Identification of cryoprotectants for cryopreservation of cells and cell aggregates

Abstract

The present disclosure provides a method of identifying an intracellular cryoprotectant expressed by a cell, the method comprising: (a) using a cell that survives in freezing and thawing, (b) modifying the used cell, (c) freezing and thawing the modified cell, and (d) obtaining a modified cell that survives after step (c), an intracellular cryoprotectant expressed by the cell is thus identified, as well as an intracellular cryoprotectant obtained by or which can be obtained by the method.

Description

Field of the Invention

[0001] The present invention relates to a method for identifying an intracellular cryoprotectant expressed by a cell, the method comprising: (a) using cells that survive freezing and thawing, (b) modifying the cells used, (c) freezing and thawing the modified cells, and (d) obtaining the modified cells that survive step (c), thereby identifying the intracellular cryoprotectant expressed by the cells. The present invention also relates to an intracellular cryoprotectant or a derivative thereof, preferably obtained by or by the method, wherein the intracellular cryoprotectant is an intracellular cryoprotectant synthesized by the cell. The present invention also relates to a method for generating an optionally modified nucleotide sequence encoding the intracellular cryoprotectant or a derivative thereof, as well as to an optionally modified nucleotide sequence encoding one or more intracellular cryoprotectants or derivatives thereof that can be obtained by or by the method, and nanoparticles comprising the optionally modified nucleotide sequence. The present invention also relates to the use of such intracellular cryoprotectants or their derivatives, the optionally modified nucleotide sequences and / or the nanoparticles for cryopreservation of cells, cell aggregates, cell cultures, tissues, organoids and / or organs, as well as a method for cryopreservation of cells, cell aggregates, cell cultures, tissues, organoids and / or organs, comprising (a) contacting cells, cell aggregates, cell cultures, tissues, organoids and / or organs with the optionally modified nucleotide sequences and / or nanoparticles, and (b) freezing the cells, cell aggregates, cell cultures, tissues, organoids and / or organs obtained from step (a), wherein freezing comprises lowering the temperature of the cells, cell aggregates, cell cultures, tissues, organoids and / or organs from above 0°C to below 0°C, preferably below -20°C, more preferably below -70°C, and even more preferably below -140°C. The invention further relates to cells, cell aggregates, cell cultures, tissues, organoids and / or organs obtained or obtainable by the method, and corresponding frozen and thawed cells, cell aggregates, cell cultures, tissues, organoids and / or organs. The invention further relates to the use of the (frozen and thawed) cells, cell aggregates, cell cultures, tissues, organoids and / or organs for drug development, active ingredient and / or drug screening, (preclinical) drug testing and / or (bio)medicine, preferably transplant medicine. Background of the Invention

[0002] Only a limited number of plant and animal species are able to respond to freezing by synthesizing cellular cryoprotectants, such as thermoprotective or antifreeze proteins, or by accumulating substances such as sugars or glycerol. In contrast, cells from other species, such as humans, are unable to produce sufficient levels of cryoprotectants to avoid cell damage caused by cooling and freezing processes. However, methods for freezing cells to maintain viability have been established.

[0003] A well-known method is called cryopreservation, and methods for (single) cells in suspension (insuspension) are well established. In the case of cryopreservation, freezing and thawing are usually carried out in the presence of a cryoprotectant, which is added to the cell suspension to prevent or inhibit the formation of ice crystals in the cells during freezing. The cryoprotectant needs to pass through the cell membrane to exert its protective effect within the cell during freezing. Since it is mainly based on diffusion processes across the cell membrane, cryopreservation methods are subject to at least three limitations. First, only relatively small molecules such as dimethyl sulfoxide (DMSO) can be transferred into the cells, because large molecules such as antifreeze proteins are not membrane permeable or cannot be introduced into the cells in sufficient quantities and within the required time due to their dependence on membrane-bound transport molecules. In addition, current methods require a considerable amount of cryoprotectant, usually in the % range, to ensure cell protection (see, for example, Mazur, P., Herole of intracellular freezing in the death of cells cooled at supraoptimal rates, Cryobiology 1977, 14: 252-272; Figure 1 (Schematically represented by "C" in the figure). However, some commonly used cryoprotectants, such as DMSO, have the disadvantage of negatively affecting cell viability due to toxic effects, especially when these cryoprotectants are present at higher concentrations in the culture medium. Finally, the transmembrane transport of any required cryoprotectant limits the applicability of cryopreservation to larger cell aggregates, especially organoids, tissues and organs. More specifically, although the required exposure time increases quadratically with the diameter, in the case of >1 mm 3 Limitations in diffusion rate and gradient formation already occur below the volume.

[0004] In order to overcome these limitations of cryopreservation, research has focused on, for example, optimizing predetermined temperature-time protocols taking into account the properties of the corresponding biomaterial to be cryopreserved (see, for example, MA Taylor et al., " New Approaches to Cryopreservation of Cells, Tissues and Organs "in " Transfus. Med. hemother. " 46: 2019, 197-215). Optimizing process parameters requires extensive experience, and when not only single cells in suspension need to be cryopreserved, but also larger cell aggregates need to be cryopreserved, the required optimization workload increases significantly. Known cryopreservation procedures include, in particular, slow freezing and vitrification, and their various improved methods and combinations. In the case of slow freezing, which can also be referred to as "slow freezing," the temperature is reduced at a low cooling rate to allow sufficient cell dehydration while minimizing the formation of ice crystals in the cell. However, methods based on slow freezing have the disadvantages of low cell viability and limited thawing cell function, such as limited growth capacity and / or the need for long re-cultivation time after thawing. On the other hand, vitrification refers to almost instantaneous freezing due to ultra-high cooling rates, which allows cells and their extracellular environment to solidify into a glass-like state without forming crystalline ice. Due to the need for ultra-high cooling rates, successful vitrification is usually limited to small volumes of cells and culture medium to ensure a relatively large surface-to-volume ratio and a relatively small distance between cells and cryoprotectants. In addition, vitrification-based methods require relatively high concentrations of cryoprotectants such as DMSO, which in turn can negatively affect cell survival and viability.

[0005] Although single cells in suspension are currently widely cryopreserved, especially using slow freezing, there is also interest in the application of cryopreservation of larger and heterogeneous cell aggregates (including organoids, tissues and organs), especially in the fields of biotechnology, pharmacology and (bio) medicine, such as transplant medicine. However, the success rate of slow freezing of three-dimensional cell aggregates is limited. Smaller cell aggregates have been preserved by vitrification. However, in order to provide a high intracellular cryoprotectant concentration and to make the temperature of all regions inside the cell aggregate reach below the glass transition point ultra-rapidly, strict technical limitations are faced. This is mainly related to sample volume, toxicity caused by the required cryoprotectant concentration and exposure time, and the increased possibility of cell damage due to thermal stress cracking as the sample size increases. Therefore, vitrification is also considered to be unsuitable for the conventional preservation of larger cell aggregates.

[0006] Therefore, there is still a need to find a solution to cryopreserve larger cell aggregates, especially organoids, tissues, and organs, without functional impairment.

[0007] The present application addresses this need by providing embodiments as described in the claims. More specifically, the present invention provides intracellular cryoprotectants that can be synthesized within cells (e.g., cells of organoids, tissues, or organs), as well as automated high-throughput methods for identifying suitable intracellular cryoprotectants and efficient methods for cryoprotecting organoids, tissues, and organs using the methods.

[0008] In particular, the present invention relates to a method for identifying an intracellular cryoprotectant expressed by a cell, the method comprising (a) using cells that survive freezing and thawing, (b) modifying the used cells, (c) freezing and thawing the modified cells, and (d) obtaining the modified cells that survived step (c), thereby identifying the intracellular cryoprotectant expressed by the cell. Summary of the Invention

[0009] A first aspect of the present invention relates to a method for identifying an intracellular cryoprotectant expressed by a cell, the method comprising (a) using cells that survive freezing and thawing, (b) modifying the used cells, (c) freezing and thawing the modified cells, and (d) obtaining a modified cell that survived step (c), thereby identifying an intracellular cryoprotectant expressed by the cell.

[0010] In some embodiments, the cells and / or modified cells are capable of being cultured in suspension and / or are cells in suspension.

[0011] In some embodiments, in step (a), the cells survive freezing and thawing in suspension, preferably comprising an added cryoprotectant at a concentration of 5% or less, preferably 1% or less, more preferably 0%, and / or wherein in step (c), the modified cells are frozen and thawed in suspension, preferably comprising an added cryoprotectant at a concentration of 5% or less, preferably 1% or less, more preferably 0%.

[0012] In some embodiments, the method further comprises: (e) Proliferating the obtained ((further) modified) cells, preferably in suspension.

[0013] In some embodiments, the method comprises the steps of: step (a), optionally followed by step (e), followed by [step (b), optionally step (e), steps (c) and (d), optionally step (e)] in i iterations, where i is preferably at least 1 and at most 1,000.

[0014] In some embodiments, the method further comprises: (f) determining the viability of the obtained (further) modified cells, preferably for each step (d).

[0015] In some embodiments, if the survival rate determined in step (f) of iteration i is at least as high as the survival rate determined in step (f) of iteration i-1, preferably 10% higher, and preferably does not exceed a given threshold, the method comprises iteration i+1.

[0016] In some embodiments, the method comprises the following steps: step (a), optionally followed by step (e), followed by i iterations of [step (b), optionally step (e), steps (c) and (d), optionally step (f), optionally step (e)], wherein i is at least 2, and preferably further comprising iteration i+1 if the survival rate determined in step (f) of iteration i is at least as high as the survival rate determined in step (f) of iteration i-1, preferably 10% higher, and preferably does not exceed a given threshold.

[0017] In some embodiments, the intracellular cryoprotectant expressed by the (further) modified cells is identified by analyzing the genome, transcriptome, epigenome, proteome and / or metabolome, preferably by using an analysis selected from gel electrophoresis, mass spectrometry, crystal structure analysis, NMR spectroscopy, DNA sequencing, (m)RNA sequencing and any combination of the foregoing.

[0018] In some embodiments, if the viability determined in step (f) exceeds a given threshold, then the intracellular cryoprotectant expressed by the (further) modified cell is identified.

[0019] In some embodiments, the method further comprises: (h) modifying said identified intracellular cryoprotectant expressed by said (further) modified cell, thereby obtaining a derivative from said identified intracellular cryoprotectant expressed by said (further) modified cell.

[0020] In some embodiments, the method further comprises the following steps: (a) using cells that survive freezing and thawing, Wherein step (a) preferably comprises the following steps: (a0) Freezing and thawing of cells in suspension, (a1) obtaining cells that survived step (a0), (f1) determining the viability of step (a0) based on the number of living cells of the cells in suspension before freezing and thawing and after freezing and thawing, (e1) growing the cells obtained in step (a1) and using the grown cells, (b) modifying the cells used in step (a), preferably the cells proliferated in step (e1) and / or the cells proliferated in step (e3), (e2) multiplying the modified cells in step (b), (c) freezing and thawing the modified cells in suspension that have proliferated in step (e2), (d) obtaining modified cells that survive step (c), thereby identifying an intracellular cryoprotectant expressed by said cells, wherein preferably, the freezing and thawing in step (a), preferably step (a0) and / or step (c) are carried out in a suspension, said suspension preferably comprising an added cryoprotectant at a concentration of 5% or less, preferably 1% or less, more preferably 0%.

[0021] In some embodiments, the intracellular cryoprotectant expressed by the modified cell is identified by: (f2) determining the viability of step (c) based on the number of modified living cells in suspension before freezing and thawing and after freezing and thawing, (e3) propagating the modified cells obtained in step (d), and (G) identifying at least one intracellular cryoprotectant based on the modified cells propagated in step (e3), preferably if the viability determined in step (f2) exceeds a given threshold, wherein if the survival rate determined in step (f2) of iteration i is at least as high as the survival rate determined in step (f2) of iteration i-1, preferably 10% higher, and preferably does not exceed the given threshold, and wherein i is at least 2, then steps [(b), (e2), (c), (d), (f2), (e3)] are iterated (iteration i+1 times).

[0022] In some embodiments, the suspension in step (c) of iteration i+1 comprises: (1) if the viability determined in step (f2) is at least as high, preferably at least 5% higher, than the viability determined in: i) step (f1) in the case of the first iteration (i=1), or ii) step (f2) of iteration i-1 in the case of iteration i, the concentration of cryoprotectant added is substantially the same as for the suspension in step (c) of iteration i; (2) If neither i) nor ii) is satisfied, the concentration of the cryoprotectant added is 0.001% to 1%, preferably 0.01% to 0.5%, higher than the concentration in the suspension in step (c) of iteration i.

[0023] The second aspect of the present invention relates to an intracellular cryoprotectant or a derivative thereof, preferably obtained or obtainable by the method of the first aspect, wherein the intracellular cryoprotectant is an intracellular cryoprotectant synthesized by cells, and is preferably an optionally glycosylated polypeptide or protein.

[0024] In another aspect, the present invention relates to a method for producing a cryoprotectant or a derivative thereof, comprising performing the method for identifying an intracellular cryoprotectant according to any embodiment of the present invention. DETAILED DESCRIPTION

[0025] It has been surprisingly found that the method according to the present invention allows the identification of intracellular cryoprotectants in an efficient, easily automated and high-throughput manner. By applying an in vitro evolution method based on cell freezing and thawing cycles (preferably repeated cycles) and introducing modifications, preferably gene mutations, during which effective intracellular cryoprotectants can be generated, isolated and identified. Therefore, compared to added cryoprotectants that need to pass through the cell membrane to show their cryoprotective effect in the cell, intracellular cryoprotectants that are less affected by size restrictions and diffusion processes can be identified. Most importantly, since the intracellular cryoprotectants are synthesized directly by the cells and directly in the cells, the limitations of added cryoprotectants due to diffusion rate and gradient formation can be overcome. Therefore, the intracellular cryoprotectants identified by the method of the present invention can, for the first time, make cryopreservation suitable for pharmaceutical and medical applications, such as ready-to-use tissue cultures for drug screening and cryopreservation of tissues and organs, such as for organ transplantation.

[0026] In the context of the present invention, the term "cryoprotectant" refers to any substance that protects cells from cold damage during freezing, such as caused by ice crystal formation within the cells. There may be different mechanisms of action for the cryoprotectants encompassed herein. For example, the cryoprotective effect may be based on the formation of hydrogen bonds with biomolecules within the cell when water molecules are displaced, allowing the cell to maintain its natural physiological structure and function without being immersed in an aqueous environment. As another example, the protective effect of some cryoprotectants may be based on lowering the glass transition temperature of the cell, thereby lowering the temperature below which the cell becomes osmotically inactivated. Thus, the cryoprotectant does not actually prevent freezing, but ensures that the cell maintains a certain flexibility in the glass phase.

[0027] "Added cryoprotectants" herein refer to cryoprotectants that are added to, for example, the cell (suspension) culture medium. Such added cryoprotectants can be any substance that prevents and / or inhibits the formation of ice crystals in cells during freezing, such as dimethyl sulfoxide (DMSO), 1,2-propylene glycol, glycols such as glycerol, ethylene glycol and propylene glycol, amino acids and / or sugar molecules such as trehalose and sucrose. Thus, added cryoprotectants are exogenously added and initially extracellular cryoprotectants that must, for example, diffuse across the cell membrane in order to exert their protective effect within the cell during freezing.

[0028] As used herein, "intracellular cryoprotectants" refer to cryoprotectants that are expressed by cells and / or synthesized within cells. Thus, the cryoprotectant is endogenously expressed and / or synthesized in the cell. This has the advantage that its presence within the cell depends, for example, on the availability of a biological template for synthesis, such as mRNA encoding a cryoprotectant such as a polypeptide or protein, and / or the availability of molecules such as amino acids for expression and / or synthesis. In some instances, the intracellular cryoprotectant obtained or obtainable by the methods of the present invention is synthesized by the cell and is preferably an optionally glycosylated polypeptide or protein. Thus, in contrast to added cryoprotectants, intracellular cryoprotectants do not need to pass through the cell membrane. Therefore, compared to added cryoprotectants, intracellular cryoprotectants are less constrained by size limitations. Furthermore, while existing methods based on added cryoprotectants are limited by the volume of the biological material to be cryopreserved due to gradient formation, intracellular cryoprotectants allow for the first time the adaptability of larger cell aggregates, organoids, tissues and organs to cryopreservation.

[0029] The method of the present invention is a method for identifying intracellular cryoprotectants expressed by cells. This "intracellular cryoprotectant expressed by cells" refers to an intracellular cryoprotectant that the cells used are inherently capable of expressing. Therefore, such cells contain endogenous, respectively required biological information to construct intracellular cryoprotectants. Therefore, cells suitable for the method of the present invention are especially cells that themselves have the ability to express intracellular cryoprotectants and / or cells that are modified in a manner that generates or enhances the ability of the cells to express intracellular cryoprotectants. For example, it is particularly advantageous to use cells that themselves have the ability to express intracellular cryoprotectants because the use of such cells can promote the identification of intracellular cryoprotectants and / or reduce the time required for the identification. Compared with unmodified wild-type cells of the same species, the use of cells modified in a manner that enhances the ability to express intracellular cryoprotectants is, for example, particularly advantageous for identifying intracellular cryoprotectants that exhibit a stronger cryoprotective effect than the corresponding intracellular cryoprotectants identified in the corresponding wild-type cells. Thus, this approach is particularly advantageous, for example, for identifying intracellular cryoprotectants that are optimized for their potency and / or the occurrence of cellular side effects on cell viability compared to the corresponding wild-type. The use of cells that have been modified in some way to confer the ability to express an intracellular cryoprotectant is particularly advantageous, for example, for ensuring the suitability of an identified intracellular cryoprotectant for a given species by ensuring that the (epi)genetic background of the cells used to identify the intracellular cryoprotectant and the cells to be cryopreserved are comparable in the sense that no interspecies adaptation and / or adjustment is required.

[0030] Therefore, in some embodiments of the present invention, the cell is obtained from vertebrates, insects, plants, algae, fungi or bacteria. For example, the cell can be obtained from terrestrial hibernating animals such as Rana sylvatica, which is a northern woodland species that hibernates on land in a location where dehydration and freezing may occur. As another example, the cell can be obtained from fish, for example, from American velvet sculpin such as Blepsias cirrhosus, from Arctic cod and / or Antarctic cod species, or from flatfish species such as Pleuronectes americanus or Limanda ferruginea. As another example, the cells may be obtained from cryospheric species such as snow algae, for example from Antarctic species of the genus Chlorominima, Arctic and / or Antarctic species of the genus Chlamydomonas, or species of the genus Chloromonas, such as Chloromonas nivalis or Chloromonas rostafinskii or Ancylonema polaris. ), or from green algae, for example from species of the genus Chlorococum, such as Chlorococum sp., or species of the genus Raphidonema, such as Raphidonema brevirostre or Raphidonema nivale.

[0031] In some embodiments of the present invention, the method is performed using cells obtained from vertebrates, insects, plants, algae, fungi, or bacteria, and the cells themselves have the ability to express an intracellular cryoprotectant.

