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, said method comprising (a) using a cell that survived freezing and thawing, (b) modifying said used cell, (c) freezing and thawing said modified cell, and (d) obtaining a modified cell, which survived step (c), whereby an intracellular cryoprotectant expressed by said cell is identified, as well as intracellular cryoprotectants obtained or obtainable by said method.

Description

Identification of cryoprotectants for cryopreservation of cells and cell aggregatesFIELD OF THE INVENTION

[0001] The present invention relates to a method of identifying an intracellular cryoprotectant expressed by a cell, said method comprising (a) using a cell that survived freezing and thawing, (b) modifying said used cell, (c) freezing and thawing modified cell, and (d) obtaining a modified cell, which survived step (c), whereby an intracellular cryoprotectant expressed by said cell is identified. The present invention also relates to an intracellular cryoprotectant, or a derivate thereof, preferably obtained by or obtainable by said method, wherein the intracellular cryoprotectant is an intracellular cryoprotectant that is synthesized by a cell. The present invention further relates to method of generating a, optionally modified, nucleotide sequence encoding said intracellular cryoprotectant, or derivate thereof, as well as to a, optionally modified, nucleotide sequence obtained by or obtainable by said method encoding one or more intracellular cryoprotectant(s) or derivative(s) thereof, and a nanoparticle comprising said, optionally modified, nucleotide sequence. The present invention also relates to the use of such an intracellular cryoprotectant or derivative thereof, said, optionally, modified nucleotide sequence, and / or said nanoparticle for cryopreserving a cell, cell aggregate, cell culture, tissue, organoid, and / or organ, as well as to a method of cryopreserving a cell, a cell aggregate, a cell culture, a tissue, an organoid, and / or an organ, comprising (a) contacting a cell, cell aggregate, cell culture, tissue, organoid and / or organ with said, optionally modified, nucleotide sequence and / or nanoparticle, and (b) freezing said cell, cell aggregate, cell culture, tissue, organoid and / or organ obtained from step (a), wherein freezing comprises a reduction from a temperature of said cell, cell aggregate, cell culture, tissue, organoid and / or organ of more than 0°C to a temperature of less than 0°C, preferably of less than -20°C, more preferably of less than -70°C, even more preferably of less than -140°C. The present invention further relates to a cell, cell aggregate, cell culture, tissue, organoid and / or organ obtained by or obtainable by said method as well as a respective frozen and thawed cell, cell aggregate, cell culture, tissue, organoid and / or organ. The present invention also relates to the use of said (frozen and thawed) cell, cell aggregate, cell culture, tissue, organoid and / or organ for drug development, active ingredient and / or drug screening, ((pre- )cl inical ) drug testing and / or (bio-)medicine, preferably transplantation medicine.1SUBSTITUTE SHEET (RULE 26)BACKGROUND OF THE INVENTION

[0002] Only a limited number of plant and animal species has the ability to respond to freezing processes by synthesizing cellular cryoprotectants, such as heat-protective proteins or antifreeze proteins, or by accumulating for example sugar or glycerol. Contrarily, cells of other species such as human cells are unable to produce cellular cryoprotectants in a sufficient extent to avoid cellular damage due to cooling and freezing processes. However, approaches have been established that pave the way to freeze cells in a vitality-preserving manner.

[0003] A commonly known method is referred to as cryopreservation and has been well established for (single) cells in suspension. In case of cryopreservation, freezing and thawing is typically performed in the presence of a cryoprotectant that is added to a cell suspension to prevent or suppress intracellular ice crystal formation during freezing. Said cryoprotectants need to pass the cell membrane to exert their protective effect within the cell during freezing. Being primarily based on diffusion processes across the cellular membrane, cryopreservation approaches suffer from at least three limitations. First, only comparatively small molecules such as dimethyl sulfoxide (DMSO) can be transferred into cells, since macromolecules such as antifreeze proteins are not membrane- permeable or cannot be introduced into cells in sufficient quantities and in the required time due to their dependency on membrane-bound transport molecules. Moreover, current approaches require considerable quantities of cryoprotectants, usually in the % range, to ensure cellular protection (see, e.g., Mazur, P., The role of intracellular freezing in the death of cells cooled at supraoptimal rates, Cryobiology 1977, 14: 252-272; schematically indicated with "C" in Figure 1). However, some commonly used cryoprotectants such DMSO have the drawback of negatively impacting cell vitality due to toxic effects, in particular in the presence of higher concentrations of these cryoprotectants in the medium. Finally, any required trans-membrane transport of a cryoprotectant limits the applicability of cryopreservation in view of larger cell aggregates, especially organoids, tissues and organs. More specifically, while required exposure times increase quadratically with diameter, limitations due to diffusion rate and gradient formation arise already at a volume of > 1 mm3.

[0004] To overcome these limitations of cryopreservation, research focused for example on optimizing predetermined temperature-time protocols in view of the properties of the respective biological material to be cryopreserved (see, e.g., review article by M. A. Taylor et al. "New Approaches to Cryopreservation of Cells, Tissues and Organs" in "Transfus. Med. hemother." 46: 2019, 197-215). Optimization of process parameters requires considerable experience and the required optimization effort increases significantly, when not only single cells in suspension are to be cryopreserved but larger cell aggregates. Known cryopreservation procedures comprise in particular slow rate freezing and vitrification, as well as several modifications and combinations thereof. In case 2SUBSTITUTE SHEET (RULE 26)of slow rate freezing, which may also be referred to as "slow freezing", temperature is reduced at low cooling rates to permit adequate cellular dehydration, while minimizing intracellular ice crystal formation. However, slow rate freezing based approaches have the disadvantage of low cell survival rates and limited functionality of the thawed cells like limited ability to grow after thawing and / or the need of long re-cultivation times. On the other side, vitrification refers to an almost instantaneous freezing due to ultra-high cooling rates that allow solidification of cells and their extracellular milieu into a glass-like state without crystal ice formation. Due to the need of ultrahigh cooling rates, successful vitrification is usually limited to small volumes of cells and medium to ensure comparatively large surface-to-volume ratios and comparatively small distances between the cells and the cryoprotectant. Moreover, vitrification based approaches require comparatively high concentrations of cryoprotectants like DMSO, which in turn negatively impact cell survival and vitality.

[0005] While cryopreservation of single cells in suspension is currently widely applied, especially using slow freezing, there is an interest in the application of cryopreservation for larger and inhomogeneous cell aggregates, including organoids, tissues and organs, especially in the fields of biotechnology, pharmacology and (bio-) medicine, such as transplantation medicine. However, the success of slow freezing three-dimensional cell aggregates is currently limited. Smaller cell assemblies have already been preserved by vitrification. However, in order to provide high intracellular concentrations of a cryoprotectant and to reach temperatures below the glass point ultrafast everywhere within the cell assembly, tight technical limits are faced. This mainly concerns sample volume, toxicity resulting from required cryoprotectant concentrations and exposure times, as well as an increasing likelihood of cellular damage due to thermal stress cracking with increasing specimen size. As a result, also vitrification is considered unsuitable for routine preservation of larger cell aggregates.

[0006] Hence, there is still a need for finding a solution for cryopreserving larger cell aggregates, especially organoids, tissues and organs, without functional impairment.

[0007] The present application addresses this need by providing the embodiments as recited in the claims. More specifically, the present invention provides intracellular cryoprotectants that can be synthesized within cells, such as cells of an organoid, tissue or organ, as well as an automatable high- throughput method for identifying suitable intracellular cryoprotectants and an efficient method for cryoprotection of organoids, tissues and organs using the same.

[0008] In particular, the present invention relates to a method of identifying an intracellular cryoprotectant expressed by a cell, said method comprising (a) using a cell that survived freezing and3SUBSTITUTE SHEET (RULE 26)thawing, (b) modifying said used cell, (c) freezing and thawing said modified cell, and (d) obtaining a modified cell, which survived step (c), whereby an intracellular cryoprotectant expressed by said cell is identified.SUMMARY OF THE INVENTION

[0009] The present invention relates in a first aspect to a method of identifying an intracellular cryoprotectant expressed by a cell, said method comprising(a) Using a cell that survived freezing and thawing,(b) Modifying said used cell,(c) Freezing and thawing said modified cell, and(d) Obtaining a modified cell, which survived step (c), whereby an intracellular cryoprotectant expressed by said cell is identified.

[0010] In some embodiments, said cell and / or modified cell is capable of being cultivated in suspension and / or is a cell in suspension.

[0011] In some embodiments, in step (a) said cell survived freezing and thawing in a suspension, which preferably comprised an added cryoprotectant in a concentration of 5% or less, preferably of 1% or less, more preferably of 0%, and / or wherein in step (c) said modified cell is frozen and thawed in a suspension, which preferably comprises an added cryoprotectant in a concentration of 5% or less, preferably of 1% or less, more preferably of 0%.

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

[0013] In some embodiments, said 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 maximal 1,000.

[0014] In some embodiments, said method further comprises:(f) Determining a survival rate of said obtained (further) modified cells for, preferably every, step (d).

[0015] In some embodiments, said method comprises an iteration i+1, if the survival rate determined in step (f) of iteration i is at least as high as, preferably by 10% higher than, the survival rate determined in step (f) of iteration i-1 and preferably not exceeding a given threshold.4SUBSTITUTE SHEET (RULE 26)

[0016] In some embodiments, said 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 (f), optionally step (e) ] in i iterations, wherein i is at least 2, and preferably further comprising an iteration i+1, if the in step (f) of iteration i determined survival rate is at least as high, preferably by 10% higher than, the survival rate determined in step (f) of iteration i-1 and preferably not exceeding a given threshold.

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

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

[0019] In some embodiments, said method further comprises(h) Modifying said identified intracellular cryoprotectant expressed by said (further) modified cell, whereby a derivate is obtained from said identified intracellular cryoprotectant expressed by said (further) modified cell.

[0020] In some embodiments, said method further comprises the following steps:(a) Using a cell that survived freezing and thawing, wherein step (a) preferably comprises the following steps:(aO) Freezing and thawing of cells in suspension,(al) Obtaining a cell that survived step (aO),(fl) Determining a survival rate for step (aO) based on the number of living cells of the cells in suspension before freezing and thawing and after freezing and thawing,(el) Proliferating said cell obtained in step (al) and using the proliferated cell,(b) Modifying said cell used in step (a), preferably said cell proliferated in step (el) and / or said cell proliferated in step (e3),(e2) Proliferating said in step (b) modified cell,(c) Freezing and thawing the in step (e2) proliferated modified cell in suspension,(d) Obtaining a modified cell, which survived step (c), whereby an intracellular cryoprotectant expressed by said cell is identified,5SUBSTITUTE SHEET (RULE 26)wherein preferably freezing and thawing in step (a), preferably in step (aO), and / or in step (c) is performed in a suspension, which preferably comprises an added cryoprotectant in a concentration of 5% or less, preferably of 1% or less, more preferably of 0%.

[0021] In some embodiments, said intracellular cryoprotectant expressed by said modified cell is identified by:(f2) Determining a survival rate for step (c) based on the number of living modified cells in suspension before freezing and thawing and after freezing and thawing,(e3) Proliferating said in step (d) obtained modified cell, and(g) Identifying at least one intracellular cryoprotectant based on the in step (e3) proliferated modified cells, preferably if the survival rate determined in step (f2) is exceeding a given threshold, wherein steps [ (b), (e2), (c), (d), (f2), (e3) ] are iterated (iteration i+1), if the survival rate determined in step (f2) of iteration i is at least as high, preferably by 10% higher than, the survival rate determined in step (f2) of iteration i-1 and preferably not exceeding said given threshold, and wherein i is at least 2.

[0022] In some embodiments, the suspension in step (c) of iteration i+1 comprises(1) Substantially the same concentration of the added cryoprotectant as the suspension in step (c) of iteration i,If the survival rate determined in step (f2) is at least as high as, preferably by at least 5% higher than, the survival rate determined in i) step (fl) in case of the first iteration (i=l), or ii) step (f2) of iteration i-1 in case of iteration i,(2) A concentration of the added cryoprotectant that is between 0.001% and 1%, preferably between 0.01% and 0.5%, higher than in the suspension in step (c) of iteration i, if neither i) nor ii) are fulfilled.

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

[0024] In a further aspect, the present invention is directed to a method of producing a cryoprotectant, or a derivate thereof, comprising conducting the method of identifying an intracellular cryoprotectants as described in any one of the embodiments of the invention.6SUBSTITUTE SHEET (RULE 26)DETAILED DESCRIPTION

[0025] It has surprisingly been found that the method according to the present invention allows the identification of intracellular cryoprotectants in an efficient, easily automatable, and high-throughput manner. By applying an in vitro evolutionary approach based on, preferably repeated, cycles of freezing and thawing of cells and introducing modifications, preferably genetic mutations, inbetween, potent intracellular cryoprotectant can be generated, isolated and identified. Thus, intracellular cryoprotectants can be identified that are less impacted by size constraints and diffusion process compared to added cryoprotectants that require passing the cellular membrane to exhibit their cryoprotective effects in a cell. Most importantly, as intracellular cryoprotectants are synthesized by and within cells directly, limitations of added cryoprotectants due to diffusion rate and gradient formation can be overcome. Hence, the intracellular cryoprotectants identified by the method according to the present invention can for the first time make cryopreservation amenable to pharmaceutical and medical applications such as ready-to-use tissue cultures for drug screening and tissue and organ cryopreservation for example for organ transplantation.

[0026] In the context of the present invention, the term "cryoprotectant" refers to any substance that prevents during a freezing process cold damage of cells, resulting, e.g., from intracellular ice crystal formation. Different mechanisms of cryoprotectants may exist that are encompassed herein. For example, a cryoprotective effect may be based on the intracellular formation of hydrogen bonds with biological molecules as water molecules are displaced, so that cells can retain their native 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 cells and thus, the temperature below which cells become osmotically inactive. Thus, said cryoprotectants do not actually prevent freezing, but ensure that cells maintain some flexibility in a glassy phase.

[0027] Herein, an "added cryoprotectant" refers to a cryoprotectant that is added to, e.g., a cell (suspension) medium. Such an added cryoprotectant can be any substance that prevents and / or suppresses ice crystal formation in cells during freezing, such as dimethyl sulfoxide (DMSO), 1,2- propanediol, glycols like glycerol, ethylene glycol and propylene glycol, amino acids and / or sugar molecules like trehalose and sucrose. Hence, an added cryoprotectant is an exogenously added and initially extracellular cryoprotectant that has to pass the membrane of a cell, for example by diffusion, in order to exert its protective effect inside the cell during freezing.

[0028] Herein, an "intracellular cryoprotectant", refers to a cryoprotectant that is expressed and / or synthesized by and within a cell. Thus, said cryoprotectant is endogenously expressed and / or7SUBSTITUTE SHEET (RULE 26)synthesized in a cell. This has the advantage that its presence within a cell depends, for example, on the availability of a biological template for synthesis, such as an mRNA that encodes a cryoprotectant being for example a polypeptide or a protein, and / or the availability of molecules like amino acids for expression and / or synthesis. In some examples, the intracellular cryoprotectant obtained or obtainable by the method of the present invention is synthesized by a cell and preferably an, optionally glycosylated, polypeptide or protein. Thus, in contrast to added cryoprotectants intracellular cryoprotectants do not require to pass a cell membrane. Hence, intracellular cryoprotectants are less constrained by size limitations compared to added cryoprotectants. Moreover, while current approaches based on added cryoprotectants suffer from volume restrictions of the biological material to be cryopreserved due to gradient formation, intracellular cryoprotectants allow for the first time the amenability of larger cell aggregates, organoids, tissues and organs to cryopreservation.

[0029] The method according to the present invention is a method of identifying an intracellular cryoprotectant expressed by a cell. Such an "intracellular cryoprotectant expressed by a cell" refers to an intracellular cryoprotectant that the used cell is intrinsically capable of expressing. Accordingly, such a cell comprises endogenously respectively required biological information to build an intracellular cryoprotectant. Suitable cells for the method according to the present invention are thus especially cells that intrinsically possess the ability to express an intracellular cryoprotectant and / or that are modified in a manner to generate or enhance the ability of said cell to express intracellular cryoprotectants. Using a cell that intrinsically possesses the ability to express an intracellular cryoprotectant is for example particularly advantageous as the use of such a cell can facilitate the identification of an intracellular cryoprotectant and / or reduce the time required for said identification. Using a cell that has been modified in a manner to enhance the ability to express an intracellular cryoprotectant compared to a non-modified, wild-type cell of the same species is for example especially advantageous for the identification of an intracellular cryoprotectant that shows a stronger cryoprotective effect compared to the respective intracellular cryoprotectant identified in a respective wild-type cell. Thus, such an approach is for example especially advantageous for identifying intracellular cryoprotectants that are optimized for example in view of their potency and / or the occurrence of cellular side effects on the cell vitality compared to their respective wildtype counterpart. Using a cell that has been modified in a manner to generate the ability of said cell to express an intracellular cryoprotectant is for example especially advantageous to ensure suitability of the identified intracellular cryoprotectant for a given species by ensuring that the (epi-) genetic background of the cell(s) used for identifying the intracellular cryoprotectant and of the cell(s) to be cryopreserved is / are comparable in the sense that no inter-species adaptation and / or adjustment is required.8SUBSTITUTE SHEET (RULE 26)

[0030] Accordingly, in some embodiments of the present invention the cell has been obtained from a vertebrate, an insect, a plant, an algae, a fungus or a bacterium. For example, said cell may be obtained from a terrestrially hibernating animal like a wood frog (Rana sylvatica), which is a northern woodland species that hibernates terrestrially in sites where dehydration and freezing may occur. As another example, said cell may be obtained from a fish, e.g. from a sea raven like a silverspotted sculpin (Blepsias cirrhosus), from an Artic and / or Antarctic species of cods, or from a flounder species like a winter flounder (Pleuronectes americanus) or a yellowtail flounder (Limanda ferruginea). As a further example, said cell may be obtained from a cryospheric species like a snow algae, e.g. from an Antarctic species of the genus Chlorominima, an Arctic and / or Antarctic species of the genus Chlamydomonas, or a species of the genus Chloromonas such as Chloromonas nivalis or Chloromonas rostafinskii, or Ancylonema nordenskibldii, or from a green algae, e.g. from a species of the genus Chlorococum such as Chlorococum sp. or a species of the genus Raphidonema such as Raphidonema brevirostre or Raphidonema nivale.

[0031] In some embodiments of the present invention, the method is carried out using a cell that has been obtained from a vertebrate, an insect, a plant, an algae, a fungus or a bacterium, and that intrinsically possess the ability to express an intracellular cryoprotectant.

