising about between about 45% w / v CPA and 61% w / v CPA, such as V3, preferably between about 59% and 60% w / v CPA, such as V3 (e.g. 59%-60% w / v V3 in LM5 carrier solution) for about 10 to 20 minutes, preferably about 20 minutes, at a temperature of about 1°C to 10°C, preferably about 4°C, followed by cooling, and optionally storage and rewarming as described herein.

[0146] Where the whole organ is a whole kidney, washout is preferably performed with a hyperoncotic solution comprising a macromolecular oncotic agent, such as dextran, hydroxyethyl starch, PVP K12 or albumin, preferably dextran, for example about 10% to 30% w / v dextran, preferably about 20 percent to 25 percent w / v dextran, in a physiologically compatible carrier solution.

[0147] In another example of shock-loading by perfusion of a whole organ, such as a whole heart, cryopreservation may be performed as follows: the organ may be perfused with a cryopreservation solution comprising at least 45% w / v CPA, such as V3, preferably at least about 55% w / v CPA, such as V3, and most preferably 59% w / v CPA, such a 59% w / v V3 in LM5 for at least 5 minutes, preferably at least 10 minutes, most preferably at least 20 minutes at a temperature of at most 20 °C, preferably at most 10 °C, most preferably about 4°C, followed by cooling, and optionally storage and rewarming as described herein.

[0148] The embodiments related to calcium depletion, calcium chelation, myosin inhibition, and gradual calcium reintroduction disclosed hereinbelow are preferred for myocardium and other contractile muscle-containing tissues, particularly whole hearts and engineered cardiac tissues. Calcium depletion, calcium chelation, myosin inhibition, and gradual calcium reintroduction may optionally also be used for other tissues such as brain, kidney, or liver.

[0149] Where the organ is a whole heart, the cryopreservation solution may further comprise a chelator, preferably a chelator with selectivity for ions with two positive charges, most preferably a chelator with selectivity for ions with two positive charges which are relevant for physiological processes, such as intracellular signaling, in particular Mg2+and / or Ca2+. The skilled artisan is aware of such chelators; Non-limiting examples compriseethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(|3-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), and l,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), or pharmaceutically acceptable salts, esters, or membrane-permeable derivatives thereof. The chelator is preferably a chelator with selectivity for Ca2+over Mg2+, such as EGTA. The skilled artisan is aware of means and methods to determine whether a given compound is a chelator and to assess selectivity of a chelator for Ca2+over Mg2+for a given chelator. In one particular example, the concentration of EGTA may be between about 0.01 and 30 mM, preferably between about 1 and 5 mM, most preferably about 2.5 mM.

[0150] It is herein envisaged that the cryopreservation solution for use in the cryopresveration of a whole heart may further comprise one or more calcium-handling modulators; i.e. one or more molecules that alter the movement, storage, release, or reuptake of calcium ions (Ca2+), preferably the movement, storage, release, or reuptake of calcium ions (Ca2+) within cells. Non-limiting examples for such calcium-handling modulators include ryanodine, thapsigargin, nifedipine, verapamil, diltiazem, dantrolene, cyclopiazonic acid, and / or ruthenium red.

[0151] Such calcium-handling modulators may be used in concentrations ranging from about l / 10thof the EC50 or IC50 value to about a 5-fold excess over their EC50 / IC50 value.

[0152] Where the organ is a whole heart, the cryopreservation solution may further comprise a myosin inhibitor. Non-limiting examples for myosin inhibitors 2,3-butanedione monoxime (BDM), blebbistatin, and mavacamten

[0153] The myosin inhibitor may be a myosin ATPase inhibitor, preferably a reversible myosin ATPase inhibitor. The reversible myosin ATPase inhibitor is preferably 2,3-Butanedione monoxime (BDM). In one particular example, BDM may be used at a concentration between about 1 and 100 mM, preferably between 10 and 50 mM, most preferably about 30 mM.

[0154] In is herein envisaged that a whole heart may be temporarily stored in a suitable buffer, such as Tyrode, Krebs-Henseleit buffer or artificial cerebrospinal fluid supplemented with sucrose (sucrose aCSF; e.g. aCSF supplemented with 75 mM sucrose) prior to being perfused, as described herein above. Such temporary storage is preferably effected by immersion in sucrose aCSF. It is herein preferred that such temporary storage does not exceed 60 minutes and is preferably carried out at about 4°C.Where electrophysiological measurements are to be conducted, the heart may be perfused with a recording aCSF solution comprising 2.5 mM Ca and 2.5 mM Mg for about 20 minutes at a temperature of 20 °C. Where electrophysiological measurements have been conducted, it is desirable to induce electromechanical arrest and myocardial relaxation prior to cryoprotectant (CPA) loading by perfusing the heart with a relax aCSF solution comprising 0 mM Ca, 4.0 mM Mg2+, 2.5 mM EGTA, and 30 mM 2,3-butanedione monoxime (BDM).

[0155] Where the organ is a whole heart, it may be desirable to deplete Ca2+in the heart cells prior to contacting with the cryoprotectant solution. Ca2+depletion may be effected by contacting the heart with a solution comprising a chelator and a myosin inhibitor, such as a physiological carrier solution comprising said chelator and myosin inhibitor, preferably tyrode, Krebs-Henseleit buffer or 75mM sucrose aCSF comprising said chelator and myosin inhibitor. The concentration considerations for the chelator and myosin inhibitor disclosed herein above also apply for the Ca2+depletion solution. In one example, the Ca2+depletion solution is referred to as "relax aCSF" (aCSF comprising 0 mM Ca, 4.0 mM Mg, 2.5 mM EGTA, and 30 mM BDM). Using a Ca2+depletion solution before contacting the heart with the cryoprotectant solution of the invention allows for the recovery of a whole heart with improved viability and physiological functionality as compared to a whole heart which has not been contacted with said Ca2+depletion solution prior to contacting with the cryoprotectant solution of the invention.

[0156] Where the organ is a whole heart, the washout step may comprise perfusing the heart with a hyperosmotic washout solution, such as 25% w / v glucose in PBS for about 20 minutes at a temperature of 4°C. Where the organ is a whole heart, it may be beneficial to re-introduce Ca2+gradually in order to reduce the risk for calcium paradox injury and allow for the recovery of a heart with improved functionality compared to a heart recovered without gradual Ca2+reintroduction. The gradual reintroduction may comprise:

[0157] (i) Perfusing the heart with the relax aCSF solution (comprising 0 mM Ca, 4.0 mM Mg, 2.5 mM EGTA, and 30 mM BDM) for 20 minutes at 4°C;

[0158] (ii) perfusing the heart with an aCSF solution comprising 0 mM Ca, 4.0 mM Mg, 0 mM EGTA, and 30 mM BDM for 10 minutes;(iii) perfusing the heart with an aCSF solution comprising 0.1 mM Ca, 4.0 mM Mg, 0 mM EGTA, and 30 mM BDM for 10 minutes; and

[0159] (iv) perfusing the heart with an aCSF solution comprising 1.0 mM Ca, 3.0 mM Mg, 0 mM EGTA, and 30 mM BDM for 20 minutes.

[0160] As shown in the appended non-limiting examples, it has surprisingly been found that whole hearts can be cryopreserved using the protocols disclosed herein. In particular, whole hearts cryopreserved in this way show successful resumption of macroscopic contractile activity and functional responsiveness, indicating preservation of the myocardial syncytium.

[0161] Where the sample is a tissue sample obtained from a heart, the cryopreservation may be carried out by shock loading (AA). For example, shock-loading may be carried out using 45% w / v V3 in LM solution at 4°C, followed by 55% w / v V3 in LM solution at -20°C. Such samples may be recovered by hyperosmotic washout, for example using 25% Glucose in PBS. The time considerations disclosed herein above for shock-loading of small biological samples apply mutatis mutandis.

[0162] The cryoprotectant solution that is used in the contacting step of the method for cryopreservation of the present invention as described herein above and below is not particularly limited as long as it comprises at least about 45 % cryoprotectant.

[0163] The term "cryoprotectant" is commonly known in the art and refers to any substance used to protect biological sample from freezing damage (i.e., damage that is due to ice formation). As used herein, the term "cryoprotectant" can refer to one or more cryoprotectant substance, i.e. includes a combination of "cryoprotectant" substances. For example, a cryoprotectant solution can refer to a solution comprising one or more of the cryoprotectant substance(s), preferably comprising one or more of DMSO, ethylene glycol, acetamide, formamide and / or polyvinylpyrrolidone K12, preferably all of DMSO, ethylene glycol, formamide and / or polyvinylpyrrolidone K12.

[0164] Cryoprotectants are commonly known to operate by increasing the solute concentration in cells. However, in order to be biologically viable they should preferably easily penetrate and should preferably not be toxic to cells.Commonly used cryoprotectants are, e.g., glycols (alcohols containing at least two hydroxyl groups), such as ethylene glycol, acetamide, propylene glycol, glycerol, dimethyl sulfoxide (DMSO) or trehalose. Preferred cryoprotectants that can be used in the method of cryopreservation of the present invention are described in more detail further below.

[0165] A preferred cryoprotectant solution (to be used herein) comprises the cryoprotectant(s) DMSO, ethylene glycol, acetamide, formamide and / or polyvinylpyrrolidone K12, preferably all of DMSO, ethylene glycol, formamide and / or polyvinylpyrrolidone K12. In a preferred embodiment the cryoprotectant solution (to be used herein) comprises the cryoprotectant(s) DMSO, ethylene glycol, formamide and / or polyvinylpyrrolidone K12 as sole cryoprotectants, preferably all of DMSO, ethylene glycol, formamide and / or polyvinylpyrrolidone K12 as sole cryoprotectants, i.e. does not comprise any other cryoprotectants.

[0166] For example, the cryoprotectant solution (to be used herein) can comprise about 20-25 % w / v DMSO), e.g. 20, 21, 22, 23, 24, 25 % DMSO, 13-18 % EG, e.g. 13, 14, 15, 16, 17 or 18 % EG, 10-15 % w / v formamide, e.g. 10, 11, 12, 13, 14, 15 % formamide and / or 5-10 % w / v polyvinylpyrrolidone K12 e.g. 5, 6, 7, 8, 9, 10 % polyvinylpyrrolidone K12, preferably all of DMSO, ethylene glycol, formamide and / or polyvinylpyrrolidone K12, in particular when provided that the cryoprotectant solution comprises in total any of the herein disclosed values of the cryoprotectant, e.g. about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85% or about 90% of the cryoprotectant, preferably 57 %, 58 %, 59 %, 60 %, or 61 % of the cryoprotectant, most preferably 59 % of the cryoprotectant. It is understood that the percentages herein refer usually to "w / v".

[0167] It is understood that the cryoprotectant solution according to the present invention may be formulated in a suitable physiological carrier solution. Non-limiting examples for such physiological carrier solutions comprise normal saline (0.9% NaCI solution), half-normal saline (0.45% NaCI solution), Ringer's solution, buffered saline (e.g. PBS) or LM5 buffer. In one particular example, the cryoprotectant is formulated in LM5 buffer.The cryoprotectant solution may comprise between about 45 % and 70 % w / v cell-permeable cryoprotectants. These cryoprotectants may be selected from the group comprising dimethyl sulfoxide, ethylene glycol, and formamide The solution may optionally further comprise between about 0% and 10% w / v polyvinylpyrrolidone (PVP), such as PVP K12.

[0168] In one example, the cryoprotectant solution according to the present invention may be a cryoprotectant solution comprising between about 48 % w / v and 68 % w / v of cryoprotectant, e.g. 48 %, 49 %, 50 %, 51 %, 52 %, 53 %, 54 %, 55 %, 56 %, 57, %, 58 %, 59 %, 60 %, 61 %, 62 %, 63 %, 64 %, 65 %, 66 %, 67 %, or 68 %, comprising or consisting of about 20-25 % w / v DMSO, 13-18 % w / v EG, 10-15 % w / v formamide, and 5-10 % w / v PVP K12 in a suitable physiological carrier solution, such as LM5; such as a cryoprotectant solution comprising between about 45 % w / v and 55 % w / v of cryoprotectant, or a cryoprotectant solution comprising between about 52 % w / v and 64 % w / v of cryoprotectant, comprising or consisting of about 21-24 % w / v DMSO, 14-17 % w / v EG, 11-14 % w / v formamide, and 6-9 % w / v PVP K12, and in particular a cryoprotectant solution comprising between about 56 % w / v and 64 % w / v of cryoprotectant, comprising or consisting of about 22-24 % w / v DMSO, 16-17 % w / v EG, 12-14 % w / v formamide, and 6-9 % w / v PVP K12.

[0169] In a preferred embodiment, the cryoprotectant solution of the present invention comprises about 59% w / v cryoprotectant.