[0032] In some embodiments of the present invention, cells can be cultured in vitro. In this article, culture in vitro refers to culturing cells in an artificial and preferably well-controlled environment comprising a cell culture medium. Cells can be cultured, for example, as adherent cell cultures and / or as suspension cultures. Alternatively or optionally, the cells can also be cells contained in cell aggregates, tissues, organoids, organs and / or their parts such as tissue sections, wherein the cell aggregates, tissues, organoids, organs and / or their parts are cultured in vitro, preferably (at least partially and / or temporarily) suspension cultures. In this article, preferably, the cells used in the method according to the present invention are cells capable of suspension culture and / or cells in suspension. Therefore, the cells can preferably be cultured (at least temporarily) in (suspension) culture medium, and optionally go down to posterity. Considering that the culture conditions of many cell types and species are available and fully studied, it is advantageous to use cells capable of suspension culture and / or cells in suspension, easy to operate and with good (upward) scalability.

[0033] Therefore, in a preferred embodiment of the present invention, the cells are cells that can be cultured in suspension and / or are cells in a suspended state.

[0034] In step (a) of the method of the present invention, cells that survive freezing and thawing are used. Therefore, it is preferred to obtain viable cells that have been exposed to temperatures below 0°C or lower, and subsequently to temperatures above 0°C. This is particularly advantageous for identifying cells that exhibit a certain degree of cold resistance, preferably cells that exhibit tolerance to freezing. Therefore, the cells used in step (a) are viable cells obtained after freezing and thawing, wherein the intracellular cold damage of the cells due to freezing is limited to at least a degree that allows the cells to survive.

[0035] In some embodiments of the present invention, freezing comprises or is a temperature drop from above 0° C. to a temperature below 0° C., preferably below -20° C., more preferably below -70° C., and even more preferably below -140° C. The latter is particularly advantageous because temperatures below -140° C. are below the glass transition temperature of water, which is approximately -135° C. Therefore, the survival of cells whose temperature is dropped from greater than 0° C. to less than the glass transition temperature of water may indicate the presence of a cryoprotectant and thus increase the ease of identification of cryoprotectants within cells according to the present invention.

[0036] In some embodiments of the present invention, thawing comprises or is increasing from a temperature below 0°C, preferably below -20°C, more preferably below -70°C, even more preferably below -140°C to a temperature above 0°C.

[0037] It should be noted that in the case of freezing and / or thawing, the temperature is preferably the temperature of a suspension in which the cells used survive freezing and thawing.

[0038] Therefore, in a particularly preferred embodiment of the present invention, the cells that survive freezing and thawing used are cells in suspension, wherein the freezing comprises or is a reduction in the temperature of the suspension from above 0°C to a temperature below 0°C, preferably below -20°C, more preferably below -70°C, even more preferably below -140°C, and / or wherein the thawing comprises or is a rise in the temperature of the suspension from below 0°C, preferably below -20°C, more preferably below -70°C, even more preferably below -140°C to a temperature above 0°C.

[0039] Furthermore, in some embodiments of the present invention, after the temperature is lowered, the lowered temperature is maintained for at least 1 second to 10 minutes, preferably at least 10 seconds to 5 minutes, before thawing. Thus, cells exposed to freezing and thawing have time to respond to the temperature change of the cell environment, preferably the suspension, during the freezing and thawing process.

[0040] It should be noted that, without being limited by theory, there may or may not be an upper limit, and thus, there may be a maximum time period during which the reduced temperature is maintained. For example, in the case of almost instantaneous freezing due to ultra-high cooling rates, where the reduced temperature is a temperature below, for example, -140°C, there may not be a (practical) upper limit to the time period during which the reduced temperature is maintained. However, in the case of reduced temperatures between, for example, less than 0°C and above (and including) -140°C, there may be some biological, physicochemical, chemical, and / or physical processes, such as migratory ice growth in cells, that may require consideration of an upper limit to the time period during which the reduced temperature is maintained. Therefore, those skilled in the art will appreciate that, depending on the reduced temperature selected for freezing, there may be an upper limit to the time period.

[0041] In addition, about freezing and thawing, it is envisaged that a predetermined temperature-time scheme is applied, which is optimized in view of the cells used, for example, in view of species and / or cell types. For example, it can be particularly ensured that the cells used are exposed to a temperature lower than but close to 0°C for a long enough time during thawing to avoid the artificial deviation caused by unsuitable rapid thawing, which may have a negative impact on the survival and viability of the cells used. For different species, cell types and / or cells, known temperature-time schemes can be obtained. Although those skilled in the art are aware of the identification of suitable temperature-time schemes and / or suitable temperature-time schemes (see, for example, Mazur, P., The role of intracellular freezing in the death of cells cooled at supraoptimal rates, Cryobiology 1977, 14: 252-272), several illustrative examples are provided in the Examples section. The present invention encompasses methods such as those based on vitrification, methods based on slow freezing and any combination and / or modifications thereof.

[0042] In addition, in some embodiments of the present invention, the cells used in step (a) survive freezing and thawing in a suspension, the suspension preferably comprising an added cryoprotectant at a concentration of 5% or less, preferably 1% or less, more preferably 0%. Thus, depending on the cells used and / or the focus of the method, the suspension may or may not comprise an added cryoprotectant. For example, in cases where the cells themselves already have the ability to express intracellular cryoprotectants and / or where the focus of the method is to identify intracellular cryoprotectants using a temperature-time protocol that initiates the naturally occurring expression of intracellular cryoprotectants in the cells to the extent that ensures cell survival. As another example, in cases where the cells are, for example, unable to express intracellular cryoprotectants to ensure cell survival when frozen and / or where the negative impact on cell viability exceeds the desired extent, it may be advantageous to add the added protectant to the suspension of cells. If the suspension contains an added cryoprotectant, it is preferred that the concentration of the cryoprotectant added to the suspension is lower than, for example, the concentration disclosed in the prior art when the corresponding cells are stored at low temperatures and / or as low as possible to obtain cells that survive freezing and thawing. Therefore, different concentrations of added cryoprotectant can be tried to study the appropriate low concentration of cryoprotectant added to the suspension to obtain cells for step (a) that survive freezing and thawing in a suspension containing an added cryoprotectant. For example, an added cryoprotectant may not be used. In the case where no cells survive freezing and thawing in a suspension to which no cryoprotectant is added (a first concentration of 0%), another experiment can be performed using a second concentration that is higher than the first concentration, for example, 0.05% or 0.01%. The experiment can be repeated with increasing concentrations of added cryoprotectant until at least one surviving cell is obtained. As another example, the concentration reported in the literature can be used as an indication of the first concentration of the added cryoprotectant. When cells that survive freezing and thawing in a suspension comprising said first concentration of added cryoprotectant are obtained, another experiment can be performed using a concentration that is, for example, 0.05% or 0.01% lower than the first concentration. The experiment can be repeated with decreasing concentrations of added cryoprotectant until no viable cells can be obtained. Preferably, cells that survive freezing and thawing according to step (a) of the method of the invention are obtained using the lowest concentration of added cryoprotectant that results in viable cells. Therefore, the person skilled in the art will appreciate that the preferred concentration depends, for example, on the cells, in particular the cell type and / or species of origin of the cells, and / or the added cryoprotectant, and that the considerations can be extended accordingly to the case of more than one added cryoprotectant.

[0043] With regard to step (b) of the method of the present invention, the cells used and / or obtained are modified. Therefore, it is preferred that cells that survive freezing and thawing are modified genetically and / or epigenetically, more preferably genetically. By modifying the cells, a modified form of the cells can be obtained, also referred to herein as a mutant. Therefore, one or more mutants can be obtained that can i) express intracellular cryoprotectants more effectively, such as intracellular cryoprotectants that result in higher intracellular concentrations, ii) can additionally express another intracellular cryoprotectant, and / or iii) can show an enhancement of the protective effect due to the newly introduced modification. Therefore, modifying cells, such as the cells used in step (a), is advantageous for obtaining one or more mutants that can exhibit better cryoprotection than the cells originally used, such as the cells of step (a).

[0044] In this article, the term "modification" encompasses spontaneous and artificially induced changes. For example, the term encompasses the modification of cells, for example, at heredity, epigenetic inheritance, transcription, translation, proteome and / or metabolome levels, preferably at the genetic level. As another example, the term also encompasses the modification of nucleotide sequences, for example, by introducing single nucleotide polymorphisms, inserting and / or lacking one or more nucleotides and / or combining one or more nucleotide sequences.

[0045] More specifically, the term "modified cell" refers herein to a cell that has been subjected to a modification that results, preferably artificially induced, in a change in the genetic, epigenetic, transcriptional, translational, proteomic and / or metabolomic levels of the cell, preferably a change at the genetic level. Preferably, the "modified cell" according to the present invention relates to a cell obtained after step (b) of the method according to the present invention, more preferably to a used cell of step (a) that has undergone step (b) once. In the case where the used cells have undergone more than one step (b), the cells are preferably referred to herein as "further modified cells". It should be noted that the terms "modified cell" and "further modified cell" both encompass i) cells that are cells used in step (a) of the method according to the present invention and ii) cells derived from and therefore derived from the used cells of step (a). Preferably, the used cells of step (a), the modified cells of step (b) and optionally the further modified cells of step (b) are therefore identical cells, except for any changes introduced by one or more rounds of step (b), and / or the cells are essentially clones, e.g., have essentially the same genetic background, except for any changes introduced by one or more rounds of step (b). Said clones can be obtained after propagating said used and / or (further) modified cells. Thus, with respect to said "further modified cells", they are the same as described herein in the context of "modified cells".

[0046] Preferably, the modification in step (b) is a modification at the genetic level of a cell (e.g., the cell used in step (a)). Therefore, a mutant showing one or more changes in the genome can be obtained compared to the cell before modification in step (b), wherein the change can be, for example, the deletion of one or more nucleotides, the insertion of one or more nucleotides, the exchange of one or more nucleotides, the multiplication of a part for a DNA sequence, the reorganization of a DNA sequence, or any combination thereof. In this article, the term "DNA" refers to a single-stranded or double-stranded deoxyribonucleotide sequence consisting of, for example, A, C, G and / or T nucleotides, i.e., adenine, guanine, cytosine and thymine as the nucleotides of each nitrogenous base. Preferably, the modification in step (b) is a modification in the regulatory and / or coding region of a DNA sequence. This is advantageous because this change can affect expression, expression intensity, expression duration and / or activity, the positioning and / or effectiveness of the expression product and / or any combination thereof. Therefore, this modification can directly affect the cryoprotective properties of a cell.

[0047] Therefore, in some embodiments of the present invention, modifying the cell in step (b) involves modification at the genetic level of the cell. Preferably, the modification is in the regulatory and / or coding region of the gene, more preferably in the coding region of the gene. The modification of the gene regulatory region can increase the transcription of the gene, which can result in a higher concentration of the intracellular cryoprotectant in the cell. The modification in the gene coding region can result in, for example, a situation in which the intracellular cryoprotectant is a polypeptide or protein with a mutant form of different function and / or activity compared to the intracellular cryoprotectant expressed in the cell before modification in step (b) (e.g., the cell used in step (a)). In addition, the modification in the gene coding region can also result in, for example, obtaining a new function, a phenomenon also referred to as gain-of-function mutation. This modification is advantageous because it can result in a mutant with improved freezing tolerance compared to their unmutated counterparts, and therefore results in the cell before being modified in the step (b).

[0048] Preferably, the modification in step (b) is an artificially induced change.

[0049] In some embodiments of the invention, the cell is modified in step (b) by i) exposure to a mutagenic agent, ii) exposure to radiation, iii) one or more modified proteins, and / or iv) any combination of the foregoing.

[0050] Therefore, in some embodiments of the present invention, in step (b), cells are modified by being exposed to mutagens. The term "mutagenic substance" as used herein refers to a chemical reagent that can induce cellular genetic and / or epigenetic levels to change, thereby causing (epi) genetic changes. Therefore, by exposing cells to mutagens, the mutation rate of cells can be increased and / or induced mutations. This is advantageous because it can result in mutant cells with increased low-temperature protection properties. This mutagenic substance is preferably selected from polycyclic aromatic hydrocarbons, nitrosamines, base analogs, peroxides and combinations thereof. Therefore, in some embodiments of the present invention, in step (b), cells are modified by being exposed to mutagens, preferably mutagenic substances selected from polycyclic aromatic hydrocarbons, nitrosamines, base analogs, peroxides and combinations thereof.

[0051] In some embodiments of the present invention, the cell is modified in step (b) by exposure to radiation. Radiation can be understood as an example of a physical mutagen. Therefore, with respect to exposure to radiation, the same applies as described above in the context of mutagenic substances. Furthermore, in the case where the mutagenic substance is a chemical agent, the other characteristics of such a physical mutagen may also be as described above. Preferably, the radiation is high-energy radiation, more preferably UV and / or X-ray radiation. Therefore, in some embodiments of the present invention, the cell is modified in step (b) by exposure to radiation, preferably high-energy radiation, more preferably UV and / or X-ray radiation.

[0052] In some embodiments of the present invention, cells are modified by one or more modified proteins in step (b). In this article, the term "modified protein" is intended to be understood as a protein that can induce changes in the genetic and / or epigenetic levels of cells and thus cause (epi) genetic changes. Therefore, with respect to the effect of the one or more modified proteins, the same applies to the content described above in the context of mutagenic substances. In this article, such modified proteins are preferably selected from TALEN, zinc finger proteins, CRISPR / Cas combinations, TET1, p300, DNMT3A, MQ1 and LSD1, wherein the one or more modified proteins more preferably include CRISPR / Cas combinations.

[0053] In the case of a method according to the invention as described below comprising more than one round of step (b), the respective methods for modifying the cells used and / or (further) modified may be identical or at least partially different between the respective steps (b). For example, it is conceivable that the cells used in step (a) are first modified in step (b) by exposure to radiation, wherein in a subsequent iteration the modified cells are further modified in the respective step (b) by exposure to a mutagenic substance.

[0054] With respect to the modification of cells, it should be noted that those skilled in the art are aware of various conventional techniques and methods for modifying cells and detecting and evaluating the resulting modifications. Corresponding methods are known in the art, and well-established experimental procedures are available. In particular, methods and techniques for modifying cells at the genetic level, thereby mutating cells to obtain modified forms of the cells, and thereby obtaining mutants of the cells, are known in the art.

[0055] As regards step (c) of the method according to the present invention, the modified cells, such as the modified cells obtained from step (b), are frozen and thawed.

[0056] In a preferred embodiment of the present invention, the modified cells obtained, for example, from step (b) and used in step (c) are cells capable of suspension culture and / or are in suspension.

[0057] Furthermore, as described above in the context of step (a), in some embodiments of the present invention, in the case of step (c), the modified cells are also frozen and thawed in a suspension, preferably comprising an added cryoprotectant at a concentration of 5% or less, preferably 1% or less, more preferably 0%. Thus, with respect to freezing and thawing (modified) cells in a suspension comprising an added cryoprotectant, the same applies as described above in the context of step (a), with the only exception that the cells of step (c) are (further) modified compared to the cells of step (a). Furthermore, other features of this freezing and thawing (modified) cells in a suspension comprising an added cryoprotectant may also be as described above in step (a).

[0058] With regard to step (d) of the method of the present invention, modified cells that survive in step (c) are obtained, thereby identifying the intracellular cryoprotectants expressed by the cells. Therefore, by obtaining (further) modified cells according to the method of the present invention, the cells preferably exhibit increased freezing tolerance due to their modification compared to the cells used in step (a) of the method. Therefore, by applying a preferred iterative combination of accelerated in vitro evolution and freezing selection pressure, (further) modified cells can be obtained for research, and therefore identify the intracellular cryoprotectants expressed by the (further) modified cells. The disclosed innovative method is well automatable and can quickly and effectively identify intracellular cryoprotectants that can be used for a variety of applications (e.g., in research, pharmaceutical industry, and (transplant) medicine) across species and / or cell types.

[0059] In some embodiments of the present invention, the method further comprises a step (e), i.e., propagating the obtained ((further) modified) cells, preferably in suspension. This is particularly advantageous because the propagation of the ((further) modified) cells enables the method of the present invention to be applied in high throughput. Thus, the ((further) modified) cells obtained, for example, from step (d) can be propagated and the method of the present invention can be performed on the propagated cells in parallel, and more specifically, the surviving cells can be subjected to one or more additional freezing and thawing repetitions and modifications. Furthermore, such parallelization of the method can increase the number of survival tests of different ((further) modified) cells, thereby increasing the likelihood of identifying a highly effective intracellular cryoprotectant and / or reducing the time required to identify such a highly effective intracellular cryoprotectant.

[0060] Thus, in some embodiments of the present invention, in step (e), the obtained ((further) modified) cells are propagated in a suspension, wherein i) the suspension is a suspension comprising an added cryoprotectant at a concentration of 5% or less, preferably 1% or less, more preferably 0%, and / or, wherein ii) the suspension comprises a conditioned (suspension) medium. Thus, the suspension in which the obtained ((further) modified) cells are propagated preferably comprises an added cryoprotectant and / or a conditioned (suspension) medium, wherein the conditioned (suspension) medium may comprise, for example, at least one growth factor to support cell viability and / or to optimize cell culture conditions, depending on the cell type and species under investigation, for example to take into account specific metabolic requirements.

[0061] In a preferred embodiment of the present invention, the method comprises the following steps: step (a), optionally followed by step (e), followed by [step (b), optionally step (e), step (c) and (d), optionally step (e)] in i iterations, wherein i is preferably at least 1 and a maximum of 1,000, preferably at least 1 and a maximum of 100, more preferably at least 2 and a maximum of 100. Thus, preferably, the cells that survive freezing and thawing in step (a) are optionally proliferated to enable parallel investigation of the modifications introduced in step (b); the modified cells in step (b) are also optionally proliferated to enable parallel investigation of the effects of a given modification on the cryoprotection of the cells and / or to exclude stochastic effects, such as cell death that is not related to a given cryoprotective effect; after freezing and thawing the optionally proliferated (further) modified cells, the cells that survived step (c) can be obtained; the surviving cells obtained from step (d) are optionally further proliferated, and then a new round of iterations of further steps (b) to (d) as described above can be initiated. Thus, using the iterative and accelerated evolution method, intracellular cryoprotectants can be efficiently identified in vitro.To further simplify cell handling, all cells described in the context of the embodiments, ie cells, modified cells and optionally further modified cells, are preferably cells in suspension.

[0062] In a preferred embodiment of the present invention, the method comprises the following steps: step (a), optionally followed by step (e), followed by [step (b), step (e), step (c) and (d), step (e)] in i iterations, wherein i is preferably at least 1 and a maximum of 1,000, preferably at least 1 and a maximum of 100, more preferably at least 2 and a maximum of 100. Thus, except that the corresponding proliferation step is performed within the i iterations, the same as described in the previous paragraph. Thus, the level of parallelization can be significantly increased, and the effects of different modifications on cell viability and / or freezing tolerance can be analyzed.

[0063] It should be noted that the maximum level of parallelization may (in practice) be limited. Therefore, it can be considered that in each subsequent step of each embodiment of the present invention, only a portion of the proliferated cells, proliferated modified cells and / or proliferated further modified cells is used.

[0064] Therefore, in some embodiments of the present invention, one (preferably each) step (b) and / or (c) after step (e) is performed on at least one, preferably at least 10%, more preferably all proliferated ((further) modified) cells obtained after step (e).