[0032] In some embodiments of the present invention the cell can be cultivated in vitro. Herein, cultivation in vitro refers to a cultivation of cells in an artificial and preferably well-controlled environment comprising a cell medium. Cells may be cultivated as adherent cell culture and / or as suspension culture for example. Alternatively or optionally, said cell may also be a cell comprised in a cell aggregate, tissue, organoid, organ, and / or a part thereof like a tissue slice, wherein said cell aggregate, tissue, organoid, organ, and / or a part thereof is cultivated in vitro and preferably (at least partially and / or temporarily) in suspension. Herein, it is preferred that a cell used in the method according to the present invention is a cell capable of being cultivated in suspension and / or is a cell in suspension. Thus, said cell can preferably be (at least temporarily) cultivated, and optionally passaged, in a (suspension) medium. Using a cell capable of being cultivated in suspension and / or in suspension is advantageous in view of available and well studied culture conditions for many cell types and species, easy handling and well (up-) scalability.

[0033] Accordingly, in preferred embodiments of the present invention the cell is a cell capable of being cultivated in suspension and / or a cell in suspension.

[0034] As regards step (a) of the method according to the present invention, a cell is used that survived freezing and thawing. Thus, a living cell is preferably obtained that was exposed to a temperature below 0°C or less, followed by an exposure to a temperature of more than 0°C. This is9SUBSTITUTE SHEET (RULE 26)especially advantageous for identifying cells that exhibit some degree of cold tolerance, preferably cells that exhibit tolerance to freezing. Hence, said cell that is used in step (a) is a living cell that is obtained after freezing and thawing, wherein intracellular cold damage of said cell due to freezing is limited to an extent that at least enables survival of the cell.

[0035] In some embodiments of the present invention, freezing comprises or is a reduction from a temperature of more than 0°C to a temperature of less than 0°C, preferably of less than -20°C, more preferably of less than -70°C, even more preferably of less than -140°C. Especially the latter is advantageous in view of the fact that a temperature below -140°C is below the glass transition temperature of water, which is about -135°C. Thus, the survival of a cell of a temperature reduction of more than 0°C to less than the glass transition temperature of water may be indicative for the presence of cryoprotectants and thus, increase ease the identification of an intracellular cryoprotectant according to the present invention.

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

[0037] It is to be noted that in case of freezing and / or thawing, said temperature is preferably the temperature of the suspension, in which the used cell survived freezing and thawing.

[0038] Hence, in particularly preferred embodiments of the present invention, the used cell that survived freezing and thawing is a cell in suspension, wherein said freezing comprises or is a reduction from the temperature of the suspension of more than 0°C to a temperature of less than 0°C, preferably of less than -20°C, more preferably of less than -70°C, even more preferably of less than -140°C, and / or wherein said thawing comprises or is an increase from the temperature of said suspension of less than 0°C, preferably of less than -20°C, more preferably of less than -70°C, even more preferably of less than -140°C, to a temperature of more than 0°C.

[0039] Furthermore, in some embodiments of the present invention, after said reduction of the temperature the reduced temperature is maintained for at least 1 sec to 10 min, preferably from at least 10 sec to 5 min, bevor thawing. Thus, a cell that was exposed to freezing and thawing had time to react to the temperature change of the cell's environment, preferably the suspension, during said process of freezing and thawing.

[0040] It is to be noted that, without being bound by theory, there may or may not be also an upper limit and thus, a maximal time period, wherein the reduced temperature is maintained. For example, in case of an almost instantaneous freezing due to ultra-high cooling rates, wherein the reduced10SUBSTITUTE SHEET (RULE 26)temperature is a temperature below, e.g., -140°C, there may not be a (practical) upper limit for the time period, wherein said reduced temperature is maintained. However, in case of a reduced temperature being between less than 0°C and above (and including) -140°C for example, there may be some biological, physico-chemical, chemical and / or physical processes like migratory ice growth in the used cell, which may require considering an upper limit of the time period, wherein the reduced temperature is maintained. Thus, the skilled person is aware that there may be an upper boundary of said time period depending on a chosen reduced temperature for freezing.

[0041] Furthermore, as regards freezing and thawing, it is envisioned that a predetermined temperature-time protocol is applied that is optimized in view of the used cell, e.g. in view of species and / or cell type. It may particularly be ensured, for example, that the used cell is sufficiently long exposed to temperatures of less than, though close to 0°C during thawing to avoid an artificial bias due to unsuitable fast thawing that may negatively impact the used cell's survival and vitality. Well- established temperature-time protocols are available for different species, cell types and / or cells. While the person skilled in the art is thus aware of suitable temperature-time protocols and / or the identification of a suitable temperature-time protocol (see, e.g., Mazur, P., The role of intracellular freezing in the death of cells cooled at supraoptimal rates, Cryobiology 1977, 14: 252-272), a few illustrative examples are given in the example section. Encompassed by the present invention are for example vitrification based approaches, slow freezing based approaches as well as any combination and / or modification thereof.

[0042] Furthermore, in some embodiments of the present invention the cell used in step (a) survived freezing and thawing in a suspension, which preferably comprised an added cryoprotectant in a concentration of 5% or less, preferably of 1% or less, more preferably of 0%. Thus, depending on the cell used and / or the focus of the approach, the suspension may comprise an added cryoprotectant or not. For example, the suspension may not comprise an added cryoprotectant in case of a cell that has already intrinsically the ability to express an intracellular cryoprotectant and / or in case the focus of the approach is to identify an intracellular cryoprotectant using a temperaturetime protocol that initiates said naturally occurring expression of the intracellular cryoprotectant in said cell in an extent that ensures the cell's survival. As another example, it may be favorable to add to the suspension of a cell an added cryoprotectant in case said cell is for example not able to express an intracellular cryoprotectant in an extent that ensures the cell's survival upon freezing and / or negatively impacts the cell's vitality more than desired. If the suspension comprises an added cryoprotectant it is preferred that the concentration of the added cryoprotectant in the suspension is lower than for example disclosed in the state of the art in the context of cryopreservation of respective cells and / or as low as possible to obtain a cell that survived freezing and thawing. Hence,11SUBSTITUTE SHEET (RULE 26)different concentrations of an added cryoprotectant may be tried for investigating a suitable low concentration of an added cryoprotectant in a suspension to obtain a cell for step (a) that survived freezing and thawing in said suspension comprising the added cryoprotectant. For example, no added cryoprotectant may be used. In case no cell survived freezing and thawing in a suspension without added cryoprotectant (first concentration is 0%), another experiment may be performed using a second concentration that is, e.g., by 0.05% or by 0.01% higher than the first concentration. Said experiment may be repeated with increasing concentrations of the added cryoprotectant until at least one surviving cell is obtained. As another example, a concentration reported in the literature may be used as an indication for a first concentration of an added cryoprotectant. When a cell is obtained that survived freezing and thawing in a suspension comprising said first concentration of the added cryoprotectant, another experiment may be performed using a concentration that is, e.g., by 0.05% or by 0.01% lower than the first concentration. Said experiment may be repeated with decreasing concentrations of the added cryoprotectant until no surviving cell may be obtained. Preferably, the lowest concentration of the added cryoprotectant that resulted in a surviving cell is used for obtaining a cell that survived freezing and thawing according to step (a) of the method of to the invention. Thus, the person skilled in the art is aware that said preferred concentration depends for example on the cell, especially the cell type and / or species the cell has been obtained from, and / or the added cryoprotectant, and that said considerations can be extended accordingly to cases of more than one added cryoprotectant.

[0043] As regards step (b) of the method according to the present invention, said used and / or obtained cell is modified. Thus, the cell, e.g. the cell of step (a), that survived freezing and thawing is modified, preferably genetically and / or epigenetically, more preferably genetically. By modifying a cell, modified versions of said cell can be obtained, herein referred to also as mutants. Thus, one or more mutants may be obtained that, compared to the cell before being modified, may i) express an intracellular cryoprotectant more efficiently, e.g. resulting in higher intracellular concentrations of the intracellular cryoprotectant, ii) may express additionally another intracellular cryoprotectant, and / or iii) may show a boost of the protective effect due to a newly introduced modification. Hence, modifying a cell, e.g. the cell used in step (a), is advantageous for obtaining one or more mutants that may exhibit a better cryoprotection than the initially used cell, e.g. the cell of step (a).

[0044] Herein, the term "modifying" encompasses both spontaneous and artificially induced alterations. For example, said term encompasses a modification of a cell, for example on genetic, epigenetic, transcriptional, translational, proteomic, and / or metabolomic level, preferably on genetic level. As another example, said term also encompasses a modification of a nucleotide sequence, for12SUBSTITUTE SHEET (RULE 26)example by introducing a single nucleotide polymorphism, inserting and / or deleting 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 resulted in a, preferably artificially induced, alteration on the cell's genetic, epigenetic, transcriptional, translational, proteomic, and / or metabolomic level, preferably on the genetic level. Preferably, a "modified cell" according to the present invention relates to the cell obtained after step (b) of the method according to the present invention, more preferably to the used cell of step (a) that has been subjected once to step (b). In case the used cell has been subjected to more than one step (b), said cell is preferably referred to herein as "further modified cell". It is to be noted that the terms "modified cell" and "further modified cell" both encompass i) a cell that is the cell used in step (a) of the method according to the present invention and ii) a cell that is derived and thus, originating from said used cell of step (a). Preferably, the used cell of step (a), the modified cell of step (b) and optionally the further modified cell of step (b) are thus either the same cell except any alteration introduced by one or more rounds of step (b) and / or cells being substantially clones, e.g. with substantially identical genetic background except any alteration introduced by one or more rounds of step (b). Said clones may be obtained after proliferating said used and / or (further) modified cell. Thus, as regards said "further modified cell", the same applies as it is described herein in the context of a "modified cell".

[0046] Preferably, the modification(s) in step (b) are modification(s) on the genetic level of a cell such as the used cell of step (a). Thus, a mutant can be obtained that exhibits one or more changes in the genome compared to the cell before being modified in step (b), wherein said change can be, e.g., a deletion of one or more nucleotides, an insertion of one or more nucleotides, an exchange of one or more nucleotides, a multiplication of a part of a DNA sequence, a reshuffling of DNA sequences, or any combination of the foregoing. Herein, the term "DNA" refers to a single- or double-stranded deoxyribonucleotide sequence built up for example of A, C, G and / or T nucleotides, i.e. nucleotides comprising adenine, guanine, cytosine, and thymine as the respective nitrogenous base. Preferably, said modification(s) in step (b) are modification(s) in a regulatory and / or coding region(s) of a DNA sequence. This is advantageous as such alteration(s) may affect expression, expression intensity, expression duration and / or activity, localization and / or effectivity of an expressed product, and / or any combination of the foregoing. Thus, such modification(s) may directly impact a cell's cryoprotective properties.

[0047] Accordingly, in some embodiments of the present invention modifying said cell in step (b) relates to a modification on the genetic level of said cell. Preferably, said modification is in a regulatory and / or coding region of a gene, more preferably in a coding region of a gene.13SUBSTITUTE SHEET (RULE 26)Modifications in a regulatory region of a gene may increase the transcription of said gene, which may lead to higher concentrations of the intracellular cryoprotectant in the cell. Modifications in a coding region of a gene may result for example in case of an intracellular cryoprotectant being a polypeptide or a protein in a mutant version with differing functionality and / or activity compared to the intracellular cryoprotectant expressed in the cell before being modified in step (b), such as the cell used in step (a). Furthermore, modifications in a coding region of a gene may also result for example in the gain of a new function, a phenomenon also known as a gain of function mutation. Such modifications are advantageous as they may lead to mutants with improved freezing tolerance compared to their non-mutated counterpart and thus, the cell before being modified in said step (b).

[0048] Preferably, the modification(s) in step (b) are artificially induced alterations.

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

[0050] Accordingly, in some embodiments of the present invention, the cell is modified in step (b) by exposure to a mutagenic substance. The term "mutagenic substance" as used herein refers to a chemical agent that is capable of inducing alterations on the genetic and / or epigenetic level of a cell and thus, of causing (epi-)genetic changes. Hence, by exposing a cell to a mutagenic substance, the cell's mutation rate may be increased and / or mutations induced. This is advantageous as it may lead to a mutant cell with increased cryoprotective properties. Such a mutagenic substance is preferably selected from the group consisting of a polycyclic aromatic hydrocarbon, a nitrosamine, a base analog, a peroxide and a combination thereof. Hence, in some embodiments of the present invention, the cell is modified in step (b) by exposure to a mutagenic substance, preferably a mutagenic substance selected from the group consisting of a polycyclic aromatic hydrocarbon, a nitrosamine, a base analog, a peroxide and a combination 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. Thus, as regards exposure to radiation the same applies as it has been described above in the context of a mutagenic substance. Moreover, also the other features of such a physical mutagen can be as described above in case of a mutagenic substance being a chemical agent. Preferably, said radiation is a high- energetic radiation, more preferably a UV- and / or X-ray radiation. Hence, in some embodiments of the present invention, the cell is modified in step (b) by exposure to radiation, preferably a high- energetic radiation, more preferably a UV- and / or X-ray radiation.14SUBSTITUTE SHEET (RULE 26)

[0052] In some embodiments of the present invention, the cell is modified in step (b) by one or more modifying proteins. Herein the term "modifying protein" is intended to be understood as a protein that is capable of inducing alterations on the genetic and / or epigenetic level of a cell and thus, of causing (epi-)genetic changes. Thus, as regards the effect of said one or more modifying proteins the same applies as it has been described above in the context of a mutagenic substance. Herein, such a modifying protein is preferably selected from the group consisting of TALEN, a zink finger protein, a CRISPR / Cas combination, TET1, p300, DNMT3A, MQ.1, and LSD1, wherein said one or more modifying proteins more preferably comprises a CRISPR / Cas combination.

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

[0054] As regards the modifications of cells, it is to be noted that the person skilled in the art is aware of various routine techniques and methods for modifying a cell as well as detecting and assessing resulting modifications. Respective methods are known in the art and well-established experimental procedures are available. In particular, methods and techniques are known in the art to modify a cell on the genetic level, thus mutating the cell for obtaining modified versions of said cell and thus, mutants of said cell.

[0055] As regards step (c) of the method according to the present invention, said modified cell, e.g. said modified cell obtained from step (b), is frozen and thawed.

[0056] In preferred embodiments of the present invention, the modified cell obtained for example from step (b) and used in step (c) is a cell capable of being cultivated in suspension and / or a cell in suspension.

[0057] Furthermore, as described above in the context of step (a), also in case of step (c) in some embodiments of the present invention the modified cell is frozen and thawed in a suspension, which preferably comprises an added cryoprotectant is comprised in a concentration of 5% or less, preferably of 1% or less, more preferably of 0%. Thus, as regards freezing and thawing of a (modified) cell in a suspension comprising an added cryoprotectant the same applies as it has been described above in the context of step (a) in this regard with the only exception being that compared to the cell of step (a) the cell of step (c) is (further) modified. Otherwise, also the other features of such a15SUBSTITUTE SHEET (RULE 26)freezing and thawing of a (modified) cell in a suspension comprising an added cryoprotectant can be as described above in case of step (a).

[0058] As regards step (d) of the method according to the present invention, a modified cell, which survived step (c), is obtained, whereby an intracellular cryoprotectant expressed by said cell is identified. Thus, a (further) modified cell is obtained by the method according to the present invention that preferably exhibits an increased freezing tolerance compared to the cell used in step (a) of the method due to its modification(s). Hence, by applying a, preferably iterative, combination of accelerated in vitro evolution and selective pressure due to freezing a (further) modified cell can be obtained for investigation and thus, identification of an intracellular cryoprotectant expressed by said (further) modified cell. The disclosed innovative method is well automatable and enables fast and efficient identification of intracellular cryoprotectants across species and / or cell types that can be used for various applications, e.g. in research, pharmaceutical industry and (transplantation) medicine.

[0059] In some embodiments of the present invention, the method comprises further the step (e), i.e. proliferating said obtained ((further) modified) cell, preferably in suspension. This is especially advantageous as proliferation of said ((further) modified) cells enables a high-throughput application of the method according to the present invention. Thus, a ((further) modified) cell, e.g. obtained from step (d), can be proliferated and the proliferated cells can be in parallel subjected to the method according to the present invention and more specifically, one or more additional repetitions of freezing and thawing and modifying surviving cells. Moreover, such a process parallelization can increase the number of survival tests of different ((further) modified) cells, thus increasing the likelihood of identifying a highly efficient intracellular cryoprotectant and / or reducing the required time to identify such a highly efficient intracellular cryoprotectant.

[0060] Accordingly, in some embodiments of the present invention, the obtained ((further) modified) cell is proliferated in step (e) in suspension, wherein i) said suspension is a suspension, wherein an added cryoprotectant is comprised in a concentration of 5% or less, preferably of 1% or less, more preferably of 0%, and / or, wherein ii) said suspension comprises a conditioning (suspension) medium. Thus, the suspension in which the obtained ((further) modified) cell is proliferated, preferably comprises an added cryoprotectant and / or a conditioning (suspension) medium, wherein said conditioning (suspension) medium may comprise for example at least one growth factor to support cell vitality and / or to optimize cell culture conditions dependent on the cell type and species under study in view of for example specific metabolic requirements.16SUBSTITUTE SHEET (RULE 26)

[0061] In preferred embodiments 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 maximal 1,000, preferably at least 1 and maximal 100, more preferably at least 2 and maximal 100. Thus, it is preferred that the cell that survived freezing and thawing of step (a) is optionally proliferated to enable parallel investigation of modification(s) introduced in step (b); modified cells of step (b) may optionally proliferated as well to enable parallel investigation of the impact of a given modification on the cryoprotection of said cell and / or to exclude stochastic effects like cell deaths independent of a given cryoprotective effect; upon having frozen and thawed the optionally proliferated (further) modified cell (s), cell(s) can be obtained, which survived step (c); said surviving cells obtained from step (d) are optionally further proliferated and then, a new iteration of further steps (b) to (d) as just described can be initiated. Thus, using said iterative and accelerated evolutionary approach intracellular cryoprotectants can be identified with high efficiency in vitro. To further ease cell handling, all cells described in the context of said embodiments, i.e. cell(s), modified cell(s) and optionally further modified cell(s), are preferably cells in suspension.

[0062] In preferred embodiments 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 maximal 1,000, preferably at least 1 and maximal 100, more preferably at least 2 and maximal 100. Thus, the same applies as described in the paragraph before except that within said i iterations, the respective proliferation steps are performed. Thus, parallelization can be substantially increased and the impact of different modifications on cell vitality and / or freezing tolerance analyzed.

[0063] It is to be noted that the maximal level of parallelization may be (practically) limited. Thus, it can be considered that only a fraction of the proliferated cells, proliferated modified cells, and / or proliferated further modified cells is used in the respective subsequent step according to a respective embodiment of the present invention.

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

[0065] Including one or more proliferation steps in the method according to the present invention offers 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 respective cell17SUBSTITUTE SHEET (RULE 26)population's freezing tolerance determined. Herein, the term "cell population" refers to cells that originate from a proliferation step of a given ((further) modified) cell and thus, cells from such a cell population are to be understood as being substantially clones, e.g. with substantially identical genetic background.