[0170] In a preferred embodiment, the cryoprotectant solution (to be used herein) comprises:

[0171] dimethylsulfoxide (DMSO) About 22 % w / v

[0172] ethylene glycol (EG) About 17 % w / v

[0173] formamide About 13 % w / v polyvinylpyrrolidone K12 About 7% w / vIn a more preferred embodiment, the cryoprotectant solution (to be used herein) comprises:

[0174] dimethylsulfoxide (DMSO) 22.3% w / v

[0175] ethylene glycol (EG) 16.84% w / v

[0176] formamide 12.86% w / v

[0177] polyvinylpyrrolidone K12 7% w / v

[0178] The cryoprotectant solution of the more preferred embodiment may also be referred to as V3 or 59% V3; i.e. V3 comprising 59% w / v cryoprotectants

[0179] Preferably, the cryoprotectant solution comprises cryoprotectant(s) (CPA), for example about 1-10 M CPA, such as about 1-10 M, 2-9 M, 3-9 M, 4-9 M, 5-9 M, 6-9 M, or7-9M. In one example the cryoprotectant solution comprises about 8 M(CPA), e.g. 7.5 M, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 M cryoprotectant, preferably 8.42 M CPA.

[0180] The CPA may be one or more cell-permeable CPAs, one or more non-cell-permeable CPAs or a mix thereof, as disclosed hereinabove in more detail. Preferably, the cryoprotectant solution comprises about 8 M permeable CPA, preferably 8.42 M permeable CPA.

[0181] It is noted that cryopreservation according to the method disclosed herein may also be achieved using cryopreservation solutions comprising or consisting of 61% Ethyleneglycol, 30% Ethylene glycol or 30% DMSO in a physiologically acceptable carrier solution, such as LM5.

[0182] The cryoprotectant solution (to be used herein) may be carrier solution as described herein below, preferably a LM5 carrier solution. In other words, the cryoprotectant(s) may be dissolved or formulated in a carrier solution, preferably a LM5 carrier solution.

[0183] As mentioned, the cryoprotectant solution that is used in the contacting step of the method for cryopreservation of the present invention as described herein above and below is not particularly limited as long as it comprises at least about 45 % cryoprotectant.

[0184] It is understood that in the context of the present invention, specifications such as "mM" or "% (w / w)" or "%" refer to the total volume or total weight of the resulting liquid aqueous solution.The term "about" (and, similarly, the term "nearly") as used herein in the context of the entire invention and its disclosure preferably refers to ±10% of the indicated numerical value, more preferably to ±5%, ±4% ±3% ±2% or ±1% of the indicated numerical value, and in particular to the exact numerical value indicated. For example, if the value is 45 %, the term "about 45%" can refer to 40.5 % to 49.5 %, if "about" refers to ±10% of the indicated numerical value, and so on. The same applies, mutatis mutandis, for any concentration given herein for cryoprotectants in cryoprotectant solution to be used herein. If the term "about" is used in connection with the endpoints of a range, it preferably refers to the range from the lower endpoint -10% of its indicated numerical value to the upper endpoint +10% of its indicated numerical value, more preferably to the range from the lower endpoint -5% to the upper endpoint +5%, and even more preferably to the range defined by the exact numerical values of the lower endpoint and the upper endpoint. If the term "about" is used in connection with the end point of an open-ended range, it preferably refers to the corresponding range starting from the lower endpoint -10% or from the upper endpoint +10%, more preferably to the range starting from the lower endpoint -5% or from the upper endpoint +5%, and even more preferably to the open-ended range defined by the exact numerical value of the corresponding endpoint.

[0185] In preferred embodiments, the concentration of the cryoprotectant in the cryoprotectant solution may be (as lower limit) at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, or at least about 60%.

[0186] In a more preferred embodiment, the concentration of the cryoprotectant in the cryoprotectant solution may be at least about 59%. In another more preferred embodiment, the concentration of the cryoprotectant in the cryoprotectant solution may be at least about 57%. It goes without saying that the cryoprotectant solution can comprise any of the herein disclosed values. For example, when it is disclosed that the cryoprotectant solution can comprise "at least about 50 % cryoprotectant" it is understood that the cryoprotectant solution can comprise "about 50 % cryoprotectant". Thus, the cryoprotectant solution can comprise about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 60%, about 65%,about 70%, about 75%, about 80%, about 85% or about 90% cryoprotectant. Preferably, the cryoprotectant solution comprises 57 %, 58 %, 59 %, 60 %, or 61 % cryoprotectant, most preferably 59 %. The cryoprotection solution can comprise ranges between any of the above disclosed values, e.g. between about 45% to about 90% cryoprotectant, for example between about 50% to about 90% cryoprotectant, preferably between 55 % to between about 70%, more preferably between 57 % to between about 65 %, even more preferably between 57 % to between about 61 %. It is understood that the percentages herein refer usually to "w / v".

[0187] In further preferred embodiments, the concentration of the cryoprotectant in the cryoprotectant solution may be (as upper limit) at most about 65%, at most about 70%, at most about 75%, at most about 80%, at most about 85% or at most about 90%.

[0188] The solution of the cryoprotectant solution is preferably an aqueous solution.

[0189] Optionally, the aqueous solution comprises a buffering agent, preferably at a concentration of about 0.1 mM to about 100 mM.

[0190] Moreover, the aqueous solution has a certain pH, i.e., a "nearly physiological pH" as defined in more detail further below.

[0191] Examples of buffering agents that will control the pH in this range include glycine, acetate, phosphate, succinate, gluconate, glutamate, histidine, citrate, glycylglycine, aspartate, MES (2-( / V-morpholino)ethanesulfonic acid) and TRIS (Tr / s(hydroxymethyl)aminomethane). Preferred buffering agents include glycine, histidine, glutamate, succinate, phosphate, acetate, gluconate and aspartate.

[0192] The term "buffering agent" as used herein also includes the salts of the above buffering agents, such as the hydrochloride salt, the acetate salt, the phosphate salt and the sulfate salt. As stated above the aqueous solution for use as defined herein, may optionally comprise the buffering agent at a concentration of about ImM to about 50mM. Thus, the concentration of the buffering agent in the aqueous solution may be about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15mM, about 16mM, about 17mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29mM, about 30 mM, about 31 mM, about 32 mM, about 33 mM, about 34 mM, about 35 mM, about 36 mM, about 37 mM, about 38 mM, about 39 mM, about 40 mM, about 41 mM, about 42 mM, about 43 mM, about 44 mM, about 45 mM, about 46 mM, about 47 mM, about 48 mM, about 49 mM or about 50 mM. The concentration of the buffering agent in the aqueous solution may preferably be about 5mM to about 40 mM, about 8 mM to about 20 mM, particularly about lOmM.

[0193] As used in the present invention, the terms "comprising", "which comprises" or equivalent expressions encompass three alternatives, namely (i) "comprising", (ii) "consisting essentially of" and (iii) "consisting of".

[0194] It is understood that in the context of the present invention, specifications such as "mM" or "% (w / v)" refer to the total volume or total weight of the resulting liquid aqueous composition / solution.

[0195] The term "water" as used herein preferably refers to distilled, demineralized water, (highly) purified water or aqua ad iniectabilia (water for injections). The use of aqua ad iniectabilia is particularly preferred.

[0196] It is understood that the term "liquid" relates to a composition that is liquid at about 20°C and about 1,01325 bar = 1 atm.

[0197] It is understood that the term "aqueous composition" or "aqueous solution" refers to a composition essentially comprising water as the solvent (i.e., more than 80% (w / v) of the total solvent). Preferably the composition is essentially free of any organic solvents, wherein essentially free means less than <l%(w / v). More preferably the composition is free of any organic solvents. Most preferably the composition is an aqueous solution.

[0198] A "physiological pH value" in general terms is a pH value or a range of pH values wherein a normal cell or component of a cell functions. As an example, in the absence of pathological states, the pH of the human body ranges between 7.35 to 7.45, with the average at 7.40.In the present invention, the term "physiological pH value" is, however, not particularly limited to this narrow range. Other pH values or pH value ranges may be used in the context of the present invention.

[0199] In preferred embodiments, the "physiological pH value" in accordance with the present invention, preferably in an aqueous solution as defined herein, has a pH in the range of about pH 4.5 to about pH 8.5. Thus, the pH may be about 4.5, about 4.6. about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.3, about 8.4, or about 8.5.

[0200] Preferably the pH is in the range of from about pH 4.5 to pH 8.5, preferably in the range of pH 5.0 to pH 7.6, more preferably in the range of pH 6.7 to 7.6 or pH 6.7 to pH 7.4. In an even more preferred embodiment, the pH is in the range from 6.5 to 7.5. In a particularly preferred embodiment, the pH is a pH of 7.3.

[0201] As regards the contacting step (AB), i.e., the contacting of the biological sample sequentially with a cryoprotectant solution comprising at least about 45 % cryoprotectant and a second solution (interleaved equilibration), the exposure to cryoprotectant solutions is intermittently alternated with exposure to a second solution. The second solution does typically not comprise a cryoprotectant (e.g. one or more of the cryoprotectants described herein). For example, the second solution may be a carrier solution, e.g. an isotonic carrier solutions or lower concentration cryoprotectant solution, thereby further reducing shrinkage and swelling of said biological samples.

[0202] Thus, in one example of contacting step (AB), the biological sample is being contacted with only one cryoprotectant solution comprising at least about 45% cryoprotectant and a second solution comprising less than 20% cryoprotectant, preferably less than 10% cryoprotectant. In one particular example of contacting step (AB), the cryoprotectant solution comprising at least about 45% cryoprotectant is 59% w / v V3 and the second solution comprising less than 20% cryoprotectant is LM5 or 8% dextran in LM5.In the context of the present invention one such alternation is considered a cycle. The number of steps within one such cycle is not particularly limited and may entail contacting the sample with two or more different solutions. For example, the sample may be brought into contact with 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different solutions, preferably 2, 3, or 4 solutions, most preferably 2 or 3 solutions.

[0203] The number of cycles, a sample may be subjected to, is not so limited. Preferably, a sample is subjected to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more cycles, more preferably 5, 7, 10 or 12 cycles, most preferably 7 cycles.

[0204] In one example, the number of cycles may also be represented as a fraction of cycles. For example, where a cycle entails contacting the sample with two different solutions, the application of each solution may be denoted as half a cycle. The same considerations apply to cycles that entail contacting the sample with three, four, five of more different solutions. In one particular example, the number of cycles is 1.5 cycles; i.e. the sample is contacted with a first solution, followed by a second solution, followed by a third solution.

[0205] The duration of steps in any such cycles is not limited. For example, the duration of a step may range from about 5 sec to about 60 minutes. Preferably, the duration of a step is about 5 sec, 6 sec, 7 sec, 8 sec, 9 sec, 10 sec, 15 sec, 20 sec, 25 sec, 30 sec, 35 sec, 40 sec, 45 sec, 50 sec, 55 sec, 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, or 60 min,

[0206] More preferably 30 sec, 1 min, 2min, or 5 min, most preferably 1 min.

[0207] Different steps of a cycle may last for identical or different time intervals.

[0208] For example, in one aspect, interleaved equilibration may be performed as follows: Contacting may be carried out using e.g. 3 to 7 cycles, preferably 5 cycles, each cycle consisting of contacting with a first cryoprotectant solution (e.g. about 59% V3 in LM5 carrier solution) for about 60 seconds followed by contacting with a second solution (e.g. about 8% Dextran in LM5 carrier solution) for about 60 seconds, followed by 1 to 3, preferably 2 cycles, each cycle consisting of contacting with a cryoprotectant solution (e.g. 59% V3 in LM5 carrier solution) for about 60 seconds followed by contacting with a second cryoprotectant solution (wherein the second cryoprotectant solution can comprise a lower % of the cryoprotectant comparedto the first solution, e.g. about 30% V3 in LM5 carrier solution) for about 60 seconds, and final contacting with a cryoprotectant solution (the final or third cryoprotectant solution can comprise the same % of the cryoprotectant as the first solution or be the same / identical to the first solution, e.g. about 59% V3 in LM5 carrier solution) for about 8 minutes.

[0209] In one particular example of interleaved equilibration by perfusion of a whole organ, such as a whole brain, cryopreservation may be performed as follows: Contacting may be carried out using e.g. 1.5 cycles; i.e. contacting the biological sample with a first cryoprotectant solution (e.g. about 45% w / v CPA in LM5 carrier solution, such as 45% V3 in LM5 carrier solution or about 61% w / v CPA in LM5 carrier solution, such as 61% V3 in LM5 carrier solution, preferably about 59% w / v CPA in LM5 carrier solution, such as 59% V3 in LM5 carrier solution) for about 5 to 10 minutes, preferably about 8 minutes followed by contacting with a second solution (e.g. LM5 carrier solution) for about 1 to 10 minutes, preferably about 3 minutes, followed by contacting the biological sample with a cryoprotectant solution (e.g. about 45% w / v CPA in LM5 carrier solution, such as 45% V3 in LM5 carrier solution or about 61% w / v CPA in LM5 carrier solution, such as 61% V3 in LM5 carrier solution, preferably about 59% w / v CPA in LM5 carrier solution, such as 59% V3 in LM5 carrier solution) for about 20 to 30 minutes, preferably about 25 minutes.

[0210] The contacting steps of this particular example are preferably carried out at 4°C.