[0065] Including one or more proliferation steps in the method of the present invention provides the opportunity to additionally determine the survival rate of the ((further) modified) cells after freezing and thawing in the presence or absence of a given added cryoprotectant. Thus, stochastic effects can be assessed and the impact of a given modification on the freezing tolerance of each cell population can be determined. In this article, the term "cell population" refers to cells derived from a proliferation step of a given ((further) modified) cell, and therefore, cells from such a cell population should be understood to be essentially clones, e.g., having essentially the same genetic background.

[0066] Therefore, in some embodiments of the present invention, the method further comprises a step (f), ie, preferably, for each step (d), a step of determining the viability of the obtained (further) modified cells.

[0067] Furthermore, in some such embodiments, if the viability determined in step (f) of iteration i is at least as high as the viability determined in step (f) of iteration i-1, preferably 10% higher, the method comprises iterating i+1 times. Thus, if the modification does not result in a viability lower than the viability of a corresponding cell population that has not undergone the modification, performing only one additional iteration may increase the efficiency of the method. It may be advantageous to apply an even higher threshold, so that, just before using one or more cell populations, they exhibit a viability that is higher than the viability of a corresponding cell population that has not undergone the modification, preferably a viability that is at least 10% higher, more preferably at least 15% higher, even more preferably at least 20% higher, than that of the corresponding cell population under study without the modification.

[0068] Furthermore, in some of the embodiments described, if the survival rate determined in step (f) at iteration i is at least as high as the survival rate determined in step (f) at iteration i-1, preferably 10% higher, and preferably does not exceed a given threshold (if the method further comprises step (g) as described elsewhere herein), the method comprises iteration i+1.

[0069] As used herein, a "given threshold" refers to a threshold value that is preferably set based on the survival rate. The advantage of doing so is that it allows the definition of a preferably pre-determined criterion, preferably without performing additional iterations, but rather continuing to identify intracellular cryoprotectants that may have a positive impact on the observed survival rate. Regarding the given threshold, it is known to those skilled in the art that such a threshold value is preferably selected based on the experimental setup under investigation, including, for example, the presence or absence of cell type, species and / or added cryoprotectants. Thus, the given threshold value can be set to, for example, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%, preferably to 15% or more and / or 75% or more, preferably to 80% or more. It should be noted that in the context of a given threshold, the term "not exceeding" refers to a parameter value, e.g. the measured survival rate is below the given threshold, whereas in this context, the term "exceeds" refers to the corresponding parameter value, e.g. the measured survival rate includes and / or is above the given threshold, preferably is above the given threshold.

[0070] In some embodiments of the invention, the method comprises the following steps: step (a), optionally followed by step (e), followed by i iterations of [step (b), optionally step (e), steps (c) and (d), optionally step (f), optionally step (e)], wherein i is at least 2, and preferably further comprising iteration i+1 if the survival rate determined in step (f) of iteration i is at least as high as the survival rate determined in step (f) of iteration i-1, preferably 10% higher, and preferably does not exceed a given threshold. More specifically, in a preferred embodiment of the present invention, the method comprises the following steps: step (a), optionally followed by step (e), followed by [step (b), step (e), steps (c) and (d), step (f), step (e)] in iteration i, wherein i is at least 2, and further comprising iteration i+1 if the survival rate determined in step (f) of iteration i is at least as high as the survival rate determined in step (f) of iteration i-1, preferably 10% higher, and preferably does not exceed a given threshold. Thus, the described embodiment represents an iterative evolutionary method for identifying intracellular cryoprotectants expressed by cells, wherein the efficiency of the method can be advantageously increased by (preferably) only operating on ((further) modified), optionally propagated cells when the survival rate determined in one iteration is not lower than the survival rate of the previous iteration. Thus, (further) modified) cells can be obtained that exhibit increased freezing tolerance due to improved cryoprotective properties from one iteration to another. Furthermore, in order to further simplify cell handling, all cells described in the context of the embodiments, ie cells, modified cells and optionally further modified cells, are preferably cells in suspension.

[0071] In a preferred embodiment of the present invention, the intracellular cryoprotectant expressed by the (further) modified cells is identified if the survival rate determined in step (f) exceeds a given threshold. Preferably, the given threshold is 75% or higher, preferably 80% or higher, more preferably 85% or higher. Thus, the costs associated with identifying the intracellular cryoprotectant can be reduced and / or intracellular cryoprotectants with equally good cryoprotective properties can be identified.

[0072] Preferably, some embodiments (including their features and advantages disclosed herein) are combined. For specific method steps, the same applies to the method steps described above, including the features and advantages mentioned in the context of the individual embodiments above. Thus, the parallelization and efficiency of the method of the present invention can be optimized, and the identification of intracellular cryoprotectants with favorable cryoprotective properties is greatly facilitated.

[0073] Therefore, in a particularly preferred embodiment of the present invention, the method comprises the following steps: (a) using cells that survive freezing and thawing, Wherein step (a) preferably comprises the following steps: (a0) Freezing and thawing of cells in suspension, (a1) obtaining cells that survived step (a0), (f1) determining the viability of step (a0) based on the number of living cells of the cells in suspension before freezing and thawing and after freezing and thawing, (e1) growing the cells obtained in step (a1) and using the grown cells, (b) modifying the cells used in step (a), preferably the cells proliferated in step (e1), (e2) multiplying the modified cells in step (b), (c) freezing and thawing the modified cells in suspension that have proliferated in step (e2), (d) obtaining modified cells that survive step (c), thereby identifying an intracellular cryoprotectant expressed by said cells, wherein preferably, the freezing and thawing in step (a), preferably step (a0) and / or step (c) are carried out in a suspension, said suspension preferably comprising an added cryoprotectant at a concentration of 5% or less, preferably 1% or less, more preferably 0%.

[0074] Furthermore, preferably, the particularly preferred embodiment of the present invention further comprises the following steps for identifying the intracellular cryoprotectant expressed by the modified cells: (f2) determining the viability of step (c) based on the number of modified living cells in suspension before freezing and thawing and after freezing and thawing, (e3) propagating the modified cells obtained in step (d), and (g) identifying at least one intracellular cryoprotectant based on the modified cells propagated in step (e3), preferably if the viability determined in step (f2) exceeds a given threshold, wherein if the survival rate determined in step (f2) of iteration i is at least as high as the survival rate determined in step (f2) of iteration i-1, preferably 10% higher, and preferably does not exceed the given threshold, and wherein i is at least 2, then steps [(b), (e2), (c), (d), (f2), (e3)] are iterated (iteration i+1 times).

[0075] Therefore, some preferred embodiments of the present invention include the following steps: (a) using cells that survive freezing and thawing, Wherein step (a) preferably comprises the following steps: (a0) Freezing and thawing of cells in suspension, (a1) obtaining cells that survived step (a0), (f1) determining the viability of step (a0) based on the number of living cells of the cells in suspension before freezing and thawing and after freezing and thawing, (e1) growing the cells obtained in step (a1) and using the grown cells, (b) (further) modifying the cells used in step (a), preferably the cells proliferated in step (e1) and / or the cells proliferated in step (e3), (e2) propagating the cells (further) modified in step (b), (c) freezing and thawing the (further) modified cells in suspension that have been proliferated in step (e2), (d) obtaining a (further) modified cell that survived step (c), thereby identifying an intracellular cryoprotectant expressed by said (further) modified cell, preferably by: (f2) determining the viability of step (c) based on the number of viable (further) modified cells in suspension i) before freezing and thawing and ii) after freezing and thawing, (e3) propagating the (further) modified cells obtained in step (d), and (g) identifying at least one intracellular cryoprotectant based on the (further) modified cells propagated in step (e3), preferably if the viability determined in step (f2) exceeds a given threshold, wherein preferably, the freezing and thawing in step (a), preferably step (a0) and / or step (c) are carried out in a suspension, said suspension preferably comprising an added cryoprotectant in a concentration of 5% or less, preferably 1% or less, more preferably 0%, and wherein if the survival rate determined in step (f2) of iteration i is at least as high as the survival rate determined in step (f2) of iteration i-1, preferably 10% higher, and preferably does not exceed said given threshold value, and wherein i is at least 2, steps [(b), (e2), (c), (d), (f2), (e3)] are iterated (iteration i+1 times).

[0076] Regarding the presence or absence of a cryoprotectant added to the suspension, preferably according to step (c) of the method of the present invention, a method of gradually reducing the concentration of the cryoprotectant added to the suspension is particularly preferred. Thus, the selection pressure on the cells under investigation can be increased. This is particularly advantageous for identifying intracellular cryoprotectants that have a cryoprotective effect, making it possible to overcome the need for any added cryoprotectant in the suspension while ensuring the survival and viability of the cells during freezing and thawing.

[0077] Therefore, it is further preferred that, in the context of said particularly preferred embodiment of the present invention, the suspension in step (c) of the above iteration i+1 comprises: (1) If the survival rate determined in step (f2) is at least as high as the survival rate determined in step (f), preferably at least 5% higher, and the survival rate is determined in the following steps: i) step (f1) in the case of the first iteration (i=1), or ii) step (f2) of iteration i-1 in the case of iteration i, the concentration of the cryoprotectant added is substantially the same as that of the suspension in step (c) of iteration i; (2) If neither i) nor ii) is satisfied, the concentration of the cryoprotectant added is 0.001% to 1%, preferably 0.01% to 0.5%, higher than the concentration in the suspension in step (c) of iteration i.

[0078] It should be noted that the term "substantially" in this context refers to deviations of 5%, 2.5%, 1%, 0.75%, 0.5%, 0.1%, 0.075%, 0.05%, 0.025%, 0.01% or even 0.001% from a given reference value. In particular, the deviations encompass deviations from the given reference value in either direction, so that the actual measured value may be lower or higher than the given reference value, with a maximum deviation from the reference value of 5%, 2.5%, 1%, 0.75%, 0.5%, 0.1%, 0.075%, 0.05%, 0.025%, 0.01% or even 0.001%. Preferably, the deviations are considered to be towards the lower value. As an illustrative example, one may consider a suspension containing an added cryoprotectant at a concentration of 15%, wherein a suspension containing substantially the same concentration of the added cryoprotectant may therefore contain, for example, a concentration of 14.999%, 14.99%, 14.975%, 14.95%, 14.925%, 14.9%, 14.5%, 14.25%, 14%, 12.5% ​​or even 10% of the added cryoprotectant.

[0079] Thus, by applying the evolutionary method of the present invention, (further) modified cells are obtained, which are preferably studied with regard to changes that may lead to an improvement in their cryoprotective properties obtained compared to their initial, unmodified counterparts, and therefore compared to the cells used in step (a), and / or compared to the (further) modified counterparts of one of the previous iterations. In the case of the first-mentioned comparison, the identification of intracellular cryoprotectants can be facilitated, since in the method disclosed herein, the initially used cells of step (a) can be considered as the cells with the lowest cryotolerance, and in step (c) of the iteration in which the cells with the highest cryotolerance are studied, the (further) modified cells survive freezing and thawing. Since the two cells or cell populations have essentially the same background, in particular genetic background, except for the modifications introduced in step (b), the comparison of the cells, for example at the genetic level, can easily identify potential intracellular cryoprotectants that differ between the cells or cell populations. The other exemplary options mentioned may be particularly suitable for situations in which a relatively strong increase in freezing tolerance is observed between cells or cell populations from a given iteration and their counterparts in the previous iteration. Thus, modifications of relatively large effect sizes on freezing tolerance and / or optimization of identified intracellular cryoprotectants can be effectively identified. However, those skilled in the art will appreciate that, depending on, for example, expected research results, economic considerations and / or technological capabilities, the two exemplary comparisons mentioned may also be combined and / or performed at different levels, for example genomes, transcriptomes and / or epigenomes.

[0080] Therefore, in a preferred embodiment of the present invention, the intracellular cryoprotectant expressed by the (further) modified, optionally proliferated, cells surviving in step (c) is identified by analyzing the genome, transcriptome, epigenome, proteome and / or metabolome, preferably by using an analysis selected from gel electrophoresis, mass spectrometry, crystal structure analysis, NMR spectroscopy, DNA sequencing, (m) RNA sequencing and any combination of the foregoing. The intracellular cryoprotectant is preferably identified by genomic analysis, preferably by DNA sequencing. Alternatively or optionally, the intracellular cryoprotectant is preferably identified by gel electrophoresis and / or mass spectrometry. The advantages of sequencing-based methods and gel electrophoresis and mass spectrometry are, in particular, that they are well established in the art and can be easily performed in an automated and high-throughput manner and at relatively low cost.

[0081] When at least one intracellular cryoprotectant is identified using the methods of the present invention, it may be advantageous to further optimize the cryoprotectant, for example taking into account the ability of the target cells to express the cryoprotectant and / or optimizing the properties of the cryoprotectant such as biological activity, localization within the cell, cell membrane passage and / or cryoprotective ability.

[0082] Therefore, in some embodiments of the present invention, the method further comprises as step (h) the step of modifying the identified intracellular cryoprotectant expressed by the (further) modified cell, thereby obtaining a derivative from the identified intracellular cryoprotectant expressed by the (further) modified cell.

[0083] The present invention also relates to an intracellular cryoprotectant or a derivative thereof, preferably obtained or obtainable by the above method, wherein the intracellular cryoprotectant is an intracellular cryoprotectant synthesized by cells.

[0084] Therefore, by applying the identification method of intracellular cryoprotectants expressed by cells of the present invention, intracellular protective agents suitable for cryopreservation can be identified efficiently and with high throughput. In addition or alternatively, other intracellular cryoprotectants may also be suitable for cryopreservation, such as nucleotide sequences, polypeptides, and proteins that are known and / or otherwise characterized. Therefore, although intracellular cryoprotectants obtained by or can be obtained by the above-mentioned method are preferred, any intracellular cryoprotectant is applicable to the method for cryopreserving cells, cell aggregates, cell cultures, tissues, organoids, and / or organs described in detail below. More specifically, it was surprisingly found that, compared to current cryopreservation methods based on added cryoprotectants, intracellular cryoprotectants can overcome corresponding limitations regarding, for example, cryoprotectant size and gradient formation. Therefore, the method of cryopreserving larger cell aggregates, tissues, organoids, and / or organs with intracellular cryoprotectants at low temperatures without substantially losing cell viability and / or tissue, organoid, and / or organ function paves the way.

[0085] The intracellular cryoprotectant expressed and / or synthesized by the cell can be a nucleotide sequence, a polypeptide or a protein. More specifically, the term "intracellular cryoprotectant" is included herein, for example, nucleotide sequences such as RNA sequences, especially mRNA sequences, preferably mRNA sequences, which optionally have known or unknown cryoprotective properties. It also includes, for example, polypeptides and / or proteins with known or unknown cryoprotective properties, such as polypeptides and proteins with structures rich in alanine and threonine. The latter is preferably similar to the structure identified in antifreeze proteins, wherein they produce a folded drum pattern in the high-level (tertiary) structure of the antifreeze protein. Antifreeze proteins are also included herein. Other examples may include, preferably small and medium-sized cytoplasmic proteins and polypeptides, glycoproteins, heat shock proteins, proteins and polypeptides with anti-apoptotic properties and / or osmotic active proteins and polypeptides. The proteins and polypeptides may be particularly advantageous for positively regulating cell survival and vitality, especially during thawing. Other examples may include albumin, globulin, histone, protamine, polyamine, kinase, growth factor, etc. Among the latter, globular proteins may be particularly advantageous, as they can be found dissolved in the cytoplasm of cells and distributed throughout the cytoplasm during cold storage. Furthermore, some globular proteins are able to bind water, thereby reducing cell damage caused by ice crystal formation during freezing. Other examples may be membrane-bound and / or superimposed proteins. These can exhibit cryoprotective properties by stabilizing organelles and limiting ice crystal formation. Other examples may be porins and other osmotically active proteins and polypeptides. Membrane transport proteins may also be optionally included. Further examples may include fibrin and polypeptides, which have a stabilizing effect on the cytoskeleton and may function in conjunction with supporting and scaffolding factors such as collagen, actin, and / or myosin. These proteins and polypeptides are particularly beneficial in supporting the temporary hardening of cells during cryopreservation. Further examples may be polypeptides and proteins involved in, and preferably driving, the synthesis of membrane lipids and fatty acids. This is advantageous for increasing cellular lipid content, including the formation of lipid vacuoles, which positively impacts cryopreservation. Additionally or optionally, an increased cellular lipid content is advantageous because the resulting fat bodies and / or fat vacuoles exert mechanical stabilization. Thus, fatty acid synthases, parts thereof, and intermediates of the cellular fatty acid cycle are advantageous. Examples may include carnitine-acyltransferase 1, fatty acid acyl-CoA desaturase, epinephrine, triglycerides, triacylglycerols, neutral fats, fats and other esters of trivalent alcohol glycerol (glycerol, propane-1,2,3-triol) with three long-chain carboxylic acids, glycerophospholipids, sphingolipids, and diacylglycerol-3-phosphate. Further examples may include membrane lipids, such as phospholipids, glycolipids, and cholesterol. Further examples may be proteins and / or polypeptides that participate in glycogen synthesis, and therefore participate in, for example, cellular sugar synthesis and / or conversion in animal and human cells.Examples include insulin, which drives glycogen accumulation, and the so-called core proteins (glycogenogens), around which glycogen structures can be built in radial chains. These are particularly advantageous for increasing the cell surface area and water-binding capacity, thus playing a hypothermic role. Other examples include the enzymes pyruvate carboxylase, phosphoenolpyruvate-carboxykinase, fructose-1,6-bisphosphatase, and glucose-6-phosphatase.

[0086] Therefore, in some embodiments of the present invention, the intracellular cryoprotectant or its derivative is a nucleotide sequence, preferably an RNA sequence, such as a miRNA sequence, a shRNA sequence, a siRNA sequence or an mRNA sequence, more preferably an mRNA sequence.

[0087] In the context of the present invention, "mRNA sequence", also abbreviated as "mRNA", should be understood as a polyribonucleotide molecule, if it enters a cell, it is suitable for the expression and / or synthesis of a protein or polypeptide and / or can be translated into a protein or polypeptide. Preferably, the mRNA contains a ribonucleotide sequence encoding a protein or polypeptide, and the function of the protein or polypeptide in or near the cell is necessary or beneficial for cryopreservation. More specifically, in the context of the present invention, an mRNA sequence refers to a single-stranded ribonucleotide sequence composed of, for example, A, C, G and / or U nucleotides, i.e., adenine, guanine, cytosine and uracil as the nucleotides of each nitrogenous base. In addition, the mRNA includes one or more coding sequences, which can be used as a template in the expression and / or synthesis of an amino acid sequence during translation. The mRNA may also include 5' and / or 3' untranslated regions (UTRs), one or more internal ribosome entry sites (IRESs), one or more additional modifications that promote translation, regulate and / or extend the duration of action, etc. Therefore, mRNA comprises at least one coding sequence, and this coding sequence can be translated into amino acid sequence, for example protein by cell, or is translated into amino acid sequence, for example protein in cell.Therefore, mRNA sequence can be translated into amino acid sequence, for example protein, and therefore described amino acid sequence, for example protein, can be expressed by cell and / or synthesized in cell.

[0088] In some embodiments of the present invention, the intracellular cryoprotectant or its derivative is or can be obtained by the method of the present invention disclosed above, wherein the intracellular cryoprotectant is an intracellular cryoprotectant synthesized by cells, and it is preferably an optionally glycosylated polypeptide or protein.