[0066] Accordingly, in some embodiments of the present invention, the method further comprises step (f), and thus, a step of determining a survival rate of said obtained (further) modified cells for, preferably every, step (d).

[0067] Moreover, in some of said embodiments, the method comprises an iteration i+1, if the survival rate determined in step (f) of iteration i is at least as high as, preferably by 10% higher than, the survival rate determined in step (f) of iteration i-1. Thus, efficiency of the method can be improved by performing only an additional iteration in case of a modification that does not result in a survival rate worse than it was observed for the respective cell population without said modification. It is advantageous to apply an even higher threshold and thus, preceding only with one or more cell population(s) that exhibited an higher survival rate than the respective cell population without the modification under study, preferably an survival rate that is by at least 10%, more preferably by at least 15%, even more preferably by at least 20% higher.

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

[0069] Herein, the term "given threshold" refers to a threshold that is preferably set in view of a survival rate. This is advantageous as it allows defining a, preferably predetermined, criterion for, preferably not performing an additional iteration but, proceeding with identifying an intracellular cryoprotectant that may positively affect the observed survival rate. As regards said given threshold, the skilled person in the art is aware that such a threshold is preferably chosen in view of the experimental setting under study including, e.g. cell type, species, and / or absence or presence of an added cryoprotectant. Hence, said given threshold may be set for example to 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 to 75% or more, preferably to 80% or more. It is to be noted that the term "not exceeding" in the context of a given threshold refers to a parameter value like a determined survival rate that is below said given threshold, while the term "exceeding" in this context refers to a respective parameter value like a determined survival rate including and / or above said given threshold, preferably above said given threshold.18SUBSTITUTE SHEET (RULE 26)

[0070] In some embodiments 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 (f), optionally step (e) ] in i iterations, wherein i is at least 2, and preferably further comprising an iteration i+1, if the in step (f) of iteration i determined survival rate is at least as high, preferably by 10% higher than, the survival rate determined in step (f) of iteration i-1 and preferably not exceeding a given threshold. More specifically, in preferred embodiments 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 (f), step (e) ] in i iterations, wherein i is at least 2, and further comprising an iteration i+1, if the in step (f) of iteration i determined survival rate is at least as high, preferably by 10% higher than, the survival rate determined in step (f) of iteration i-1 and preferably not exceeding a given threshold. Thus, said embodiments represent an iterative evolutionary approach to identify intracellular cryoprotectants expressed by a cell, wherein efficiency of the method is advantageously increased by (preferably) proceeding only with ((further) modified), optionally proliferated, cells when the determined survival rate is not lower in an iteration compared to the one of the previous iteration. Thus, ((further) modified) cells can be obtained that show increasing freezing tolerance through improved cryoprotective properties from iteration to iteration. Moreover, to further ease cell handling, all cells described in the context of said embodiments, i.e. cell(s), modified cell(s) and optionally further modified cell (s), are preferably cells in suspension.

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

[0072] Preferably, some of the embodiments, including their features and advantages disclosed herein, are combined. As regards a particular method step the same applies as stated above including features and advantages mentioned in the context of the respective embodiment described above. Thus, parallelization and efficiency of the method according to the present invention can be optimized and the identification of intracellular cryoprotectants with advantageous cryoprotective properties substantially facilitated.

[0073] Accordingly, in particularly preferred embodiments of the present invention, the method comprises the steps of:(a) Using a cell that survived freezing and thawing, wherein step (a) preferably comprises the following steps:19SUBSTITUTE SHEET (RULE 26)(aO) Freezing and thawing of cells in suspension,(al) Obtaining a cell that survived step (aO),(fl) Determining a survival rate for step (aO) based on the number of living cells of the cells in suspension before freezing and thawing and after freezing and thawing,(el) Proliferating said cell obtained in step (al) and using the proliferated cell,(b) Modifying said cell used in step (a), preferably said cell proliferated in step (el),(e2) Proliferating said in step (b) modified cell,(c) Freezing and thawing the in step (e2) proliferated modified cell in suspension,(d) Obtaining a modified cell, which survived step (c), whereby an intracellular cryoprotectant expressed by said cell is identified, wherein preferably freezing and thawing in step (a), preferably in step (aO), and / or in step (c) is performed in a suspension, which preferably comprises an added cryoprotectant in a concentration of 5% or less, preferably of 1% or less, more preferably of 0%.

[0074] Moreover, it is preferred that said particularly preferred embodiments of the present invention described above further comprise the following steps for identifying an intracellular cryoprotectant expressed by said modified cell:(f2) Determining a survival rate for step (c) based on the number of living modified cells in suspension before freezing and thawing and after freezing and thawing,(e3) Proliferating said in step (d) obtained modified cell, and(g) Identifying at least one intracellular cryoprotectant based on the in step (e3) proliferated modified cells, preferably if the survival rate determined in step (f2) is exceeding a given threshold, wherein steps [ (b), (e2), (c), (d), (f2), (e3) ] are iterated (iteration i+1), if the survival rate determined in step (f2) of iteration i is at least as high, preferably by 10% higher than, the survival rate determined in step (f2) of iteration i-1 and preferably not exceeding said given threshold, and wherein i is at least 2.

[0075] Thus, some preferred embodiments of the present invention comprise the following steps:(a) Using a cell that survived freezing and thawing, wherein step (a) preferably comprises the following steps:(aO) Freezing and thawing of cells in suspension,(al) Obtaining a cell that survived step (aO),(fl) Determining a survival rate for step (aO) based on the number of living cells of the cells in suspension before freezing and thawing and after freezing and thawing,(el) Proliferating said cell obtained in step (al) and using the proliferated cell,20SUBSTITUTE SHEET (RULE 26)(b) (Further) Modifying said cell used in step (a), preferably said cell proliferated in step (el) and / or said cell proliferated in step (e3),(e2) Proliferating said in step (b) (further) modified cell,(c) Freezing and thawing the in step (e2) proliferated (further) modified cell in suspension,(d) Obtaining a (further) modified cell, which survived step (c), whereby an intracellular cryoprotectant expressed by said (further) modified cell is identified, preferably by:(f2) Determining a survival rate for step (c) based on the number of living (further) modified cells in suspension i) before freezing and thawing and ii) after freezing and thawing,(e3) Proliferating said in step (d) obtained (further) modified cell, and(g) Identifying at least one intracellular cryoprotectant based on the in step (e3) proliferated (further) modified cells, preferably if the survival rate determined in step (f2) is exceeding a given threshold, wherein preferably freezing and thawing in step (a), preferably in step (aO), and / or in step (c) is performed in a suspension, which preferably comprises an added cryoprotectant in a concentration of 5% or less, preferably of 1% or less, more preferably of 0%, and wherein steps [ (b), (e2), (c), (d), (f2), (e3) ] are iterated (iteration i+1), if the survival rate determined in step (f2) of iteration i is at least as high, preferably by 10% higher than, the survival rate determined in step (f2) of iteration i-1 and preferably not exceeding said given threshold, and wherein i is at least 2.

[0076] As regards the presence or absence of an added cryoprotectant in the suspension, preferably of step (c) according to the method of the present invention, it is particularly preferred to apply an approach with a step-wise reduction of the concentration of an added cryoprotectant in the suspension from iteration to iteration. Thus, selective pressure can be increased on the cell (s) under study. This is especially advantageous for an identifying intracellular cryoprotectant that exhibits a cryoprotective effect that allows overcoming the requirement of any added cryoprotectant in the suspension while ensuring cell survival and vitality upon freezing and thawing.

[0077] Accordingly, it is further preferred that the suspension in step (c) of iteration i+1 recited above in the context of said particularly preferred embodiments of the present invention comprises(1) Substantially the same concentration of the added cryoprotectant as the suspension in step (c) of iteration i, if the survival rate determined in step (f2) is at least as high as, preferably by at least 5% higher than, the survival rate determined in i) step (fl) in case of the first iteration (i=l), or21SUBSTITUTE SHEET (RULE 26)ii) step (f2) of iteration i-1 in case of iteration i,(2) A concentration of the added cryoprotectant that is between 0.001% and 1%, preferably between 0.01% and 0.5%, higher than in the suspension in step (c) of iteration i, if neither i) nor ii) are fulfilled.

[0078] It is to be noted that the term "substantially" refers herein to a deviation of the given 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%. In particular, said deviation encompasses a deviation from a given reference value in either direction and thus, an actually measured value may be lower or higher compared to the given reference value with a deviation of maximal 5%, 2.5%, 1%, 0.75%, 0.5%, 0.1%, 0.075%, 0.05%, 0.025%, 0.01% or even 0.001% from said reference value. Preferably, said deviation is observed towards a lower value. As an illustrative example, a concentration of 15% of an added cryoprotectant comprised in a suspension may be considered, wherein a suspension comprising substantially the same concentration of said added cryoprotectant may comprise thus, for example, said added cryoprotectant in 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%.

[0079] Thus, by applying the evolutionary approach according to the present invention, (further) modified cells are obtained that are preferably investigated in view of alterations that may cause the obtained improvement of their cryoprotective properties compared to their initial, non-modified counterpart, and thus, the used cell of step (a), and / or to the (further) modified counterpart of one of the previous iteration(s). In case of the first mentioned comparison, identification of an intracellular cryoprotectant may be facilitated as the initially used cell of step (a) can be seen as the cell with the lowest freezing tolerance and the (further) modified cell that survived freezing and thawing in step (c) of the iteration under study the cell with the highest freezing tolerance within the approach disclosed herein. As said two cells or cell populations have a substantially identical, especially genetic, background, except the modification(s) introduced during step(s) (b), a comparison of said cells on, e.g. a genetic level, may ease identification of the underlying intracellular cryoprotectant that differs between said cells or cell populations. The other exemplarily mentioned option may be especially suitable in cases, in which a comparably strong increase of freezing tolerance is observed between cells or cell populations from a given iteration and their counterpart one iteration before. Thus, modifications of comparably large effect size concerning freezing tolerance and / or optimizations of already identified intracellular cryoprotectants can be identified efficiently. However, the person skilled in the art is aware that depending for example on the intended study outcome, financial considerations and / or technical capabilities, said two exemplarily22SUBSTITUTE SHEET (RULE 26)mentioned comparisons may also be combined and / or performed on different levels such as genome, transcriptome and / or epigenome.

[0080] Accordingly, in preferred embodiments of the present invention, an intracellular cryoprotectant expressed by said (further) modified, optionally proliferated, cell (s) that survived step (c) is identified by analyzing the genome, transcriptome, epigenome, proteome and / or metabolome, preferably by using an analysis selected from the group consisting of gel electrophoresis, mass spectrometry, crystal structure analysis, NMR-spectroscopy, DNA sequencing, (m)RNA sequencing and any combination of the foregoing. It is preferred that an intracellular cryoprotectant is identified by genome analysis, preferably by DNA sequencing. Alternatively or optionally, it is preferred that an intracellular cryoprotectant is identified by gel electrophoresis and / or mass spectrometry. Advantages especially of sequencing based approaches as well as gel electrophoresis and mass spectrometry are, for example, the fact that they are already well established in the art, can be easily performed in an automated and high-throughput manner and at comparatively low cost.

[0081] When at least one intracellular cryoprotectant is identified using the method according to the present invention, it may be advantageous to further optimize said cryoprotectant, for example in view of the ability of a target cell to express said cryoprotectant and / or to optimize properties of said cryoprotectant like biological activity, localization within the cell, cell membrane passaging, and / or cryoprotective effect.

[0082] Accordingly, in some embodiments of the present invention the method further comprises as step (h) the step of modifying said identified intracellular cryoprotectant expressed by said (further) modified cell, whereby a derivate is obtained from said identified intracellular cryoprotectant expressed by said (further) modified cell.

[0083] The present invention further relates to an intracellular cryoprotectant, or a derivate thereof, preferably obtained by or obtainable by the method described above, wherein the intracellular cryoprotectant is an intracellular cryoprotectant that is synthesized by a cell.

[0084] Thus, by applying the method for identifying an intracellular cryoprotectant expressed by a cell according to the present invention, intracellular protectants can be identified efficiently and in high-throughput that are well suited for cryopreservation. Additionally or alternatively, also other intracellular cryoprotectants may be suitable for cryopreservation such as already known and / or otherwise characterized nucleotide sequences, polypeptides and proteins. Hence, while intracellular cryoprotectants obtained by or obtainable by the method described above are preferred, any intracellular cryoprotectant may be suitable for the method of cryopreserving a cell, cell aggregate, cell culture, tissue, organoid and / or organ as described herein in the following in detail. More 23SUBSTITUTE SHEET (RULE 26)specifically, it was surprisingly found that in contrast to current cryopreservation approaches that are based on added cryoprotectants, intracellular cryoprotectants can overcome respective limitations as regards for example cryoprotectant size and gradient formation. Thus, intracellular cryoprotectants pave the way to cryopreserve larger cell aggregates, tissues, organoids and / or organs without substantial loss of cell vitality and / or tissue, organoid and / or organ functionality.

[0085] An intracellular cryoprotectant expressed and / or synthesized by a cell may be a nucleotide sequence, a polypeptide or a protein. More specifically, encompassed by the term "intracellular cryoprotectant" are herein for example nucleotide sequences like RNA sequences and especially mRNA sequence, preferably mRNA sequences that may optionally have known or not yet known cryoprotective properties. Further encompassed are, for example, polypeptides and / or proteins with known or not yet known cryoprotective properties, such as for example polypeptides and proteins with alanine- and threonine-rich structures. The latter are preferably analogous to structures identified in anti-freeze proteins, in which they result in folded drum patterns in the superordinate (tertiary) structure of said anti-freeze proteins. Also anti-freeze proteins are encompassed herein. Other examples may include, preferably small- and medium-sized, cytoplasmatic proteins and polypeptides, glycoproteins, heat shock proteins, proteins and polypeptides with anti-apoptotic properties and / or osmotically active proteins and polypeptides. Said proteins and polypeptides may be especially advantageous for positively modulating cell survival and vitality, especially during thawing. Other examples may include albumins, globulins, histones, protamines, polyamines, kinases, growth factors and other. Of the latter, in particular globulins may be advantageous as they can be found dissolved in the cytoplasm of a cell and distributed throughout said cytoplasm during cryopreservation. Moreover, some globulins are capable of binding water, thus reducing cellular damage due to ice crystal formation during freezing. Further examples may be membrane-bound and / or superimposed proteins. These may exhibit cryoprotective properties by stabilizing organelles and limiting ice crystal formation. Further examples may be porins and other osmotically active proteins and polypeptides. Membrane transporters are optionally encompassed as well. Further examples may include fibrillary proteins and polypeptides that have stabilizing effects on the cytoskeleton and that may act together with supporting and scaffolding factors like collagen, actin and / or myosin. Said proteins and polypeptides are in particular advantageous for supporting temporary stiffening of cells during cryopreservation. Further examples may be polypeptides and proteins that are involved, and preferably drive, membrane lipid and fatty acid synthesis. This is advantageous for increasing the cellular lipid content, including the formation of lipid vacuoles, which positively impacts cryopreservation. Additionally or optionally, increased cellular lipid content is advantageous as the resulting fat bodies and / or fat vacuoles exert mechanic stabilization effects. Thus, advantageous are fatty acid synthase(s), parts thereof as well intermediates of the cellular fatty 24SUBSTITUTE SHEET (RULE 26)acid cycle. Examples may comprise carnitin-acyltransferase 1, fatty acid acyl-CoA desaturase, adrenaline, triglycerides, triacylglycerols, neutral fats, fats, and other esters of trivalent alcohol glycerol (glycerol, propane-1, 2, 3-triol) and three long-chain carboxylic acids, glycerophospholipids, sphingolipids, and diacylglycerin-3-phosphat. Further examples may comprise membrane lipids, such as phospholipids, glycolipids and cholesterol. Even further examples may be proteins and / or polypeptides that are involved in the glycogen synthesis and thus, in the cellular carbohydrate synthesis and / or conversion for example in animal and human cells. Respective example may include insulin, which drives glycogen build-up, and so-called core proteins (glycogenin), around which the glycogen structure can be build in radiating chains. These are in particular advantageous for increasing cellular surface areas and water binding capabilities and thus may act cryoactive. Further examples may refer to pyruvatcarboxylase phosphoenolpyruvat-carboxykinase, fructose-1,6- bisphosphatase, and glucose-6-phosphatase.

[0086] Accordingly, in some embodiments of the present invention, the intracellular cryoprotectant, or a derivate thereof, is a nucleotide sequence, preferably an RNA sequence like 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, an "mRNA sequence", also abbreviated as "mRNA", should be understood as a polyribonucleotide molecule which, if it comes into the cell, is suitable for the expression and / or synthesis of a protein or polypeptide and / or is translatable into a protein or polypeptide. Preferably, said mRNA contains a ribonucleotide sequence which encodes a protein or polypeptide, whose function in a cell or in the vicinity of a cell is needed or beneficial for cryopreservation. More specifically, an mRNA sequence refers in the context of the present invention to a single-stranded ribonucleotide sequence built up for example of A, C, G and / or U nucleotides, i.e. nucleotides comprising adenine, guanine, cytosine, and uracil as the respective nitrogenous base. Furthermore, said mRNA comprises one or more coding sequences that can be used as a template during expression and / or synthesis of an amino acid sequence during translation. Said mRNA may further comprise a 5' and / or 3' untranslated region (UTR), one or more internal ribosome entry site(s) (IRES), one or more additional modifications to promote translation, adjust and / or extend duration of action etc. Hence, an mRNA comprises at least one coding sequence that is translatable into an amino acid sequence such as a protein by and within a cell. Thus, an mRNA sequence can be translated into an amino acid sequence such as a protein and hence, said amino acid sequence like a protein can be expressed und / or synthesized by and within a cell.

[0088] In some embodiments of the present invention, the intracellular cryoprotectant, or a derivate thereof, is obtained by or obtainable by the method according to the present invention25SUBSTITUTE SHEET (RULE 26)disclosed herein above, wherein the intracellular cryoprotectant is an intracellular cryoprotectant that is synthesized by a cell and that is preferably an, optionally glycosylated, polypeptide or protein.