[0211] In this context, the second cryoprotectant solution (to be used herein) can comprise: dimethylsulfoxide (DMSO) About 22 % w / v

[0212] ethylene glycol (EG) About 17 % w / v

[0213] formamide About 13 % w / v polyvinylpyrrolidone K12 About 7% w / v

[0214] In a more preferred embodiment, the cryoprotectant solution (to be used herein) comprises: dimethylsulfoxide (DMSO) 22.3% w / v

[0215] ethylene glycol (EG) 16.84% w / v

[0216] formamide 12.86% w / v

[0217] polyvinylpyrrolidone K12 7% w / vThe 30% V3 solution with additional 300 mM Mannitol comprises:

[0218] dimethylsulfoxide (DMSO) 11.15% w / v

[0219] ethylene glycol (EG) 8.42% w / v

[0220] formamide 6.43% w / v

[0221] polyvinylpyrrolidone K12 4% w / v

[0222] A carrier solution in terms of the present invention is any solvent, solution, buffer, or mixture thereof suitable to formulate a stable cryoprotectant solution. Suitable carrier solutions are known in the art.

[0223] The carrier solution preferably comprises one or more of glucose mannitol, lactose, KCI, K2HPO4, glutathione, adenine and / or NaHCCh, preferably all of these components.

[0224] In preferred embodiments, the carrier solution may have a composition as shown in the following Table.

[0225] Carrier solution LM5.

[0226] chemical concentration

[0227] glucose About 90 mM

[0228] mannitol About 45 mM

[0229] lactose About 45 mM

[0230] KCI About 8.2 mM

[0231] K2HPO4 About 7.2 mM

[0232] glutathione About 5 mM

[0233] adenine About 1 mM

[0234] NaHCCh About 10 mM

[0235] Regarding the second major step which is common to both alternative steps (AA) and (AB), i.e., the step of lowering the temperature, the biological sample, after the contacting step, is exposed to a low temperature. This lowering of the temperature is also referred to herein as a cooling step.

[0236] Thus, both alternative steps (AA) and (AB) have in common that subsequent to the contacting step, there is a step of "lowering the temperature of the biological sample".In preferred embodiments, said "lowering the temperature of the biological sample"-step is immediately following the contacting step without any further steps in between.

[0237] In preferred embodiments, the lowering the temperature / cooling is performed as rapid and uniform as possible to prevent crystallization and cracking, respectively.

[0238] As an example, this can be performed by the transfer on copper block in liquid nitrogen as described in [3], In one embodiment, lowering the temperature may be achieved by mounting a sample onto a mesh or membrane and transferring said membrane onto a copper block precooled with liquid nitrogen to about -196°C. For consistent results it may be advisable to keep the copper block partially submerged in liquid nitrogen to maintain the target temperature.

[0239] In case of organs, there are multiple options for lowering the temperature available which are all known to the skilled person. As non-limiting examples, conductive cooling or volumetric cooling with refrigerant-perfusion can be used (see, e.g., Alexander German, Perfusion quenching, Cryobiology, Volume 117,2024).

[0240] Subsequently, for rewarming, conduction, refrigerant perfusion, nanowarming, dielectric warming or HIFUS can be employed.

[0241] Without being bound by theory, the temperature should be lowered below the glass transition temperature of the employed vitrification solution, ranging from -105 to -140°C.

[0242] The glass-liquid transition, or glass transition, is known in the art and is commonly understood as the gradual and reversible transition in amorphous materials (or in amorphous regions within semicrystalline materials) from a hard and relatively brittle "glassy" state into a viscous or rubbery state as the temperature is increased. An amorphous solid that exhibits a glass transition is called a glass. The reverse transition, achieved by supercooling a viscous liquid into the glass state, is called vitrification.

[0243] The step of lowering the temperature can, thus, be performed to achieve a temperature ranging from -105 to -140°C, preferably below about -130°C.

[0244] Thus, in more preferred embodiments, the temperature may be below about -105°C, below about -110°C, below about -115°C, below about -120°C, below about -125°C, below about -130°C, below about -135°C, below about -140°C, below about -145°C, below about -150°C, below about -155°C, below about -160°C, below about -160°C, or below about -165°C.In one example, the step of lowering the temperature may be carried out by immersion in a further solution. Said further solution for use in lowering the temperature may be a further cryoprotectant solution. Where the solution for use in lowering the temperature is a cryoprotectant solution, said solution may comprise the same or a higher percentage of cryoprotectants as the cryoprotectant solution used in the contacting step. In one particular example, the solution for use in lowering the temperature comprises between 55% w / v and 70% w / v cryoprotectant, preferably between about 60% w / v and 65% w / v cryoprotectant. In a non-limiting example, the solution for use in lowering the temperature comprises 65% V3. Lowering the temperature may involve one or more steps, such as a first step wherein the temperature of the sample is lowered to < 0°C, preferably below -15°C. In one example, said first step of lowering the temperature lowers the temperature of the sample to about -20°C. Said lowering may be followed by a further step of lowering the temperature, e.g. by immersing the sample into liquid isopentane, preferably liquid isopentane at a temperature between about -150°C and -130°C.

[0245] In a preferred embodiment, there may be a step prior to step A (i.e., prior to the alternative step AA or prior to the alternative step AB as described above) wherein the biological sample is contacted with a „cooldown solution". This initial step prior to step A may be termed "cooldown step". The temperature of this cooldown step may be effected at a temperature from above 0°C to about 20°C. Thus, in more preferred embodiments, the temperature is in the range of about 1°C to about 20°C. Thus, the temperature may be about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, bout 15°C, about 16°C, about 17°C, about 18°C, about 19°C, or about 20°C.

[0246] In a more preferred embodiment, the temperature may be about 4°C.

[0247] In a preferred embodiment, said "step prior to step A" is immediately followed by the contacting step, i.e. without any further steps in between.

[0248] The composition of the "cooldown solution" is not particularly limited and may be any physiological solution known to the skilled person. As an Example, 4°C PBS with 3% Dextran may be used.In a preferred embodiment, the present invention relates to a method for cryopreservation of a biological sample as defined herein, wherein the method further comprises a step B of storing the biological sample.

[0249] Accordingly, in a preferred embodiment, subsequent to the step AA or AB (i.e., the contacting the biological sample with a cryoprotectant solution comprising at least about 45 % cryoprotectant (shock loading) as described herein; or the contacting the biological sample sequentially with a cryoprotectant solution comprising at least about 45 % cryoprotectant and a second solution (interleaved equilibration) which are both followed by a step of "lowering the temperature of the biological sample" as described herein, the biological sample is stored for a certain period of time.

[0250] Thus, once obtained the cryopreserved biological sample after the lowering the temperature step as described herein, the biological sample can be stored for an extended period of time until the biological sample is needed for further purposes and, for this, is warmed up again and / or the cryoprotectant solution is removed from the biological sample.

[0251] The warming up and / or the removal of the cryoprotectant solution from the biological sample is described in more detail further below.

[0252] The duration / period of time of the storage duration is not particularly limited.

[0253] For storage, the biological sample should, in preferred embodiments, be stored at a temperature below the glass transition temperature of the employed cryoprotectant solution. Further preferred temperatures for storage can be chosen as already described for the "lowering the temperature step". These preferred temperatures and temperature ranges apply, mutatis mutandis, to the storage step B.

[0254] In preferred embodiments, the biological sample may be stored below -150°C.

[0255] In another preferred embodiment, the biological sample may be stored 15°C below the glass transition temperature of the employed cryoprotectant solution.

[0256] Generally, in a preferred embodiment, the herein described steps immediately follow each other, i.e. without any further steps in between. For example, the "storing step" can immediately follow the "lowering the temperature step". The "warming up step" can immediately follow the "storing step". The "wash out step" " can immediately follow the "warming up step", and so on.In a preferred embodiment, the present invention relates to a method for cryopreservation of a biological sample as defined herein, wherein the method further comprises a step C of removing the cryoprotectant solution from the biological sample and, optionally, recovering the biological sample.

[0257] The removal of the cryoprotectant solution from the biological sample is described in more detail further below.

[0258] Prior to step C, there may be an additional step of rewarming said biological sample.

[0259] In preferred embodiments, warming is performed as rapid and uniform as possible to prevent crystallization and cracking, respectively. Without being bound by theory, the warming may be performed by immersion in -10°C cryoprotectant as described in

[0015] . In a preferred embodiment, rewarming is carried out by immersion in -10 °C cryoprotectant solution. More preferably said rewarming solution is based on the vitrification solution used, but characterized by a cryoprotectant concentration that is not higher than the one used for vitrification or storage.

[0260] In one particular example, the rewarming solution may be a vitrification solution comprising between about 50% w / v and 70% w / v cryoprotectant, such as 59% V3 or 65% V3. The sample and rewarming solution may be moved in relation to each other to ensure uniform temperature dissipation. In one particular example, the rewarming solution may be stirred. The rewarming solution may have a temperature above 0°C, preferably between about 4°C and about 37°C, such as between about 4°C and 20°C.

[0261] As a further example, laser-warming may be used which has, in particular, been proven to be useful for small samples. In case of organs several options are available like, e.g., conduction, refrigerant perfusion, nanowarming, dielectric warming and / or HIFUS.

[0262] In a preferred embodiment, the present invention relates to a method for cryopreservation of a biological sample as defined herein, wherein said cryoprotectant solution comprises at least about 45% w / v cryoprotectant selected from the group consisting of dimethyl sulfoxide, ethylene glycol, propylene glycol, glycerol, formamide, N-methylformamide,dimethylformamide, 3-methoxy-l,2-propanediol, and any combination thereof, optionally supplemented with non-cell-permeating sugars or macromolecules.

[0263] In a preferred embodiment, the present invention relates to a method for cryopreservation of a biological sample as defined herein, wherein cryoprotectant solution comprises 61% w / v ethylene glycol or 59% w / v "V3".

[0264] The solution "V3" that is referred to herein and, in particular, also used in the Examples of the present invention has the following composition as shown in the following Table.

[0265] Modified standard vitrification solution (59% w / v V3 = 8.42 M permeable CPA*). chemical Concentration

[0266] dimethylsulfoxide (DMSO) 22.3% w / v

[0267] ethylene glycol (EG) 16.84% w / v

[0268] formamide 12.86% w / v

[0269] polyvinylpyrrolidone K12 7% w / v

[0270] *As used herein, 8.42 M permeable CPA relates to the amount of permeable CPAs in V3; i.e. DMSO, EG and formamide. PVP K12 is a non-permeable CPA, and its molarity is negligible here.

[0271] In a preferred embodiment, the present invention relates to a method for cryopreservation of a biological sample as defined herein, wherein the cryoprotectants are removed from said biological sample by contacting the sample with hyperoncotic solutions (hyperoncotic washout), preferably with colloidal solutions of oncotic pressure greater than physiological levels.

[0272] As used herein, the term "hyperoncotic washout solution" refers to a colloidal solution (; i.e. a solution comprising one or more macromolecular oncotic agents) having an oncotic pressure greater than physiological levels, which is employed for the removal of cryoprotectant agents from a biological sample following cryopreservation by vitrification. The hyperoncotic washout solution is characterized by its ability to significantly reduce cellular and tissue swelling during cryoprotectant removal, compared to conventional osmotic buffers or isotonic solutions.

[0273] As used herein, the term "macromolecular oncotic agent" refers to a water-soluble polymeric or proteinaceous solute that remains predominantly extracellular during the washout intervaland, at the concentration used, raises oncotic pressure so as to oppose tissue swelling during cryoprotectant removal. Non-limiting examples include dextran, polyvinylpyrrolidone, albumin, gelatin, and hydroxyethyl starch.

[0274] A given substance, such as dextran or polyvinylpyrrolidone, may function as a cryoprotective additive when present in a vitrification solution and as a macromolecular oncotic agent when present in a washout solution; the skilled person understands that the designation depends on the stage of use and principal function.

[0275] In preferred embodiments, the hyperoncotic washout solution is a colloidal solution comprising about 10% to 30% w / v dextran, or an equivalent concentration of another colloid, in a physiologically compatible carrier solution.

[0276] In other examples, the washout solution may comprise 10-30% PVP, such as PVP K12.

[0277] It is herein envisaged that a hyperosmotic washout solution may be used instead of a hyperoncotic washout solution to effect the removal of cryoprotectant agents from a biological sample following cryopreservation by vitrification and significantly reduce cellular and tissue swelling during cryoprotectant removal. In one example, the hyperosmotic washout solution may comprise a non-cell permeable carbohydrate, such as glucose, trehalose, sucrose and / or mannitol. The concentration of said non-cell permeable carbohydrate is not particularly limited, as long as it is above 5% and below the concentration at which said carbohydrate becomes toxic and the solution remains a true solution without precipitation and acceptable viscosity. The skilled artisan is aware of means and methods to determine such concentration ranges and their toxicity. Where the carbohydrate is glucose, the hyperosmotic solution may comprise between 10-30% w / v glucose or an equivalent concentration of another osmotic agent, in a physiologically compatible carrier solution. In one particular example, the hyperosmotic washout solution comprises 25% w / v glucose in a physiologically acceptable carrier solution. The physiologically acceptable carrier solution may be any physiologically acceptable solution known in the art, such as LM5 buffer, PBS, normal saline (0.9% NaCI solution), Ringer's solution, half-normal saline (0.45% NaCI solution), or the like. In one particular example, the physiologically acceptable carrier solution may be PBS.Where the washout solution is a hyperosmotic washout solution, such as 25% glucose, between 1 and 20%, preferably 1 to 10% more preferably 5 to 10%, such as 5%, 6%, 7%, 8%, 9% or 10% of a colloid forming agent, such as dextran and / or PVP K12, may be added to provide "oncotic support".