[0089] In some embodiments of the present invention, the intracellular cryoprotectant or its derivative is preferably an optionally glycosylated polypeptide or protein and is characterized by a size, weight, and / or structure suitable for providing the cryoprotectant with cell membrane impermeability. The term "cell membrane impermeability" must be understood as the characteristic of the intracellular cryoprotectant that the cell membrane cannot be passively permeated. Thus, the intracellular cryoprotectant itself cannot diffuse through the cell membrane. This cell permeability occurs passively in known cryoprotectants such as DMSO, urea, or small molecule sugars such as trehalose. In addition, the cell membrane impermeability of the intracellular cryoprotectant of the present invention does not exclude the possibility that the cryoprotectant may be actively introduced into the cell through the cell membrane via naturally occurring membrane transporters, which may be located within the cell membrane. However, this active transport of the cryoprotectant from the outside to the inside of the cell by membrane transporters of the present invention does not result in a sufficient amount of the cryoprotectant to provide an appropriate effect as a cryoprotectant. According to the present invention, it is foreseeable that, given the unique characteristics of the intracellular cryoprotectant, the intracellular cryoprotectant is produced and retained within the cell. Therefore, this allows the cryoprotective ability of the cryoprotectant of the present invention to be exerted within the cell because diffusion through the cell membrane is avoided or at least minimized. Therefore, the intracellular cryoprotectant of the present invention is preferably synthesized within the cell and has a significantly large size and structure, which allows for sufficient concentration within the cell. This provides the advantage of no dependence on transmembrane transport, which is only applicable to smaller molecules as potential cryoprotectants. In addition, such smaller molecule cryoprotectants may not be introduced into the cell at sufficient concentrations, in particular not into larger cell aggregates, such as organoids, tissues or organs. Therefore, the intracellular cryoprotectant of the present invention has the advantage of achieving a useful cryoprotectant concentration within the cell, and because the cryoprotectant is retained within the cell, the desired cryoprotective effect is achieved within the cell.

[0090] In some embodiments of the present invention, the intracellular cryoprotectant is preferably an optionally glycosylated polypeptide or protein, and preferably has an average molecular weight (Mw) greater than 9800 Da, preferably greater than 900 kDa or greater. According to the present invention, it is foreseeable that the intracellular cryoprotectant is an optionally glycosylated polypeptide or protein having a large molecular weight, wherein the large size is only limited by the production capacity of the cell. Preferably, the molecular weight, preferably the upper limit of the molecular weight, is determined by the existing possibility of the cell to synthesize polypeptides or proteins of different large sizes.

[0091] In another aspect, the present invention relates to a method for producing a cryoprotectant or a derivative thereof, comprising carrying out the method for identifying an intracellular cryoprotectant as described above in any embodiment of the present invention. Thus, the method for producing a cryoprotectant or a derivative thereof comprises identifying a cryoprotectant and further producing the cryoprotectant in an amount up to a desired amount.

[0092] In this article, the term "amino acid sequence" includes any kind of amino acid sequence chain comprising two or more amino acids connected by peptide bonds. Preferably, the amino acid sequence length is at least 5 amino acids, more preferably at least 10 amino acids or at least 20, 50, 75, 100, 125, 150, 175 or 200 amino acids. Therefore, the term includes short peptides, oligopeptides, polypeptides, fusion proteins, proteins and fragments thereof, such as functional parts of known proteins. In this article, the term "polypeptide" refers to an amino acid sequence of at least 10 amino acids and up to and including 100 amino acids. In addition, the term "protein" refers to an amino acid sequence of more than 100 amino acids. There are no restrictions on the function of the amino acid sequence. Preferably, the function is (directly) related to the cryoprotective effect, preferably within the cell.

[0093] As used herein, the term "glycosylated" refers to the process of glycosylation, and thus, preferably the controlled, preferably enzymatic, modification of an amino acid sequence, such as a polypeptide or protein, by the addition of at least one sugar moiety. Glycosylation thus preferably involves the modification of an amino acid sequence, wherein the modification may be a post-translational modification within a cell. Glycosylated proteins may also be referred to herein as glycoproteins. Glycosylation is relevant to many biological processes, as it can affect intracellular trafficking, cell attachment to the extracellular matrix, and / or protein-ligand interactions.

[0094] In some embodiments of the present invention, the intracellular cryoprotectant or its derivative is selected from antifreeze proteins, proteins or polypeptides having alanine- and / or threonine-rich structures and tertiary structures similar to antifreeze proteins, glycoproteins, cytoplasmic proteins or polypeptides, heat shock proteins, albumins, globulins, histones, protamines, kinases, growth factors, globular proteins or polypeptides, membrane-bound and / or superimposed proteins or polypeptides, membrane pores, osmotically active proteins or polypeptides, fibrous proteins or polypeptides, polypeptides or proteins involved in the synthesis of membrane lipids and / or fatty acids, phospholipids, glycolipids, cholesterol, polypeptides or proteins involved in glycogen synthesis, insulin and core proteins.

[0095] In this document, it should be noted that the term "intracellular cryoprotectant synthesized by a cell" includes the term "intracellular cryoprotectant expressed by a cell". In addition, the term "intracellular cryoprotectant synthesized by a cell" also includes the situation where the cell produces the intracellular cryoprotectant but is intrinsically unable to express the intracellular cryoprotectant. Therefore, the cell that synthesizes the intracellular cryoprotectant may or may not endogenously contain the corresponding biological information required for the construction of the intracellular cryoprotectant. The cell can be modified by introducing the respective required biological information so as to become at least temporarily capable of producing the intracellular cryoprotectant and / or its derivatives. Therefore, it may be necessary to introduce the corresponding biological information into the cell, such as an optionally modified nucleotide sequence encoding the intracellular cryoprotectant, the intracellular cryoprotectant being a protein or polypeptide. This method provides the advantage that cells can be made suitable for cryopreservation, which do not have any or at least sufficient cryopreservation properties endogenously to ensure cell survival and proper cell function during freezing and thawing. In addition, it provides the advantage of allowing cells to be permanently and / or temporarily adapted to cryopreservation. Furthermore, the term "intracellular cryoprotectant synthesized by cells" also includes the case where cells that are already intrinsically capable of expressing an intracellular cryoprotectant are modified in such a way that the cells are able to (newly) synthesize derivatives of the intracellular cryoprotectant and / or to alter the amount of the intracellular cryoprotectant and / or its derivatives expressed and / or synthesized by the cells. Thus, cells that synthesize intracellular cryoprotectants may or may not endogenously contain (at least partially) the corresponding biological information required for the construction of the intracellular cryoprotectant.

[0096] Therefore, in a preferred embodiment of the present invention, the intracellular cryoprotectant is an intracellular cryoprotectant that is capable of cryopreserving cells, preferably cell aggregates, cell cultures, tissues, organoids and / or organs.

[0097] As used herein, the terms "cell," "cell aggregate," and "cell culture" have their usual meanings in the art. In addition, as used herein, the cells, cell aggregates, and cell cultures are preferably capable of being cultured in vitro and / or at least temporarily cultured in vitro. The term "tissue" also has its usual meaning in the art and can therefore be considered, for example, a collection of structurally and functionally equivalent cells, optionally with intercellular material, organized to perform a specific function. As used herein, the term "organoid" has its usual meaning in the art and can therefore be understood to refer to a self-organized three-dimensional tissue culture preferably derived from stem cells. The organoid can be cultured in vitro, thereby providing the possibility of replicating selected and / or most features of other relatively complex organs. Organoids are advantageous, for example, for studying the effects of drug candidates and / or drugs on a given organ in vitro and / or for gaining an understanding of evolutionary processes associated with, for example, organ formation. As used herein, the term "organ" has its usual meaning in the art and can therefore be understood to refer to a collection of cell types and / or tissues connected in a structural unit that can perform a common function, including, for example, kidney, heart, lung, and liver. Tissues and / or organs are particularly advantageous for (bio)medical purposes such as transplantation. In addition, with respect to the terms, it is contemplated that the corresponding terms herein for cells, cell aggregates, cell cultures, tissues, organoids and / or organs encompass both artificial and natural origin. For example, a tissue can be a tissue obtained from an animal and / or an in vitro engineered tissue.

[0098] Herein, cryopreservation capacity relates to the ability of cells to survive freezing and thawing, wherein freezing preferably comprises or is a reduced temperature below -140° C., and accordingly, the ability of an intracellular cryoprotectant to cryopreserve cells relates to the ability of said intracellular cryoprotectant to ensure cell survival, viability, vitality and / or functionality during freezing and thawing, wherein said freezing preferably comprises or is a reduced temperature below -140° C. More specifically, the intracellular cryoprotectant of the present invention preferably exhibits cryoprotective properties, wherein said cryoprotective properties are preferably characterized by a level of viability, vitality and / or functionality of cells, cell aggregates, cell cultures, tissues, organoids and / or organs after freezing and thawing of at least 70%, preferably at least 80%, more preferably at least 90% compared to said levels before freezing and thawing. Thus, in the case of cell aggregates, cell cultures, tissues, organoids and / or organs, cryopreservation of said cell aggregates, cell cultures, tissues, organoids and / or organs may be accompanied by cell death and / or reduced cell viability of a small fraction of the cells comprised in said cell aggregates, cell cultures, tissues, organoids and / or organs during freezing and thawing, wherein said small fraction is a fraction that is so small that the overall viability, viability and / or functionality of said cell aggregates, cell cultures, tissues, organoids and / or organs is preferably reduced by no more than 30%, more preferably by no more than 20%, even more preferably by no more than 10% after freezing and thawing, compared to the corresponding viability, viability and / or functionality before said freezing and thawing.

[0099] As used herein, the term "survival rate", which may also be referred to as "viability", is intended to be understood as the percentage of living cells in a cell population such as a cell culture or tissue at a given time point. In addition to cell death, cell damage due to freezing and thawing can negatively affect cells through many cellular changes that may impair the ability of cells to (usually) function normally. Therefore, the term "cell vitality" herein refers to the physiological capacity of a cell. It should be noted that both cell viability and cell viability are intended to represent two different aspects of cell function and are preferably considered when assessing the physiological state of a cell before and after freezing and thawing. In addition, the term "functionality" is intended to be understood as a measurement related to a specific function, wherein the measurement is preferably obtained and / or assessed at a multicellular level. Therefore, the term preferably refers to a functional property of a cell aggregate, tissue, organoid and / or organ, for example, taking into account the contractility of a muscle cell. Thus, for example, a functionality of 100% may represent a corresponding healthy tissue, organoid and / or tissue, organoid and / or organ before freezing and thawing, whereas, for example, a functionality of 50% after freezing and thawing may indicate that a given tissue, organoid and / or organ, for example in the case of muscle cells, may only exhibit half the contractile force and / or frequency compared to a healthy reference and / or said tissue, organoid and / or organ before freezing and thawing.

[0100] Therefore, in some embodiments of the present invention, the intracellular cryoprotectant, preferably obtained or obtainable by the above-mentioned method of the present invention, is an intracellular cryoprotectant, which is capable of cryopreserving cells, cell aggregates, cell cultures, tissues, organoids and / or organs, preferably by ensuring and / or maintaining the viability, vitality and / or functionality of the cells, cell aggregates, cell cultures, tissues, organoids and / or organs after freezing and thawing at a level of at least 70%, preferably at least 80%, more preferably at least 90% compared to the level before freezing and thawing.

[0101] As described above, it may be necessary to introduce and thus transfer the biological information into cells in order to enable the cells to synthesize the intracellular cryoprotectant of the present invention. For the introduction of biological information into cells, nucleotide sequences are particularly advantageous, taking into account the conventionally available methods known and established in the art regarding preparation, modification, optimization, application, safety, and efficiency.

[0102] Therefore, the present invention also relates to a method for producing an optionally modified nucleotide sequence encoding an intracellular cryoprotectant according to the invention or a derivative thereof.

[0103] More specifically, in some embodiments of the present invention, the method comprises the steps of (a) identifying a nucleotide sequence encoding the intracellular cryoprotectant or a derivative thereof, (b) optionally modifying the identified nucleotide sequence, and (c) synthesizing the identified, optionally modified nucleotide sequence in vitro.

[0104] Therefore, in some embodiments of the present invention, a nucleotide sequence encoding an intracellular cryoprotectant or a derivative thereof is identified. The nucleotide sequence is preferably an RNA sequence, preferably an mRNA sequence, comprising a coding sequence encoding an intracellular cryoprotectant, wherein the intracellular cryoprotectant is optionally obtained or can be obtained by the method for identifying an intracellular cryoprotectant of the present invention.

[0105] As used herein, the term "encoding" in the context of a nucleotide sequence, such as a DNA sequence or an mRNA sequence, refers to a nucleotide sequence that contains a sequence that encodes biological information. For example, an mRNA sequence can encode a protein, meaning that the nucleotide sequence contains a coding region that encodes an amino acid sequence. Thus, the mRNA sequence can be translated into an amino acid sequence, such as a protein, which can then be expressed by a cell and / or synthesized within the cell.

[0106] Regarding step (a), the nucleotide sequence encoding the intracellular cryoprotectant or its derivative can be identified, for example, using a corresponding method according to the present invention. In the case where the intracellular cryoprotectant identified according to the method is a polypeptide or protein, the skilled person is aware of methods and techniques for deriving the respective nucleotide sequence encoding the intracellular cryoprotectant from the obtained amino acid sequence information.

[0107] Thus, the identified nucleotide sequences encoding intracellular cryoprotectants or derivatives thereof, preferably RNA sequences, more preferably mRNA sequences, comprise one or more coding sequences encoding one or more intracellular cryoprotectants, wherein the at least one coding sequence may be a naturally occurring sequence, a modified sequence such as a partially or fully codon-optimized sequence derived from a naturally occurring sequence, or an artificial sequence. Codon optimization refers to a technique for maximizing protein expression, for example by increasing translation efficiency, for example when species exhibit differences in their preferred codon usage for a given amino acid.

[0108] With regard to step (b), i.e., modification of the nucleotide sequence, the same applies to the case of modifying cells at the genetic and / or transcriptome level as described above. More specifically, the nucleotide sequence can be modified, for example, by introducing single nucleotide polymorphisms, inserting and / or deleting one or more nucleotides, and / or combining one or more nucleotide sequences. In addition, the term "modifying the nucleotide sequence" also includes codon optimization, adding, removing or changing one or more sequences selected from 5' caps, 3' polyadenylic acid (poly A) tails, IRES, 3'UTR, 5'UTR and other regulatory and / or translation promoting sequences, as well as adding, removing or changing at least one other nucleotide sequence encoding at least one other intracellular cryoprotectant. Therefore, modifying the nucleotide sequence encoding the intracellular cryoprotectant, such as the mRNA sequence, also includes, for example, a combination of coding sequences of one or more intracellular cryoprotectants. More specifically, the combination may also include a combination of one or more coding sequences encoding one or more intracellular cryoprotectants, wherein preferably, at least one of the one or more intracellular cryoprotectants is or can be obtained by the method for identifying the intracellular cryoprotectant according to the present invention. Thus, the nucleotide sequence encoding the intracellular cryoprotectant can be modified, for example to include other sequences encoding the same intracellular cryoprotectant and / or to include a sequence encoding an additional alternative intracellular cryoprotectant. In any of these cases, the respective nucleotide sequence, preferably an RNA sequence, more preferably an mRNA sequence, can be a partially or fully modified sequence. Thus, by modifying the nucleotide sequence encoding the intracellular cryoprotectant according to step (b), localization and duration of action can be optimized, and in the case where the nucleotide sequence is an mRNA sequence, translation efficiency and / or duration can also be optimized.

[0109] Regarding step (c), in vitro synthesis methods are well established in the art and are available to those skilled in the art. Therefore, step (c) provides the advantage of in vitro amplification of the identified, optionally modified nucleotide sequence obtained, for example, from step (b). Therefore, it is envisioned that the nucleotide sequence may be applied on a large scale to cryopreservation of cells, cell aggregates, cell cultures, tissues, organoids and / or organs, preferably using the methods described in detail below.

[0110] In some embodiments of the present invention, the method for generating an optionally modified nucleotide sequence encoding an intracellular cryoprotectant or a derivative thereof is a method for generating an optionally modified RNA sequence, preferably an mRNA sequence, encoding an intracellular cryoprotectant or a derivative thereof. Thus, in some embodiments, the optionally modified nucleotide sequence is an RNA sequence, preferably an mRNA sequence.

[0111] The present invention also relates to optionally modified nucleotide sequences obtained or obtainable by a method of generating an optionally modified nucleotide sequence of the present invention encoding one or more intracellular cryoprotectants of the present invention or derivatives thereof. The optionally modified nucleotide sequence facilitates the introduction of biological information into cells to render the cells suitable for cryopreservation, preferably using a corresponding method of the present invention as described in more detail below.

[0112] In some embodiments of the present invention, the optionally modified nucleotide sequence of the present invention is an RNA sequence, preferably an mRNA sequence.

[0113] The present invention also relates to nanoparticles comprising an optionally modified nucleotide sequence according to the present invention as described above, wherein the nanoparticles are preferably lipid nanoparticles. Encapsulating the nucleotide sequence in the nanoparticles can advantageously facilitate the introduction of the nucleotide sequence into cells. In addition, corresponding methods and techniques for preparing nanoparticles, loading nanoparticles with nucleotide sequences, using the nanoparticles to introduce the nucleotide sequence contained therein into cells, and / or evaluating the safety and efficacy of such methods are well known and established in the art.

[0114] In some embodiments of the present invention, the nanoparticle comprises an optionally modified nucleotide sequence according to the present invention, wherein the nucleotide sequence is an RNA sequence, preferably an mRNA sequence, and wherein the nanoparticle is preferably a lipid nanoparticle.

[0115] The present invention further relates to the use of i) an intracellular cryoprotectant according to the invention or a derivative thereof, ii) an optionally modified nucleotide sequence according to the invention, and / or iii) a nanoparticle according to the invention for cryopreservation of cells, cell aggregates, cell cultures, tissues, organoids and / or organs.

[0116] The use of optionally modified nucleotide sequences encoding intracellular cryoprotectants according to the present invention can facilitate the synthesis of intracellular cryoprotectants by cells, which may or may not endogenously possess cryoprotective properties, and thus be suitable for cryopreservation. In addition, the at least one intracellular cryoprotectant can even be initially identified in a species different from the cells to be cryopreserved according to the method of the present invention. This is particularly important considering that many species, including humans, are unable to express functional intracellular cryoprotectants at all, or the expression levels are insufficient to allow, for example, cryopreservation of tissues or organs without the addition of cryoprotectants. Therefore, biological information regarding the synthesis of the intracellular cryoprotectants of the present invention is advantageously transferred to the cells to be cryopreserved before freezing and thawing. Preferably, the biological information is provided in the form of an optionally modified mRNA sequence, optionally contained in nanoparticles, which can be transferred into and passed through the cells and translated into the intracellular cryoprotectant within the cells.

[0117] Therefore, the present invention also relates to a method for cryopreserving cells, cell aggregates, cell cultures, tissues, organoids and / or organs, comprising the following steps: (a) contacting cells, cell aggregates, cell cultures, tissues, organoids and / or organs with optionally modified nucleotide sequences and / or nanoparticles according to the present invention, and (b) freezing the cells, cell aggregates, cell cultures, tissues, organoids and / or organs obtained from step (a), wherein freezing comprises or is lowering the temperature of the cells, cell aggregates, cell cultures, tissues, organoids and / or organs from above 0°C to below 0°C, preferably below -20°C, more preferably below -70°C, and even more preferably below -140°C.