[0089] In some embodiments of the present invention, the intracellular cryoprotectant, or derivate thereof, is preferably a, optionally glycosylated, polypeptide or protein, and is characterized by a size, weight, and / or structure which is suitable to provide the cryoprotectant the capability of cell membrane impermeability. The term cell "cell membrane impermeability" has to be understood as the characteristic of the intracellular cryoprotectant to be not cell membrane permeable in a passive way. Thus, the intracellular cryoprotectant is not able to diffuse through the cell membrane per se. Such a cell permeability in a passive way occurs with known cryoprotectants like DMSO, urea or small sugar, like trehalose. Moreover, the capability of cell membrane impermeability of the intracellular cryoprotectant according to the present invention does not exclude the possibility that the cryoprotectant may be actively introduced in a cell across the cell membrane via a naturally occurring membrane transporter which may be located within the cell membrane. However, such an active transport of the cryoprotectant according to the present invention via a membrane transporter from outside to the inside of the cell does not allow that sufficient amounts of the cryoprotectant are achieved to provide a proper effect as cryoprotectant. According to the present invention, it is foreseen that the intracellular cryoprotectant is produced within the cells and remains within the cell in view of distinct characteristics of said intracellular cryoprotectant. Thus, this allows that the cryoprotective ability of the cryoprotectant according to the invention is exerted within the cell, since diffusion through the cell membrane is avoided or at least minimized. Accordingly, the intracellular cryoprotectant of the present invention is preferably synthesized in the cell with a distinct large size and structure which allows a sufficient concentration within the cell. This provides the advantage that there is no dependency on trans-membrane transport which is only applicable with smaller molecules serving as potential cryoprotectant. Further, such smaller molecule cryoprotectants may not be introduced in a sufficient concentration within the cell and in particular not in larger cell aggregates, such as organoids, tissues or organs. Therefore, the intracellular cryoprotectant of the present invention provides the advantages of achieving a beneficial concentration of the cryoprotectant inside the cell, and achieving the desired cryoprotective effect intracellularly since the cryoprotectant is kept within the cell.

[0090] In some embodiments of the present invention, the intracellular cryoprotectant is preferably a, optionally glycosylated, polypeptide or protein, and preferably is having an average molecular weight (Mw) of greater than 9800 Da, preferably greater than 900 kDa, or more. According to the present invention it is foreseen that the intracellular cryoprotectant is a, optionally glycosylated, polypeptide or protein, which has a large molecular weight, wherein the large size is only limited by26SUBSTITUTE SHEET (RULE 26)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 a cell to be able to synthesize a polypeptide or protein of a distinct large size.

[0091] In a further aspect, the present invention is directed to a method of producing a cryoprotectant, or a derivate thereof, comprising conducting the method of identifying an intracellular cryoprotectant as described in any one of the embodiments of the invention above. Accordingly, the method of producing a cryoprotectant, or derivate thereof, comprises the identification of the cryoprotectant and further producing the cryoprotectant up to a desired amount.

[0092] Herein, the term "amino acid sequence" encompasses any kind of amino acid sequence chain comprising two or more amino acids linked via peptide bonds. Preferably, the amino acid sequence is at least 5 amino acids long, more preferably at least 10 amino acids or at least 20, 50, 75, 100, 125, 150, 175 or 200 amino acids. Thus, said term encompasses short peptides, oligopeptides, polypeptides, fusion proteins, proteins as well as fragments thereof such as functional parts of a known protein. Herein, the term "polypeptide" refers to an amino acid sequence of at least 10 amino acids and up to and including 100 amino acids. Furthermore, herein the term "protein" refers to an amino acid sequence of more than 100 amino acids. As regards the function of an amino acid sequence, there is no limitation. Preferably, said function is (directly) linked to a cryoprotective effect, preferably within a cell.

[0093] Herein, the term "glycosylated" refers to the process of glycosylation and thus, a controlled, preferably enzymatic, modification, preferably of an amino acid sequence such as a polypeptide or a protein, by addition of at least one sugar moiety. Hence, glycosylation preferably relates to a modification of an amino acid sequence, wherein said modification can be a post-translational modification within a cell. A glycosylated protein may also be referred to as glycoprotein herein. Glycosylation is relevant for many biological processes as glycosylation can affect intracellular trafficking, cell attachment to the extracellular matrix and / or protein-ligand interaction.

[0094] In some embodiments of the present invention, the intracellular cryoprotectant, or a derivate thereof, is selected from the group consisting of an anti-freeze protein, a protein or polypeptide with an alanine- and / or threonine-rich structure analogous to anti-freeze proteins and tertiary structure, a glycoprotein, a cytoplasmatic protein or polypeptide, a heat shock protein, an albumin, a globulin, a histone, a protamine, a kinase, a growth factor, a globular protein or polypeptide, a membrane-bound and / or superimposed protein or polypeptide, a porine, an osmotically active protein or polypeptide, a fibrillary protein or polypeptide, a polypeptide or protein 1SUBSTITUTE SHEET (RULE 26)involved in membrane lipid and / or fatty acid synthesis, a phospholipid, a glycolipid, cholesterol, a polypeptide or protein involved in glycogen synthesis, insulin, and a core protein.

[0095] Herein, it is to be noted that the term "intracellular cryoprotectant that is synthesized by a cell" encompasses the term "intracellular cryoprotectant expressed by a cell". Moreover, the term "intracellular cryoprotectant that is synthesized by a cell" encompasses further the situation that a cell generates an intracellular cryoprotectant without being intrinsically capable of expressing said intracellular cryoprotectant. Thus, a cell synthesizing an intracellular cryoprotectant may or may not comprise endogenously the respective required biological information to build said intracellular cryoprotectant. Said cell may be modified by introducing the respective required biological information, thus becoming at least transiently capable of generating an intracellular cryoprotectant and / or a derivative thereof. Thus, respective biological information may have to be introduced into a cell like a, optionally modified, nucleotide sequence encoding an intracellular cryoprotectant being a protein or a polypeptide. Such an approach offers the advantage that a cell can be made amenable to cryopreservation that would not have had endogenously any or at least sufficient cryoprotective properties to ensure cell survival and proper cell function after freezing and thawing. Moreover, it offers the advantage to make a cell both long-term and / or transiently amenable to cryopreservation. Furthermore, said term "intracellular cryoprotectant that is synthesized by a cell" encompasses also the scenario that a cell that has already been intrinsically capable of expressing an intracellular cryoprotectant is modified in a manner that enables said cell to (newly) synthesize a derivate of said intracellular cryoprotectant and / or alters the amount of the intracellular cryoprotectant and / or its derivative expressed and / or synthesized by said cell. Thus, a cell synthesizing an intracellular cryoprotectant may or may not comprise (at least partially) endogenously the respective required biological information to build said intracellular cryoprotectant.

[0096] Accordingly, in preferred embodiments of the present invention, the intracellular cryoprotectant is an intracellular cryoprotectant that is capable of cryopreserving a cell, preferably a cell aggregate, a cell culture, a tissue, an organoid, and / or an organ.

[0097] Herein, the terms "cell", "cell aggregate" and "cell culture" have their usual meaning in the art. Moreover, herein said cells, cell aggregates and cell cultures are preferably capable of being cultivated in vitro and / or cultivated at least temporarily in vitro. Also the term "tissue" has its usual meaning in the art and may thus, be seen for example as a collection of structurally and functionally comparable cells, optionally with intercellular material, that are organized as to perform a specific function. Herein, the term "organoid" has its usual meaning in the art and may thus be understood as referring to self-organized, three-dimensional tissue cultures that are preferably derived from stem cells. Said organoids can be cultured in vitro, thus offering the possibility to replicate selected 28SUBSTITUTE SHEET (RULE 26)and / or most features of an otherwise comparatively complex organ. Organoids are advantageous for example for studying effects of drug candidates and / or drugs on a given organ in vitro and / or obtaining insights on evolutionary processes associated with organ formation for example. Herein, the term "organ" has its usual meaning in the art and may thus be understood as referring to a collection of cell types and / or tissues joined in a structural unit that may serve a common function, with examples including kidney, heart, lung and liver. Tissues and / or organs are for example particularly advantageous for (bio-) medical purposes such as transplantation. Moreover, as regards said terms, it is envisioned that herein the respective terms for cells, cell aggregates, cell cultures, tissues, organoids, and / or organs encompass both an artificial and a natural origin. For example, a tissue may be a tissue obtained from an animal and / or an in vitro engineered tissue.

[0098] Herein, the ability of cryopreservation relates to the ability of a cell to survive freezing and thawing, wherein freezing preferably comprises or is a reduced temperature of less than -140°C. Thus, the capability of an intracellular cryoprotectant to cryopreserve a cell 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 of less than -140°C. More specifically, an intracellular cryoprotectant according to the present invention preferably exhibits a cryoprotective property, wherein said cryoprotective property is preferably characterized by a level of viability, vitality and / or functionality of a cell, cell aggregate, cell culture, tissue, organoid and / or organ of at least 70%, preferably of at least 80%, more preferably of at least 90%, after freezing and thawing compared to said level before freezing and thawing. Hence, in case of a cell aggregate, cell culture, tissue, organoid and / or an organ cryopreservation of said cell aggregate, cell culture, tissue, organoid and / or organ may be accompanied during freezing and thawing by cell death and / or reduced cell vitality of a minor portion of cells comprised in said cell aggregate, cell culture, tissue, organoid and / or organ, wherein said minor portion is a portion that is so minor that the overall viability, vitality and / or functionality of said cell aggregate, cell culture, tissue, organoid and / or organ is preferably not reduced by more than 30%, more preferably not by more than 20%, even more preferably not by more than 10%, after freezing and thawing compared to the respective viability, vitality and / or functionality before said freezing and thawing.

[0099] Herein, the term "survival rate", which may also be referred to as "viability", is intended to be understood as a percentage of living cells in a cell population like a cell culture or a tissue at a given point in time. Besides cell death, cellular damages due to freezing and thawing may exert negative effects on a cell through a number of cellular alterations that may compromise a cell's ability to (generally) function properly. Hence, herein, the term "cell vitality" refers to a cell's physiological capability. It is to be noted that both cell viability and cell vitality are intended to29SUBSTITUTE SHEET (RULE 26)represent two different aspects of cell functions, and are preferably both considered when estimating a cell's physiological state before and after freezing and thawing. Furthermore, the term "functionality" is intended to be understood as a measure relating to a specific function, wherein said measure is preferably obtained and / or assessed on a multi-cell level. Thus, said term preferably refers to a functional characteristic of a cell aggregate, tissue, organoid and / or organ, for example in view of contractility in case of muscle cells. Hence, for example 100% functionality may represent a respective healthy tissue, organoid and / or organ and / or a tissue, organoid and / or organ before freezing and thawing, whereas for example 50% functionality after freezing and thawing may indicate that a given tissue, organoid and / or organ may exhibit in case of, for example, muscle cells only half the contraction force and / or frequency compared to either a healthy reference and / or said tissue, organoid and / or organ before freezing and thawing.

[0100] Thus, in some embodiments of the present invention, said intracellular cryoprotectant, preferably obtained by or obtainable by the method according to the present invention described above, is an intracellular cryoprotectant that is capable of cryopreserving a cell, a cell aggregate, a cell culture, a tissue, an organoid, and / or an organ, preferably by ensuring and / or maintaining a level of viability, vitality and / or functionality of said cell, cell aggregate, cell culture, tissue, organoid and / or organ of at least 70%, preferably of at least 80%, more preferably of at least 90%, after freezing and thawing compared to said level before freezing and thawing.

[0101] As indicated above, it may be required to introduce and thus, to transfer biological information into a cell to enable said cell to synthesize an intracellular cryoprotectant according to the present invention. For introducing biological information into a cell, a nucleotide sequence is particularly advantageous in view of well known and established routine methods available in the art as regards preparation, modification, optimization, application, safety and efficiency.

[0102] Hence, the present invention further relates to a method of generating a, optionally modified, nucleotide sequence encoding an intracellular cryoprotectant, or derivate thereof, according to the present invention.

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

[0104] Thus, in some embodiments of the present invention, a nucleotide sequence encoding an intracellular cryoprotectant, or derivate thereof, is identified. Said nucleotide sequence is preferably an RNA sequence, preferably an mRNA sequence, that comprises a coding sequence encoding an 30SUBSTITUTE SHEET (RULE 26)intracellular cryoprotectant, wherein said intracellular cryoprotectant is optionally obtained by or obtainable by the method for identifying in an intracellular cryoprotectant according to the present invention.

[0105] Herein, the term "encoding" refers to in the context of a nucleotide sequence like a DNA sequence or an mRNA sequence to a nucleotide sequence that comprises a sequence which encodes biological information. For example, an mRNA sequence may encode a protein in the sense that said nucleotide sequence contains a coding region which encodes a sequence of amino acids. Thus, an mRNA sequence can be translated into an amino acid sequence such as a protein and hence, said amino acid sequence like a protein can be expressed und / or synthesized by and within a cell.

[0106] As regards step (a), nucleotide sequence encoding an intracellular cryoprotectant or derivative thereof may be identified for example using the respective method according to the present invention. In case the intracellular cryoprotectant identified according to said method is a polypeptide or protein, the skilled person in the art is aware of methods and techniques to derive a respective nucleotide sequence encoding said intracellular cryoprotectant from the obtained amino acid sequence information.

[0107] Accordingly, an identified nucleotide sequence, preferably RNA sequence, more preferably mRNA sequence, encoding an intracellular cryoprotectant or derivate thereof comprises one or more coding sequence(s) encoding one or more intracellular cryoprotectant(s), wherein said at least one coding sequence comprised can be a naturally occurring sequence, a modified sequence like a partially or fully codon optimized sequence derived from a naturally occurring sequence, or an artificial sequence. Codon optimization refers to a technique which is applied for example to maximize protein expression by increasing translation efficiency for example as species exhibit differences in their preferential codon use for a given amino acid.

[0108] As regards step (b), modification of a nucleotide sequence, the same applies as described above in the context of modifying a cell on a genetic and / or transcriptomic level. More specifically, a nucleotide sequence may be modified, for example by introducing a single nucleotide polymorphism, inserting and / or deleting one or more nucleotides, and / or combining one or more nucleotide sequences. Moreover, the term "modifying said nucleotide sequence" further encompasses codon optimization, addition, removal or alteration of one or more of the group consisting of 5' cap, 3' poly A tail, IRES, 3' UTR, 5' UTR, and further regulatory and / or translation promoting sequence(s), as well as addition, removal or alteration of at least one additional nucleotide sequence encoding at least one other intracellular cryoprotectant. Thus, modifying a nucleotide sequence like an mRNA sequence that encodes an intracellular cryoprotectant encompasses for example also the31SUBSTITUTE SHEET (RULE 26)combination of coding sequences for one or more intracellular cryoprotectants. More specifically, said combination may also encompass the combination of one or more coding sequence(s) encoding one ore more intracellular cryoprotectant(s), wherein preferably at least one of said one or more intracellular cryoprotectant(s) is obtained by or obtainable by the method for identifying an intracellular cryoprotectant according to the present invention. Thus, a nucleotide sequence encoding an intracellular cryoprotectant may be modified for example to comprise a further sequence encode the same intracellular cryoprotectant and / or to comprise a sequence encoding additionally another intracellular cryoprotectant. In any of these cases, the respective nucleotide sequence, preferably RNA sequence, more preferably mRNA sequence, may be a partially or fully modified sequence. Hence, by modifying a nucleotide sequence encoding an intracellular cryoprotectant according to step (b) localization and duration of action can be optimized as well as in case of the nucleotide sequence being an mRNA sequence also translation efficiency and / or duration.

[0109] As regards step (c), in vitro synthesis methods are well established in the art and available to the skilled artisan. Thus, step (c) offers the advantage to amplify in vitro said identified, optionally modified, nucleotide sequence obtained for example from step (b). Hence, a large scale application of said nucleotide sequence can be envisioned for cryopreservation of cells, cell aggregates, cell cultures, tissue, organoids and / or organs, preferably using the method described below in detail.

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

[0111] The present invention further relates to a, optionally modified, nucleotide sequence obtained by or obtainable by the method of generating a, optionally modified, nucleotide sequence according to the present invention, encoding one or more intracellular cryoprotectant(s) or derivative(s) thereof according to the present invention. Said, optionally modified, nucleotide sequence is advantageous for the introducing biological information into a cell for making said cell amenable for cryopreservation, preferably using the respective method according to the present invention described herein in the following in more detail.

[0112] In some embodiments of the present invention, said, optionally modified, nucleotide sequence according to the present invention is an RNA sequence, preferably an mRNA sequence.32SUBSTITUTE SHEET (RULE 26)

[0113] The present invention relates further to a nanoparticle comprising a, optionally modified, nucleotide sequence according to the present invention as described above, wherein said nanoparticle is preferably a lipid-nanoparticle. Encapsulating a nucleotide sequence in a nanoparticle is advantageously facilitates introduction of said nucleotide sequence into a cell. Moreover, respective methods and techniques for preparing nanoparticles, loading nanoparticles with a nucleotide sequence, using said nanoparticles for introducing nucleotide sequence(s) comprised therein into a cell and / or assessing safety and efficacy of such approaches are well-known and established in the art.

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

[0115] The present invention further relates to the use i) of an intracellular cryoprotectant, or derivative thereof, according to the present invention, ii) of a, optionally modified, nucleotide sequence according to the present invention, and / or iii) of a nanoparticle according to the present invention for cryopreserving a cell, cell aggregate, cell culture, tissue, organoid, and / or organ.

[0116] Using a, optionally modified, nucleotide sequence encoding an intracellular cryoprotectant according to the present invention a cell can be made amenable for synthesis of an intracellular cryoprotectant, and thus for cryopreservation, that may or may not endogenously have had or has cryoprotective properties. Moreover, said at least one intracellular cryoprotectant may even be originally identified in a species other than that of the cell that is to be made amenable to cryopreservation according to the method of the present invention. This is especially relevant in view of the fact that several species including humans are not capable of expressing functional intracellular cryoprotectants at all or in an amount that is sufficient for allowing for example tissue or organ cryopreservation in the absence of added cryoprotectants. Hence, biological information as regards synthesis of an intracellular cryoprotectant according to the present invention is advantageously transferred into a cell to be cryopreserved before freezing and thawing. Preferably, said biological information is provided in form of a, optionally modified, mRNA sequence, optionally comprised in a nano-particle, that can be transferred into a cell and translated into an intracellular cryoprotectant by and within said cell.

[0117] Accordingly, the present invention further relates to a method of cryopreserving a cell, a cell aggregate, a cell culture, a tissue, an organoid, and / or an organ, comprising the following steps of (a) contacting a cell, cell aggregate, cell culture, tissue, organoid and / or organ with a, optionally33SUBSTITUTE SHEET (RULE 26)modified, nucleotide sequence and / or a nanoparticle according to the present invention, and (b) freezing said cell, cell aggregate, cell culture, tissue, organoid and / or organ obtained from step (a), wherein freezing comprises or is a reduction from a temperature of said cell, cell aggregate, cell culture, tissue, organoid and / or organ of more than 0°C to a temperature of less than 0°C, preferably of less than -20°C, more preferably of less than -70°C, even more preferably of less than -140°C.