[0278] Where the sample to be contacted with the washout solution according to the present invention is a whole organ, such as a whole heart or a whole brain, hyperosmotic washout solutions are preferred due to their lower viscosity compared to hyperoncotic washout solutions that achieve a comparable reduction in tissue swelling.

[0279] Where the sample to be contacted with the washout solution according to the present invention is a whole organ, such as a whole kidney, it is preferred that a hyperoncotic washout solution comprising colloids greater than the renal filtration threshold (approx. 60-70 kDa) are used.

[0280] The hyperoncotic washout solution is typically used at hypothermic temperatures (e.g., 4°C) and may be applied by perfusion, immersion, or superperfusion, with flow rates adjusted according to the viscosity of the solution. The solution may be used alone or in sequential steps with varying concentrations to optimize cryoprotectant removal and minimize tissue edema. The superior effectiveness of hyperoncotic washout solutions compared to small-molecule osmotic buffers is attributed to reduced permeability, increased viscosity, steric hindrance, excluded volume, and macromolecular crowding effects, resulting in improved preservation of tissue integrity and viability during post-cryopreservation recovery.

[0281] As outlined above, once obtained the cryopreserved biological sample after the lowering the temperature step as described herein, the biological sample can be stored for an extended period of time until the biological sample is needed for further purposes. In order to prepare the biological sample for further purposes, the biological sample is warmed up again and / or the cryoprotectant solution is removed from the biological sample.

[0282] Thus, in a preferred embodiment, during the recovery step, the biological sample may be warmed up again. This warming up has already been described above.

[0283] In one example, said warming may be performed by contacting the sample with a hyperoncotic washout solution at a temperature above 0°C, preferably between 4°C and 37°C. Atemperature between 4°C and 10°C is particularly preferred, such as e.g. 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C.

[0284] In other preferred embodiments, the cryoprotectants can be removed from said biological sample by different means and methods known to the skilled person.

[0285] In a more preferred embodiment, as also illustrated in the Examples of the present application, the cryoprotectants can be removed from said biological sample by contacting the sample with hyperoncotic solutions. This step of contacting the biological sample with a hyperoncotic solution is also termed herein "hyperoncotic washout".

[0286] The term "oncotic" refers to the osmotic pressure caused by the presence of colloids (which is a mixture in which one substance consisting of microscopically dispersed insoluble particles is suspended throughout another substance) while the term "hyperoncotic" refers to a higher than normal oncotic pressure.

[0287] This step of contacting the biological sample with a hyperoncotic solution ("hyperoncotic washout") is particularly preferred in methods of cryoprotection of a biological sample of the present invention wherein the biological sample is a brain or a tissue derived from a brain.

[0288] In further more preferred embodiments, the step of contacting the biological sample is performed with colloidal solutions of oncotic pressure greater than physiological levels.

[0289] The term "colloidal suspension" is known in the art and refers to the overall mixture of colloids while a colloid is a mixture in which one substance consisting of microscopically dispersed insoluble particles is suspended throughout another substance. A colloid has a dispersed phase (the suspended particles) and a continuous phase (the medium of suspension).

[0290] Under physiological conditions, the oncotic pressure depends on a variety of parameters, including membrane permeability and concentration gradients, which may differ between individuals and / or tissues. For example, the physiological oncotic pressure in a capillary may range from 20 to 40 mmHg, but significantly higher values may be found in tissues with high concentration gradients, such as renal tissues. Within tissues, the physiological oncotic pressure is about 5 mmHg.The nature of the colloid and / or the colloid solution is not particularly limited. In the context of the present invention any bio-compatible colloidal or colloid-forming agent may be employed, including but not limited to proteins, peptides, or sugars. As examples, albumin, dextran, gelatin, starch, casein, sodium carboxymethylcellulose (NaCMC), or hydroxyethyl starch (HES) solutions may be used. As further examples, Polyvinylpyrrolidone (PVP), Polyethylene glycol (PEG), Polyvinyl alcohol (PVA), Polygeline, Gelofusine, Chitosan, Collagen, Liposome-based colloids, Hydroxypropyl methylcellulose (HPMC), Polysucrose (Ficoll®), Polyamidoamine (PAMAM) dendrimers, Methylcellulose, Pullulan, Alginate or Carrageenan may be used.

[0291] In one particular example, the colloidal solution (i.e. the solution comprising one or more macromolecular oncotic agents) comprises dextran or PVP; i.e. the hyperoncotic washout solution comprises dextran and / or PVP, such as PVP K12.

[0292] In a preferred embodiment, the present invention relates to a method for cryopreservation of a biological sample as defined herein, wherein the cryoprotectants are removed from said biological sample by contacting the sample with said colloidal solutions of oncotic pressure greater than physiological levels. The colloidal solution can, for example, comprise about 10% to 30% w / v dextran, for example 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 % w / v dextran.

[0293] While dextran is preferred, also other polysaccharides, also termed polycarbohydrates, may be used. Any polysaccharide, i.e., any long-chain polymeric carbohydrate (which is composed of monosaccharide units bound together by glycosidic linkages) may be used. Non-limiting examples of polysaccharides are starch, glycogen and galactogen.

[0294] It is envisaged that contacting the sample with said colloidal solution(s) may be performed as a single step (e.g. contacting with solely one colloidal solution) or as multiple steps (e.g. contacting with two or more different colloidal solution, e.g. colloidal solutions having a different oncotic pressure. For example, the sample may be contacted with a first hyperoncotic solution comprising for example about 30 % w / v dextran, followed by contacting with a second hyperoncotic solution comprising a lower % of dextran, e.g. about 10 % w / v dextran.In one example, the colloidal solution (i.e. the hyperoncotic washout solution) comprises 30% w / v dextran. In another example, the colloidal solution (i.e. the hyperoncotic washout solution) comprises 10% w / v dextran.

[0295] Alternatively, the washout solution may be a hyperosmotic washout solution.

[0296] Where the washout solution is a hyperosmotic washout solution, the solution may comprise 10-30% w / v glucose, preferably 25% w / v glucose; i.e. in one example, the washout solution may comprise 10-30% w / v glucose, preferably 25% w / v glucose. In one particular example, the washout solution comprises 25% glucose in a suitable carrier solution, such as LM5 or PBS.

[0297] Without being bound by theory, as a non-limiting example, the hyperonoctic washout step may be performed 4 min 30% Dextran with 5% perfusion flow, 10 min 10% Dextran with 25% perfusion flow, all steps at 4°C

[0298] In a preferred embodiment, the present invention relates to a method for cryopreservation of a biological sample as defined herein, wherein the biological sample is / are or comprises cells, cellular systems, tissues, tissue-engineered constructs, organoids, organs, or organisms.

[0299] In a preferred embodiment, the present invention relates to a method for cryopreservation of a biological sample as defined herein, wherein the biological sample is a sample from a vertebrate, preferably a mammal.

[0300] In a preferred embodiment, the present invention relates to a method for cryopreservation of a biological sample as defined herein, wherein the biological sample is a brain, preferably the whole brain, and / or wherein said contacting with said cryoprotectant solution and / or removing of said cryoprotectant solution do not require deliberate opening of the blood-brain barrier.

[0301] In a preferred embodiment, the present invention relates to a method for cryopreservation of a biological sample as defined herein, wherein contacting with the cryoprotectant solutionsand / or removing of the cryoprotectant solutions are performed via perfusion, superperfusion, immersion, or combinations thereof.

[0302] The term "perfusion" has already been defined above and the above definition applies, mutatis mutandis, to the contacting with the cryoprotectant solutions and / or removing of the cryoprotectant solutions as described herein while the term "immersion" generally refers to all kinds of immerging or dipping of a biological sample into a solution or vice versa.

[0303] The term "superperfusion" is known in the art and refers to an alternative form of superfusion used in neurosurgery as it is, e.g., described in Gaddum JH. The technique of superfusion. Br J Pharmacol. 1997 Feb;120(4 Suppl):82-7.

[0304] In a preferred embodiment, the present invention relates to a method for cryopreservation of a biological sample as defined herein, wherein the method does not comprise contacting the biological sample with a cryoprotectant solution comprising less than about 45 % cryoprotectant.

[0305] As explained above, as regards the contacting step (AA) the contacting of the biological sample "solely" with a cryoprotectant solution comprising at least about 45 % cryoprotectant (shock loading), the term "solely" is to be understood in a way that the contacting of the biological sample with a cryoprotectant solution comprising at least about 45 % cryoprotectant is performed in a single step only without contacting the biological sample in two or more subsequent steps wherein, e.g., the biological sample is contacted with different cryoprotectant solutions (having, e.g., different concentrations) and / or the biological sample is contacted with a gradient of cryoprotectant solutions. Thus, as explained, in preferred embodiments, the contacting of the biological sample "solely" with a cryoprotectant solution does not include (a) step(s) of changing the cryoprotectant solution (e.g., by using one or more cryoprotectant solutions having a different concentration than a solution comprising at least about 45 % cryoprotectant). Thus, in preferred embodiments, the contacting of the biological sample "solely" with a cryoprotectant solution does not include (a) step(s) of using (a) gradient(s) of cryoprotectant solutions.

[0306] In further preferred embodiments, the contacting step (AA) does not comprise contacting the biological sample with a cryoprotectant solution comprising less than about 45 %cryoprotectant. In one example, the contacting step (AA) does not comprise contacting the biological sample with any further solution(s) prior to cooling; i.e. the cryoprotectant solution is the only solution with which the biological sample is being contacted prior to cooling. As used herein, a contacting step is considered to be prior to cooling if said contacting step does not involve lowering the temperature of the biological sample.

[0307] The same also applies, in preferred embodiments, to the contacting step (AB) as described herein. Accordingly, in preferred embodiments, the contacting step (AB) does not comprise contacting the biological sample with a cryoprotectant solution comprising less than about 45 % cryoprotectant.

[0308] In a preferred embodiment, the present invention relates to a method for cryopreservation of a biological sample as defined herein, wherein the biological sample is the whole mammalian brain and the method comprises the following steps:

[0309] A. performing blood washout and connecting said brain to a perfusion system;

[0310] AA. perfusing the brain with vitrification solution (also termed "cryoprotectant solution" as described herein) containing at least 45% w / v cryoprotectant at approximately half of physiological baseline flow rates; or

[0311] AB. perfusing the brain sequentially with a vitrification solution (also termed "cryoprotectant solution" as described herein) containing at least 45% w / v cryoprotectant and a carrier solution by intermittently alternating perfusion between said vitrification solution and carrier solution;

[0312] B. vitrifying and storing said brain below the glass transition temperature;

[0313] BB. rewarming said brain above the melting temperature without causing crystallization;

[0314] and

[0315] C. perfusing colloidal solutions at reduced flow rates adjusted to solution viscosity.

[0316] Alternatively to the above preferred embodiment, in another preferred embodiment, the present invention relates to a method for cryopreservation of a biological sample as defined herein, wherein the method comprises the steps of:

[0317] AA. immersing said biological sample in vitrification solution containing at least 50% w / v cryoprotectant until said samples sink or equilibrate;

[0318] B. vitrifying and storing said biological samples below the glass transition temperature;BB. rewarming said biological samples above the melting temperature without crystallization;

[0319] C. immersing said biological samples in a colloidal solution; and

[0320] D. subsequent immersion in isotonic carrier solutions.

[0321] The blood washout can be carried out by methods known in the art and by applying means available to the skilled person. Correspondingly, the skilled person knows how to connect a brain to a perfusion system while the term "perfusion" has already been described above.

[0322] The terms "vitrification solution" (also termed "cryoprotectant solution" as described herein), "carrier solution", "intermittently alternating", "vitrification", "glass transition temperature", "rewarming" and "colloidal solutions" have above been described above. As regards these features as well as preferred embodiments of these features, the same applies, mutatis mutandis, as has been set forth above.

[0323] In the above step AA of perfusing the brain with vitrification solution containing at least 45% w / v cryoprotectant, the perfusion is preferably effected at a rate that is approximately half of physiological baseline flow rate.

[0324] A "physiological baseline flow rate" is known to depend on the physiological compartment in question and methods to determine such flow rates are known in the art. For blood vessels, physiological baseline flow rates may range from 1.2 ml / min to 4.8 ml / min in vein and 3.0 ml / min to 26 ml / min in arteries. Forthe lymphatic system, physiological baseline flow rates have been reported to range from 4 L / day to 8 L / day. For cerebrospinal fluids physiological baseline flow rates ranging from 200 ml / day to 4 l / day have been reported.

[0325] A physiological base flow rate stays close to approx. 100 mmHg perfusion pressure for transaortic perfusion. As a non-limiting example, a value of approx. 0.7 ml / min / g mouse body weight is appropriate.

[0326] In the above step C wherein the perfusion of colloidal solutions is effected "at reduced flow rates adjusted to solution viscosity", the reduced flow rate is below the above-defined "physiological baseline flow rate". A concrete value cannot be determined as it largely depends on the viscosity of the solution. However, the skilled person can adjust, i.e., reduce, the flowrate in relation the viscosity of the solution. If the viscosity is high, the flow rate should be lowered to keep the pefusion pressure approximately constant.