[0118] In this article, the term "contacting" can be understood as "bringing into contact", for example, cells to be cryopreserved are contacted with nucleotides encoding at least one intracellular cryoprotectant according to the present invention. In this case, the cells can be contacted with the nucleotides by adding the nucleotides to a (suspension) culture medium (at least temporarily) containing the cells. The skilled person is aware of methods and techniques for evaluating and determining suitable methods and parameters for contacting cells, cell aggregates, cell cultures, tissues, organoids and / or organs with nucleotide sequences and / or nanoparticles according to the present invention to ensure that the corresponding biological information is fully and / or effectively introduced into the cells, cell aggregates, cell cultures, tissues, organoids and / or organs. Examples of suitable methods may include adding a nucleotide sequence encoding at least one intracellular cryoprotectant to a suspension comprising cells to be cryopreserved, perfusing an organ with a solution comprising a nucleotide sequence according to the present invention, and / or a vaccine-based vaccination method based on clinical safety and perfection, such as using mRNA lipid nanoparticles. Related parameters can include contact time, for example, in a suspension and / or culture medium comprising cells to be cryopreserved, cell aggregates, cell cultures, tissues, organoids and / or organs, and / or the concentration of the nucleotide sequence according to the present invention and / or nanoparticles, and / or the duration between contact and freezing, optionally the duration between the start and / or end of contact and the start of freezing. Therefore, it is preferred to ensure that enough intracellular cryoprotectants are synthesized by the cells or in the cells to limit or even avoid cell damage, for example, cell damage caused by crystalline ice formation during freezing. In addition, the contact is advantageous because it allows the method according to the present invention to be applied in vitro to different cell systems, such as cell suspensions, tissue sections and organs.

[0119] Therefore, in some embodiments of the present invention, the method for cryopreserving cells, cell aggregates, cell cultures, tissues, organoids and / or organs at low temperatures is an in vitro method. In addition, embodiments of the method for non-isolated cryopreservation of cells, cell aggregates, cell cultures, tissues, organoids and / or organs are also encompassed herein. Therefore, in some embodiments of the present invention, cells, cell aggregates, cell cultures, tissues, organoids and / or organs can be cryopreserved, wherein the cells, cell aggregates, cell cultures, tissues, organoids and / or organs are at least partially and / or temporarily embedded in a physiological environment. In this article, "physiological environment" refers to an artificial and / or natural environment that constitutes or is similar to a physiological condition, and the artificial and / or natural environment is suitable with the physiological condition that can be observed (naturally) in the species and / or cells, cell aggregates, cell cultures, tissues, organoids and / or organs studied. Therefore, included herein can also be cells, cell aggregates, cell cultures, tissues, organoids and / or organs contained in mammals with irreversible brain failure. Described brain failure should be measured according to perfect measuring method, method and / or standard (preferably neurological method), and is optionally measured by qualified personnel.Therefore, for the purpose of illustration, it can be considered that the ovarian tissue comprising oocyte obtained from people before chemotherapy, by using the solution comprising described nucleotide sequence to carry out in vitro perfusion, this ovarian tissue can be contacted with nucleotide sequence of the present invention, so that not only cryopreservation ovarian tissue, and particularly the oocyte therein comprised.As another example, it can be considered that after measuring the irreversible failure of brain function, mammals are contacted with nucleotide sequence according to the present invention using the method based on vaccination.This will be consistent with the step (a) of the method for the cryopreservation of, for example, mammalian kidney.In addition, described contact can be carried out for example between 12 hours to 72 hours before stopping any life-supporting (medical treatment) measure and / or method of described mammal. In this context, the intracellular cryoprotectants according to the present invention and their use in the innovative cryopreservation methods disclosed herein are particularly advantageous for adapting (mammalian) cells, cell aggregates, cell cultures, tissues, organoids and / or organs to cryopreservation in the context of (bio)medical applications (particularly transplantation), while reducing the cell damage associated with current methods and / or overcoming logistical limitations in the context of transplant medicine.

[0120] With regard to freezing, and possible and / or suitable freezing (and thawing) temperature-time protocols, the same applies to the methods described above for identifying intracellular cryoprotectants expressed by the cells of the invention. In addition, it should be noted that, for example, the freezing process can be adjusted to optimize the cryoprotection conferred by the intracellular cryoprotectant with which the cells, cell aggregates, cell cultures, tissues, organoids and / or organs are in contact before freezing. For example, by initially lowering the temperature of the cells, cell aggregates, cell cultures, tissues, organoids and / or organs to a first temperature between 30°C and -20°C and maintaining it for an appropriate time, cellular processes can be slowed down while still allowing the synthesis of intracellular cryoprotectants, thereby allowing synchronization of the levels of intracellular cryoprotectants synthesized by the cells of a given organ or tissue, for example, and then further lowering the temperature to a second temperature between -20°C and below -140°C. In particular, in the case of animal (especially mammalian) and human cells, at least temporary in vitro cultivation, for example in vitro cultivation of cells, can be carried out at a temperature of 30° C. to 40° C., preferably about 35° C., whereupon for synchronization the temperature can be lowered to a first temperature, for example, between 30° C. and 0° C., preferably between 20° C. and 15° C. or between 4° C. and 0° C. Thus, the temperature-time regime can be optimized taking into account the cell system used and / or, for example, actual needs.

[0121] In some embodiments of the invention, the cells, cell aggregates, cell cultures, tissues, organoids and / or organs to be cryopreserved are contacted with optionally modified nucleotide sequences encoding one or more intracellular cryoprotectants according to the invention, and are optionally encapsulated and / or contained in nanoparticles. Alternatively or optionally, the cells, cell aggregates, cell cultures, tissues, organoids and / or organs to be cryopreserved can be contacted with more than one nucleotide sequence species, for example, a first nucleotide sequence encoding a first intracellular cryoprotectant and a second nucleotide sequence encoding a second intracellular cryoprotectant.

[0122] Thus, in some embodiments of the present invention, step (a) of the method for cryopreserving cells, cell aggregates, cell cultures, tissues, organoids and / or organs according to the present invention comprises the steps of: (a1) contacting the cells, cell aggregates, cell cultures, tissues, organoids and / or organs with an optionally modified nucleotide sequence encoding a first intracellular cryoprotectant or a derivative thereof and / or nanoparticles comprising an optionally modified nucleotide sequence encoding a first intracellular cryoprotectant or a derivative thereof, and (a2) contacting the cells, cell aggregates, cell cultures, tissues, organoids and / or organs with another optionally modified nucleotide sequence encoding a second intracellular cryoprotectant or a derivative thereof and / or with another nanoparticle comprising an optionally modified nucleotide sequence encoding a second intracellular cryoprotectant or a derivative thereof. It should be noted that the method is not limited to the use of two different types of nucleotide sequences and / or nanoparticles and is therefore not limited to the introduction of biological materials for two intracellular cryoprotectants. Therefore, the description of the embodiments should be understood as illustrative and the corresponding rationale can be extended accordingly to the use of more than two nucleotide sequences and / or nanoparticles as needed. Furthermore, it should be noted that steps (a1) and (a2) can be performed in parallel, temporarily at least partially overlapping, and / or sequentially. A person skilled in the art will appreciate that appropriate methods are determined depending on the specific requirements of the cells, cell aggregates, cell cultures, tissues, organoids, and / or organs to be cryopreserved, the nucleotide sequences and / or nanoparticles used, and / or the respectively selected intracellular cryoprotectants.

[0123] In some embodiments of the present invention, the method for cryopreserving cells, cell aggregates, cell cultures, tissues, organoids and / or organs further comprises the steps of: (c) thawing the frozen cells, cell aggregates, cell cultures, tissues, organoids and / or organs, and / or (d) determining the viability, activity and / or functionality level of the cells, cell aggregates, cell cultures, tissues, organoids and / or organs before step (a), between steps (a) and (b) and / or after step (c), wherein the viability, activity and / or functionality level after freezing and thawing is at least 70%, preferably at least 80%, more preferably at least 90% compared to the level before freezing and thawing.

[0124] With regard to thawing, and possible and / or suitable temperature-time protocols for (freezing and) thawing, the same applies as stated above in the method according to the invention for identifying an intracellular cryoprotectant expressed by a cell.

[0125] About viability, vigor and / or functionality, the same applies to the situation of the intracellular cryoprotectant of the present invention described above. In addition, about step (d), those skilled in the art know the method and technology of assessing the survival, viability, viability and / or functionality of cells, cell aggregates, cell cultures, tissues, organoids and / or organs. The selection of corresponding methods can depend on, for example, sample type, such as cell aggregates or tissues, and / or cell types. For the purpose of illustration, cardiomyocytes can be considered as an example. In this case, the suitable test of assessing viability and / or functionality can be based on inducing contraction by electric pulse stimulation and characterizing the resulting contraction, for example, considering contractility, contraction frequency and / or time delay. Optionally or alternatively, especially in the case of tissues, organoids and / or organs, particularly organs, quantitative measurement of a given function can be assessed. Exemplarily, in the case of kidneys, urea accumulation can be assessed, and in the case of liver, the presence and / or amount of degradation products of one or more substances can be assessed. In the case of larger tissues, organoids and particularly organs, it is preferred to capture the parameters of the overall function and / or performance of the tissues, organoids and / or organs. Alternatively or optionally, indirect measurements can be used, for example based on a combination of specific biomarkers. Other examples can include measurements based on the detection of certain test substances as indicators of cellular synthesis and / or degradation, for example, by quantitative fluorescence measurements, and / or methods based on live / dead cell staining.

[0126] Preferably, the method for cryopreserving cells, cell aggregates, cell cultures, tissues, organoids and / or organs at low temperatures is applied to cells, cell aggregates, cell cultures, tissues, organoids and / or organs of plant or vertebrate origin, preferably mammalian and / or human origin. This is particularly advantageous for (bio)medical applications such as transplant medicine and / or pharmaceutical applications such as "ready-to-use" cells, cell aggregates, cell cultures, tissues, organoids and / or organs.

[0127] Therefore, in some embodiments of the present invention, the cells, cell aggregates, cell cultures, tissues, organoids and / or organs are cells, cell aggregates, cell cultures, tissues, organoids and / or organs from vertebrates or plants, preferably from vertebrates, more preferably from mammals and / or humans.

[0128] Furthermore, the present invention relates to cells, cell aggregates, cell cultures, tissues, organoids and / or organs obtained or obtainable by the above-described method of the present invention for cryopreserving cells, cell aggregates, cell cultures, tissues, organoids and / or organs.

[0129] In some embodiments of the present invention, the cells, cell aggregates, cell cultures, tissues, organoids and / or organs obtained by or by the cryopreservation method according to the present invention preferably have cryoprotective properties at least temporarily. With respect to the cells, cell aggregates, cell cultures, tissues, organoids and / or organs and the cryoprotective properties, the same applies to the intracellular cryoprotectants according to the present invention as described above and the cells, cell aggregates, cell cultures, tissues, organoids and / or organs obtained by or by the cryopreservation method according to the present invention, respectively. Therefore, by using the intracellular cryoprotectants or derivatives thereof of the present invention (preferably identified using the corresponding methods of the present invention), a nucleotide sequence encoding at least the intracellular cryoprotectant, preferably an optionally modified mRNA sequence, can be generated and contacted with cells, cell aggregates, cell cultures, tissues, organoids and / or organs according to the cryopreservation method of the present invention. It is particularly advantageous to enable the cells to synthesize the intracellular cryoprotectant using mRNA because it makes the cells suitable for cryopreservation without changing their genetic background. Therefore, the cells preferably only obtain the short-term cryopreservation properties required for cryopreservation. This is particularly advantageous for medical applications such as tissue and / or organ transplantation (biological) because no exogenously added biological information will remain in the tissue and / or organ after prolonged thawing. Depending on the specific purpose, cells, cell aggregates, cell cultures, tissues, organoids, and / or organs can be cryopreserved for more than one cycle by repeating the cryopreservation method of the present invention, preferably without affecting the cell characteristics and / or genetic background.

[0130] In some embodiments of the present invention, the cells, cell aggregates, cell cultures, tissues, organoids and / or organs obtained or obtainable by the cryopreservation method according to the present invention are at least temporarily provided with cryoprotective properties, wherein the cryoprotective properties are characterized in that the cells, cell aggregates, cell cultures, tissues, organoids and / or organs have a level of viability, vitality and / or functionality of at least 70%, preferably at least 80%, more preferably at least 90% after freezing and thawing compared to the level before freezing and thawing. With respect to the cryoprotective properties and the levels of viability, vitality and / or functionality and their corresponding measures, the same applies in the context of the intracellular cryoprotectants according to the present invention and the freezing and cryogenic method according to the present invention, respectively, as described herein.

[0131] The present invention also relates to frozen and thawed cells, cell aggregates, cell cultures, tissues, organoids and / or organs. As described above with respect to the frozen and thawed cells, cell aggregates, cell cultures, tissues, organoids and / or organs, the same applies to the cells, cell aggregates, cell cultures, tissues, organoids and / or organs of the present invention that are or can be obtained by the cryopreservation method of the present invention. Therefore, in addition, the frozen and thawed cells, cell aggregates, cell cultures, tissues, organoids and / or organs are or can be obtained by the cryopreservation method according to the present invention and preferably have at least temporarily cryoprotective properties, wherein the cryoprotective properties are preferably characterized by a viability, vitality and / or functionality level of at least 70%, preferably at least 80%, more preferably at least 90% after freezing and thawing compared to the level before freezing and thawing.

[0132] In some embodiments of the present invention, the (frozen and thawed) cells, cell aggregates, cell cultures, tissues, organoids and / or organs are suitable for transplanting (frozen and thawed) cells, cell aggregates, cell cultures, tissues, organoids and / or organs. Therefore, corresponding (frozen and thawed) cells, cell aggregates, cell cultures, tissues, organoids and / or organs preferably have the viability, vitality and / or functional level that meet national requirements and (bio) medical background, particularly transplanting. More specifically, in some embodiments of the present invention, the (frozen and thawed) cells, cell aggregates, cell cultures, tissues, organoids and / or organs are suitable for transplanting (frozen and thawed) cells, cell aggregates, cell cultures, tissues, organoids and / or organs, wherein the (frozen and thawed) cells, cell aggregates, cell cultures, tissues, organoids and / or organs are suitable for transplanting and are of artificial origin. The example of artificial origin includes, for example, in vitro engineered tissues etc. In some embodiments of the present invention, the (frozen and thawed) cells, cell aggregates, cell cultures, tissues, organoids and / or organs are (frozen and thawed) cells, cell aggregates, cell cultures, tissues, organoids and / or organs suitable for transplantation obtained by contacting the cells, cell aggregates, cell cultures, tissues, organoids and / or organs with nucleotide sequences encoding intracellular cryoprotectants according to the present invention and / or nanoparticles comprising nucleotide sequences encoding intracellular cryoprotectants according to the present invention at least briefly and / or partially in a physiological environment. Alternatively or optionally, in some embodiments of the present invention, the (frozen and thawed) cells, cell aggregates, cell cultures, tissues, organoids and / or organs are (frozen and thawed) cells, cell aggregates, cell cultures, tissues, organoids and / or organs to be transplanted. This is particularly advantageous because the present disclosure paves the way for cryopreservation of cells, cell aggregates, cell cultures, tissues, organoids and / or organs by temporarily enabling them to synthesize intracellular cryoprotectants according to the present invention without interfering with their long-term physiological properties. Furthermore, while well-established vaccination-based approaches may be used, (bio)medical needs and / or logistic limitations in transplant medicine may be overcome.

[0133] In some embodiments of the present invention, the (frozen and thawed) cells, cell aggregates, cell cultures, tissues, organoids and / or organs are "ready-to-use" (frozen and thawed) cells, cell aggregates, cell cultures, tissues, organoids and / or organs, preferably (frozen and thawed) "ready-to-use" "organ-on-a-chip". Thus, using the cryopreservation method according to the present invention, cells, cell aggregates, cell cultures, tissues, organoids and / or organs can be cryopreserved in a manner that maintains their initial viability, vitality and / or functionality, stored as needed and ready to use after thawing. The "ready-to-use" (frozen and thawed) cells, cell aggregates, cell cultures, tissues, organoids and / or organs open up new avenues for a range of (bio)medical applications, including highly relevant applications in the context of transplant medicine. In addition, the (frozen and thawed) "ready-to-use" "organ chips" are advantageous because they make cryopreservation easy for pharmaceutical and (bio)medical applications, such as ready-to-use tissue culture for drug screening. In particular, such "ready-to-use" cell systems, and in particular "ready-to-use" "organ chip" systems, represent a promising tool, for example, for screening the efficacy and toxicity of drug candidates at very early stages and / or (pre-)clinical stages in the drug development process. "Organ chip" technology can culture human cells in 3D in vitro while representing organs under physiological conditions. Thus, "ready-to-use" "organ chips" representing specific patients and / or reflecting a given patient population can be established, and can therefore be used, for example, for personalized medicine, where drug screening can be performed when needed simply by thawing the "organ chip" cryopreserved according to the corresponding method of the present invention disclosed herein.

[0134] The present invention also relates to the use of (frozen and thawed) cells, cell aggregates, cell cultures, tissues, organoids and / or organs according to the invention in drug development, active ingredient and / or drug screening, (preclinical) drug testing and / or (bio) medicine. In some embodiments of the invention, the use is for (bio) medicine, preferably for transplantation medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0135] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and accompanying drawings.

[0136] Figure 1: Schematic diagram of the effect of an added cryoprotectant on cell viability (y-axis) in a cell suspension comprising the (suspension) medium as a function of concentration in the (suspension) medium (x-axis). The ranges A (left), B (center), and C (right) indicated by the corresponding arrows are to be understood as follows: A) a range characterized by a low concentration of the added cryoprotectant, B) a range characterized by a concentration of the added cryoprotectant that has been experimentally determined to be a concentration of the added cryoprotectant that is suitable for cryopreserving at least a portion of the cells in suspension, and C) a range characterized by a high concentration of the added cryoprotectant.

[0137] Figure 2 : Schematic representation of the expected survival of cells in suspension, wherein the (suspension) culture medium contains added cryoprotectants at concentrations encompassed at higher resolution Figure 1 "Process 1" shows a hypothetical example of a process without an added cryoprotectant, and thus, an example in which cells in suspension are frozen in a (suspension) culture medium in the absence of an added cryoprotectant, wherein approximately 1 cell survives freezing and thawing of an initial 10^6 cells. "Process 2" shows another hypothetical example of a process in which surviving cells are found when freezing and thawing at least in the presence of some cryoprotectant added to the (suspension) culture medium, and thus, the added cryoprotectant is included in the (suspension) culture medium, for example, at a concentration of 0.5%.

[0138] Figure 3 : Schematic representation of three temperature-time protocols for cryopreservation, comprising freezing and subsequent thawing of cells in suspension (x-axis: time; y-axis: temperature). Solid lines: Examples of temperature-time protocols, wherein the freezing and thawing steps are characterized in that the reduction and increase in the temperature of the (suspension) culture medium, respectively, have a step-like profile, with the temperature drop or increase being relatively sudden, as in the case of rapid freezing or sudden heating. Dashed lines: Examples of temperature-time protocols, wherein freezing is characterized by a slower temperature reduction compared to the temperature-time protocol shown by the solid lines, as in the case of the "slow freezing" method, and wherein thawing is characterized by an abrupt increase in temperature. Dashed lines: Examples of temperature-time protocols, wherein the freezing and thawing steps are characterized in that the reduction and increase in the temperature of the (suspension) culture medium, respectively, have a relatively slow reduction and increase in the temperature of the (suspension) culture medium.