[0118] Herein, the term "contacting" can be understood as "bringing in contact", for example a cell to be cryopreserved with a nucleotide according to the present invention encoding at least one intracellular cryoprotectant. In this case said cell may be brought in contact with said nucleotide by adding the nucleotide to a (suspension) medium that comprises (at least temporarily) said cell. The skilled person is aware of methods and techniques to assess and determine suitable approaches and parameters for contacting a cell, cell aggregate, cell culture, tissue, organoid and / or organ with a nucleotide sequence and / or nano-particle according to the present invention to ensure sufficient and / or efficient introduction of the respective biological information into said cell, cell aggregate, cell culture, tissue, organoid and / or organ. Examples of suitable approaches may include addition of a nucleotide sequence encoding at least one intracellular cryoprotectant to a suspension comprising a cell to be cryopreserved, perfusion of an organ with a solution comprising a nucleotide sequence according to the present invention, and / or clinically safe and well established vaccination based approaches, for example using mRNA lipid nano-particles. Relevant parameters may comprise contacting times, concentrations of a nucleotide sequence and / or nano-particle according to the present invention for example in a suspension and / or medium that comprises the cell, cell aggregate, cell culture, tissue, organoid and / or organ to be cryopreserved, and / or duration between contacting and freezing, optionally between contacting start and / or end and initiation of freezing. Thus, it is preferably assured that sufficient intracellular cryoprotectant(s) is synthesized by and within a cell to limit or even avoid cellular damage, for example resulting from crystal ice formation, during freezing. Moreover, said contacting is advantageous as it allows applying the method according to the present invention ex vivo to different cell systems like cell suspensions, tissue slices, and organs.

[0119] Accordingly, in some embodiments of the present invention, the method of cryopreserving a cell, a cell aggregate, a cell culture, a tissue, an organoid, and / or an organ is an ex vivo method. Furthermore, encompassed herein may also be embodiments relating to a method of cryopreserving a cell, a cell aggregate, a cell culture, a tissue, an organoid, and / or an organ non ex vivo. Thus, in some embodiments of the present invention, a cell, a cell aggregate, a cell culture, a tissue, an organoid, and / or an organ may be cryopreserved, wherein said cell, cell aggregate, cell culture, tissue, organoid, and / or organ is at least partially and / or transiently embedded in a physiological environment. Herein, a "physiological environment" refers to an artificial and / or natural34SUBSTITUTE SHEET (RULE 26)environment that constitutes or resembles physiological conditions comparable to physiological conditions that can (naturally) be observed in the species and / or cell, cell aggregate, cell culture, tissue, organoid, and / or organ under study. Encompassed herein may thus also be a cell, cell aggregate, cell culture, tissue, organoid, and / or organ comprised in a mammal with irreversible failure of brain functions. Said failure of brain functions is to be determined according to well established, preferably neurological, measurements, methods and / or criteria and optionally by a qualified person. Thus, for illustrative purposes, an ovarian tissue obtained from a human before chemotherapy and comprising an oocyte may be considered that may be contacted with a nucleotide sequence according to the present invention by in vitro perfusion using a solution comprising said nucleotide sequence for cryopreserving not only the ovarian tissue but particularly the oocyte comprised therein. As another example, a mammal may be considered that is contacted after determination of irreversible failure of brain functions with a nucleotide sequence according to the present invention using a vaccination based approach. This would be in line with step (a) of the method for cryopreserving for example the mammal's kidney. Further, said contacting may be done for example between 12 hrs and 72 hrs before stopping any life sustaining (medical) measures and / or methods for said mammal. In such a scenario, the intracellular cryoprotectants according to the present invention as well as their use in the innovative method of cryopreserving disclosed herein, is particularly advantageous for making (mammalian) cells, cell aggregates, cell cultures, tissues, organoids, and / or organs amenable for cryopreservation in the context of (bio)medical applications, in particular for transplantation, while reducing cellular damage associated with current approaches and / or overcoming logistic limitations in the context of transplantation medicine.

[0120] As regards freezing, as well as potential and / or suitable temperature-time protocols for freezing (and thawing), the same applies as described herein above in the context of the method of identifying an intracellular cryoprotectant expressed by a cell according to the present invention. Additionally, it is to be noted that for example the freezing process may be modulated to optimize cryoprotection conferred by an intracellular cryoprotectant said cell, cell aggregate, cell culture, tissue, organoid and / or organ is contacted with before freezing. For example, by initially reducing the temperature of said cell, cell aggregate, cell culture, tissue, organoid and / or organ to a first temperature between 30°C and -20°C cellular for a suitable period of time, cellular processes may be slowed down while still allowing the intracellular cryoprotectant(s) to be synthesized, thus allowing to synchronize for example the level of synthesized intracellular cryoprotectant(s) across cells of for example a given organ or tissue, before further reducing 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 culture, e.g., of cells can take place at a temperature of 30°C to 40°C, preferably around 35°C, so that for synchronization the temperature can be reduced, for 35SUBSTITUTE SHEET (RULE 26)example, to a first temperature between 30°C and 0°C, preferably between 20°C and 15°C or between 4°C and 0°C. Thus, temperature-time protocols may be optimized in view of the cell system used and / or for example practical requirements.

[0121] In some embodiments of the present invention, said cell, cell aggregate, cell culture, tissue, organoid and / or organ to be cryopreserved is contacted with a, optionally modified, nucleotide sequence encoding one or more intracellular cryoprotectants according to the present invention and optionally encapsulated and / or comprised in a nano-particle. Alternatively or optionally, said cell, cell aggregate, cell culture, tissue, organoid and / or organ to be cryopreserved may be contacted with more than one nucleotide sequence species, for example with a first nucleotide sequence encoding a first intracellular cryoprotectant and a second nucleotide sequence encoding a second intracellular cryoprotectant.

[0122] Accordingly, in some embodiments of the present invention, said step (a) of the method of cryopreserving a cell, a cell aggregate, a cell culture, a tissue, an organoid, and / or an organ according to the present invention comprises the steps of (al) contacting said cell, cell aggregate, cell culture, tissue, organoid and / or organ with a, optionally modified, nucleotide sequence encoding a first intracellular cryoprotectant or derivative thereof, and / or a nanoparticle comprising a, optionally modified, nucleotide sequence encoding a first intracellular cryoprotectant or derivative thereof, and (a2) contacting said cell, cell aggregate, cell culture, tissue, organoid and / or organ with a further, optionally modified, nucleotide sequence encoding a second intracellular cryoprotectant or derivative thereof, and / or with a further nanoparticle comprising a, optionally modified, nucleotide sequence encoding a second intracellular cryoprotectant, or derivative thereof. It is to be noted that said method is not limited to the use of two different species of nucleotide sequences and / or nanoparticles, and thus, the introduction of biological material as regards two intracellular cryoprotectants. Hence, the description of said embodiments is to be understood as illustrative and the respective rational can be extended accordingly to the use of more than two species of nucleotide sequences and / or nano-particles as required. Furthermore, it is to be noted that said steps (al) and (a2) may be performed in parallel, temporarily at least partially overlapping and / or sequentially. The skilled person in the art is aware of identify suitable approaches in view of requirements specific to the cell, cell aggregate, cell culture, tissue, organoid and / or organ to be cryopreserved, the nucleotide sequence(s) and / or nano-particle(s) used and / or respectively chosen intracellular cryoprotectant(s).

[0123] In some embodiments of the present invention, the method of cryopreserving a cell, a cell aggregate, a cell culture, a tissue, an organoid, and / or an organ, comprises further the steps of (c) thawing said frozen cell, cell aggregate, cell culture, tissue, organoid and / or organ, and / or (d) 36SUBSTITUTE SHEET (RULE 26)determining a level of viability, vitality and / or functionality of said cell, cell aggregate, cell culture, tissue, organoid and / or organ before step (a), between steps (a) and (b), and / or after step (c), wherein said level of viability, vitality and / or functionality is at least 70%, preferably of at least 80%, more preferably of at least 90%, after freezing and thawing compared to said level before freezing and thawing.

[0124] As regards thawing, as well as potential and / or suitable temperature-time protocols for (freezing and) thawing, the same applies as described herein above in the context of the method of identifying an intracellular cryoprotectant expressed by a cell according to the present invention.

[0125] As regards viability, vitality and / or functionality the same applies as described above in the context of the intracellular cryoprotectant according to the present invention. Furthermore, as regards step (d), the person skilled in the art is aware of methods and techniques to assess survival, viability, vitality and / or functionality of a cell, cell aggregate, cell culture, tissue, organoid and / or organ. The choice of a respective method may depend for example on the sample type, like cell aggregate or tissue, and / or cell type. For illustrative purposes, cardiac muscle cells may be considered as an example. In this case, a suitable test to assess vitality and / or functionality may be based on the induction of contractions by electrical pulse stimulation and characterization of resulting contractions, for example in view of force of contraction, contraction frequency and / or time delay. Optionally or alternatively, especially in case of tissues, organoids and / or organs, in particular organs, quantitative measures of a given function may be assessed. Exemplarily in case of a kidney urea accumulation may be assessed as well as in case of a liver presence and / or amount of degradation products of one or more substances. In particular in case of larger tissues, organoids and especially organs, parameters are preferred, which capture the overall function and / or performance of said tissue, organoid and / or organ. Alternatively or optionally, indirect measurements may be used, for example based on combinations of specific biomarkers. Further examples may include measures based on detecting certain test substances as indicator of cellular synthesis and / or degradation, e.g., via quantitative fluorescence measurements, and / or vital / lethal cell staining based methods.

[0126] Preferably, the method of cryopreserving a cell, a cell aggregate, a cell culture, a tissue, an organoid, and / or an organ is applied to a cell, cell aggregate, cell culture, tissue, organoid and / or organ of plant or vertebrate origin, preferably of mammalian and / or human origin. This is especially advantageous for (bio-) medical applications like in transplantation medicine and / or pharmaceutical applications like "ready-to-use" cells, cell aggregates, cell cultures, tissues, organoids and / or organs.37SUBSTITUTE SHEET (RULE 26)

[0127] Accordingly, in some embodiments of the present invention, said cell, cell aggregate, cell culture, tissue, organoid and / or organ is a cell, cell aggregate, cell culture, tissue, organoid and / or organ from a vertebrate or a plant, preferably from a vertebrate, more preferably from a mammal and / or a human.

[0128] Furthermore, the present invention relates to a cell, cell aggregate, cell culture, tissue, organoid and / or organ obtained by or obtainable by the method of cryopreserving a cell, a cell aggregate, a cell culture, a tissue, an organoid, and / or an organ according to the present invention described above.

[0129] In some embodiments of the present invention, said cell, cell aggregate, cell culture, tissue, organoid and / or organ, obtained by or obtainable by the method of cryopreserving according to the present invention, has, preferably at least transiently, a cryoprotective property. As regards said cell, cell aggregate, cell culture, tissue, organoid and / or organ as well as said cryoprotective property the same applies as stated before in the context of the intracellular cryoprotectant according to the present invention and the cell, cell aggregate, cell culture, tissue, organoid and / or organ obtained by or obtainable by the method of cryopreserving according to the present invention, respectively. Thus, by using an intracellular cryoprotectant, or derivate thereof, according to the present invention, preferably identified using the respective method according to the present invention, a nucleotide sequence, preferably an, optionally modified, mRNA sequence encoding at least said intracellular cryoprotectant may be generated and brought in contact with a cell, cell aggregate, cell culture, tissue, organoid and / or organ according to the method of cryopreserving according to the present invention. Using an mRNA to make said cell(s) capable of synthesizing said intracellular cryoprotectant(s) is especially advantageous as it makes cells amendable for cryopreservation without altering their genetic background. Thus, cells gain preferably only transiently cryoprotective properties required for cryopreservation. This is especially advantageous for (bio)medical applications like tissue and / or organ transplantation as no exogenously added biological information will remain in said tissue and / or organ after thawing on a long-term. Depending on the specific purpose, cells, cell aggregates, cell cultures, tissues, organoids and / or organs may also be subjected to more than one round of cryopreservation by repeating the method of cryopreserving according to the present invention, preferably without affecting cell characteristics and / or genetic backgrounds.

[0130] In some embodiments of the present invention, said cell, cell aggregate, cell culture, tissue, organoid and / or organ, obtained by or obtainable by the method of cryopreserving according to the present invention, has, at least transiently, a cryoprotective property, wherein said cryoprotective property is characterized by a level of viability, vitality and / or functionality of said cell, cell aggregate, cell culture, tissue, organoid and / or organ of at least 70%, preferably of at least 80%, more 38SUBSTITUTE SHEET (RULE 26)preferably of at least 90%, after freezing and thawing compared to said level before freezing and thawing. As regards said cryoprotective property and said levels of viability, vitality and / or functionality as well as their respective measures, the same applies as stated herein in the context of the intracellular cryoprotectant according to the present invention and the method of cryopreserving according to the present invention, respectively.

[0131] The present invention further relates to a frozen and thawed cell, cell aggregate, cell culture, tissue, organoid and / or organ. As regards said frozen and thawed cell, cell aggregate, cell culture, tissue, organoid and / or organ the same applies as stated above in the context of the cell, cell aggregate, cell culture, tissue, organoid and / or organ according to the present invention, obtained by or obtainable by the method of cryopreserving according to the present invention. Accordingly, also said frozen and thawed cell, cell aggregate, cell culture, tissue, organoid and / or organ are obtained by or obtainable by the method of cryopreserving according to the present invention, and preferably have, at least transiently, a cryoprotective property, wherein said cryoprotective property is preferably characterized by a level of viability, vitality and / or functionality of said cell, cell aggregate, cell culture, tissue, organoid and / or organ of at least 70%, preferably of at least 80%, more preferably of at least 90%, after freezing and thawing compared to said level before freezing and thawing.

[0132] In some embodiments of the present invention, said (frozen and thawed) cell, cell aggregate, cell culture, tissue, organoid and / or organ is a (frozen and thawed) cell, cell aggregate, cell culture, tissue, organoid and / or organ suitable for transplantation. Thus, a respective (frozen and thawed) cell, cell aggregate, cell culture, tissue, organoid and / or organ has preferably a level of viability, vitality and / or functionality in line with national requirements and stipulations in the context of (bio)medicine, and in particular transplantation. More specifically, in some embodiments of the present invention, said (frozen and thawed) cell, cell aggregate, cell culture, tissue, organoid and / or organ is a (frozen and thawed) cell, cell aggregate, cell culture, tissue, organoid and / or organ suitable for transplantation, wherein said (frozen and thawed) cell, cell aggregate, cell culture, tissue, organoid and / or organ is suitable for transplantation and is of artificial origin. Examples of an artificial origin include for example in vitro engineered tissues and the like. In some embodiments of the present invention, said (frozen and thawed) cell, cell aggregate, cell culture, tissue, organoid and / or organ is a (frozen and thawed) cell, cell aggregate, cell culture, tissue, organoid and / or organ suitable for transplantation obtained by contacting said cell, cell aggregate, cell culture, tissue, organoid and / or organ with a nucleotide sequence encoding an intracellular cryoprotectant according to the present invention and / or a nanoparticle comprising a nucleotide sequence encoding an intracellular cryoprotectant according to the present invention at least transiently and / or partially39SUBSTITUTE SHEET (RULE 26)in a physiological environment. Alternatively or optionally, in some embodiments of the present invention said (frozen and thawed) cell, cell aggregate, cell culture, tissue, organoid and / or organ is a (frozen and thawed) cell, cell aggregate, cell culture, tissue, organoid and / or organ to be transplanted. This is especially advantageous as the present disclosure paves the way to cryopreserve cells, cell aggregates, cell cultures, tissues, organoids and / or organs by transiently making them capable of synthesizing an intracellular cryoprotectant according to the present invention without intervening with their physiological properties on a longer term. Moreover, while in particular well-established vaccination based approaches may be used, (bio)medical needs and / or logistic limitations in transplantation medicine may be overcome.

[0133] In some embodiments of the present invention, said (frozen and thawed) cell, cell aggregate, cell culture, tissue, organoid and / or organ is a "ready-to-use" (frozen and thawed) cell, cell aggregate, cell culture, tissue, organoid and / or organ, preferably a (frozen and thawed) "ready-to- use" "organ-on-a-chip". Thus, using the method of cryopreserving according to the present invention, cells, cell aggregates, cell cultures, tissues, organoids and / or organs can be cryopreserved in a manner that preserves their initial viability, vitality and / or functionality, stored as required and that are ready to use upon thawing. Said "ready-to-use" (frozen and thawed) cell, cell aggregate, cell culture, tissue, organoid and / or organ open new avenues for a range of (bio-) medical applications including highly relevant applications in the context of transplantation medicine. Moreover, said (frozen and thawed) "ready-to-use" "organs-on-a-chip" are advantageous as they make cryopreservation amenable to pharmaceutical and (bio-) medical applications such as ready-to-use tissue cultures for drug screening. In particular, such "ready-to-use" cell systems and especially "ready-to-use" "organs-on-a-chip" systems represent a promising tool, for example, for screening drug candidates in a very early stage during the drug developmental process and / or (pre-) clinical stage for efficacy and toxicity. The "organs-on-a-chip" technology allows, for example, human cells cultivation in 3D in vitro, while representing organs under physiological conditions. Hence, "ready-to- use" "organs-on-a-chip" may be established that represent a specific patient and / or reflect a given patient group and can thus be used, for example in the context of personalized medicine, for drug screening when required just by thawing a "organ-on-a-chip" cryopreserved according to the respective method according to the present invention disclosed herein.

[0134] The present invention further relates to the use of a (frozen and thawed) cell, cell aggregate, cell culture, tissue, organoid and / or organ according to the present invention for drug development, active ingredient and / or drug screening, ((pre-)clinical) drug testing and / or (bio-) medicine. In some embodiments of the present invention, said use is a use for (bio-) medicine and preferably for transplantation medicine.40SUBSTITUTE SHEET (RULE 26)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 the drawings.

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

[0137] Figure 2: Schematic illustration of expected survival rates of cells in suspension, wherein the (suspension) medium comprises an added cryoprotectant in a concentration encompassed in the range indicated with "A" in Figure 1 in higher resolution. "Course 1" shows an hypothetical example of a course without added cryoprotectant and thus, an example in which cells in suspension are frozen in a (suspension) medium in the absence of an added cryoprotectant, wherein approximately 1 cell survives freezing and thawing of initially 10A6 cells. "Course 2" shows another hypothetical example of a course, wherein surviving cells are found when freezing and thawing are performed at least in the presence of some cryoprotectant added to the (suspension) medium and thus, in a (suspension) medium comprising said added cryoprotectant in a concentration of e.g. 0.5%.

[0138] Figure 3: Schematic illustration of three temperature-time protocols for cryopreservation comprising freezing followed by thawing of cells in suspension (x-axis: time; y-axis: temperature). Solid line: example of a temperature-time protocol, wherein both freezing and thawing steps are characterized in that both the reduction and the increase of the temperature of the (suspension) medium, respectively, have a step-like profile with a comparatively abrupt drop or rise in temperature as in case of shock freezing or shock heating. Dashed line: example of a temperaturetime protocol, wherein freezing is characterized by a slower reduction of temperature compared to the temperature-time protocol indicated by the solid line as in case of a "slow rate freezing" approach, and wherein thawing is characterized by an abrupt rise in temperature. Dotted line: example of a temperature-time protocol, wherein both freezing and thawing steps are characterized in that both the reduction and the increase of the temperature of the (suspension) medium,41SUBSTITUTE SHEET (RULE 26)respectively, have a comparably slow reduction and increase of the temperature of the (suspension) medium, respectively.