[0327] As a non-limiting example, viscosity of 59% V3 is 3.6x water, 10% Dextran is 6x water, 20% Dextran is 19x water viscosity.

[0328] In the above step D wherein a subsequent immersion in isotonic carrier solutions may be carried out, a carrier solution as described herein above can be used. As regards this "carrier solution" as well as preferred embodiments thereof, the same applies, mutatis mutandis, as has been set forth above.

[0329] "Isotonic" means in this context that the solution has the same as the average osmotic pressure of human blood (which is about 7.8 bar at 37°C).

[0330] In a preferred embodiment, the present invention relates to a method for cryopreservation of a biological sample as defined herein, wherein said biological samples are viable after removal of the cyroprotectant solution and, optionally, are assessed after recovery, by methods such as, but not limited to, electrophysiological, structural, biochemical, immunohistochemical, functional, or molecular assessment post-cryopreservation.

[0331] Viability of the biological sample can be assessed by many different means and methods known in the art. Non-limiting examples are electrophysiological, structural, biochemical, immunohistochemical, functional, or molecular assessments.

[0332] In a preferred embodiment, the present invention relates to a method for cryopreservation of a biological sample as defined herein, wherein, said biological samples include human neurospheres, organotypic brain slices, acute brain slices, mammalian including human organs and organisms, natural and engineered cells, stem cells, oocytes, sperm, embryos, ovarian and testicular tissue, pancreatic islets, and tissue-engineered constructs such as skin, cartilage, vascular grafts, nerves, bladder augmentation, and corneas.

[0333] The present invention also relates to a biological sample cryopreserved by any of the methods for cryopreservation as defined herein.The thus obtained biological sample can then be subjected to any desired purpose. Nonlimiting examples of desired purposes have already been outlined in the above introduction and can be summarized as follows:

[0334] oocytes, sperm, embryos, ovary and testicular tissue in reproductive medicine and animal breeding.

[0335] cells, cellular systems, tissues, tissue-engineered constructs, and organoids in research, personalized and regenerative medicine, including, but not limited to, organotypic slices, as well as replacements and transplants for cartilage, skin, pancreatic islets, vascular grafts, nerves, bladder augmentation, and corneas.

[0336] mammalian organs for transplantation medicine and research.

[0337] the central nervous system, including the mammalian and human brain, for structural brain preservation, cryonics and research.

[0338] organisms, including the mammalian and human organism, for deep space exploration, cryonics and research.

[0339] The Figures show:

[0340] Figure 1. Upper panel: Numerical integration of the two-parameter formalism of solutesolvent flux from equations (1) and (2) for conventional single-step loading of 10% w / v dimethyl sulfoxide, interleaved and gradual loading protocols of cryoprotectant (CPA) for 100 seconds. Parameters from

[0016] for temperature of 4°C, and cell radius of 2 micrometer. The interleaved protocol involves repeated short-durations alternating between the cryoprotectant solution and an isotonic carrier solution. The minimum normalized cell volumes reached by the single-step, gradual, and interleaved protocols were approximately 0.8211, 0.8561, and 0.8687, respectively, demonstrating reduced shrinkage using the interleaved and protocols under idealized conditions. Lower panel illustrates the external DMSO concentration profiles for each protocol.

[0341] Figure 2. Pumping system for shock-loading, interleaved equilibration, and hyperoncotic washout of cryoprotectants in biological samples by perfusion or superperfusion. Solution 1: full-concentration vitrification solution, e.g., 59% w / v V3, solution 2: initial full concentration hyperoncotic washout solution, e.g. 30% w / v dextran, solution 3: reduced concentrationhyperoncotic washout solution, e.g. 10% w / v dextran, solution 4: carrier solution, e.g. LM5 (as disclosed e.g. in US8679735B2), solution 5: reduced-concentration vitrification solution, e.g., 30% w / v V3.

[0342] Figure 3. Cryopreservation of mammalian brain. Macroscopic aspect of the whole mouse brain in-situ after multi-step loading (A), shock-loading with 59% w / v V3 (B) and interleaved equilibration (C) with 59% w / v V3. Multi-step loading (A) was performed according to the protocol in

[0015] , Extracellular electrophysiological recordings after shock-loading and hyperoncotic washout of the whole mouse brain demonstrate intact synaptic transmission (D; 50-200 pA stimulation of mPP-DG granule cell synapses) and long-term-potentiation (E, CAI). Whole-cell patch-clamp recordings demonstrate intact spontaneous synaptic events (F; at resting membrane potential of -80 mV) and evoked action potential discharge (G; 0-100 pA depolarizing ramp with membrane potential pre-set to -70 mV) in a DG granule cell. Flow cytometric analysis demonstrates survival of cells isolated from mouse brain in three biological replicates, immune cell isolation for flow cytometric analysis from mouse brain was conducted with subsequent staining for the immune cell marker CDllb and DAPI (4',6-diamidino-2-phenylindole), the latter is a reagent taken up by dead cells, DAPI- CDllb+ are alive cells that express the immune cell marker CDllb. (H).

[0343] Figure 4. Thermally controlled container for shock-loading, and hyperoncotic washout of cryoprotectants in biological samples by immersion. (A): Top view of thermally controlled container with solutions cooled to 4°C. (B): Side view with mesh insert carrying the biological sample. Solution 1: full-concentration vitrification solution, e.g. 59% w / v V3, solution 2: same as 1, solution 3: same as 1, solution 4: hyperoncotic washout solution, e.g. 30% w / v dextran, solution 5: carrier solution, solution 6: same as 5. Immersions are performed in the order of solutions. Cryopreservation is performed after sinking of the sample in solution 3 to the bottom of the mesh insert .

[0344] Figure 5. Extracellularly recorded field postsynaptic potentials (field PSPs, fPSPs) in acute mouse brain slices without treatment (A, control), and after shock-loading with 59% w / v V3and hyperoncotic washout (B) using the thermally controlled container in Figure 4 and the protocol from Table 2. The input-output response curves (to single stimulus with varied intensities from 50 to 200 pA; traces on the left) and short-term plasticity (to train stimuli; traces on the right) of hippocampal SC-CA1 pyramidal cell synapses are indistinguishable between recordings.

[0345] Figure 6. Whole-cell current-clamp recordings in mouse hippocampal slice of a DG granule cell (A) and in a human prefrontal neocortex slice of a pyramidal cell (B), recovered from vitrification with CPA shock-loading and hyperoncotic washout (as in Figure 5). Action potentials were evoked by depolarizing ramp from 0 to 100 pA in 2s.

[0346] Figure 7. Organoid size post vitrification: Two iPSC Iines-Sojd3 and UKERi82A were differentiated into cortical brain organoids following protocol from Lancaster et al 2014. On day 35 of differentiation, organoids were vitrified (shock-loading). Non-vitrified and vitrified organoids were monitored in culture for 21 days post vitrification. Number of organoids for Sojd3, N=5 (non-vitrified), N=4 (vitrified); for82A, N=5 (non-vitrified), N=3 (vitrified).

[0347] Figure 8. Hippocampal cellular excitability and synaptic function after whole brain CPA perfusion, vitrification and hyperoncotic washout in situ. (A) Photographs of the murine brain in situ, showing lethal cerebral dehydration after gradual CPA equilibration (representative brain, reduced to 203 mg) (i), intermediate volume reduction after successful interleaved equilibration (representative brain, reduced to 313 mg) (ii), insufficient volume reduction after interleaved equilibration with no visual gap between brain and skull base (iii), and lethal cerebral edema after gradual CPA washout instead of hyperoncotic washout causing marked swelling beyond the skull base (iv). Functional evaluations in (B-G) were performed in acute hippocampal slices prepared from adult murine brains after vitrification in condition (Aii), storage at -140 °C for 1-8 days, and rewarming followed by hyperoncotic washout. (B) Mitochondrial activity assessed using a Seahorse XFe96 metabolic analyzer across defined respiratory states: basal (0-20 min), oligomycin-inhibited (20-40 min), FCCP-stimulated (40— 60 min), and rotenone / antimycin A-induced non-mitochondrial respiration (60-80 min).Hippocampi recovered from whole-brain vitrification with 59% w / v CPA showed a significant reduction in OCRs compared to controls. Data from the post-vitrification hippocampal slices were replicated here for comparison. Statistical analysis: one-way ANOVA with Fisher's LSD; n.s. not significant, *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001. (C) Whole-cell current-clamp recordings of hippocampal granule cells in slices from brains after in situ vitrification show comparable rheobase (indicated with arrowhead in current trace, inset) and enhanced excitability. As illustrated from voltage traces above, depolarizing currents were rectangular steps from membrane potential adjusted to -70 mV. (D) Occurrence of spontaneous postsynaptic potentials (spPSPs), monitored at resting membrane potential under whole-cell current-clamp mode, did not differ between control and post-vitrification brains. (E) Whole-cell voltage-clamp recordings were performed to assess the balance of excitatory and inhibitory drives (E / l balance) onto control and post-vitrification DG cells. Membrane potential of DG granule cells was clamped either at -80 mV to monitor excitatory postsynaptic currents (EPSCs) or at 0 mV for inhibitory PSC (IPSC), as shown on the left with traces from a control slice. Histogram with E / l ratio, calculated with the frequency of PSCs in individual cells, indicated no appreciable alteration after cryopreservation. (F-G) mPP-DG granule cell synaptic transmission and plasticity after in situ vitrification with interleaved equilibration. Compared to controls, extracellularly recorded responses of mPP-DG synapses to electrical stimulation show well-preserved 1-0 curve for basic synaptic transmission after vitrification and rewarming (F, left panel). In contrast to significant reduction in STP (upon 20 Hz train stimuli; F, right panel), HFS-induced LTP of mPP-DG granule cell synapses was not affected after vitrification, as shown by example of time course for field PSP from a postvitrification brain (G, left) and group data of normalized magnitude of change 51-60 min post tetanus (G, right). Statistical comparisons were performed using unpaired, two-tailed student's t-test or one-way ANOVA followed by Tukey's post-hoc test. n.s. not significant; * p < 0.05; ** p < 0.01; *** p < 0.001

[0348] Figure 9. Perfusion strategy for cephalothoracic specimens and failure modes. The button cannula was inserted via the left ventricle and secured with small hemostatic forceps (A-C). For perfusion steps prior to vitrification, the equator of the button cannula was not advanced beyond the atrioventricular plane (A). Post-rewarming, the button was fully advanced into theascending aorta (B). Quality of perfusion was monitored visually at the internal thoracic arteries via addition of 0.01% w / v methylene blue to LM5 during interleaved equilibration and to the initial 10% dextran washout perfusate (C). Several failure modes of the protocol were visually detectable (D-G). Apparent crystallization of the left cerebral hemisphere (asterisk) vs. apparent vitrification of the right brain hemisphere after immersion in cryogenic isopentane (D). Pulmonary edema (asterisk) due to reflux in the pulmonary veins during CPA washout via addition of 0.01% w / v methylene blue to the initial 10% dextran washout perfusate (E). Successful cerebral perfusion was confirmed via staining of small pial vessels with methylene blue, as present in (F). Absence of this cerebral vessel staining indicated insufficient cerebral perfusion. Cerebral edema was detectable via bulging from the skull base and tissue whitening (G).

[0349] Figure 10. Macroscopic changes of the same brain during interleaved equilibration and hyperoncotic washout. The brain was exposed during the entire perfusion protocol via craniectomy after incision along the sagittal suture from the foramen magnum (A-F), showing the non-dehydrated native initial state (A), the dehydrated state after the first 8-minute CPA-perfusion step (B), the rehydrated state after the 3-minute LM5-perfusion step (C), the fully CPA-loaded state after the 25-minute CPA-perfusion step (D), the state during the first hyperoncotic washout step (E), the normalization of brain volume after completion of the hyperoncotic washout (F). Note that due to craniectomy in the non-dehydrated native state (A), median-sagittal damage to the cerebral vasculature and parenchyma is evident in (D-F).

[0350] This could be prevented by delaying craniectomy to state (B).

[0351] Figure 11. Protocol for cerebral vitrification in situ.Time course of vitrification protocol, with varied temperature (top), perfusate (middle) and perfusion flow rate (bottom). Arrows indicate when images (A-F) in Fig. 12 were taken. Dotted line indicates when perfusion was terminated.

[0352] Figure 12. Properties of dentate gyrus granule cells after in situ brain vitrification. (A-C) Summary of RMP, Rmand Cmin DG granule cells post in situ vitrification. (D) Voltage trace froma granule cell from a brain after vitrification (8 days at -150 °C and 5 h post rewarming), illustrating how the initial and the last firing rate were determined for characterization of AP frequency adaptation. (E-F) Adaptation ratio for AP frequency (E) and amplitude (F), obtained with repetitive discharge (around 20 APs) in response to depolarization step (pulse duration 1 s). (G) Superimposed phase plots of granule cell APs from a control brain (black plot) and a post-vitrification brain (red plot). (H-L) Summary of peak amplitude (H), voltage threshold (I), maximum rising slope (J), half-width (K) and fAHP (L) of evoked AP. Statistical comparisons were performed using an unpaired, two-tailed t-test. n.s. not significant; * p < 0.05; ** p < 0.01.