[0139] Figure 4: Schematic representation of the method of the invention, wherein one or more initially unknown intracellular cryoprotectants expressed by the cells of interest are identified by applying evolutionary methods (1-14), and then optionally characterized and used (15-20). The latter is exemplified by the following steps: identification of a nucleotide sequence encoding the identified cryoprotectant, generation of a nucleotide sequence encoding the cryoprotectant, packaging and transfer thereof into cells, wherein the information contained in the generated nucleotide sequence is translated into one or more intracellular cryoprotectants synthesized by the cells.

[0140] Figure 5 : Figure 4 Examples of modifications of the evolutionary method described in , wherein in steps 5 and 10, the cells under investigation are (epi-)genetically modified by modulating epigenetic masking (“EPI”) and / or applying CRISPR / Cas (“CRIS”).

[0141] Figure 6 : Results from frozen and thawed L929 cells in suspension. "EK" refers to a single cell colony (upper panel), while "MK" refers to a cell colony from different cells (lower panel). Results for sample 2A obtained on day 1, day 3, day 5, and day 7 (from left to right) are shown.

[0142] Figure 7 : Results from frozen and thawed L929 cells in suspension. "EK" refers to a single cell colony (upper panel), while "MK" refers to a cell colony from different cells (lower panel). Results for sample 2B obtained on day 1, day 3, day 5, and day 7 (from left to right) are shown.

[0143] Figure 8 : Results from frozen and thawed L929 cells in suspension. "EK" refers to a single cell colony (upper panel), while "MK" refers to a cell colony from different cells (lower panel). Results for sample 2C obtained on day 1, day 3, day 5, and day 7 (from left to right) are shown. After day 1, no viable cells were obtained for the 2C MK-2 sample.

[0144] Various aspects of the invention may be independent inventions that may also be claimed.

[0145] Other aspects and advantages of the present invention will be described in the following examples, which are provided for illustrative purposes only and not for limiting purposes. Example

[0146] The methods and materials used in the present disclosure are described herein; other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not limiting. Effects of added cryoprotectants on cell survival

[0147] Figure 1 Illustrative examples of the effect of an added cryoprotectant on the viability of cells in suspension as a function of its concentration are shown, assuming that the cells are unable to express the intracellular cryoprotectant.

[0148] Region A (illustrated by an arrow pointing to the lower left) relates to an area characterized by a relatively low concentration of the added cryoprotectant. Concentrations within the region are considered unfavorable because the positive protective effect of the added cryoprotectant cannot outweigh the negative effects of freezing and thawing on cell survival and vitality. Therefore, concentrations of cryoprotectants added within the range result in low cell survival rates. In contrast, region C (illustrated by an arrow pointing to the lower right) refers to an area characterized by a relatively high concentration of the added cryoprotectant. Concentrations within the region are also considered unfavorable because such concentrations typically have a negative impact on cell survival and may even be toxic to cells. Therefore, concentrations of cryoprotectants added within the range also result in low cell survival rates.

[0149] Region B (illustrated by an arrow pointing to the upper left) relates to a region characterized by an intermediate concentration of the added cryoprotectant. Within this range, an optimum is generally observed because the negative effects of the concentration of the added cryoprotectant, and therefore the stress on the cells associated with its presence, are outweighed by the positive effects of the added cryoprotectant, which limits or prevents freezing damage in the cells. Therefore, previous studies have focused on region B to test the cryoprotective properties of added cryoprotectants such as DMSO. In addition, previous studies have generally had the goal of optimizing region B, i.e., the lowest possible concentration of added cryoprotectant that still provides sufficient cryoprotection for cells in suspension. Therefore, studies to date have focused on optimizing the concentration of the added cryoprotectant in region B, toward low concentrations of added cryoprotectant in region B.

[0150] In contrast, the present disclosure focuses on the concentration of the added cryoprotectant in region A and relates to a method according to the present invention for identifying intracellular cryoprotectants expressed by and within cells. This method has the advantage that cryoprotection of cells (preferably in suspension) can be optimized in the presence of very low concentrations of the added cryoprotectant, preferably even in its absence. Thus, cell stress due to the addition of the cryoprotectant can be minimized and limitations associated with the use of added cryoprotectants, such as size limitations and gradient formation, can be overcome.

[0151] When considering Figure 1The following hypothetical example can be considered to illustrate the expected survival of cells in suspension when adding cryoprotectant concentrations within the relatively low range shown in Figure A. Figure 2 In the figures, the possible results are indicated by the solid line and are referred to as "Course 2". In this context, no or only a very limited number of cells are obtained after freezing and thawing in the presence of at least some concentration of added cryoprotectant in the suspension. However, the situation is expected to change when the frozen and thawed cells are capable of expressing and / or synthesizing at least a certain amount of intracellular cryoprotectant. Therefore, the dotted line, also referred to as "Course 1" illustrates the results when cells capable of expressing and / or synthesizing intracellular cryoprotectants are frozen and thawed in suspension in the absence of added cryoprotectant. Therefore, although Course 1 is most likely to be observed in the initial stages of the method of the present invention for identifying intracellular cryoprotectants, it is expected that the method of repeating freezing and thawing while modifying cells will result in cells displaying behavior consistent with Course 1. Therefore, by increasing the selection pressure by reducing the concentration of the added cryoprotectant, modified cells can be obtained that display improved cryoprotection during freezing and thawing even in the absence of an added cryoprotectant, such as DMSO, in the suspension. Illustrative Temperature-Time Protocol

[0152] Temperature-time protocols for cryopreservation of cells are well known in the art, and the skilled person knows that the parameters of such protocols are selected according to the cells under investigation, for example according to the cell type and species. Figure 3 Three illustrative temperature-time protocols are schematically depicted, which may be particularly advantageous for cryopreservation of cells in suspension. The protocols are characterized by at least two temperature changes.

[0153] More specifically, the temperature, preferably the temperature of the suspension containing the cells, can be lowered from a first value to a second value, wherein the lowering can be gradual or stepwise, preferably gradual. Then, preferably, the temperature is raised from the second value to a third value, wherein the increase can be gradual or stepwise. Thus, in a first step, the cells are cooled, preferably frozen, and subsequently thawed in a second step. After the second step, the viability of the cells can be determined, and thus the number of cells that survived the freezing and thawing.

[0154] The first value is preferably higher than the second value, particularly preferably at least 20°C, 70°C, or 140°C higher. For example, the first value may be greater than 0°C, for example, between 25°C and 30°C, and the second value may be less than 0°C, for example, between -10°C and -160°C. The third value is preferably higher than the second value, particularly preferably at least 20°C, 70°C, or 140°C higher. For example, the third value may be greater than 0°C, for example, between 25°C and 30°C. Furthermore, the first and third values ​​may be substantially the same. For example, the first value may be 21°C, the second value may be -140°C, and the third value may be 21°C.

[0155] Preferably, the first value is substantially maintained for at least 1 minute, more preferably at least 5 minutes. Preferably, the second value is substantially maintained for at least 10 seconds to 60 minutes, more preferably at least 30 seconds to 30 minutes. Preferably, the second value is substantially maintained for a period of 1 minute to 100 years, more preferably for a period of 1 minute to 50 years. Preferably, the third value is substantially maintained for at least 5 minutes.

[0156] Preferably, the temperature is lowered from the first value to the second value within 0.1 seconds to 60 minutes, preferably between about 0.25 seconds and 20 minutes, particularly preferably between about 0.5 seconds and 15 minutes. Preferably, the temperature is increased from the second value to the third value within 0.1 seconds to 60 minutes, preferably between 0.25 seconds and 20 minutes, particularly preferably between 0.5 seconds and 15 minutes.

[0157] For example, Figure 3 Three possible temperature-time schemes for cryopreservation are shown in FIG. With reference to the temperature-time scheme represented by the solid line, a step-by-step approach can be envisaged. In this case, the temperature-time scheme is characterized by a relatively sudden drop and rise in temperature, such as in the case of rapid freezing or rapid heating. In particular, such relatively sudden freezing methods are also known in the context of vitrification, in which temporary freezing is performed due to an ultra-high cooling rate to allow cells to solidify without forming crystalline ice. In contrast, in the case of the temperature-time scheme shown by the dotted line, the temperature is lowered and raised relatively slowly, respectively. This allows cells to adapt to temperature changes.

[0158] Combinations and modifications of the two embodiments are also included herein. Specifically, such slow cooling is well known in the context of a "slow freezing" method, in which the temperature is reduced at a low cooling rate to allow sufficient cell dehydration while minimizing the formation of ice crystals within the cells. In addition, a combination of the two exemplary temperature-time schemes is indicated by a dotted line as an illustration of potential modifications and / or combinations of the aforementioned schemes. In the scheme shown in the dotted line, freezing is characterized by a relatively slow decrease in temperature, as in a "slow freezing" method, while thawing is characterized by a relatively sudden increase in temperature.

[0159] It should be noted that the method for identifying intracellular cryoprotectants according to the present invention can be carried out using a temperature-time scheme. Alternatively or optionally, the temperature-time scheme can be varied. More specifically, the temperature-time scheme can be varied between two or more iterations. This may be advantageous for assessing the dependency between the modification and the selected temperature-time scheme and its effect on cell survival and viability. Alternatively or optionally, the temperature-time scheme can be varied between the units of a given iteration studied in parallel. In particular, when the method according to the present invention includes a proliferation step, a portion of the proliferating cells can be further used according to the method of the present invention using a first temperature-time scheme, while another portion of the proliferating cells can be used using a second temperature-time scheme. Therefore, the potential differences in the effects of modifications taking into account different temperature-time schemes on cryoprotection can also be assessed by efficient throughput methods. Exemplary schematic diagram of the method of the present invention for identifying intracellular cryoprotectants

[0160] The following will refer to Figure 4 and 5 The method according to the present invention for identifying an intracellular cryoprotectant is exemplarily described in more detail.

[0161] The cells can be obtained, for example, from cold-tolerant species such as Rana sylvatica and cultured in suspension. Preferably, the cells are propagated to increase the probability of obtaining cells that survive the initial freezing and thawing cycles. Thus, the cells can be cultured in suspension until, without the addition of cryoprotectants, e.g. 100 to 100,000 (microplate) wells are obtained, each well containing 10^5 to 10^6 suspension cells (1). The cells can be frozen and thawed according to step (a0) of the method of the invention by applying a predetermined temperature-time regime, e.g. reflecting e.g. Figure 3 (2) Slow freezing method shown.

[0162] In the event that no viable cells are obtained after freezing and thawing (3a), new cells can be obtained and the method is restarted from (1), but the first concentration x% of the added cryoprotectant contained in the suspension is lower, for example 0.1% DMSO (4a), and the cells are then frozen and thawed in the suspension containing the added cryoprotectant (5a). In the event that no viable cells are obtained after freezing and thawing (6b), new cells are again obtained and the method is restarted from (1), but the second concentration of the added cryoprotectant is slightly increased compared to the first concentration.

[0163] In the case where at least one cell (3b or 6a) that survives freezing and thawing is obtained according to step (a1) of the method according to the invention, preferably, the at least one surviving cell is proliferated in a suspension according to step (e1) of the method according to the invention (4b). The suspension may contain a conditioned medium to promote the proliferation of a single cell or a small number of cells. Preferably, the survival rate of the cells resulting from the freezing and thawing cycle is determined according to step (f1) of the method according to the invention.

[0164] The cells thus obtained are used according to the invention, cf. step (a).

[0165] The preferably proliferating cells that survive freezing and thawing are then modified (5) according to step (b) of the method of the invention. Figure 5 As shown, the modification can be epigenetic modification (EPI) and / or genetic modification using, for example, CRISPR / Cas (CRIS). Other examples of modified cells can be exposure to radiation or the addition of mutagenic substances. Preferably, the modified cells (6) are propagated according to step (e2) of the inventive method. Preferably, the propagated modified cells are then frozen and thawed (7), for example according to the same temperature-time scheme as used in (2) and / or (5a). This can represent step (c) of the method according to the present invention.

[0166] Thus, according to step (c) of the method (8) according to the invention, which is consistent with step (d), at least one modified cell that survives freezing and thawing can be obtained. Preferably, according to step (f2) of the method according to the invention, the survival rate y% (8) of the freezing and thawing cycle of the modified cells is determined. In the case where the survival rate y% (8) does not exceed the survival rate determined for (3b), the method can be continued at (4b) or (1). This can be iterated until the survival rate y% (8) is determined to exceed the survival rate determined for (3b). If this is the case, the at least one modified cell (9) obtained from (8) can be propagated according to step (e3) of the method according to the invention. The suspension can contain a conditioning medium to promote the proliferation of a single or a small number of modified cells.

[0167] According to step (b) of the inventive method, the modified cells that are preferably proliferated and "survival" after freezing and thawing are further modified (10). As in the case of (5), the modification can be, for example, epigenetic modification (EPI) and / or genetic modification using, for example, CRISPR / Cas (CRIS). The method used to modify the cells in (5) can be the same or different from the method selected for further modification of the modified cells in (10). Preferably, the further modified cells (11) are proliferated according to step (e2) of the inventive method. The further modified cells that are preferably proliferated are then frozen and thawed (12), for example according to the same temperature-time scheme as used in (2), (5a) and / or (7). This can represent step (c) of the method according to the invention.

[0168] Thus, according to step (c) of the method (13) according to the present invention, which is consistent with step (d), at least one further modified cell that survives freezing and thawing can be obtained. Preferably, according to step (f2) of the method according to the present invention, the survival rate (y+z)% (13) of the freezing and thawing cycle of the further modified cells is determined. In the case where the survival rate (y+z)% (13) does not exceed the survival rate determined for (8), the method can be continued at (9), (4b) or (1). This can be iterated until the survival rate (y+z)% (13) is determined to exceed the survival rate determined for (8). If this is the case, the at least one further modified cell obtained from (13) can be propagated according to step (e3) of the method according to the present invention. The suspension can contain a conditioning medium to promote the proliferation of a single or a small number of modified cells.

[0169] When the survival rate (y+z)% is determined to be greater than, for example, 80% (14), the further modified cells can be considered to be able to express an intracellular cryoprotectant suitable for cryopreservation. This can be understood as an example of step (d) of the method according to the invention. Preferably, if the survival rate determined in step (f2) is 80% or higher (14), the at least one intracellular cryoprotectant is identified (15), optionally based on the (further) modified cells proliferated in step (e3). The identification of the at least one intracellular cryoprotectant (15) can be performed, for example, using genome sequencing and / or in the case of polypeptide or protein 2-SDS-PAGE gel electrophoresis. The identified intracellular cryoprotectant is preferably isolated and further characterized, for example, in terms of structure, function and / or cellular localization (16). For exemplary applications (17-20), the mRNA sequence can be synthesized in vitro (17) and optionally optimized, for example taking into account the codon usage preference of the target species. The generated mRNA sequence can be encapsulated (18) and introduced into the target cells (19) according to known techniques, for example in the case of inoculation. The target cells can then synthesize (20) the intracellular cryoprotectants identified using the methods of the present invention and are therefore able to survive cryopreservation without significant effects on cell survival and viability.

[0170] Preferred target species may be vertebrates such as humans, and corresponding target cells such as human kidney cells. Thus, by applying, for example, well-established vaccination methods to introduce an mRNA sequence encoding at least one intracellular cryoprotectant identified using the methods of the present invention, cells, cell aggregates, organoids, tissues, and even organs can be cryopreserved, for example for organ transplantation or for the preparation of ready-to-use tissue sections or organoids for drug screening methods in a pharmaceutical setting. Furthermore, as shown in the example of vaccination-based methods, the target cells may only temporarily possess the ability to synthesize the intracellular cryoprotectant, since the methods preferably do not alter the properties of the target cells, such as their genetic and epigenetic makeup. Cryopreservation of cells – Experiment I

[0171] L929 cells were harvested by aspirating the culture medium, washed once with PBS, and 2.5 ml of trypsin was added to the cell lawn (T75), aspirated again after 20 seconds, and then incubated in an incubator at 37°C and 5% CO2 for 3 minutes. The cells were then rinsed with CGM (CGM: complete growth medium = RPMI 1640 + glutamine + Pen / Strep + 10% FCS). The cell number was determined, and 2 × 10^6 cells were removed from 1.0 ml of CGM and transferred to a cryogenic tube and stored at -85°C for 24 hours using the cold box method.

[0172] On the 1st day, the cells were transferred to -150°C and kept at -150°C for 2-8 days. On the 9th day, the cells (2 × 10^6 cells / ml / sample) were thawed as follows: 24-well cloning plates, each containing 1ml KCGM (conditional CGM, sterile filtered 0.2 μm), 10 wells for 1 sample, followed by 4.0ml KCGM added to each 1ml cell suspension, and this 0.5ml was added to each well of the cloning plate (submitting 1.0ml KCGM) = 10 wells, 2 × 10^5 cells per well. In addition, 5 wells containing only KCGM were used as controls. Microscopic characterization and photography were performed on the 10th, 12th, 14th and 16th days. On the 15th-16th day, the colonies were counted according to colony growth, and single colonies were picked, cultured and refrozen for another thawing test.

[0173] A total of 9 samples were studied, and the corresponding numbers of cell colonies successfully obtained after freezing and thawing L929 cells are summarized in Table 1 . sample Number of colonies grown after freezing and thawing 1A 293 1B 262 1C 139 2A 218 2B 184 2C 196 3A 406 3B 97 3C 288 average value 231.44 Standard Deviation 92.66 Table 1: Number of cell colonies obtained from 2×10^6 frozen and thawed L929 cells. Cryopreservation of cells - Experiment II

[0174] Subsequently, a subset of samples obtained from Experiment 1 was further investigated following the same experimental setup as Experiment 1 above.

[0175] like Figure 6-8 As shown, multiple cell colonies can be obtained from L929 cells that survived freezing and thawing as described above. More specifically, the number of cell colonies successfully obtained in each sample is summarized in Table 2. It is worth noting that "EK" refers to individual cell colonies, and therefore refers to individual colonies obtained from surviving and growing colonies that were initially "picked" from the corresponding culture dishes of Experiment 1. Even more noteworthy is that "MK" refers to cell colonies generated from different cells obtained from Experiment 1. In the latter case, no sorting was performed, but all corresponding surviving and growing colonies were further investigated. sample Number of colonies grown after freezing and thawing 2A EK-2 295 2A MK-2 367 2B EK-1 380 2B MK-2 501 2C EK-1 94 2C MK-2 0 Table 2: Number of cell colonies obtained from 2×10^6 frozen and thawed L929 cells.

[0176] It can be seen that the experimental method, the single colony-based method, and the mixed colony-based method all showed comparable results. Identification of intracellular cryoprotectants, generation of mRNA sequences encoding them, packaging and use thereof - Example 1

[0177] As shown in Experiments I and II described above, it was surprisingly found that a portion of L929 cells that were cryopreserved according to a temperature-time protocol commonly used in the art survived freezing and thawing without the addition of a cryoprotectant such as DMSO. These cells, i.e., cells that survived freezing and thawing, are suitable for use in step (a) of the method for identifying intracellular cryoprotectants expressed by the cells of the present invention, such as Figure 4 and 5 As shown. According to the method, experiments I and II can alternatively or optionally also be performed with very low concentrations of an added cryoprotectant such as DMSO, for example less than 1% DMSO of the cell suspension medium. This would have the advantage that more viable cells can be expected compared to the same experiment without the added cryoprotectant, as is the case with experiments I and II.