[0139] Figure 4: Schematic illustration of a method according to the present invention, wherein one or more initially unknown intracellular cryoprotectants expressed by the cells under study are identified by applying an evolutionary approach (1 to 14), optionally followed by their characterization and their use (15 to 20). The latter is shown exemplarily by the steps of identifying nucleotide sequences encoding said identified cryoprotectant(s), generating nucleotide sequences encoding the same, and encapsulating and transferring them into a cell, where the information comprised in the generated nucleotide sequences is translated into one or more intracellular cryoprotectant(s) synthetized by said cell.

[0140] Figure 5: Example of a modification of the evolutionary approach depicted in Figure 4, wherein in steps 5 and 10 investigated cells are (epi-) genetically modified by modulating epigenetic masking ("EPI") and / or applying CRISPR / Cas ("CRIS").

[0141] Figure 6: Results from L929 cells in suspension that were frozen and thawed. "EK" refers to individual cell colonies (upper panel), whereas "MK" refers to cell colonies originating from different cells (lower panel). Shown are results from sample 2A obtained on day 1, day 3, day 5, and day 7 (from left to right).

[0142] Figure 7: Results from L929 cells in suspension that were frozen and thawed. "EK" refers to individual cell colonies (upper panel), whereas "MK" refers to cell colonies originating from different cells (lower panel). Shown are results from sample 2B obtained on day 1, day 3, day 5, and day 7 (from left to right).

[0143] Figure 8: Results from L929 cells in suspension that were frozen and thawed. "EK" refers to individual cell colonies (upper panel), whereas "MK" refers to cell colonies originating from different cells (lower panel). Shown are results from sample 2C obtained on day 1, day 3, day 5, and day 7 (from left to right). No living cells could be obtained for the 2C MK-2 sample after day 1.

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

[0145] 0ther aspects and advantages of the invention will be described in the following examples, which are given for purposes of illustration and not by way of limitation.42SUBSTITUTE SHEET (RULE 26)Examples

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

[0147] Figure 1 shows an illustrative example of the effect of an added cryoprotectant as a function of its concentration on the survival rate of cells in suspension, assuming that the cells are not capable of expressing an intracellular cryoprotectant.

[0148] Area A, graphically indicated by an arrow pointing downwards to the left, relates to an area characterized by a comparatively low concentration of an added cryoprotectant. Concentrations within said area are considered disadvantageous as the positive protective effect of the added cryoprotectant cannot outweigh the negative effects of freezing and thawing on cell survival and vitality. Consequently, concentrations of an added cryoprotectant within said range results in low cell survival rates. In contrast, area C, graphically indicated with an arrow pointing downwards to the right refers to an area characterized by a comparatively high concentration of an added cryoprotectant. Concentrations within said area are also considered disadvantageous, since such concentrations usually have a negative effect on cell survival and may even be toxic for the cells. Consequently, also concentrations of an added cryoprotectant within said range result in low cell survival rates.

[0149] Area B, graphically indicated by an arrow pointing upwards to the left, relates to an area characterized by intermediate concentrations of an added cryoprotectant. In said range, an optimum can usually be observed, as the negative effects of the concentration of the added cryoprotectant, and thus the stress on the cell associated with its presence, are outweighed by the positive effects of the added cryoprotectant, which limits or prevents freezing damage in the cells. Consequently, previous studies focused on area B for testing cryoprotective properties of added cryoprotectants like DMSO. Furthermore, previous investigations usually had the goal of optimizing area B with regard to the lowest possible concentration of the added cryoprotectant that still provides sufficient cryoprotection of the cells in suspension. Thus, investigations focused so far on optimizing the concentration of an added cryoprotectant within area B towards low concentrations of the added cryoprotectant within area B.43SUBSTITUTE SHEET (RULE 26)

[0150] In contrast, the present disclosure focuses on concentrations of an added cryoprotectant within region A concerning the method according to the present invention for identifying an intracellular cryoprotectant that is expressed by and within a cell. This has the advantage that cryoprotection for cells, preferably in suspension, can be optimized in the presence of unprecedentedly low concentrations of an added cryoprotectant, preferably even in its absence. Thus, cellular stress through added cryoprotectants can be minimized and limitations overcome that are associated with the used of added cryoprotectants like size restrictions and gradient formation.

[0151] When considering a concentration of an added cryoprotectant in a comparatively low range as indicated by A in Figure 1, the following hypothetical examples may be considered to illustrate expected survival rates of cells in suspension. In Figure 2, a likely result is indicated by the solid line and referred to as "Course 2". Here, no or only a very limited number of cells is obtained after freezing and thawing in the presence of at least some added cryoprotectant concentration in the suspension. However, the situation is expected to change, when the cells that are frozen and thawed are capable of expressing and / or synthesizing at least some amount of an intracellular cryoprotectant. Hence, the dashed line, referred to also as "Course 1" illustrates the outcome, when cells capable of expressing and / or synthetizing intracellular cryoprotectants are frozen and thawed in suspension in the absence of an added cryoprotectant. Thus, while Course 1 will be observed most likely in the initial phase of the method according to the invention for identifying intracellular cryoprotectants, the approach of repeated freezing and thawing while modifying the cells is expected to result in cells that show a behavior in line with Course 1. Hence, by increasing selective pressure due to decreasing concentrations of added cryoprotectant modified cells can be obtained that show improved cryoprotection even in the absence of an added cryoprotectant like DMSO in the suspension during freezing and thawing.Illustrative temperature-time protocols

[0152] Temperature-time protocols for cryopreservation of cells are well known in the art and the skilled person is aware that parameters of such a regime are to be chosen in view of the cell under study, for example in view of cell type and species. Figure 3 depicts schematically three illustrative temperature-time protocols, which may be particularly advantageous for the cryopreservation of cells in suspension. Said protocols are characterized by at least two temperature changes.

[0153] More specifically, the temperature, preferably the temperature of the suspension comprising the cells, may be reduced from a first value to a second value, wherein said reduction may be gradual or stepwise, preferably gradual. From this second value, the temperature is then preferably 44SUBSTITUTE SHEET (RULE 26)increased to a third value, wherein such increase may be gradual or stepwise. Thus, the cells are cooled, preferably frozen, in a first step and subsequently thawed in a second step. After said second step, the survival rate of the cells may be determined and thus, the number of cells that survived freezing and thawing.

[0154] The first value is preferably higher than the second value, particularly preferably by at least 20°C, 70°C, or 140°C. For example, the first value may be more 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 by at least 20°C, 70°C, or 140°C. For example, the third value may be more than 0°C, for example between 25°C and 30°C. Also, the first value and third value may be substantially identical. 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 min, preferably for at least 5 min. Preferably, the second value is substantially maintained for at least 10 sec to 60 min, more preferably for at least 30 sec to 30 min. Preferably, the second value is substantially maintained for a time period of 1 min to 100 years, preferably of 1 min to 50 years. Preferably, the third value is substantially maintained for at least 5 min.

[0156] Preferably, the decrease in temperature from said first value to said second value is performed within 0.1 sec to 60 min, preferably between circa 0.25 sec to 20 min, particularly preferably between circa 0.5 sec to 15 min. Preferably, the increase in temperature from said second value to said third value is performed within 0.1 sec to 60 min, preferably between 0.25 sec to 20 min, particularly preferably between 0.5 sec to 15 min.

[0157] Exemplarily, three potential temperature-time protocols for cryopreservation are illustrated in Figure 3. Referring to the temperature-time protocol indicated by the solid line, a step-wise approach can be envisioned. In this case, the temperature-time protocol is characterized by a comparatively abrupt drop and rise in temperature, respectively, as for example in case of shock freezing or shock heating. In particular, such a comparatively abrupt freezing approach is commonly known also in the context of vitrification, wherein an instantaneous freezing due to ultra-high cooling rates is performed to allow solidification of cells without crystal ice formation. Contrarily, in case of the temperature-time protocol indicated by the dotted line, both reduction and increase of the temperature, respectively, are performed comparatively slowly. This may allow a cell to adapt to the temperature change.

[0158] Combinations and modifications of said two examples are encompassed herein as well. In particular, such a slow cooling is commonly known in the context of "slow freezing" approaches, 45SUBSTITUTE SHEET (RULE 26)wherein temperature is reduced at low cooling rates to permit adequate cellular dehydration, while minimizing intracellular ice crystal formation. Furthermore, a combination of said two exemplarily temperature-time protocols is indicated by the dashed line as an illustration of a potential modification and / or combination of the foregoing protocols. In case of the protocol indicated by the dotted line, freezing is characterized by a comparatively slow reduction of temperature as in case of a "slow rate freezing" approach, whereas thawing is characterized by a comparatively abrupt rise in temperature.

[0159] It is to be noted that the method according to the present invention for identifying an intracellular cryoprotectant may be performed using one temperature-time protocol. Alternatively or optionally, the temperature-time protocol may vary. More specifically, the temperature-time protocol may be varied between two or more iterations. This may be advantageous to assess dependencies between modifications and a chosen temperature-time protocol and their effect on cell survival and vitality. Alternatively or optionally, temperature-time protocols may be varied between cells of a given iteration that are investigated in parallel. Especially, when a proliferation step is comprised in the method according to the invention, a portion of the proliferated cells may be further used according to the method of the present invention using a first temperature-time protocol, while another portion of the proliferated cells may be used using a second temperaturetime protocol. Thus, also potential differences in the cryoprotective effect of modifications considering different temperature-time protocols can be assessed in a highly efficient throughput approach.Illustrative schematic representation of the method according to the present invention for identifying intracellular cryoprotectants

[0160] In the following the method according to the present invention for identifying an intracellular cryoprotectant will exemplarily be described in more detail with reference to Figures 4 and 5.

[0161] Cells may be obtained for example from a cold tolerant species like the wood frog and cultured in suspension. Preferably, said cells are proliferated to increase probability of obtaining cells that survived the initial freezing and thawing cycle. Thus, said cells may be cultivated in suspension until, for example, 100 to 100,000 (microplate) wells each comprising 10A5 to 10A6 cells in suspension without added cryoprotectant can be obtained (1). Said cells may be frozen and thawed in accordance with step (oO) of the method according to the present invention by applying a predetermined temperature-time protocol, for example reflecting a slow freezing approach as indicated e.g. in Figure 3 (2).46SUBSTITUTE SHEET (RULE 26)

[0162] In case no surviving cells are obtained after freezing and thawing (3a), new cells may be obtained and the method re-initiated from (1), though with a low first concentration x% of an added cryoprotectant comprised in the suspension, for example 0.1% DMSO (4a), before said cells are frozen and thawed in the suspension comprising the added cryoprotectant (5a). In case no surviving cells are obtained after said freezing and thawing (6b), another time new cells are obtained and the method re-initiated from (1), though with a slightly increased second concentration of the added cryoprotectant compared to the first concentration.

[0163] In case at least one cell is obtained that survived freezing and thawing (3b or 6a) in line with step (al) of the method according to the present invention, said at least one surviving cell is preferably proliferated (4b) in suspension in line with step (el) according to the method of the present invention. Said suspension may comprise a conditioning medium to facilitate proliferation of single or few cells. Preferably, the survival rate of the cells from said freezing and thawing cycle is determined in line with step (fl) of the method according to the present invention.

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

[0165] The preferably proliferated cells that survived freezing and thawing are then modified (5) according to step (b) of the method according to the present invention. As indicated in Figure 5, said modification may be an epigenetic modification (EPI) and / or a genetic modification using for example CRISPR / Cas (CRIS). Other examples for modifying cells may be exposure to radiation or addition of mutagenic substances. Preferably, the modified cells are proliferated (6) in line with step (e2) of the method according to the present invention. The preferably proliferated modified cells are then frozen and thawed (7), for example according to the identical temperature-time protocol as used in (2) and / or (5a). This may represent step (c) of the method according to the present invention.

[0166] Thus, at least one modified cell may be obtained that survived freezing and thawing according to step (c) in line with step (d) of the method according to the present invention (8). Preferably, the survival rate y% (8) of said freezing and thawing cycle of the modified cell(s) is determined in line with step (f2) of the method according to the present invention. In case said survival rate y% (8) is not exceeding the survival rate determined for (3b), the method may be continued at (4b) or (1). This may be iterated until a survival rate y% (8) is determined that exceeds the survival rate determined for (3b). If this is the case, said at least one modified cell obtained from (8) may be proliferated (9) in line with step (e3) the method according to the present invention. Said suspension may comprise a conditioning medium to facilitate proliferation of single or few modified cells.47SUBSTITUTE SHEET (RULE 26)

[0167] The preferably proliferated modified cells that survived "again" freezing and thawing are further modified (10) according to step (b) of the method according to the present invention. As in case of (5), said modification may be for example an epigenetic modification (EPI) and / or a genetic modification using for example CRISPR / Cas (CRIS). The approach used for modifying the cell(s) in (5) may be the same or another one than the approach chosen for further modifying the modified cell(s) in (10). Preferably, the further modified cells are proliferated (11) in line with step (e2) of the method according to the present invention. The preferably proliferated further modified cells are then frozen and thawed (12), for example according to the identical temperature-time protocol as used in (2), (5a) and / or (7). This may represent step (c) of the method according to the present invention.

[0168] Thus, at least one further modified cell may be obtained that survived freezing and thawing according to step (c) in line with step (d) of the method according to the present invention (13). Preferably, the survival rate (y+z)% (13) of said freezing and thawing cycle of the further modified cell (s) is determined in line with step ( / 2) of the method according to the present invention. In case said survival rate (y+z)% (13) is not exceeding the survival rate determined for (8), the method may be continued at (9), (4b) or (1). This may be iterated until a survival rate (y+z)% (13) is determined that exceeds the survival rate determined for (8). If this is the case, said at least one further modified cell obtained from (13) may be proliferated in line with step (e3) the method according to the present invention. Said suspension may comprise a conditioning medium to facilitate proliferation of single or few modified cells.

[0169] When a survival rate (y+z)% exceeding, e.g., 80% is determined (14), it can be assumed that said further modified cell(s) are capable of expressing an intracellular cryoprotectant that is suitable for cryopreservation. This may be understood as an example of step (d) of the method according to the present invention. Preferably, said at least one intracellular cryoprotectant is then identified (15), optionally based on the in step (e3) proliferated, (further) modified cells, if the survival rate determined in step ( / 2) is 80% or higher (14). Identification of said at least one intracellular cryoprotectant (15) may be done for example using genome sequencing and / or in case of a polypeptide or protein 2-SDS-Page gel electrophoresis. An identified intracellular cryoprotectant is preferably isolated and further characterized, for example as regards structure, function and / or cellular localization (16). For an exemplarily application (17-20), mRNA sequences may be in vitro synthesized (17) and optionally optimized, for example in view of codon usage preferences of the target species. Said generated mRNA sequences may be encapsulated (18) and introduced into target cell(s) (19) according to known techniques, for example in the context of vaccination. The target cell(s) may then synthetize (20) the intracellular cryoprotectant identified using the method48SUBSTITUTE SHEET (RULE 26)according to the present invention and are thus capable of surviving cryopreservation without substantial impact of the cell's survival and vitality.

[0170] A preferred target species may be a vertebrate like a human, and a respective target cell for example a human kidney cell. Thus, by applying for example a well-established vaccination approach to introduce mRNA sequences encoding at least one intracellular cryoprotectant identified using the method according to the present invention, cells, cell aggregates, organoids, tissues and even organs may be cryopreserved for example for organ transplantation or the preparation of ready-to-use tissue slices or organoids for drug screening approaches in a pharmaceutical context. Furthermore, as shown in the example of a vaccination based approach, the target cell(s) may only transiently possess the ability of synthetizing an intracellular cryoprotectant as the approach preferably does not alter the target cell's properties like genetic and epigenetic constitution.Cryopreservation of cells - Experiment I

[0171] L929 cells were harvested by aspirating the medium, washing 1 x with PBS, adding 2.5 ml trypsin to the cell lawn (T75), which was aspirated again after 20 sec, followed by 3 min in the incubator at 37°C and 5% CO2, before rinsing the cells with CGM (CGM: complete growth medium = RPMI 1640 + glutamine + Pen / Strep + 10% FCS). Cell number was determined and 2 x 10A6 cells were picked up in 1.0 ml CGM and transferred to cryotubes for storage using the cool box method for 24 hr at -85°C.

[0172] Cells were transferred to -150°C on day 1 and remained at -150°C for days 2-8. On day 9, cells were thawed (2 xlOA6 cells / ml per sample) as follows: 24 well cloning plates with 1 ml KCGM (conditioned CGM, sterile filtered 0.2pm) per well for 1 sample 10 wells, followed by the addition of 4.0 ml KCGM per 1 ml cell suspension and of this 0.5 ml to each 1 well of the cloning plate (with 1.0 ml KCGM submitted) = 10 wells with 2 xlOA5 cells each. In addition, 5 wells with KCGM only were used as controls. Microscopic characterization and photographs followed on days 10, 12, 14 and 16. On day 15 - 16, colonies were counted depending on colony growth, and individual colonies were picked, grown and refrozen for another thawing assay.

[0173] In total 9 samples were investigated with respective numbers of cell colonies that could be successfully obtained after freezing and thawing of L929 cells being summarized in Table 1.49SUBSTITUTE SHEET (RULE 26)Table 1: Number of cell colonies obtained from 2xlOA6 L929 cells each that were frozen and thawed.Cryopreservation of cells - Experiment II

[0174] Subsequently, a subset of samples obtained from Experiment I were investigated further following the same experimental setup as described above for Experiment I.

[0175] As shown in Figures 6 to 8, several cell colonies could be obtained from L929 cells that survived freezing and thawing as described above. More specifically, the numbers of cell colonies that could be successfully obtained per sample are summarized in Table 2. Of note, "EK" refers to individual cell colonies, and thus to single colonies obtained from surviving and grown colonies that were initially "picked" from the respective Petri dish of Experiment I. Of further note, "MK" refers to cell colonies that originated from different cells obtained from Experiment I. In the latter case no sorting took place, but all respective surviving and grown colonies were further investigated.Table 2: Number of cell colonies obtained from 2xlOA6 L929 cells each that were frozen and thawed.SUBSTITUTE SHEET (RULE 26)

[0176] As it can be seen, both experimental approaches and thus, single colony based approach as well as mixed colony based approach, showed comparable results.Identification of an intracellular cryoprotectant, generation of an mRNA sequence encoding the same, packaging and use thereof - Example I

[0177] As shown in Experiments I and II described above, it was surprisingly found that a portion of L929 cells subjected to cryopreservation following a temperature-time protocols commonly used in the art survived freezing and thawing in the absence of an added cryoprotectant like DMSO. Such cells, i.e. cells that survived freezing and thawing, are cells suitable for use in step (a) of the method of identifying an intracellular cryoprotectant expressed by a cell according to the present invention that is schematically illustrated in Figures 4 and 5. In line with said method, Experiments I and II may be done alternatively or optionally also with a very low concentration of an added cryoprotectant like DMSO, for example of less than 1% of the cell suspension medium. This would have the advantage that more surviving cells could be expected compared to the identical experiment without said added cryoprotectant and thus, as in case of Experiments I and II.