[0353] Figure 13. Ibal microglia staining of acute mouse brain slices after vitrification compared to non-vitrified controls. Mouse brains were sectioned via vibratome and either subjected to cryopreservation or kept as fresh controls. Slices from both groups were then cultured for 1 hour at 24°C, followed by an additional 3 hours at 34°C. The lower panels present representative micrographs of Ibal-stained microglia for each condition to assess morphological preservation and activation state. (A, B) Microglial morphology after 1 hour of recovery at 24°C in vitrified (A) and fresh control (B) slices. (C, D) Microglial morphology after 3 hours of subsequent incubation at 34°C in vitrified (C) and fresh control (D) slices. The staining demonstrates comparable preservation of microglial structure and comparable morphological responses to physiological incubation temperatures between the cryopreserved and fresh tissue. Scale bars 20 urn.

[0354] Figure 14. Kidney tissue section stained with Periodic acid— Schiff (PAS ) stain post-vitrification. Renal morphology is intact with tubuli, glomeruli and capillaries. Scale bars 100 pm.

[0355] Figure 15. Atrial and ventricular contractility was assessed for mouse hearts after 20 minutes of Langendorff perfusion with carbogenated Ringer solution for (A) untreated control (B) 30%w / v V3 vitrification solution loading and hyperosmotic washout with EGTA and BDM and (C) 59% w / v V3 vitrification solution shock-loading and hyperosmotic washout with EGTA and BDM. Hearts were assessed during an interval shorter than one minute without Langendorffperfusion to exclude motion artifacts with a stereomicroscope from above (left images). Myocardial contractility was documented via quantification of optical flow in stereomicroscope video recordings (middle) and plotted over time (right). Contractility and frequency is reduced, but retained in (B) and (C).

[0356] Figure 16. Extracellular recording in adult mouse left ventricular myocardial slice (300 pm) after CPA shock-loading at 45% V3 at 4°C, followed by 55% V3 at -20°C, followed by hyperosmotic washout with 25% Glucose in PBS, showing raw trace of spontaneous action potential of a cardiomyocyte (left) and averaged waveform of discharge (right).

[0357] Figure 17. Recordings of cutaneous C-nociceptors were conducted in hairy skin of Black 6 mouse using the skin saphenouse nerve preparation. (A) electrical stimulation within the receptive field of the nociceptor to determine response latency which was at about 50 ms. Thereafter the receptive field was stimulated by mechanical and heat stimuli to identify the respective fiber subtype: polymodal C-mechano-heat (C-MH) -sensitive with a mechanical v. Frey threshold of 5.7 mN. Chemical responsiveness (B) was tested by superfusion of the receptive field with Capsaicin at lOpM concentration. The instantaneous discharge frequency (1 / s) is depicted, every dot represents a capcaicin-evoked C-fiber action potential / spike).The present invention is illustrated by the following examples. The skilled person will understand that cryopreservation of brain-derived biological samples, such as brain slices, and whole brains, illustrates cryopreservation of biological samples that are difficult to cryopreserve.

[0358] Example 1 (Cryopreservation of whole mammalian brains)

[0359] To study cryopreservation of whole mammalian brains, adult male C57BL6 / J mice were sacrificed, blood was washed out by transcardial perfusion and craniectomy was performed. Here, cryopreservation was carried out by perfusion. To this end, the descending aorta was clamped, the left cardiac ventricle opened, a button cannula inserted into the ascending aorta, and connected to the pumping system depicted in Figure 2. Importantly, no additional steps were performed to permeabilize the blood brain barrier prior to perfusion with cryoprotectants.Here, three different cryopreservation protocols are being compared: i) multi-step loading, ii) shock loading and iii) interleaved equilibration. All procedures were carried out using the LM5 carrier solution and vitrification solution V3 (Tables 4 and 5). Multi-step loading was performed as published

[0015] , In brief, perfusion was initiated using LM5 carrier solution followed by increasing concentration of cryoprotectant in the LM5 carrier solution (0% w / v V3 (corresponding to pure LM5 carrier solution) for 2 min, 2% for 2 min, 4% for 2 min, 8% for 2 min, 16% for 2 min and 30% w / v V3 for 5 min each at 10 °C, followed by 45% w / v V3 for 2 minutes and three iterations of 59% w / v V3 for 5 minutes each at -10 °C).

[0360] For shock loading, perfusion was performed as shown in Table 1.

[0361] Interleaved equilibration was performed by alternating between a first perfusion solution (59% w / v V3 in LM5 carrier solution) and a second perfusion solution (8% Dextran in LM5 carrier solution). Here, perfusion was exemplarily carried out using 5 cycles, each cycle consisting of perfusing with a first perfusion solution (59% V3 in LM5 carrier solution) for 60 seconds followed by perfusing with a second perfusion solution (8% Dextran in LM5 carrier solution) for 60 seconds, followed by 2 cycles, each cycle consisting of perfusing with a first perfusion solution (59% V3 in LM5 carrier solution) for 60 seconds followed by perfusing with a second perfusion solution (30% V3 in LM5 carrier solution) for 60 seconds, and final perfusion with a perfusion solution (59% V3 in LM5 carrier solution) for 8 minutes. All steps were carried out at 4°C.

[0362] Samples may be cooled by external heat conduction, e.g., via immersion in a cryogenic refrigerant cooled to -150°C (e.g. Isopentane) and stored below the glass transition temperature for arbitrary durations, e.g. in liquid nitrogen, liquid nitrogen vapor phase, or a -150 °C freezer. For rewarming, several techniques are appropriate, including external heat conduction by immersion in 4°C vitrification solution, and volumetric warming techniques perfusion with a 4°C refrigerant which stays liquid in the cryogenic range, nanowarming as in [2], dielectric , or HIFUS warming. Here, samples were rewarmed by immersion and perfusion with 4°C vitrification solution and then subjected to washout and downstream utilization. Samples were recovered from cryopreservation by either multi-step washout as described in [3], or hyperoncotic washout using 30% w / v dextran and 10% w / v dextran in LM5 carrier solution.Use of the established cryopreservation protocol led to lethal shrinkage of the brain (Fig. 3A), recovery of viable cells was not possible and, thus, also no electrophysiological measurements could be conducted.

[0363] In contrast, both shock loading and interleaved equilibration allowed for cryopreservation of murine brains. Murine brains cryopreserved using said approaches maintained their structure (Fig. 3 B-C) and physiological function was preserved upon recovery. Specifically, the data reveal intact synaptic transmission (Fig. 3D) and long-term-potentiation (Fig. 3E). Additionally, whole-cell patch-clamp recordings demonstrate intact spontaneous synaptic events (Fig. 3F) and evoked action potential discharge (Fig. 3G) in DG granule cells. Additionally, flow cytometry analyses demonstrate survival of cells isolated from said murine brain samples (Fig.

[0364] 3H, n=3). To this end, the brain was extracted from the skull, homogenized and strained through a 70-pm nylon filter (Fisher Scientific). After centrifugation, cells were resuspended in 37% isotonic Percoll and centrifuged at 800g for 30 min at room temperature without breaks. Cells were collected and washed with ice cold PBS before being stained. For flow cytometry, cells were stained for 30 min at 4 °C with DAPI and CDllb antibodies (BD Biosciences, Franklin Lakes, NJ, USA). After being washed, data was acquired using a FACS BD Fortessa X20 (BD Biosciences) and data was analyzed using FlowJo.

[0365] In a similar but independent experiment, whole murine brains were cryopreserved using the shock-loading method, rewarmed, sliced in 30% V3 in LM carrier solution supplemented with 300 mM Mannitol and subjected to multi-step washout as described in

[0015] , Downstream analyses revealed preservation of fEPSP (field excitatory postsynaptic potentials) responses indicative of physiological brain function.

[0366] However, the use of hyperonctotic washout solutions is preferable for certain tissues, including whole mammalian brains or tissue samples derived therefrom.

[0367] These data support that the disclosed protocols are the first to enable cryopreservation of the whole brain by vitrification [1],Table 1: Perfusion protocol for organs and organisms.

[0368] Perfusate Flow rate Duration Temperature Cooldown solution, Increase until arterial 10 min 4 °C e.g., phosphate-buffered saline with pressure reaches 100

[0369] 3% w / v dextran mmHg (baseline flow)

[0370] Vitrification solution, e.g., 59% w / v 50% of baseline flow 10 min 4 °C

[0371] V3 in LM5 carrier solution

[0372] Cryopreservation

[0373] storage

[0374] rewarming

[0375] Full-concentration hyperoncotic 5% of baseline flow 4 min 4 °C washout solution, e.g., 30% w / v

[0376] dextran in LM5 carrier solution

[0377] Reduced-concentration 25% of baseline flow 10 min 4 °C hyperoncotic washout solution, e.g.,

[0378] 10% w / v dextran in LM5 carrier

[0379] solution

[0380] downstream utilization

[0381]

[0382] Example 2 (Cryopreservation of acute and organotypic brain slices)

[0383] Acute or organotypic brain slices were prepared as described in

[0015] , In brief, 350 pm thick horizontal slices containing the hippocampus were prepared from mice anesthetized with isoflurane using a Leica VT1200 Vibratome (Leica Biosystems, Germany) in ice-cold sucrose-based artificial cerebrospinal fluid (aCSF) containing 75 mM sucrose, 87 mM NaCI, 3 mM KCI, 0.5 mM CaCI2, 7 mM MgCI2, 1.25 mM NaH2PO4, 25 mM NaHCO3 and 10 mM D-glucose. Slices were incubated in the same solution for 10 min at 35 °C and then maintained in aCSF containing 125 mM NaCI, 3 mM KCI, 1 mM CaCI2, 3 mM MgCI2, 1.25 mM NaH2PO4, 25 mM NaHCO3 and 10 mM D-glucose at room temperature (RT). All solutions were constantly gassed with 95% 02 - 5% CO2 to keep pH constant (7.4). After a 30-minute recovery phase, the slices were transferred into a Netwell polyester mesh insert (Corning, USA) for 6-well plates with mesh size 74 pm.Multi-step loading and washout have previously been reported for brain slices

[0015] , However, these data show that PSP and STP signals were reduced as compared to non-cryopreserved controls.

[0384] In light of these data, it was tested whether shock loading and hyperoncotic washout could also be performed by immersion, and whether improved viability and physiological functionality could be achieved (Fig. 4, Table 2).

[0385] Here, exposure of the samples to the vitrification solution was repeated to ensure the local target concentration is reached. Next, samples were directionally cooled from below, by placing them on a copper cylinder cooled to -196 °C with liquid nitrogen. The mesh insert carrying the samples is first transferred onto the top of the cylinder. After one minute, the mesh insert was slowly transferred into liquid nitrogen. This allows to avoid physical cracking of vitreous brain slices due to thermomechanical stress, as would occur during direct immersion into liquid nitrogen. After arbitrary duration of storage below the glass transition temperature, e.g. in liquid nitrogen, liquid nitrogen vapor phase, or a -150 °C freezer, the samples are rewarmed in 4°C vitrification solution and then subjected to washout and downstream utilization.

[0386] Importantly, said simplified and accelerated protocol allowed for superior recovery compared to methods known in the art

[0015] , Electrophysiological measurements obtained for brain slices that underwent cryopreservation and recovery were indistinguishable from measurements obtained from fresh samples (Fig. 5).

[0387] Importantly, similar results were obtained by whole-cell current-clamp recordings in human prefrontal neocortex slice of a pyramidal cells and interneurons, as in mouse hippocampal slice of a DG granule cell. (Fig. 6).Table 2: Immersion protocol for tissue slices

[0388] Immersion solution Duration Temperature Vitrification solution, e.g., 59% w / v V3 5 min 4 °C Vitrification solution, e.g., 59% w / v V3 5 min 4 °C Vitrification solution, e.g., 59% w / v V3 until the sample fully 4 °C submerges, at least 5 min

[0389]

[0390] cryopreservation

[0391] storage

[0392] rewarming

[0393] Hyperoncotic washout solution, 5 min 4 °C

[0394] e.g., 30% w / v dextran in LM5 carrier

[0395] solution

[0396] Carrier solution, e.g., LM5 carrier 5 min 4 °C

[0397] solution

[0398] Carrier solution, e.g., LM5 carrier 10 min 4 °C

[0399] solution

[0400] downstream utilization

[0401]

[0402] Example 3 (Cryopreservation of neurospheres):

[0403] Finally, it was tested whether cryopreservation using shock loading and hyperoncotic washout could also be used to preserve human neurospheres. To this end, human neurospheres were generated as described in

[0022] , In brief, neurospheres were generated from human embryonic stem cells (hESC) or induces pluripotent stem cells (iPSC). Cells were harvested, resuspended in low-bFGF hESC medium with ROCK inhibitor, seeded in low-attachment 96-well U-bottom plates at a density of approximately 9.000 cells per well. Medium changes were performed as needed to support formation and growth of viable embryoid bodies (EB). When EBs reached a size of approximately 500 pm, neuroepithelial differentiation was initiated. Forthis purpose, EBs were transferred to low attachment 24-well plates containing 500 pl neural induction medium (DMEM-F12 supplemented with 1% v / v N2 supplement, 1% v / v GlutaMAX, 1% v / v MEM-NEAA, and 1 pg / ml heparin). Medium changes were performed as needed anddifferentiation was confirmed microscopically on day 4 to 5.