[0178] Then, the cells that survive freezing and thawing (e.g., fibroblasts and / or stem cells) can be subjected to multiple iterations of in vitro proliferation, modification, freeze-thaw, etc., and used in accordance with the present invention to identify intracellular cryoprotectants. When a survival rate, for example, greater than 80%, is observed after a given repeated freezing and thawing, the modified cells obtained that survive freezing and thawing are studied with respect to intracellular cryoprotectants that provide cryoprotection to the cells in the presence of very low concentrations in the suspension or even in the absence of added cryoprotectants during the freezing and thawing of the cells. Although a variety of methods and techniques are available and well established in the art, gel electrophoresis and / or mass spectrometry may be particularly suitable for identifying one or more intracellular cryoprotectants expressed by the cells. Exemplarily, using SDS gel electrophoresis, the proteomes of wild-type and obtained cells can be compared, where the wild-type is preferably the cell initially used. By comparing the proteomes of the wild-type, preferably initially used, cell and the (further) modified cell finally obtained, modifications can be identified by two-dimensional mapping of the two proteomes in the form of separate spots in a gel, so that the modified polypeptides and / or proteins can be easily identified, for example due to the presence of spots only in the SDS map of the proteome of the (further) modified cell and not in the corresponding SDS map of the wild-type proteome, or visually different spot intensities indicating different concentrations of the corresponding polypeptides or proteins. SDS gel electrophoresis is an example of a conventional method that is commonly used to determine polypeptide and / or protein differences, for example indicative of altered gene activity in a cell, which can easily be used to identify intracellular cryoprotectants expressed by the (further) modified cell, in contrast to the initially used cell and / or expressed at a different intensity compared to the initially used cell.

[0179] The polypeptide or protein of this identification can be a globular protein such as protamine, and is therefore an animal mixture of strongly basic peptides belonging to the protamine group, which can be found in, for example, sperm or eggs of certain salmon species, or a common cellular protein such as histone. After preliminary identification of the intracellular cryoprotectant using the SDS gel electrophoresis described above, the intracellular cryoprotectant can be further characterized based on its structure and / or reference amino acid sequence, wherein the reference amino acid sequence is obtained from a commonly used prior art protein database (e.g., the National Center for Biotechnology Information (NCBI), SwissProt, Protein Information Resource (PIR) or Protein Data Bank (PDB)). Based on the obtained amino acid sequence, the corresponding mRNA sequence (hereinafter also referred to as cryo-mRNA) encoding at least the at least one identified intracellular cryoprotectant can be designed and synthesized in vitro. Optionally, the corresponding gene and / or mRNA sequence can also be obtained from a commonly used prior art protein database such as the NIH National Library of Medicine.

[0180] As demonstrated by the prior art of mRNA-COVID vaccines, the production of suitable cryo-mRNA can be carried out in a cell system or fully synthesized (see, for example, Javier T. Granados-Riveron and Guillermo Aquino-Jarquin; Biomed Pharmacother. 2021 Oct; 142: 111953. Engineering of the current nucleoside-modified mRNA-LNP vaccines against SARS-CoV-2). Similar to the COVID vaccine, cryo-mRNA can be packaged to ensure efficient cellular uptake by fusion with the cell membrane and appropriate stability of the cryo-mRNA. According to the prior art, lipid nanoparticles can be used for this purpose, which contain a mixture of components to achieve this function. Since the mixture is ideally optimized according to the target cell system, this optimization is preferably also performed on the cryo-mRNA. According to the prior art, the following components of the nanoparticles are expected to show a high probability of successfully introducing mRNA sequences into cells: a mixture of cationic and ionizable lipids, embedded structural auxiliary lipids, polyethylene glycol (PEG) lipids, and cholesterol, with the corresponding proportions adjusted according to the respective cryo-mRNA and target cell system.

[0181] In order to preserve cells at low temperatures according to the corresponding methods of the nanoparticles containing cryo-mRNA produced in the embodiments of the present invention, the nanoparticles can be added to the (cell) suspension and / or (cell) culture medium at a suitable concentration and for a suitable time, depending on the target cell system. Alternatively or optionally, the organoid or organ can be contacted with the nanoparticles by injecting the nanoparticles into the organoid or organ in vitro. Then, in the corresponding cells, the cryo-mRNA can be translated, and thus, the above-mentioned intracellular cryoprotectants such as globulins such as protamine, protein mixtures and / or histones can be synthesized in the cytoplasm of the corresponding cells. When the concentration of the cryoprotectant in the cell is high enough, the cells are expected to have increased and / or improved cryoprotective properties during subsequent freezing and thawing. After thawing, the cryo-mRNA introduced into the cells is expected to degrade according to conventional biological processes in the cells. Therefore, the expected cryoprotective properties are only temporarily obtained and / or improved without modifying the genetic background of the target cells. Therefore, when applying the method of the present invention to cryopreserve single cells and, for example, cells contained in organs, it is expected that the corresponding cells or, for example, organs are very suitable for medical and / or pharmaceutical purposes after freezing and thawing. Identification of intracellular cryoprotectants, generation of mRNA sequences encoding them, packaging and use thereof - Example II

[0182] By applying the evolutionary method of the method for identifying intracellular cryoprotectants according to the invention, (further) modified cells can be obtained which are characterized by a relatively higher survival rate than the initially used cells and have improved cryoprotective properties due to an enrichment (preferably iterative) of modifications which have a positive influence on the cryoprotective properties of the (further) modified cells compared to the initially used cells.

[0183] Example II extends Example I by the following steps: After identifying at least one intracellular cryoprotectant and a corresponding nucleotide sequence (preferably a corresponding mRNA sequence), the nucleotide sequence can be modified and synthesized in vitro according to standard state-of-the-art methods for the synthesis of engineered mRNA sequence fragments (see, for example, Javier T. Granados-Riveron and Guillermo Aquino-Jarquin; Biomed Pharmacother. 2021 Oct; 142: 111953. Engineering of the current nucleoside-modified mRNA-LNP vaccines against SARS-CoV-2).

[0184] Optionally or alternatively, more than one form of this synthetically engineered mRNA sequence can be studied. Therefore, preferably in parallel, multiple identical cell systems can be contacted with a form of each of the nucleotide sequence encoding the at least one intracellular cryoprotectant. The form can be generated by targeted or non-targeted modification of the identified and / or modified nucleotide sequence, thus following a systemic effect evaluation method or a random effect evaluation method. Therefore, a high-throughput effect screening of different modifications of the mRNA sequence encoding the identified intracellular cryoprotectant can be achieved, and the form with the best performance of the synthetically engineered mRNA sequence in terms of cryoprotection of the corresponding cell system is identified for further application. Identification of intracellular cryoprotectants, production of mRNA encoding them, their packaging and use - Example III

[0185] Example III represents an improvement of Example I and / or II, extending the corresponding example by the following steps: In the method of identifying intracellular cryoprotectants according to the present invention, different temperature-time protocols are preferably evaluated in parallel. More specifically, for example, one can try to use ultrafast cooling rates to achieve vitrification-like conditions, or apply a more classical temperature gradient, where the temperature change is in the range of seconds to minutes per degree. Both temperature-time protocols can be tested, but may lead to (slightly) different results regarding the identification of intracellular cryoprotectants. Specifically, relatively slow freezing and thawing rates compared to ultrafast cooling and thawing rates can allow larger ice crystals to form, due to the migratory ice growth effect at the expense of smaller ice crystals; whereas in the case of vitrification-based methods, due to the higher cooling and thawing rates, respectively, this migratory ice growth effect may not be observed. Therefore, the identified modifications and / or their cryoprotective effects can vary between these two settings. Therefore, by identifying intracellular cryoprotectants using different temperature-time protocols, different modes of action of cellular cryoprotectants and / or the identification of highly effective intracellular cryoprotectants for a given temperature-time protocol can be elucidated. Illustrative examples of parameters related to survivability, vitality and / or functionality

[0186] According to the cryopreservation method of the present invention, (frozen and thawed) cells, cell aggregates, cell cultures, tissues, organoids and / or organs can be obtained, which are also included in the present invention. These preferably show cryoprotective properties at least temporarily, so that after freezing and thawing, for example, at least 70% of the viability, vitality and / or functional levels of cells, cell aggregates, cell cultures, tissues, organoids and / or organs can be observed compared to the corresponding levels before freezing and thawing. Exemplary methods and techniques for this are exemplarily mentioned below.

[0187] For example, in the case of cardiac organoids, there are established methods known to technicians for determining the viability, vitality and / or function of corresponding organoids based on various parameters. For an overview of the method for determining relevant parameters, reference can be made to publications such as Ergir et al. ((Scientific Reports, 2022, 12: 17409; https: / / doi.org / 10.1038 / s41598-022-22225-w; Generation and maturation of human iPSC-derived 3D organotypic cardiac microtissues in long-term culture); Lewis-Israeli et al. publications (Nature Communications, 2021, 12: 5142; https: / / doi.org / 10.1038 / s41467-021-25329-5; Self-assembling human heart organoids for the modeling of cardiac development and congenital heart disease); Kim et al. publications (Nano Lett.2022,22,7892-7901; https: / / doi.org / 10.1021 / acs.nanolett.2c02790; MultimodalCharacterization of Cardiac Organoids Using Integrations of Pressure-Sensitive Transistor Arrays with Three Dimensional Liquid Metal Electrodes); Publications by Richards et al. (Nature Biomedical Engineering, Volume 4, pages 446-462, 2020; https: / / www.nature.com / articles / s41551-020-0539-4; Human cardiacorganoids for the modeling of myocardial infarction and drugcardiotoxicity); and publications by Zhao et al. (Stem Cell Research & Therapy, 2021, 12:272; https: / / doi.org / 10.1186 / s13287-021-02340-7; Cardiac organoid - a promising perspective of preclinical model). For example, functional assays can be performed based on calcium flux and / or electrophysiological parameters, by characterizing beating frequency, RNA sequencing, metabolic evidence (e.g., through hippocampal-based assays), oxygen consumption rate (OCR), and / or transmission electron microscopy (TEM)-based assays. Microscopic and immunohistological parameters and assays can also be used and / or considered.

[0188] Well-known procedures and methods can also be found for many other examples of cells, cell aggregates, cell cultures, tissues, organoids and / or organs. Thus, as examples, reference is made herein to the publication by Marthaler et al. (StemCell Research 16 (2016) 202–205; Generation of an isogenic, gene-corrected control cell line of the spinocerebellar ataxia type 2 patient-derived iPSCline H266; http: / / dx.doi.org / 10.1016 / j.scr.2015.12.048), which lists relevant parameters, in particular for demonstrating the pluripotency of stem cells, and to the publication by Lancaster et al. (Nature, 2013, 501 (7467); doi: 10.1038 / nature12517; Cerebral organoids model human brain development and microcephaly), which lists relevant parameters in the context of cerebral organoids, in particular in the context of disease models and / or models (e.g., microcephaly).