[0178] A cell, like a fibroblast and / or a stem cell, which survived freezing and thawing, can then be used in line with the method of identifying an intracellular cryoprotectant of the present invention by subjecting said cell in vitro to preferably several iterations of proliferation, modification, freezing and thawing. When a survival rate of for example more than 80% has been observed after a given iteration of freezing and thawing, the obtained modified cell that survived freezing and thawing is investigated in view of an intracellular cryoprotectant that provided cryoprotection to said cell in the presence of very low concentrations or even absence of an added cryoprotectant in the suspension during freezing and thawing of the cell. While various methods and techniques are available and well established in the art, gel electrophoresis and / or mass spectrometry may be particularly suited for identifying one ore more intracellular cryoprotectants expressed by said cell(s). Taking exemplarily an SDS gel electrophoresis, the proteome of the wildtype and the obtained cell can be compared, wherein said wildtype is preferably the initially used cell. By comparing the proteome of the wildtype, preferably the initially used cell, and the finally obtained (further) modified cell, modifications can be identified by mapping the two proteomes two-dimensionally, in the form of separate spots, in the gel so that modified polypeptides and / or proteins can be easily identified, e.g. due to a spot appearing only in the proteome SDS plot of the (further) modified cell while being absent in the respective SDS plot of the wildtype proteome or in case of visually differing spot intensities indicating differing concentrations of the respective polypeptide or protein. SDS gel51SUBSTITUTE SHEET (RULE 26)electrophoresis is an example of a routine method standardly used to determine polypeptide and / or protein differences, e.g. indicative for altered gene activities in cells, which can be easily used for identifying an intracellular cryoprotectant expressed by said (further) modified cell in contrast to the initially used cell and / or in a differing intensity compared to said initially used cell.

[0179] Such an identified polypeptide or protein can be a globular protein such as protamine and thus, an animal mixture of strongly basic peptides belonging to the protamine group that may be found, e.g., in the sperm or roe of certain salmon species, or a common cellular protein, such as a histone. Upon its initial identification using in as stated above SDS gel electrophoresis, an intracellular cryoprotectant can be further characterized in view of its structure and / or a reference amino acid sequence can be obtained from a commonly used, state-of-the-art protein databases, e.g. the National Center for Biotechnology Information (NCBI), SwissProt, Protein Information Resource (PIR) or Protein Data Base (PDB)). Based on the obtained amino acid sequence, a respective mRNA sequence encoding at least said at least one identified intracellular cryoprotectant (hereafter also referred to as cryo-mRNA) can be designed and in vitro synthesized. Optionally, also respective gene and / or mRNA sequences may be obtained from a commonly used, state-of-the-art protein database like the NIH National Library of Medicine.

[0180] As evidenced by the state-of-the-art in mRNA-COVID vaccines, the production of a suitable cryo-mRNA can be done in cell systems or fully synthetically (see, e.g., 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). Analogous to COVID vaccines, packaging of the cryo-mRNA may be done to ensure both effective cell uptake via fusion with the cell membrane as well as and suitable stability of the cryo-mRNA. According to the state-of-the-art, lipid nano-particles may be used for this purpose, which comprise a mixture of components in order to fulfill this function. As said mixture is ideally optimized in view of the target cell system, such an optimization is preferably done for the cryo-mRNA as well. According to the state-of-the-art, the following components of nano-particles are expected to show a high probability of success of introducing an mRNA sequence into a cell: a mixture of cationic and ionizable lipids, embedded structural helper lipids, polyethylene glycol (PEG) lipids as well as cholesterol, with the respective ratios to be adjusted according to the respective cryo-mRNA and target cell system.

[0181] For cryopreserving a cell in accordance with the respective method according to the present invention using in said Example the generated nano-particles comprising the cryo-mRNA, said nanoparticles may be added to a (cell) suspension and / or (cell) culture medium in a suitable concentration and for a suitable period of time depending on the target cell system. Alternatively or optionally, an organoid or organ can be contacted with said nano-particles via injecting said nano-particles into the 52SUBSTITUTE SHEET (RULE 26)organoid or organ in vitro. Within the respective cell, the cryo-mRNA may then then translated and thus, the intracellular cryoprotectant mentioned above like the globular protein such as protamine, a protein mixture and / or histone synthesized in the respective cell's cytoplasm. When the intracellular cryoprotectant's concentration is sufficiently high, the cell is expected to have gained and / or improved cryoprotective properties during subsequent freezing and thawing. Upon thawing, the cryo-mRNA that has been introduced into the cell is expected to be degraded in accordance with regular biological processes within cells. Thus, cryoprotective properties are expected to be gained and / or improved only transiently without modifying the target cell's genetic background. Hence, when applying the inventive method of cryopreserving a single cell as well as a cell comprised in for example an organ, the respective cell or for example organ is expected to be well suitable for medical and / or pharmaceutical purposes after freezing and thawing.Identification of an intracellular cryoprotectant, generation of an mRNA sequence encoding the same, packaging and use thereof - Example II

[0182] By applying the evolutionary approach of the method of identifying an intracellular cryoprotectant according to the present invention, (further) modified cells can be obtained that are characterized by a comparatively higher survival rate than the initially used cells and with improved cryoprotective properties resulting from an, preferably iterative, enrichment of modifications that positively affect the (further) modified cell's cryoprotective properties compared to the initially used cell.

[0183] Example II extends Example I by the following step: upon having identified at least one intracellular cryoprotectant and a respective nucleotide sequence, preferably a respective mRNA sequence, said nucleotide sequence can be modified and in vitro synthesized according to standard state-of-the-art methods as regards the synthesis of synthetically engineered mRNA sequence fragments (see, e.g., 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] 0ptionally or alternatively, more than one version of such a synthetically engineered mRNA sequence may be investigated. Thus, preferably in parallel, several identical cell systems may be contacted with one version each of nucleotide sequences that encode said at least one intracellular cryoprotectant. Said versions may be generated by targeted or untargeted modification of the identified and / or modified nucleotide sequence and thus, following a systemic effect evaluation approach or a random effect evaluation approach. Hence, high throughput effect screening of 53SUBSTITUTE SHEET (RULE 26)different modifications of an mRNA sequence encoding an identified intracellular cryoprotectant can be realized and the best performing version of said synthetically engineered mRNA sequence as regard cryoprotection of the respective cell system identified for further applications.Identification of an intracellular cryoprotectant, generation of an mRNA encoding the same, packaging and use thereof - Example III

[0185] Example III represents a modification of Example I and / or II by extending the respective Example by the following step of a, preferably parallel, evaluation of different temperature-time protocols during the method of identifying an intracellular cryoprotectant according to the present invention. More specifically, it may be attempted, for example, either to achieve vitrification-like conditions using ultrafast cooling rates or to apply a more classical temperature gradient with temperature changes in the range of seconds to minutes per degree. Both temperature-time protocols may be tested, but may a lead to (slightly) different results with respect to the identification of intracellular cryoprotectants. In particular, compared to ultrafast cooling and thawing rates comparatively slow freezing and thawing rates may allow larger ice crystal formation due to migratory ice growth effects at the expense of smaller ice crystals, while such migratory ice growth effects may not be seen in case of vitrification based approaches due to the respectively high cooling and thawing rates. Consequently, identified modifications and / or their cryoprotective effect may to vary between these two settings. Thus, by identifying intracellular cryoprotectants using different temperature-time protocols, different modes of action of cellular cryoprotection may be unraveled and / or highly effective intracellular cryoprotectants identified for a given temperaturetime protocol.Illustrative examples on relevant parameters regarding survivability, vitality and / or functionality

[0186] According to the method of cryopreserving of the present invention, (frozen and thawed) cells, cell aggregates, cell cultures, tissues, organoids and / or organs can be obtained, which are also encompassed by the present invention. These preferably exhibit, at least temporarily, a cryoprotective property such that a level of viability, vitality and / or functionality of the cells, cell aggregates, cell cultures, tissues, organoids and / or organs of, for example, at least 70% can be observed after freezing and thawing compared to the corresponding level before freezing and thawing. Exemplary methods and techniques for this are mentioned illustratively below.54SUBSTITUTE SHEET (RULE 26)

[0187] In the case of organoids of the heart, for example, there are established methods known to the skilled person for determining viability, vitality and / or functionality of a corresponding organoid on the basis of various parameters. For an overview of relevant parameter procedures for their determination, reference is made, for example, to the publications by 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. (Nature Communications, 2021, 12:5142; https: / / doi.org / 10.1038 / s41467-021-25329-5; Selfassembling human heart organoids for the modeling of cardiac development and congenital heart disease), Kim et al. (Nano Lett. 2022, 22, 7892-7901; https: / / doi.org / 10.1021 / acs.nanolett.2c02790; Multimodal Characterization of Cardiac Organoids Using Integrations of Pressure-Sensitive Transistor Arrays with Three Dimensional Liquid Metal Electrodes), Richards et al. (Nature Biomedical Engineering, Volume 4, pages 446-462, 2020; https: / / www.nature.com / articles / s41551-020-0539-4; Human cardiac organoids for the modelling of myocardial infarction and drug cardiotoxicity), and Zhao et al. (Stem Cell Research & Therapy, 2021, 12:272; https: / / doi.org / 10.1186 / sl3287-021- 02340-7; Cardiac organoid - a promising perspective of preclinical model). For example, a determination of the functionality can be made on the basis of calcium flux and / or electrophysiological parameters, by means of characterization of the beat frequency, RNA sequencing, metabolic evidence e.g. by means of Seahorse based analyses, oxygen consumption rate (OCR, oxygen consumption rate) and / or transmission electron microscopy (TEM, transmission electron microscopy) based analyses, but also microscopically and immune-histologically relevant parameters and analyses can be used and / or taken into account.

[0188] Known procedures and methods can also be found well established for numerous other examples of cells, cell aggregates, cell cultures, tissues, organoids and / or organs. Thus, by way of example, mentioned herein are the publication by Marthaler et al. (Stem Cell Research 16 (2016) 202-205; Generation of an isogenic, gene-corrected control cell line of the spinocerebellar ataxia type 2 patient-derived iPSC line H266; http: / / dx.doi.Org / 10.1016 / j.scr.2015.12.048), which lists relevant parameters, particularly for demonstrating pluripotency of stem cells, and the publication by Lancaster et al. (Nature, 2013, 501(7467); doi:10.1038 / naturel2517; Cerebral organoids model human brain development and microcephaly), which lists relevant parameters in the case of brain organoids, particularly also in the context of disease patterns and / or models such as microcephalitis.