[0404] Neurospheres were pipetted onto a mesh insert with a pore width of 75 urn and processed according to the immersion protocol illustrated in Figure 4 and Table 3. Here, meshes were transferred to 6-well plates and immersed in 59% w / v V3 solution at 4°C for 8 min, followed by cryopreservation, storage and rewarming as described in Example 2. Using said protocol recovery of viable human neurospheres was achieved, which could be maintained ex vivo for timeframes comparable to non-cryopreserved neurosphere.

[0405] As shown in figure 7, both vitrified and fresh neurospheres also exhibited similar viability and growth kinetics.

[0406] The protocol is the first to enable cryopreservation of neurospheres by vitrification

[0021] ,

[0407] Table 3: Immersion protocol for small biological samples

[0408] Immersion solution Duration Temperature Vitrification solution, e.g., 59% w / v V3 in LM5 8 min 4 °C

[0409] carrier solution

[0410] cryopreservation

[0411] storage

[0412] rewarming

[0413] Hyperoncotic washout solution, e.g., 30% w / v 3 min 4 °C

[0414] dextran in LM5 carrier solution

[0415] Carrier solution, e.g., LM5 carrier solution 1 min 4 °C

[0416] Carrier solution, e.g., LM5 carrier solution 10 min 4 °C downstream utilizationTable 4: Carrier solution LM5

[0417] chemical concentration

[0418] glucose 90 mM

[0419] mannitol 45 mM

[0420] lactose 45 mM

[0421] KCI 8.2 mM

[0422] K2HPO4 7.2 mM

[0423] glutathione 5 mM

[0424] adenine I mM

[0425] NaHCO3 10 mM

[0426] Table 5: Modified standard vitrification solution (59% w / v V3 = 8.42 M permeable CPA) in LM5 carrier solution.

[0427] chemical concentration

[0428] dimethylsulfoxide (DMSO) 22.3% w / v

[0429] ethylene glycol (EG) 16.84% w / v

[0430] formamide 12.86% w / v

[0431] polyvinylpyrrolidone K12 7% w / v

[0432] Example 4 (whole-brain vitrification in situ)

[0433] 1. Physiological assessment

[0434] Here, the vitrification protocols disclosed in Example 1 (gradual loading, shock-loading and interleaved equilibration were compared in detail. Unless specifically indicated, the perfusion protocols of Example 1 and the buffers of Example 3 (Table 4 and 5) were used.

[0435] Gradual CPA during transaortic vascular perfusion led to extensive cerebral dehydration and macroscopic darkening (Fig. 8Ai), with reduction in cerebral mass (mean ± SD) to 44.5 ± 1.8% (209 ± 9 mg, N = 2) relative to a PBS-perfused reference brain (469 mg). Brains exposed to gradual CPA loading were macroscopically dehydrated to the extent that they became concave, with no synaptic response elicitable. Shock-loading via perfusion with full-strength vitrification solution (59% w / v V3) improved total cerebral mass retention to 55.7 ± 3.0% (261±14 mg, N = 5) of reference (Table 6) To harness the mismatch between the permeability for CPA and water within the BBB, interleaved equilibration was performed by alternating perfusion between full-strength vitrification solution and LM5 to rehydrate the partly CPA-loaded brain. Repeated cycles of interleaved equilibration enabled vitrification at normal cerebral mass and volume (Fig. 8Aii i), but resulted in cerebral edema during CPA washout (Fig.

[0436] 8Aiv). Therefore a final interleaved equilibration protocol that resulted in intermediate cerebral dehydration and maintained brain convexity was chosen (Fig. 8Aii), corresponding to a cerebral mass of 69.8 ± 13.5% (327 ± 63 mg, N = 5) of reference. Detailed information regarding the protocol for whole-brain cryopreservation is provided in table 6 and Fig. 9 and 10. The brains remained in situ and were stored at -140°C for 1-8 days. After rewarming, CPA was washed out via transaortic vascular perfusion at reduced flow rates using a hyperoncotic solution (see table 6 and figure 11).

[0437] Next, physiological evaluation was performed using brain slices obtained after cryopreservation with the interleaved equilibration protocol and hyperoncotic whashout using in the Seahorse metabolic analysis (N = 3, Fig. 8) and electrophysiological recordings (N = 10; Fig. 8). Tissue demonstrated viability and physiological function after cryopreservation. Basal respiration and spare respiratory capacity of the hippocampi after whole brain vitrification (138.2 ± 4.45 pmol / min and 58.4 ± 3.99 pmol / min, respectively) was compared to those of slice vitrification, and found no significant differences between the two conditions (Fig. 8B).

[0438] Next, electrophysiological experiments were conducted on hippocampal granule cells, given their observed viability in cryopreserved slices. Whole-cell recordings were performed in current-clamp mode and in voltage-clamp mode to characterize their intrinsic excitability and the excitatory and inhibitory synaptic drives, respectively. DG granule cells in acute slices prepared from brains post in situ vitrification had slightly depolarized RMP (-82.15 ± 1.98 mV, n = 12 from 6 mice; p = 0.013 vs control GCs) and reduced Cm (fig. S12C). Compared to control cells, granule cells of post-vitrification brains had comparable input resistance (at -70 mV, fig. S12B), and rheobase (-45.95 ± 4.45 pA) with evoked action potentials via ramp-like depolarization (Fig. 8C, left, inset). However, when rectangular depolarization (1 s) was delivered at larger steps (100 - 200 pA), increased discharge in GCs from post-vitrificationbrains was observed (Fig. 8C, right), suggesting an elevation in intrinsic excitability. Detailed analysis of AP parameters revealed faster and narrower APs in granule cells after in situ vitrification (fig. S12G-L).

[0439] The functional synaptic network after in situ vitrification was manifested in spontaneously occurring postsynaptic potentials (spPSPs, 1.14 ± 0.014 Hz, n = 12 from 6 mice; Fig. 8D). When the recordings were switched to voltage-clamp mode, glutamatergic excitatory drive and GABAergic inhibitory drive could be distinguished with voltage clamped at -80 mV and 0 mV, respectively (Fig. 8E). Such simultaneous monitoring enables a measurement of the overall balance between excitation and inhibition (E / l) in each cell, ex-pressed as E / l ratio using the frequency of postsynaptic currents (PSCs). The E / l balance was unchanged after in situ vitrification (post-vitrification 2.14 ± 0.33, n = 7 from 3 mice; control 1.80 ± 0.13, n = 19 from 4 mice; p = 0.255; Fig. 8E). In field potential recordings from mPP-GC synapses of postvitrification brains (n = 13 from 6 mice; Fig. 8F-G), basic synaptic transmission to mPP activation (1-0 curve; Fig. 8F, left) was affected only at low stimulus intensity. STP of mPP-GC synapses was markedly reduced after in situ vitrification (Fig. 8F, right), LTP of the mPP-GC synapse was operational (51-60 min post-HFS, 125.39 ± 6.10 %, n = 9 from 4 mice; p = 0.949 vs controls; Fig. 8G). Functional measurements are summarized in Table 8.

[0440] Table 6. Cerebral mass retention after different CPA loading approaches (reference brain mass: 469 mg).

[0441] Loading approach Cerebral mass (mg) Mass (% of Qualitative outcome reference)

[0442] Gradual loading 209 ± 9 (N=2) 44.5 ± 1.8% Marked dehydration / concavity; no synaptic response Shock-loading 261 ± 14 (N=5) 55.7 ± 3.0% Improved mass retention (59% (w / v) V3) vs gradual loading Final interleaved 327 ± 63 (N=5) 69.8 ± 13.5% Intermediate dehydration; equilibration maintained convexity; (selected protocol) used for vitrification

[0443]

[0444] Table 7 - Exemplary protocol for cerebral vitrification in situ.

[0445] Perfusate Flow rate duration temperature Thoracotomy and incision of the right atrium 4 min 36 °C PBS 10 ml / min 2 min 4 °C Aortic cannulation of cephalothoracic specimen 2 min 4 °C 59% V3 1 ml / min 8 min 4 °C Craniectomy 4 min 4°C LM5 + 0.01% methylene blue 3 ml / min 3 min 4 °C 59% V3 1 ml / min 25 min 4 °C Immersion in 65% V3 2 min -20 °C Immersion in isopentane >1 d -140 °C Stirring in 65% V3 1 min 20 °C Immersion in 59% V3 5 min 4 °C 10% dextran + 0.01% 0.3 ml / min 10 min 4 °C methylene blue

[0446] 10% dextran 0.4 ml / min 5 min 4 °C 10% dextran 0.5 ml / min 10 min 4 °C Continue perfusion until normal brain size 5-10 min 4 °C Brain extraction and immersion in LM5 5 min 4 °C Slicing in sucrose-aCSF 20 min 4 °C Incubation in aCSF 1 h 20 °C Incubation in sucrose-aCSF 10 min 35°C Incubation in aCSF >30 min 20 °CTable 8. Representative functional measures obtained from hippocampal tissue after in situ whole-brain vitrification.

[0447] Measure Post-vitrification value Notes / comparison

[0448]

[0449] Dentate gyrus granule cell -82.15 ± 1.98 mV (n=12 Slight depolarization vs resting membrane potential cells, 6 mice) controls (p=0.013);

[0450] (RMP) capacitance reduced -45.95 ± 4.45 pA Comparable to controls; Rheobase (ramp

[0451] enhanced firing to 100-200 depolarization)

[0452] pA rectangular steps Spontaneous postsynaptic 1.14 ± 0.014 Hz (n=12 cells, Comparable to controls potentials (spPSPs) 6 mice)

[0453] Excitation / inhibition (E / l) 2.14 ± 0.33 (n=7 cells, 3 Control 1.80 ± 0.13 (n=19 ratio from spPSCs mice) cells, 4 mice), p=0.255 mPP-DG input-output (l-O) Near-normal; affected only Extracellular field recordings relationship at low stimulus intensity

[0454] Short-term plasticity (STP) Reduced Reduction observed after in at mPP-DG synapse (20 Hz situ vitrification train)

[0455] Long-term potentiation 125.39 ± 6.10% (51-60 min Not different from controls (LTP) at mPP-DG synapse post-HFS; n=9 slices, 4 mice) (p=0.949)

[0456]

[0457] 2. Microcilia assessment:

[0458] Adult C57BL / 6J mice mice (age 3.5 months) were housed under standard conditions. Horizontal 350-pm brain slices of the whole forebrain hemispheres were prepared in ice-cold sucrose-aCSF, allowed to recover in aCSF at room temperature (RT). All slices derived from the right hemisphere underwent cryopreservation by vitrification as described in Table 2. Both post-vitrification slices from the entire right hemispheres, as well as non-cryopreserved slices from the entire left hemispheres were subsequently incubated for 3 hours at 32°C to elicit an early microglial response to the slicing injury. All samples were subsequently fixated, stained with Ibal-Antibodies and inspected under a fluorescence microscope. No difference in microglial morphology at the different time points was apparent. Representative images are shown in Figure 13.Example 5 (Cryopreservation of kidneys by shock-loading and hyperoncotic washout)

[0459] 1. Materials and methods

[0460] i) Cryopreservation and washout protocol

[0461] Murine kidneys were ex vivo perfused via the aorta using the shock-loading protocol disclosed in Example 1. Briefly, the organs were perfused with a 60% w / v V3 cryoprotectant solution to facilitate cooling and subsequent cryopreservation. Following rewarming, a hyperoncotic washout was performed using a 20% w / v polyvinylpyrrolidone (PVP K12) solution.

[0462] ii) PAS staining

[0463] Post-cryopreservation, Periodic acid-Schiff (PAS) staining was performed according to standard histological protocols. Tissue sections were oxidized in a 0.5% periodic acid solution for 5 minutes. After rinsing, sections were treated with Schiff's reagent for 15 minutes. The sections were subsequently washed, counterstained with hematoxylin to visualize nuclei, dehydrated through graded alcohols, cleared in xylene, and mounted.

[0464] 2. Results

[0465] Figure 14 presents representative PAS-stained micrographs of murine kidney tissue following the shock-loading cryopreservation and hyperoncotic washout protocol, shown at increasing magnifications. The histological evaluation reveals a high degree of structural preservation. The cortical architecture is intact, with glomeruli showing normal capillary loop structures. The renal tubules demonstrate intact epithelial linings with distinctly visible, PAS-positive basement membranesand brush borders.

[0466] Example 6 (cryopreservation of hearts by shock-loading, hyperosmotic washout, calcium control and myosin inhibition)

[0467] 1. Materials and Methods

[0468] Adult murine hearts were excised and immediately immersed in a first solution comprising ice-cold (4 °C) sucrose artificial cerebrospinal fluid (75 mM sucrose in aCSF) for a duration ofless than 60 minutes. Said first solution comprised 0.5 mM calcium (Ca) and 7.0 mM magnesium (Mg) to minimize ischemic injury.