[0189] The present invention is also characterized by the following items. 1. A method for identifying an intracellular cryoprotectant expressed by a cell, the method comprising (a) using cells that survive freezing and thawing, (b) modifying the used cells, (c) freezing and thawing the modified cells, and (d) obtaining a modified cell that survived step (c), thereby identifying an intracellular cryoprotectant expressed by the cell. 2. The method according to item 1, wherein the cells are obtained from vertebrates, insects, plants, algae, fungi or bacteria. 3. The method according to item 1 or 2, wherein the cells and / or modified cells can be cultured in suspension and / or are cells in a suspension state. 4. A method according to any one of the preceding items, wherein in step (a), the cells survive freezing and thawing in a suspension, the suspension preferably comprising an added cryoprotectant at a concentration of 5% or less, preferably 1% or less, more preferably 0%. 5. A method according to any of the preceding items, wherein in step (c), the modified cells are frozen and thawed in a suspension, the suspension preferably comprising an added cryoprotectant at a concentration of 5% or less, preferably 1% or less, more preferably 0%. 6. The method according to any of the preceding items, wherein in steps (a) and / or (c) i) the freezing comprises lowering the temperature from above 0°C to a temperature below 0°C, preferably below -20°C, more preferably below -70°C, even more preferably below -140°C, and / or ii) the thawing comprises raising the temperature from below 0°C, preferably below -20°C, more preferably below -70°C, even more preferably below -140°C to a temperature above 0°C, wherein preferably, the temperature is the temperature of a suspension in which the cells used survive freezing and thawing, and / or the modified cells are frozen and thawed, and / or It is preferred that, after the temperature is lowered, the lowered temperature is maintained for at least 1 second to 10 minutes, preferably at least 10 seconds to 5 minutes, and then thawed. 7. The method according to any one of the preceding items, wherein in step (b), the cell is modified by the following steps i) exposure to a mutagenic substance, preferably a mutagenic substance selected from the group consisting of polycyclic aromatic hydrocarbons, nitrosamines, base analogs, peroxides and combinations thereof, ii) exposure to radiation, preferably high-energy radiation, more preferably UV- and / or X-ray radiation, iii) one or more modifying proteins, preferably selected from TALEN, zinc finger protein, CRISPR / Cas combination, TET1, p300, DNMT3A, MQ1 and LSD1, wherein the one or more modifying proteins more preferably comprise a CRISPR / Cas combination, and / or iv) any combination of the above. 8. The method according to any of the preceding items, further comprising (e) propagating the obtained ((further) modified) cells, preferably in suspension. 9. The method according to item 8, wherein in step (e) i) the suspension is a suspension containing an added cryoprotectant at a concentration of 5% or less, preferably 1% or less, more preferably 0%, and / or ii) The suspension comprises a conditioned (suspension) medium. 10. The method according to any one of the preceding items, comprising the steps of: step (a), optionally followed by step (e), followed by [step (b), optionally step (e), steps (c) and (d), optionally step (e)] in i iterations, wherein i is preferably at least 1 and a maximum of 1,000. 11. The method according to any one of the preceding items, comprising the following steps: step (a), optionally followed by step (e), followed by [step (b), step (e), step (c) and (d), step (e)] in i iterations, wherein i is preferably at least 1 and a maximum of 1,000. 12. The method according to item 10 or 11, wherein one (preferably each) step (b) and / or (c) after step (e) is performed on at least one, preferably at least 10%, more preferably all, of the proliferated ((further) modified) cells obtained after step (e). 13. The method according to any one of items 10 to 12, further comprising: (f) determining the viability of the obtained (further) modified cells, preferably for each step (d). 14. The method according to claim 13, comprising iteration i+1 if the survival rate determined in step (f) of iteration i is at least as high as the survival rate determined in step (f) of iteration i-1, preferably 10% higher, and preferably does not exceed a given threshold. 15. A method according to any one of items 10 or 14, comprising the following steps: step (a), optionally followed by step (e), followed by [step (b), optionally step (e), steps (c) and (d), optionally step (f), optionally step (e)] in i iterations, where i is at least 2, and preferably also comprising iteration i+1, if the survival rate determined in step (f) of iteration i is at least as high as the survival rate determined in step (f) of iteration i-1, preferably 10% higher, and preferably does not exceed a given threshold. 16. A method according to any one of items 10 to 15, comprising the following steps: step (a), optionally followed by step (e), followed by [step (b), step (e), steps (c) and (d), step (f), step (e)] in i iterations, where i is at least 2, and further comprising iteration i+1 if the survival rate determined in step (f) in iteration i is at least as high as the survival rate determined in step (f) in iteration i-1, preferably 10% higher, and preferably does not exceed a given threshold. 17. The method according to any of the preceding items, wherein the intracellular cryoprotectant expressed by the (further) modified cells is identified by analyzing the genome, transcriptome, epigenome, proteome and / or metabolome, preferably by using an analysis selected from gel electrophoresis, mass spectrometry, crystal structure analysis, NMR spectroscopy, DNA sequencing, (m)RNA sequencing and any combination of the foregoing. 18. The method according to any of the preceding items, wherein an intracellular cryoprotectant expressed by the (further) modified cell is identified if the viability determined in step (f) exceeds a given threshold. 19. The method according to any of the preceding items, further comprising (h) modifying the identified intracellular cryoprotectant expressed by the (further) modified cell, thereby obtaining a derivative from the identified intracellular cryoprotectant expressed by the (further) modified cell. 20. The method according to any one of the preceding items, comprising the following steps: (a) using cells that survive freezing and thawing, Wherein step (a) preferably comprises the following steps: (a0) Freezing and thawing of cells in suspension, (a1) obtaining cells that survived step (a0), (f1) determining the viability of step (a0) based on the number of living cells of the cells in suspension before freezing and thawing and after freezing and thawing, (e1) growing the cells obtained in step (a1) and using the grown cells, (b) modifying the cells used in step (a), preferably the cells proliferated in step (e1) and / or the cells proliferated in step (e3), (e2) multiplying the modified cells in step (b), (c) freezing and thawing the modified cells in suspension that have proliferated in step (e2), (d) obtaining modified cells that survive step (c), thereby identifying an intracellular cryoprotectant expressed by said cells, wherein preferably, the freezing and thawing in step (a), preferably step (a0) and / or step (c) are carried out in a suspension, said suspension preferably comprising an added cryoprotectant at a concentration of 5% or less, preferably 1% or less, more preferably 0%. 21. The method according to item 20, wherein the intracellular cryoprotectant expressed by the modified cell is identified by: (f2) determining the viability of step (c) based on the number of modified living cells in suspension before freezing and thawing and after freezing and thawing, (e3) propagating the modified cells obtained in step (d), and (g) identifying at least one intracellular cryoprotectant based on the modified cells propagated in step (e3), preferably if the viability determined in step (f2) exceeds a given threshold, wherein if the survival rate determined in step (f2) of iteration i is at least as high as the survival rate determined in step (f2) of iteration i-1, preferably 10% higher, and preferably does not exceed the given threshold, and wherein i is at least 2, then steps [(b), (e2), (c), (d), (f2), (e3)] are iterated (iteration i+1 times). 22. The method according to item 21, wherein the suspension in step (c) of iteration i+1 comprises (1) If the survival rate determined in step (f2) is at least as high as the survival rate determined in step (f), preferably at least 5% higher, and the survival rate is determined in the following steps: i) step (f1) in the case of the first iteration (i=1), or ii) step (f2) of iteration i-1 in the case of iteration i, the concentration of the cryoprotectant added is substantially the same as that of the suspension in step (c) of iteration i; (2) If neither i) nor ii) is satisfied, the concentration of the cryoprotectant added is 0.001% to 1%, preferably 0.01% to 0.5%, higher than the concentration in the suspension in step (c) of iteration i. 23. An intracellular cryoprotectant or a derivative thereof, preferably obtained or obtainable by a method according to any one of the preceding claims, wherein the intracellular cryoprotectant is an intracellular cryoprotectant synthesized by a cell and is preferably an optionally glycosylated polypeptide or protein. 24. The intracellular cryoprotectant or derivative thereof according to item 23, wherein the intracellular cryoprotectant is selected from antifreeze proteins, proteins or polypeptides having an alanine- and / or threonine-rich structure and a tertiary structure similar to antifreeze proteins, glycoproteins, cytoplasmic proteins or polypeptides, heat shock proteins, albumins, globulins, histones, protamines, kinases, growth factors, globular proteins or polypeptides, membrane-bound and / or superimposed proteins or polypeptides, porins, osmotically active proteins or polypeptides, fibrous proteins or polypeptides, polypeptides or proteins involved in the synthesis of membrane lipids and / or fatty acids, phospholipids, glycolipids, cholesterol, polypeptides or proteins involved in glycogen synthesis, insulin, and core protein. 25. Intracellular cryoprotectant according to item 23 or 24, wherein the intracellular cryoprotectant is capable of cryopreserving cells, preferably cell aggregates, cell cultures, tissues, organoids and / or organs. 26. A method for producing an optionally modified nucleotide sequence encoding an intracellular cryoprotectant according to any one of items 23 to 25 or a derivative thereof. 27. The method according to item 26, wherein generating the optionally modified nucleotide sequence comprises the steps of (a) identifying a nucleotide sequence encoding the intracellular cryoprotectant or a derivative thereof, (b) optionally modifying said identified nucleotide sequence, and (c) synthesizing said identified, optionally modified, nucleotide sequence in vitro. 28. An optionally modified nucleotide sequence, which is or is obtainable by a method according to item 26 or 27, encoding one or more intracellular cryoprotectants according to any one of items 23 to 25 or derivatives thereof. 29. A nanoparticle comprising the optionally modified nucleotide sequence according to item 28, preferably wherein the nanoparticle is a lipid nanoparticle. 30. The method according to item 26 or 27, the optionally modified nucleotide sequence according to item 28 and / or the nanoparticle according to item 29, wherein the optionally modified nucleotide sequence is an RNA sequence, preferably an mRNA sequence. 31. Use of the intracellular cryoprotectant or derivative thereof according to any one of items 23 to 25, the optionally modified nucleotide sequence according to item 28 or 30, or the nanoparticle according to item 29 or 30 for cryopreservation of cells, cell aggregates, cell cultures, tissues, organoids and / or organs. 32. A method for cryopreserving cells, cell aggregates, cell cultures, tissues, organoids and / or organs, comprising the steps of (a) contacting cells, cell aggregates, cell cultures, tissues, organoids and / or organs with an optionally modified nucleotide sequence according to item 28 or 30 and / or nanoparticles according to item 29 or 30, and (b) freezing the cells, cell aggregates, cell cultures, tissues, organoids and / or organs obtained from step (a), wherein freezing comprises lowering the temperature of the cells, cell aggregates, cell cultures, tissues, organoids and / or organs from above 0°C to below 0°C, preferably below -20°C, more preferably below -70°C, even more preferably below -140°C. 33. The method according to item 32, wherein step (a) comprises the following steps: (a1) contacting the cells, cell aggregates, cell cultures, tissues, organoids and / or organs with an optionally modified nucleotide sequence according to item 28 or 30 encoding a first intracellular cryoprotectant or a derivative thereof and / or with nanoparticles according to item 29 or 30 comprising an optionally modified nucleotide sequence encoding a first intracellular cryoprotectant or a derivative thereof, and (a2) contacting the cells, cell aggregates, cell cultures, tissues, organoids and / or organs with another optionally modified nucleotide sequence according to item 28 or 30 encoding a second intracellular cryoprotectant or a derivative thereof and / or with another nanoparticle according to item 29 or 30 comprising an optionally modified nucleotide sequence encoding a second intracellular cryoprotectant or a derivative thereof. 34. The method according to any one of items 32 or 33, further comprising (c) thawing the frozen cells, cell aggregates, cell cultures, tissues, organoids and / or organs, and / or (d) determining the viability, activity and / or functionality level of the cells, cell aggregates, cell cultures, tissues, organoids and / or organs before step (a), between steps (a) and (b) and / or after step (c), wherein the viability, activity and / or functionality level after freezing and thawing is at least 70%, preferably at least 80%, more preferably at least 90% compared to the level before freezing and thawing. 35. The method according to any one of items 32 to 34, wherein the cells, cell aggregates, cell cultures, tissues, organoids and / or organs are preferably cells, cell aggregates, cell cultures, tissues, organoids and / or organs from vertebrates or plants, preferably from vertebrates, more preferably from humans. 36. Cells, cell aggregates, cell cultures, tissues, organoids and / or organs obtainable or obtainable by a method according to any one of items 32 to 35. 37. Cells, cell aggregates, cell cultures, tissues, organoids and / or organs according to item 36, wherein the cells, cell aggregates, cell cultures, tissues, organoids and / or organs preferably have at least temporarily cryoprotective properties. 38. Cells, cell aggregates, cell cultures, tissues, organoids and / or organs according to item 36 or 37, wherein the cryoprotective properties are characterized in that the viability, activity and / or functionality of the cells, cell aggregates, cell cultures, tissues, organoids and / or organs after freezing and thawing are at least 70%, preferably at least 80%, more preferably at least 90% compared to the levels before freezing and thawing. 39. Frozen and thawed cells, cell aggregates, cell cultures, tissues, organoids and / or organs according to any one of items 36 to 38. 40. The (frozen and thawed) cells, cell aggregates, cell cultures, tissues, organoids and / or organs according to any one of items 36 to 39, wherein the (frozen and thawed) cells, cell aggregates, cell cultures, tissues, organoids and / or organs are "ready-to-use" (frozen and thawed) cells, cell aggregates, cell cultures, tissues, organoids and / or organs, preferably (frozen and thawed) "ready-to-use" "organ-on-a-chip". 41. The (frozen and thawed) cells, cell aggregates, cell cultures, tissues, organoids and / or organs of any one of items 36 to 40, wherein the (frozen and thawed) cells, cell aggregates, cell cultures, tissues, organoids and / or organs are suitable for transplantation and / or are (frozen and thawed) cells, cell aggregates, cell cultures, tissues, organoids and / or organs to be transplanted. 42. Use of the (frozen and thawed) cells, cell aggregates, cell cultures, tissues, organoids and / or organs according to any one of items 36 to 41 for drug development, active ingredient and / or drug screening, (preclinical) drug testing and / or (bio)medicine, preferably transplant medicine. The present invention is also characterized by the following items: 1. A method for identifying an intracellular cryoprotectant expressed by a cell, the method comprising (a) using cells that survive freezing and thawing, (b) modifying the used cells, (c) freezing and thawing the modified cells, and (d) obtaining a modified cell that survived step (c), thereby identifying an intracellular cryoprotectant expressed by the cell. 2. The method according to item 1, wherein the cells and / or modified cells are capable of being cultured in suspension and / or are cells in a suspension state. 3. The method according to item 1 or 2, wherein in step (a), the cells survive freezing and thawing in a suspension, the suspension preferably comprising an added cryoprotectant at a concentration of 5% or less, preferably 1% or less, more preferably 0%, and / or wherein in step (c), the modified cells are frozen and thawed in a suspension, the suspension preferably comprising an added cryoprotectant at a concentration of 5% or less, preferably 1% or less, more preferably 0%. 4. The method according to any one of the preceding items, further comprising (e) Proliferating the obtained ((further) modified) cells, preferably in suspension. 5. The method according to any one of the preceding items, comprising the following steps: step (a), optionally followed by step (e), followed by [step (b), optionally step (e), steps (c) and (d), optionally step (e)] in i iterations, wherein i is preferably at least 1 and a maximum of 1,000. 6. The method according to item 5, further comprising: (f) determining the viability of the obtained (further) modified cells, preferably for each step (d). 7. The method according to claim 6, comprising iteration i+1 if the survival rate determined in step (f) of iteration i is at least as high as the survival rate determined in step (f) of iteration i-1, preferably 10% higher, and preferably does not exceed a given threshold. 8. A method according to any one of items 6 or 7, comprising the following steps: step (a), optionally followed by step (e), followed by i iterations of [step (b), optionally step (e), steps (c) and (d), optionally step (f), optionally step (e)], wherein i is at least 2, and preferably also comprising iteration i+1, if the survival rate determined in step (f) of iteration i is at least as high as the survival rate determined in step (f) of iteration i-1, preferably 10% higher, and preferably does not exceed a given threshold. 9. The method according to any of the preceding items, wherein the intracellular cryoprotectant expressed by the (further) modified cells is identified by analyzing the genome, transcriptome, epigenome, proteome and / or metabolome, preferably by using an analysis selected from gel electrophoresis, mass spectrometry, crystal structure analysis, NMR spectroscopy, DNA sequencing, (m)RNA sequencing and any combination of the foregoing. 10. The method according to any one of items 6 to 9, wherein an intracellular cryoprotectant expressed by the (further) modified cell is identified if the viability determined in step (f) exceeds a given threshold value. 11. The method according to any of the preceding items, further comprising (h) modifying the identified intracellular cryoprotectant expressed by the (further) modified cell, thereby obtaining a derivative from the identified intracellular cryoprotectant expressed by the (further) modified cell. 12. The method according to any one of the preceding items, comprising the following steps: (a) using cells that survive freezing and thawing, Wherein step (a) preferably comprises the following steps: (a0) Freezing and thawing of cells in suspension, (a1) obtaining cells that survived step (a0), (f1) determining the viability of step (a0) based on the number of living cells of the cells in suspension before freezing and thawing and after freezing and thawing, (e1) growing the cells obtained in step (a1) and using the grown cells, (b) modifying the cells used in step (a), preferably the cells proliferated in step (e1) and / or the cells proliferated in step (e3), (e2) multiplying the modified cells in step (b), (c) freezing and thawing the modified cells in suspension that have proliferated in step (e2), (d) obtaining modified cells that survive step (c), thereby identifying an intracellular cryoprotectant expressed by said cells, wherein preferably, the freezing and thawing in step (a), preferably step (a0) and / or step (c) are carried out in a suspension, said suspension preferably comprising an added cryoprotectant at a concentration of 5% or less, preferably 1% or less, more preferably 0%. 13. The method according to item 12, wherein the intracellular cryoprotectant expressed by the modified cell is identified by: (f2) determining the viability of step (c) based on the number of modified living cells in suspension before freezing and thawing and after freezing and thawing, (e3) propagating the modified cells obtained in step (d), and (g) identifying at least one intracellular cryoprotectant based on the modified cells propagated in step (e3) if the viability determined in step (f2) exceeds a given threshold, wherein if the viability determined in step (f2) at iteration i is at least as high as the viability determined in step (f2) at iteration i-1, preferably 10% higher, and preferably does not exceed the given threshold, and wherein i is at least 2, then iterating steps [(b), (e2), (c), (d), (f2), (e3)] (iteration i+1 times). 14. The method according to claim 13, wherein the suspension in step (c) of iteration i+1 comprises (1), If the survival rate determined in step (f2) is at least as high as the survival rate determined in step (f), preferably at least 5% higher, and the survival rate is determined in the following steps: i) step (f1) in the case of the first iteration (i=1), or ii) step (f2) of iteration i-1 in the case of iteration i, the concentration of the cryoprotectant added is substantially the same as that of the suspension in step (c) of iteration i; (2) If neither i) nor ii) is satisfied, the concentration of the cryoprotectant added is 0.001% to 1%, preferably 0.01% to 0.5%, higher than the concentration in the suspension in step (c) of iteration i. 15. An intracellular cryoprotectant or a derivative thereof, which is obtained or can be obtained by any of the methods described above, wherein the intracellular cryoprotectant is an intracellular cryoprotectant synthesized by cells, and is preferably an optionally glycosylated polypeptide or protein.

[0190] It will be apparent to those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0191] All patents, patent applications, and publications mentioned in this specification are indicative of the levels of ordinary skill in the art to which this invention pertains. All patents, patent applications, and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0192] The invention exemplarily described herein can be suitably practiced in the absence of any element or combination of elements, restriction or combination of restrictions not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. should be understood broadly and not restrictively. In addition, the terms and expressions used herein have been used as terms of description rather than limitation, and the use of these terms and expressions is not intended to exclude any equivalents or portions thereof of the features shown and described, but it should be recognized that various modifications are possible within the scope of the claimed invention. Therefore, it should be understood that although the present invention has been specifically disclosed through preferred embodiments and optional features, those skilled in the art may make modifications and variations to the embodiments of the invention disclosed herein, and these modifications and variations are considered to be within the scope of the present invention. The invention has been described broadly and generally herein. Each narrower class and subclass grouping that falls within the generally disclosed scope also forms part of this disclosure. This includes a general description of the invention, with its proviso or negative limitation removing any subject matter from that genus, regardless of whether the deleted material is specifically described herein. In addition, when features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. Other embodiments of the invention will become apparent from the following claims.

Claims

1. A method for identifying an intracellular cryoprotectant expressed by a cell, the method comprising (a) obtaining cells that survive freezing and thawing, (b) genetically modifying the cell of step (a), (c) freezing and thawing the modified cells, and (d) obtaining modified cells that survive step (c) to thereby identify the intracellular cryoprotectant expressed by said cells by an appropriate method.

2. The method according to claim 1 , wherein the cell is selected from the group consisting of prokaryotes, eukaryotes, vertebrates, insects, plants, algae, fungi and bacteria; preferably, the cell is selected from the group consisting of frogs, preferably Rana temporaria; and fish, preferably beaver, such as silverspotted sculpin - Blepsias cirrhosus; Arctic cod and / or Antarctic cod species; flatfish species such as Pleuronectes americanus or Limanda ferruginea; preferably, the algal cell is selected from cryosphere species, such as snow algae, preferably Antarctic species of the genus Chlorominima, Arctic and / or Antarctic species of Chlamydomonas, or species of the genus Chloromonas, preferably Chloromonas nivalis, Chloromonas rostavskyi, and / or Chloromonas spp. rostafinskii), polar snow algae (Ancylonema ); selected from green algae, preferably species of the genus Chlorococum, preferably species of the genus Chlorococum and species of the genus Raphidonema, preferably Raphidonema brevirostre or Raphidonema nivale.

3. The method according to claim 1 or 2, wherein the cells and / or modified cells can be cultured in suspension and / or are cells in a suspension state.

4. The method according to any one of the preceding claims, wherein in step (a), the cells survive freezing and thawing in suspension, the suspension preferably comprising an added cryoprotectant at a concentration of 5% or less, preferably 1% or less, more preferably 0%, and / or wherein in step (c), the modified cells are frozen and thawed in suspension, the suspension preferably comprising an added cryoprotectant at a concentration of 5% or less, preferably 1% or less, more preferably 0%.

5. The method according to any one of the preceding claims, further comprising (e) Proliferating the obtained modified cells, preferably further modified cells, preferably in suspension.

6. The method according to any one of the preceding claims, comprising the steps of: Step (a), optionally followed by step (e), followed by [step (b), optionally step (e), step (c) and (d), optionally step (e)] in i iterations, wherein i is preferably at least 1 and at most 1,000.

7. The method according to claim 6, further comprising: (f) determining the viability of the obtained (further) modified cells, preferably for each step (d).

8. The method according to claim 7, comprising iteration i+1 if the survival rate determined in step (f) of iteration i is at least as high as the survival rate determined in step (f) of iteration i-1, preferably 10% higher, and preferably does not exceed a given threshold.

9. The method according to any one of claims 7 or 8, comprising the steps of: Step (a), optionally followed by step (e), followed by i iterations of [step (b), optionally step (e), steps (c) and (d), optionally step (f), optionally step (e)], wherein i is at least 2, and preferably also includes iteration i+1, if the survival rate determined in step (f) of iteration i is at least as high as the survival rate determined in step (f) of iteration i-1, preferably 10% higher, and preferably does not exceed a given threshold.

10. The method according to any of the preceding claims, wherein the intracellular cryoprotectant expressed by the (further) modified cells is identified by analyzing the genome, transcriptome, epigenome, proteome and / or metabolome, preferably by using an analysis selected from gel electrophoresis, mass spectrometry, crystal structure analysis, NMR spectroscopy, DNA sequencing, (m)RNA sequencing and any combination of the foregoing.

11. The method according to any one of claims 7 to 10, wherein the intracellular cryoprotectant expressed by the (further) modified cell is identified if the viability determined in step (f) exceeds a given threshold.

12. The method according to any one of the preceding claims, further comprising (h) genetically modifying said identified intracellular cryoprotectant expressed by said (further) modified cell, thereby obtaining a derivative from said identified intracellular cryoprotectant expressed by said (further) modified cell.

13. The method according to any one of the preceding claims, comprising the steps of: (a) obtaining cells that survive freezing and thawing, Wherein step (a) preferably comprises the following steps: (a0) Freezing and thawing of cells in suspension, (a1) obtaining cells that survived step (a0), (f1) determining the viability of step (a0) based on the number of living cells of the cells in suspension before freezing and thawing and after freezing and thawing, (e1) proliferating the cells obtained in step (a1) and obtaining the proliferated cells, (b) modifying the cells of step (a), preferably the cells proliferated in step (e1), and / or the cells proliferated in step (e3) at the genetic level, (e2) multiplying the modified cells of step (b), (c) freezing and thawing the modified cells in suspension that have proliferated in step (e2), (d) obtaining modified cells that survive step (c), thereby identifying an intracellular cryoprotectant expressed by said cells, wherein preferably, the freezing and thawing in step (a), preferably step (a0) and / or step (c) are carried out in a suspension, said suspension preferably comprising an added cryoprotectant at a concentration of 5% or less, preferably 1% or less, more preferably 0%.

14. The method of claim 13, wherein the intracellular cryoprotectant expressed by the modified cell is identified by: (f2) determining the viability of step (c) based on the number of modified living cells in suspension before freezing and thawing and after freezing and thawing, (e3) growing the modified cells obtained in step (d), and (g) identifying at least one intracellular cryoprotectant based on the modified cells proliferated in step (e3) if the viability determined in step (f2) exceeds a given threshold, wherein if the survival rate determined in step (f2) of iteration i is at least as high as the survival rate determined in step (f2) of iteration i-1, preferably 10% higher, and preferably does not exceed the given threshold, and wherein i is at least 2, then steps [(b), (e2), (c), (d), (f2), (e3)] are iterated (iteration i+1 times).

15. The method of claim 14, wherein the suspension in step (c) of iteration i+1 comprises (1) If the survival rate determined in step (f2) is at least as high, preferably at least 5% higher, as the survival rate determined in step: i) step (f1) in the case of the first iteration (i=1), or ii) iterating step (f2) for i-1 times in the case of iteration i, the concentration of the cryoprotectant added is substantially the same as that of the suspension in step (c) of iteration i; (2) If neither i) nor ii) is satisfied, the concentration of the cryoprotectant added is 0.001% to 1%, preferably 0.01% to 0.5%, higher than the concentration in the suspension in step (c) of iteration i.

16. An intracellular cryoprotectant or a derivative thereof, which is or is obtainable by the method according to any one of the preceding claims, wherein the intracellular cryoprotectant is an intracellular cryoprotectant synthesized by cells and is preferably an optionally glycosylated polypeptide or protein.

17. The intracellular cryoprotectant according to claim 16, wherein the optionally glycosylated polypeptide or protein is characterized by a size, weight and / or structure suitable for providing the cryoprotectant with cell membrane impermeability capabilities.

18. The intracellular cryoprotectant according to claim 16 or 17, wherein the optionally glycosylated polypeptide or protein has an average molecular weight (Mw) of greater than 9800 Da, preferably greater than 900 kDa or more.

19. A method of producing a cryoprotectant or a derivative thereof, the method comprising performing the method of any one of claims 1 to 15.

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