[0189] The invention is also characterized by the following items.55SUBSTITUTE SHEET (RULE 26)1. A method of identifying an intracellular cryoprotectant expressed by a cell, said method comprising(a) Using a cell that survived freezing and thawing,(b) Modifying said used cell,(c) Freezing and thawing said modified cell, and(d) Obtaining a modified cell, which survived step (c), whereby an intracellular cryoprotectant expressed by said cell is identified.2. Method according to item 1, wherein said cell has been obtained from a vertebrate, an insect, a plant, an algae, a fungus or a bacterium.3. Method according to item 1 or 2, wherein said cell and / or modified cell is capable of being cultivated in suspension and / or is a cell in suspension.4. Method according to any one of the preceding items, wherein in step (a) said cell survived freezing and thawing in a suspension, which preferably comprised an added cryoprotectant in a concentration of 5% or less, preferably of 1% or less, more preferably of 0%.5. Method according to any one of the preceding items, wherein in step (c) said modified cell is frozen and thawed in a suspension, which preferably comprises an added cryoprotectant in a concentration of 5% or less, preferably of 1% or less, more preferably of 0%.6. Method according to any one of the preceding items, wherein in step (a) and / or (c) i) Said freezing comprises a reduction from a temperature of more than 0°C to a temperature of less than 0°C, preferably of less than -20°C, more preferably of less than -70°C, even more preferably of less than -140°C, and / or ii) Said thawing comprises an increase from a temperature of less than 0°C, preferably of less than -20°C, more preferably of less than -70°C, even more preferably of less than - 140°C, to a temperature of more than 0°C, wherein preferably said temperature is the temperature of the suspension, in which the used cell survived freezing and thawing, and / or the modified cell is frozen and thawed, and / or wherein preferably after said reduction of the temperature the reduced temperature is maintained for at least 1 sec to 10 min, preferably from at least 10 sec to 5 min, before thawing.7. Method according to any one of the preceding items, wherein in step (b) said cell is modified by56SUBSTITUTE SHEET (RULE 26)i) Exposure to a mutagenic substance, preferably a mutagenic substance selected from the group consisting of a polycyclic aromatic hydrocarbon, a nitrosamine, a base analog, a peroxide and a combination thereof, ii) Exposure to radiation, preferably a high-energetic radiation, more preferably a UV- and / or X-ray radiation, iii) One or more modifying proteins, preferably selected from the group consisting of TALEN, a zink finger protein, a CRISPR / Cas combination, TET1, p300, DNMT3A, MQ.1, and LSD1, wherein said one or more modifying proteins more preferably comprises a CRISPR / Cas combination, and / or iv) Any combination of the foregoing.8. Method according to any one of the preceding items, further comprising(e) Proliferating said obtained ((further) modified) cell, preferably in suspension.9. Method according to item 8, wherein in step (e) i) Said suspension is a suspension, wherein an added cryoprotectant is comprised in a concentration of 5% or less, preferably of 1% or less, more preferably of 0%, and / or ii) Said suspension comprises a conditioning (suspension) medium.10. 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), step (c) and (d), optionally step (e) ] in i iterations, wherein i is preferably at least 1 and maximal 1,000.11. 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 maximal 1,000.12. Method according to item 10 or 11, wherein one, preferably every, step (b) and / or (c) following a step (e) is performed for at least one, preferably for at least 10%, more preferably for all proliferated ((further) modified) cells obtained after step (e).13. Method according to any one of items 10 to 12, further comprising:(f) Determining a survival rate of said obtained (further) modified cells for, preferably every, step (d).14. Method according to item 13, comprising an iteration i+1, if the survival rate determined in step (f) of iteration i is at least as high as, preferably by 10% higher than, the survival rate determined in step (f) of iteration i-1 and preferably not exceeding a given threshold.57SUBSTITUTE SHEET (RULE 26)Method according to any one of items 10 to 14, comprising the following steps: step (a), optionally followed by step (e), followed by [step (b), optionally step (e), step (c) and (d), optionally step (f), optionally step (e)] in i iterations, wherein i is at least 2, and preferably further comprising an iteration i+1, if the in step (f) of iteration i determined survival rate is at least as high, preferably by 10% higher than, the survival rate determined in step (f) of iteration i-1 and preferably not exceeding a given threshold. 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), step (c) and (d), step (f), step (e)] in i iterations, wherein i is at least 2, and further comprising an iteration i+1, if the in step (f) of iteration i determined survival rate is at least as high, preferably by 10% higher than, the survival rate determined in step (f) of iteration i-1 and preferably not exceeding a given threshold. Method according to any one of the preceding items, wherein an intracellular cryoprotectant expressed by said (further) modified cell is identified by analyzing the genome, transcriptome, epigenome, proteome and / or metabolome, preferably by using an analysis selected from the group consisting of gel electrophoresis, mass spectrometry, crystal structure analysis, NMR- spectroscopy, DNA sequencing, (m)RNA sequencing and any combination of the foregoing. Method according to any one of the preceding items, wherein an intracellular cryoprotectant expressed by said (further) modified cell is identified, if the survival rate determined in step (f) is exceeding a given threshold. Method according to any one of the preceding items, further comprising(h) Modifying said identified intracellular cryoprotectant expressed by said (further) modified cell, whereby a derivate is obtained from said identified intracellular cryoprotectant expressed by said (further) modified cell. Method according to any one of the preceding items, comprising the following steps:(a) Using a cell that survived freezing and thawing, wherein step (a) preferably comprises the following steps:(aO) Freezing and thawing of cells in suspension,(al) Obtaining a cell that survived step (aO),(fl) Determining a survival rate for step (aO) based on the number of living cells of the cells in suspension before freezing and thawing and after freezing and thawing,(el) Proliferating said cell obtained in step (al) and using the proliferated cell,58SUBSTITUTE SHEET (RULE 26)(b) Modifying said cell used in step (a), preferably said cell proliferated in step (el) and / or said cell proliferated in step (e3),(e2) Proliferating said in step (b) modified cell,(c) Freezing and thawing the in step (e2) proliferated modified cell in suspension,(d) Obtaining a modified cell, which survived step (c), whereby an intracellular cryoprotectant expressed by said cell is identified, wherein preferably freezing and thawing in step (a), preferably in step (aO), and / or in step (c) is performed in a suspension, which preferably comprises an added cryoprotectant in a concentration of 5% or less, preferably of 1% or less, more preferably of 0%.21. Method according to item 20, wherein an intracellular cryoprotectant expressed by said modified cell is identified by:(f2) Determining a survival rate for step (c) based on the number of living modified cells in suspension before freezing and thawing and after freezing and thawing,(e3) Proliferating said in step (d) obtained modified cell, and(g) Identifying at least one intracellular cryoprotectant based on the in step (e3) proliferated modified cells, preferably if the survival rate determined in step (f2) is exceeding a given threshold, wherein steps [ (b), (e2), (c), (d), (f2), (e3) ] are iterated (iteration i+1), if the survival rate determined in step (f2) of iteration i is at least as high, preferably by 10% higher than, the survival rate determined in step (f2) of iteration i-1 and preferably not exceeding said given threshold, and wherein i is at least 2.22. Method according to item 21, wherein the suspension in step (c) of iteration i+1 comprises(1) Substantially the same concentration of the added cryoprotectant as the suspension in step (c) of iteration i,If the survival rate determined in step (f2) is at least as high as, preferably by at least 5% higher than, the survival rate determined in i) step (fl) in case of the first iteration (i=l), or ii) step (f2) of iteration i-1 in case of iteration i,(2) A concentration of the added cryoprotectant that is between 0.001% and 1%, preferably between 0.01% and 0.5%, higher than in the suspension in step (c) of iteration i, if neither i) nor ii) are fulfilled.23. An intracellular cryoprotectant, or a derivate thereof, preferably obtained by or obtainable by the method according to any one of the preceding items, wherein the intracellular59SUBSTITUTE SHEET (RULE 26)cryoprotectant is an intracellular cryoprotectant that is synthesized by a cell and that is preferably an, optionally glycosylated, polypeptide or protein. Intracellular cryoprotectant, or derivate thereof, according to item 23, wherein the intracellular cryoprotectant is selected from the group consisting of an anti-freeze protein, a protein or polypeptide with an alanine- and / or threonine-rich structure analogous to antifreeze proteins and tertiary structure, a glycoprotein, a cytoplasmatic protein or polypeptide, a heat shock protein, an albumin, a globulin, a histone, a protamine, a kinase, a growth factor, a globular protein or polypeptide, a membrane-bound and / or superimposed protein or polypeptide, a porine, an osmotically active protein or polypeptide, a fibrillary protein or polypeptide, a polypeptide or protein involved in membrane lipid and / or fatty acid synthesis, a phospholipid, a glycolipid, cholesterol, a polypeptide or protein involved in glycogen synthesis, insulin, and a core protein. Intracellular cryoprotectant of item 23 or 24, wherein the intracellular cryoprotectant is capable of cryopreserving a cell, preferably a cell aggregate, a cell culture, a tissue, an organoid, and / or an organ. A method of generating a, optionally modified, nucleotide sequence encoding an intracellular cryoprotectant, or derivate thereof, according to any one of items 23 to 25. Method of according to item 26, wherein generating said, optionally modified, nucleotide sequence comprises the steps of(a) Identifying a nucleotide sequence encoding said intracellular cryoprotectant or derivate thereof,(b) Optionally modifying said identified nucleotide sequence, and(c) In vitro synthesizing said identified, optionally modified, nucleotide sequence. A, optionally modified, nucleotide sequence obtained by or obtainable by the method according to item 26 or 27, encoding one or more intracellular cryoprotectant(s) or derivative(s) thereof according to any one of items 23 to 25. A nanoparticle comprising a, optionally modified, nucleotide sequence according to item 28, preferably wherein said nanoparticle is a lipid-nanoparticle. The method according to item 26 or 27, the optionally modified nucleotide sequence according to item 28, and / or nanoparticle according to item 29, wherein said, optionally modified, nucleotide sequence is an RNA sequence, preferably an mRNA sequence.60SUBSTITUTE SHEET (RULE 26)31. Use of an intracellular cryoprotectant or derivative thereof according to any one of items 23 to 25, of a, optionally, modified nucleotide sequence according to item 28 or 30, a nanoparticle according to item 29 or 30 for cryopreserving a cell, cell aggregate, cell culture, tissue, organoid, and / or organ.32. A method of cryopreserving a cell, a cell aggregate, a cell culture, a tissue, an organoid, and / or an organ, comprising the following steps of(a) Contacting a cell, cell aggregate, cell culture, tissue, organoid and / or organ with a, optionally modified, nucleotide sequence according to item 28 or 30 and / or a nanoparticle according to item 29 or 30, and(b) Freezing said cell, cell aggregate, cell culture, tissue, organoid and / or organ obtained from step (a), wherein freezing comprises a reduction from a temperature of said cell, cell aggregate, cell culture, tissue, organoid and / or organ of more than 0°C to a temperature of less than 0°C, preferably of less than -20°C, more preferably of less than -70°C, even more preferably of less than -140°C.33. Method according to item 32, wherein step (a) comprises the steps of(al) Contacting said cell, cell aggregate, cell culture, tissue, organoid and / or organ with a, optionally modified, nucleotide sequence according to item 28 or 30 encoding a first intracellular cryoprotectant or derivative thereof and / or with a nanoparticle according to item 29 or 30 comprising a, optionally modified, nucleotide sequence encoding a first intracellular cryoprotectant or derivative thereof, and(a2) Contacting said cell, cell aggregate, cell culture, tissue, organoid and / or organ with a further, optionally modified, nucleotide sequence according to item 28 or 30 encoding a second intracellular cryoprotectant or derivative thereof and / or with a further nanoparticle according to item 29 or 30 comprising a, optionally modified, nucleotide sequence encoding a second intracellular cryoprotectant or derivative thereof.34. Method according to any one of items 32 or 33, further comprising(c) Thawing said frozen cell, cell aggregate, cell culture, tissue, organoid and / or organ, and / or(d) Determining a level of viability, vitality and / or functionality of said cell, cell aggregate, cell culture, tissue, organoid and / or organ before step (a), between steps (a) and (b), and / or after step (c), wherein said level of viability, vitality and / or functionality is at least 70%, preferably of at least 80%, more preferably of at least 90%, after freezing and thawing compared to said level before freezing and thawing.61SUBSTITUTE SHEET (RULE 26)35. The method according to any one of the items 32 to 34, wherein said cell, cell aggregate, cell culture, tissue, organoid and / or organ is preferably a cell, cell aggregate, cell culture, tissue, organoid and / or organ from a vertebrate or a plant, preferably from a vertebrate, more preferably from a human.36. Cell, cell aggregate, cell culture, tissue, organoid and / or organ obtained by or obtainable by the method according to any one of items 32 to 35.37. Cell, cell aggregate, cell culture, tissue, organoid and / or organ according to item 36, wherein said cell, cell aggregate, cell culture, tissue, organoid and / or organ has, preferably at least transiently, a cryoprotective property.38. Cell, cell aggregate, cell culture, tissue, organoid and / or organ according to item 36 or 37, wherein said cryoprotective property is characterized by a level of viability, vitality and / or functionality of said cell, cell aggregate, cell culture, tissue, organoid and / or organ of at least 70%, preferably of at least 80%, more preferably of at least 90%, after freezing and thawing compared to said level before freezing and thawing.39. A frozen and thawed cell, cell aggregate, cell culture, tissue, organoid and / or organ according to any one of items 36 to 38.40. (Frozen and thawed) Cell, cell aggregate, cell culture, tissue, organoid and / or organ according to any one of items 36 to 39, wherein said (frozen and thawed) cell, cell aggregate, cell culture, tissue, organoid and / or organ is a "ready-to-use" (frozen and thawed) cell, cell aggregate, cell culture, tissue, organoid and / or organ, preferably a (frozen and thawed) "ready-to-use" "organ-on-a-chip".41. (Frozen and thawed) Cell, cell aggregate, cell culture, tissue, organoid and / or organ according to any one of items 36 to 40, wherein said (frozen and thawed) cell, cell aggregate, cell culture, tissue, organoid and / or organ is suitable for transplantation and / or a (frozen and thawed) cell, cell aggregate, cell culture, tissue, organoid and / or organ to be transplanted.42. Use of a (frozen and thawed) cell, cell aggregate, cell culture, tissue, organoid and / or organ according to any one of items 36 to 41 for drug development, active ingredient and / or drug screening, ((pre-)clinical) drug testing and / or (bio-)medicine, preferably transplantation medicine.The invention is also characterized by the following items:62SUBSTITUTE SHEET (RULE 26)1. A method of identifying an intracellular cryoprotectant expressed by a cell, said method comprising(a) Using a cell that survived freezing and thawing,(b) Modifying said used cell,(c) Freezing and thawing said modified cell, and(d) Obtaining a modified cell, which survived step (c), whereby an intracellular cryoprotectant expressed by said cell is identified.2. Method according to item 1, wherein said cell and / or modified cell is capable of being cultivated in suspension and / or is a cell in suspension.3. Method according to item 1 or 2, wherein in step (a) said cell survived freezing and thawing in a suspension, which preferably comprised an added cryoprotectant in a concentration of 5% or less, preferably of 1% or less, more preferably of 0%, and / or wherein in step (c) said modified cell is frozen and thawed in a suspension, which preferably comprises an added cryoprotectant in a concentration of 5% or less, preferably of 1% or less, more preferably of 0%.4. Method according to any one of the preceding items, further comprising(e) Proliferating said obtained ((further) modified) cell, preferably in suspension.5. 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), step (c) and (d), optionally step (e) ] in i iterations, wherein i is preferably at least 1 and maximal 1,000.6. Method according to item 5, further comprising:(f) Determining a survival rate of said obtained (further) modified cells for, preferably every, step (d).7. Method according to item 6, comprising an iteration i+1, if the survival rate determined in step (f) of iteration i is at least as high as, preferably by 10% higher than, the survival rate determined in step (f) of iteration i-1 and preferably not exceeding a given threshold.8. Method according to any one of items 6 or 7, comprising the following steps: step (a), optionally followed by step (e), followed by [ step (b), optionally step (e), step (c) and (d), optionally step (f), optionally step (e) ] in i iterations, wherein i is at least 2, and preferably further comprising an iteration i+1, if the in step (f) of iteration i determined survival rate is at least as high, preferably by 10% higher than, the survival rate determined in step (f) of iteration i-1 and preferably not exceeding a given threshold.63SUBSTITUTE SHEET (RULE 26)9. Method according to any one of the preceding items, wherein an intracellular cryoprotectant expressed by said (further) modified cell is identified by analyzing the genome, transcriptome, epigenome, proteome and / or metabolome, preferably by using an analysis selected from the group consisting of gel electrophoresis, mass spectrometry, crystal structure analysis, NMR- spectroscopy, DNA sequencing, (m)RNA sequencing and any combination of the foregoing.10. Method according to any one of items 6 to 9, wherein an intracellular cryoprotectant expressed by said (further) modified cell is identified, if the survival rate determined in step (f) is exceeding a given threshold.11. Method according to any one of the preceding items, further comprising(h) Modifying said identified intracellular cryoprotectant expressed by said (further) modified cell, whereby a derivate is obtained from said identified intracellular cryoprotectant expressed by said (further) modified cell.12. Method according to any one of the preceding items, comprising the following steps:(a) Using a cell that survived freezing and thawing, wherein step (a) preferably comprises the following steps:(aO) Freezing and thawing of cells in suspension,(al) Obtaining a cell that survived step (aO),(fl) Determining a survival rate for step (aO) based on the number of living cells of the cells in suspension before freezing and thawing and after freezing and thawing,(el) Proliferating said cell obtained in step (al) and using the proliferated cell,(b) Modifying said cell used in step (a), preferably said cell proliferated in step (el), and / or said cell proliferated in step (e3),(e2) Proliferating said in step (b) modified cell,(c) Freezing and thawing the in step (e2) proliferated modified cell in suspension,(d) Obtaining a modified cell, which survived step (c), whereby an intracellular cryoprotectant expressed by said cell is identified, wherein preferably freezing and thawing in step (a), preferably in step (aO), and / or in step (c) is performed in a suspension, which preferably comprises an added cryoprotectant in a concentration of 5% or less, preferably of 1% or less, more preferably of 0%.13. Method according to item 12, wherein an intracellular cryoprotectant expressed by said modified cell is identified by:(f2) Determining a survival rate for step (c) based on the number of living modified cells in suspension before freezing and thawing and after freezing and thawing,64SUBSTITUTE SHEET (RULE 26)(e3) Proliferating said in step (d) obtained modified cell, and(g) Identifying at least one intracellular cryoprotectant based on the in step (e3) proliferated modified cells, if the survival rate determined in step (f2) is exceeding a given threshold, wherein steps [ (b), (el), (c), (d), (f2), (e3) ] are iterated (iteration i+1), if the survival rate determined in step (f2) of iteration i is at least as high, preferably by 10% higher than, the survival rate determined in step (f2) of iteration i-1 and preferably not exceeding said given threshold, and wherein i is at least 2.14. Method according to item 13, wherein the suspension in step (c) of iteration i+1 comprises(1) Substantially the same concentration of the added cryoprotectant as the suspension in step (c) of iteration i,If the survival rate determined in step (f2) is at least as high as, preferably by at least 5% higher than, the survival rate determined in i) step (fl) in case of the first iteration (i=l), or ii) step (f2) of iteration i-1 in case of iteration i,(2) A concentration of the added cryoprotectant that is between 0.001% and 1%, preferably between 0.01% and 0.5%, higher than in the suspension in step (c) of iteration i, if neither i) nor ii) are fulfilled.15. An intracellular cryoprotectant, or a derivate thereof, obtained by or obtainable by the method according to any one of the preceding items, wherein the intracellular cryoprotectant is an intracellular cryoprotectant that is synthesized by a cell and that is preferably an, optionally glycosylated, polypeptide or protein.

[0190] It will be readily apparent to a person skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0191] AII patents, patent applications and publications mentioned in the specification are indicative of the levels of those of ordinary skill in the art to which the 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.65SUBSTITUTE SHEET (RULE 26)

[0192] The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention. The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. In addition, where 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. Further embodiments of the invention will become apparent from the following claims.SUBSTITUTE SHEET (RULE 26)

Claims

Claims A method of identifying an intracellular cryoprotectant expressed by a cell, said method comprising(a) Obtaining a cell that survived freezing and thawing,(b) Modifying the cell of step (a) on a genetic level,(c) Freezing and thawing said modified cell, and(d) Obtaining a modified cell, which survived step (c), whereby an intracellular cryoprotectant expressed by said cell is identified by suitable methods. Method according to claim 1, wherein the cell is selected from a prokaryote, a eukaryote, an vertebrate, an insect, a plant, an algae, a fungus, and a bacterium; preferably the cell is selected from a frog, preferably Rana sylvatica, and a fish, preferably sea raven like a silverspotted sculpin - Blepsias cirrhosis -, from an Artic and / or Antarctic species of cods, from a flounder species like a winter flounder -Pleuronectes americanus - and a yellowtail flounder - Limanda ferruginea preferably the algae cell is selected from cryospheric species like a snow algae, preferably from an Antarctic species of the genus Chlorominima, an Arctic and / or Antarctic species of the genus Chlamydomonas, a species of the genus Chloromonas, preferably Chloromonas nivalis, Chloromonas rostafinskii, Ancylonema nordenskibldii, from a green algae, preferably from a species of the genus Chlorococum, preferably Chlorococum sp., and a species of the genus Raphidonema, preferably Raphidonema brevirostre, or Raphidonema nivale. Method according to claim 1 or 2, wherein said cell and / or modified cell is capable of being cultivated in suspension and / or is a cell in suspension. Method according to any one of the preceding claims, wherein in step (a) said cell survived freezing and thawing in a suspension, which preferably comprised an added cryoprotectant in a concentration of 5% or less, preferably of 1% or less, more preferably of 0%, and / or wherein in step (c) said modified cell is frozen and thawed in a suspension, which preferably comprises an added cryoprotectant in a concentration of 5% or less, preferably of 1% or less, more preferably of 0%. Method according to any one of the preceding claims, further comprising(e) Proliferating said obtained modified cell, preferably further modified cell, preferably in suspension.67SUBSTITUTE SHEET (RULE 26)Method according to any one of the preceding claims, comprising 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 maximal 1,000. Method according to claim 6, further comprising:(f) Determining a survival rate of said obtained (further) modified cells for, preferably every, step (d). Method according to claim 7, comprising an iteration i+1, if the survival rate determined in step (f) of iteration i is at least as high as, preferably by 10% higher than, the survival rate determined in step (f) of iteration i-1 and preferably not exceeding a given threshold. Method according to any one of claims 7 or 8, comprising the following steps: step (a), optionally followed by step (e), followed by [ step (b), optionally step (e), step (c) and (d), optionally step (f), optionally step (e) ] in i iterations, wherein i is at least 2, and preferably further comprising an iteration i+1, if the in step (f) of iteration i determined survival rate is at least as high, preferably by 10% higher than, the survival rate determined in step (f) of iteration i-1 and preferably not exceeding a given threshold. Method according to any one of the preceding claims, wherein an intracellular cryoprotectant expressed by said (further) modified cell is identified by analyzing the genome, transcriptome, epigenome, proteome and / or metabolome, preferably by using an analysis selected from the group consisting of gel electrophoresis, mass spectrometry, crystal structure analysis, NMR- spectroscopy, DNA sequencing, (m)RNA sequencing and any combination of the foregoing. Method according to any one of claims 7 to 10, wherein an intracellular cryoprotectant expressed by said (further) modified cell is identified, if the survival rate determined in step (f) is exceeding a given threshold. Method according to any one of the preceding claims, further comprising(h) Modifying said identified intracellular cryoprotectant expressed by said (further) modified cell on a genetic level, whereby a derivate is obtained from said identified intracellular cryoprotectant expressed by said (further) modified cell. Method according to any one of the preceding claims, comprising the following steps:(a) Obtaining a cell that survived freezing and thawing, wherein step (a) preferably comprises the following steps:(aO) Freezing and thawing of cells in suspension,(al) Obtaining a cell that survived step (aO),68SUBSTITUTE SHEET (RULE 26)(fl) Determining a survival rate for step (aO) based on the number of living cells of the cells in suspension before freezing and thawing and after freezing and thawing,(el) Proliferating said cell obtained in step (al) and obtaining the proliferated cell,(b) Modifying said cell of step (a) on a genetic level, preferably said cell proliferated in step (el), and / or said cell proliferated in step (e3),(e2) Proliferating said modified cell of step (b),(c) Freezing and thawing the in step (e2) proliferated modified cell in suspension,(d) Obtaining a modified cell, which survived step (c), whereby an intracellular cryoprotectant expressed by said cell is identified, wherein preferably freezing and thawing in step (a), preferably in step (aO), and / or in step (c) is performed in a suspension, which preferably comprises an added cryoprotectant in a concentration of 5% or less, preferably of 1% or less, more preferably of 0%. Method according to claim 13, wherein an intracellular cryoprotectant expressed by said modified cell is identified by:(f2) Determining a survival rate for step (c) based on the number of living modified cells in suspension before freezing and thawing and after freezing and thawing,(e3) Proliferating said obtained modified cell of step (d), and(g) Identifying at least one intracellular cryoprotectant based on the in step (e3) proliferated modified cells, if the survival rate determined in step (f2) is exceeding a given threshold, wherein steps [ (b), (e2), (c), (d), (f2), (e3) ] are iterated (iteration i+1), if the survival rate determined in step (f2) of iteration i is at least as high, preferably by 10% higher than, the survival rate determined in step (f2) of iteration i-1 and preferably not exceeding said given threshold, and wherein i is at least 2. Method according to claim 14, wherein the suspension in step (c) of iteration i+1 comprises(1) Substantially the same concentration of the added cryoprotectant as the suspension in step (c) of iteration i,If the survival rate determined in step (f2) is at least as high as, preferably by at least 5% higher than, the survival rate determined in i) step (fl) in case of the first iteration (i=l), or ii) step (f2) of iteration i-1 in case of iteration i,(2) A concentration of the added cryoprotectant that is between 0.001% and 1%, preferably between 0.01% and 0.5%, higher than in the suspension in step (c) of iteration i,69SUBSTITUTE SHEET (RULE 26)if neither i) nor ii) are fulfilled. An intracellular cryoprotectant, or a derivate thereof, obtained by or obtainable by the method according to any one of the preceding claims, wherein the intracellular cryoprotectant is an intracellular cryoprotectant that is synthesized by a cell and that is preferably an, optionally glycosylated, polypeptide or protein. The intracellular cryoprotectant according to claim 16, wherein the, optionally glycosylated, polypeptide or protein is characterized by a size, weight, and / or structure which is suitable to provide the cryoprotectant the capability of cell membrane impermeability. The intracellular cryoprotectant according to claim 16 or 17, wherein the, optionally glycosylated, polypeptide or protein is having an average molecular weight (Mw) of greater than 9800 Da, preferably greater than 900 kDa, or more. A method of producing a cryoprotectant, or a derivate thereof, comprising conducting the method according to any one of claims 1 to 15.70SUBSTITUTE SHEET (RULE 26)

Images