[0469] The aorta of each murine heart was cannulated and attached to a Langendorff perfusion apparatus. To establish a functional baseline, the heart was perfused with a recording aCSF solution comprising 2.5 mM Ca and 2.5 mM Mg for 20 minutes at a flow rate of 1.8 ml / min and a temperature of 20 °C.

[0470] To induce electromechanical arrest and myocardial relaxation prior to cryoprotectant (CPA) loading, the heart was subsequently perfused with a relax aCSF solution for 20 minutes at a flow rate of 1.8 ml / min and a temperature of 4 °C. Said relax aCSF solution comprised 0 mM Ca, 4.0 mM Mg, 2.5 mM EGTA, and 30 mM 2,3-butanedione monoxime (BDM).

[0471] Shock-loading of CPA was performed by perfusing the heart with a vitrification solution comprising 30% w / v V3 in LM5 carrier solution at a flow rate of 0.8 ml / min and a temperature of 4 °C. Alternatively, this step was performed by perfusing the heart with a vitrification solution comprising 59% w / v V3 in LM5 carrier solution for 20 minutes at a flow rate of 0.5 ml / min and a temperature of 4 °C. Both the vitrification solutions comprised 0 mM Ca, 0 mM Mg, 2.5 mM ethylene glycol-bis(|3-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), and 30 mM BDM.

[0472] CPA washout was performed by perfusing the heart with a hyperosmotic washout solution comprising 25% w / v glucose in phosphate-buffered saline (PBS) for 10 minutes at a flow rate of 0.8 ml / min and a temperature of 4 °C. Said hyperosmotic washout solution further comprised 0 mM Ca, 0 mM Mg, 2.5 mM EGTA, and 30 mM BDM.

[0473] The heart was subsequently perfused with the relax aCSF solution (comprising 0 mM Ca, 4.0 mM Mg, 2.5 mM EGTA, and 30 mM BDM) for 20 minutes at a flow rate of 1.8 ml / min and a temperature of 4 °C. To mitigate calcium paradox injury, calcium was reintroduced in a stepwise manner at a temperature of 20 °C and a flow rate of 1.8 ml / min. The stepwise reintroduction comprised:

[0474] (i) perfusing the heart with an aCSF solution comprising 0 mM Ca, 4.0 mM Mg, 0 mM EGTA, and 30 mM BDM for 10 minutes;

[0475] (ii) perfusing the heart with an aCSF solution comprising 0.1 mM Ca, 4.0 mM Mg, 0 mM EGTA, and 30 mM BDM for 10 minutes; and(iii) perfusing the heart with an aCSF solution comprising 1.0 mM Ca, 3.0 mM Mg, 0 mM EGTA, and 30 mM BDM for 20 minutes.

[0476] Finally, the heart was perfused with the recording aCSF solution comprising 2.5 mM Ca, 2.5 mM Mg, 0 mM EGTA, and 0 mM BDM for 20 minutes at a flow rate of 1.8 ml / min and a temperature of 20 °C to evaluate post-vitrification functional recovery. These steps are provided in Table 9.

[0477] 2. Results

[0478] Use of the disclosed method comprising shock-loading, hyperosmotic washout, calcium control, and myosin inhibition allowed for the first functional preservation of whole murine hearts after perfusion with verifiable CPA concentration. Perfusion with the hyperosmotic washout solution comprising 25% w / v glucose successfully prevented macroscopic myocardial edema during CPA removal, maintaining normal organ volume and tissue compliance. Furthermore, the utilization of 0 mM Ca, combined with the calcium chelator EGTA and the myosin ATPase inhibitor BDM during the cooling, CPA loading, and hyperosmotic washout steps prevented irreversible hypercontracture.

[0479] The stepwise reintroduction of calcium while maintaining BDM in the perfusate (progressing from 0 mM to 0.1 mM, to 1.0 mM, to 2.5 mM Ca) effectively mitigated calcium paradox injury. Upon final perfusion with the recording aCSF solution comprising 2.5 mM Ca, the hearts demonstrated successful resumption of macroscopic contractile activity and functional responsiveness, indicating preservation of the myocardial syncytium (Figure 15).

[0480] As shown in Figure 16, extracellular recordings for murine left ventricular myocardial slices (300 pm) showed spontaneous action potential of cardiomyocytes. Myocardial slices were cryopreserved by immersion shock-loading at 45% w / v V3 in LM5 solution at 4°C, followed by 55% w / v V3 in LM5 solution at -20°C and hyperosmotic washout using 25% Glucose in PBS,Table 9. Heart Vitrification protocol via Langendorff perfusion

[0481] Step Solution Duration Flow Temperature Ca Mg EGTA BDM (mM) (mM) (mM) (mM) 1 Sucrose <60min Immersion 4°C 0.5 7 0 0 aCSF

[0482] 2 Recording 20 min 1.8ml / min 20°C 2.5 2.5 0 0 aCSF

[0483] 3 Relax aCSF 20min 1.8ml / min 4°C 0 4 2.5 30 4a 30% w / v 20min 0.8ml / min 4°C 0 0 2.5 30 V3 VS in

[0484] LM5

[0485] 4b 59% w / v 20min 0.5ml / min 4°C 0 0 2.5 30 V3 VS in

[0486] LM5

[0487] Optionally: cooling, vitrification and storage

[0488] 45% w / v 10 min 4°C

[0489] V3

[0490] 55% w / v 10 min -20°C

[0491] V3

[0492] Vitrification <ld <-140°C

[0493] 5 25% w / v lOmin 0.8ml / min 4°C 0 0 2.5 30 Glucose in

[0494] PBS

[0495] 6 Relax aCSF 20min 1.8ml / min 4°C 0 4 2.5 30 7 aCSF 0 Ca lOmin 1.8ml / min 20°C 0 4 0 30 8 aCSF 0.1 lOmin 1.8ml / min 20°C 0.1 4 0 30 Ca

[0496] 9 aCSF 1 Ca 20min 1.8ml / min 20°C 1 3 0 30 10 Recording 20 min 1.8ml / min 20°C 2.5 2.5 0 0

[0497]

[0498] aCSF

[0499] Krebs-Henseleit Buffer (KHB) may be used instead of aCSF. In certain examples, the solution in step 4a was replaced with 45% w / v V3 in LM5. Step 4b may alternatively be performed at a temperature of -20°C. In certain examples, there may be an additional step between "vitrification" and step 5, wherein washout with 45% w / v V3 + 300mM Mannitol in LM5 buffer is performed for 5 min at -20°C, before proceeding to step 5.Example 7 (cryopreservation of skin and cutaneous nerves)

[0500] Recordings of cutaneous C-nociceptors were conducted in hairy skin of Black 6 mouse using the skin saphenouse nerve preparation (Babes A. et al 2010 in Methods Mol. Biol. 617, 237-259) about 30 minutes after completion of the cryopreservation procedure (shock-loading via immersion with 45%V3 at 4°C for 10 min, followed by 59%V3 for 20 min at -20°C; rewarming and washout by 45%V3 +300mM Mannitol at -20°C, followed by hyperosmotic washout with 25% Glucose in PBS at 4°C for 10 min, followed by LM5 at 4°C for 20 min).

[0501] Fig. 17A shows electrical stimulation within the receptive field of the nociceptor to determine response latency which was at about 50 ms. Thereafter the receptive field was stimulated by mechanical and heat stimuli to identify the respective fiber subtype: polymodal C-mechano-heat (C-MH) -sensitive with a mechanical v. Frey threshold of 5.7 mN. Chemical responsiveness (Fig. 17B) was tested by superfusion of the receptive field with Capsaicin at lOpM concentration. The instantaneous discharge frequency (1 / s) is depicted, every dot represents a capsaicin-evoked C-fi be r action potential / spike).

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[0529] All references cited herein are fully incorporated by reference. Having now fully described the invention, it will be understood by a person skilled in the art that the invention may be practiced within a wide and equivalent range of conditions, parameters and the like, without affecting the spirit or scope of the invention or any embodiment thereof.

Claims

New PCT-Patent ApplicationFriedrich-Alexander-Universitat Erlangen-NurnbergVossius Ref.: AK1615 PCT S3Claims1. A method for cryopreservation of a biological sample comprising the step:AA. contacting the biological sample with a cryoprotectant solution comprising at least about 45 % cryoprotectant, andlowering the temperature of the biological sample,wherein the cryoprotectant solution comprising at least about 45 % cryoprotectant is the sole cryoprotectant solution with which the biological sample is contacted;orwherein the method comprises contacting the biological sample with a cryoprotectant solution comprising at least about 50 % and does not comprise contacting the biological sample with a cryoprotectant solution comprising less than about 50 % cryoprotectant; orAB. contacting the biological sample sequentially with a cryoprotectant solution comprising at least about 45 % cryoprotectant and a second solution wherein the cryoprotectant solution comprising at least about 45 % cryoprotectant is the sole cryoprotectant solution with which the biological sample is contacted, andlowering the temperature of the biological sample.

2. The method of claim 1, wherein said cryoprotectant solution comprises at least about 45% w / v cryoprotectant selected from the group consisting of dimethyl sulfoxide, ethylene glycol, propylene glycol, glycerol, formamide, N-methylformamide, dimethylformamide, 3-methoxy-l,2-propanediol, and any combination thereof, optionally supplemented with non-cell-permeating sugars or macromolecules.

3. The method of any one of claims 1 to 2, wherein said cryoprotectant solution comprises at least about 50 % cryoprotectant.

4. The method of any one of claims 1 to 3, wherein said cryoprotectant solution comprises at least about 55 % cryoprotectant.

5. The method of any one of claims 1 to 3, wherein said cryoprotectant solution comprises 59% w / v cryoprotectant.

6. The method of any one of claims 1 to 3, wherein said cryoprotectant solution comprises 59% w / v V3.

7. The method of any one of claims 1 to 6, wherein the method comprises contacting the biological sample with a cryoprotectant solution comprising at least about 50 % and does not comprise contacting the biological sample with a cryoprotectant solution comprising less than about 50 % cryoprotectant.

8. The method of any one of claims 1 to 7, wherein the method does not comprise contacting the biological sample with a cryoprotectant solution comprising 2 %, 4 %, 8 %, 16 % and / or 30 % cryoprotectant.

9. The method of any one of claims 1 to 8, wherein the method further comprises a step B of storing the biological sample and a step C of removing the cryoprotectant solution from the biological sample, and wherein the cryoprotectants are removed from said biological sample by contacting the sample with one or more hyperoncotic solution(s), preferably with colloidal solutions of oncotic pressure greater than physiological levels.

10. The method of claim 9, wherein said colloidal solution comprises about 10% to 30% w / v dextran.

11. The method of any one of claims 1 to 10, wherein the biological sample is / are or comprises cells, cellular systems, tissues, tissue-engineered constructs, organoids, organs, or organisms.

12. The method of any one of claims 1 to 11, wherein the biological sample is a sample from a vertebrate, preferably a mammal.

13. The method of any one of claims 1 to 11, wherein the biological sample is a brain, preferably the whole brain, and / or wherein said contacting with said cryoprotectant solution and / or removing of said cryoprotectant solution do not require deliberate opening of the blood-brain barrier.

14. The method of any one of claims 1 to 13, wherein contacting with the cryoprotectant solutions and / or removing of the cryoprotectant solutions are performed via perfusion, superperfusion, immersion, or combinations thereof.

15. The method of any one of claims 13 and 14, wherein when the biological sample is the whole mammalian brain, the method comprises the following steps:A. performing blood washout and connecting said brain to a perfusion system; AA. perfusing the brain with vitrification solution containing at least 45% w / v cryoprotectant at approximately half of physiological baseline flow rates; or AB. perfusing the brain sequentially with a vitrification solution containing at least 50% w / v cryoprotectant and a carrier solution by intermittently alternating perfusion between said vitrification solution and carrier solution B. vitrifying and storing said brain below the glass transition temperature;BB. rewarming said brain above the melting temperature without causing crystallization; andC. perfusing colloidal solutions at reduced flow rates adjusted to solution viscosity.

16. The method of any one of claims 1 to 13, comprising the steps of:AA. immersing said biological sample in vitrification solution containing at least 45% w / v cryoprotectant until said samples sink or equilibrate;B. vitrifying and storing said biological samples below the glass transitiontemperature;BB. rewarming said biological samples above the melting temperature without crystallization;C. immersing said biological samples in a colloidal solution; andD. subsequent immersion in isotonic carrier solutions.

17. The method of any one of claims 1 to 16, wherein said biological samples are viable after removal of the cyroprotectant solution and, optionally, are assessed after recovery, by methods such as, but not limited to, electrophysiological, structural, biochemical, immunohistochemical, functional, or molecular assessment postcryopreservation.

18. The method of any one of claims 1 to 17, wherein said biological samples include human neurospheres, organotypic brain slices, acute brain slices, mammalian including human organs and organisms, natural and engineered cells, stem cells, oocytes, sperm, embryos, ovarian and testicular tissue, pancreatic islets, and tissue- engineered constructs such as skin, cartilage, vascular grafts, nerves, bladder augmentation, and corneas.

19. A biological sample cryopreserved by the method of any one of claims 1 to 18.

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