Cryopreserved formulation comprising collagen hydrolysate

Abstract

The present invention relates to a cryopreserved formulation comprising a collagen hydrolysate and preferably comprising dimethyl sulfoxide. The invention also relates to a method for cryopreservation of one or more biological substances, said method comprising the step of providing one or more biological substances in a cryopreserved formulation comprising a collagen hydrolysate and preferably dimethyl sulfoxide. The invention also relates to the use of a cryopreservation formulation comprising a collagen hydrolysate and preferably dimethyl sulfoxide for cryopreservation of one or more biological substances selected from the group consisting of eukaryotic cells, prokaryotic cells, organelles, extracellular vesicles, organoids, tissues and organs. The invention also relates to the use of the collagen hydrolysate, preferably the collagen hydrolysate combined with dimethyl sulfoxide, in the preparation of a cryopreservation preparation.

Description

Technical Field

[0001] The present invention relates to the cryopreservation of biological materials containing cells and tissues, and cryoprotective agents therefor. Background Art

[0002] Cryopreservation

[0003] Cryopreservation is a process in which biological materials (most commonly single cell suspensions, but also including cell tissues, etc.) are preserved by freezing. By bringing the biological material to a frozen state, the enzymatic and chemical activities of the cells are halted. At any desired time, the biological material can be thawed and cell activity can be restored. Cryopreservation gives biological materials an indefinite lifespan, and in the frozen state, biological materials can also be easily transported between different (distant) laboratories. In particular, the cryopreservation of cells has opened up a variety of new possibilities for medical treatment, where cells can be amplified and preserved on a large scale until they are needed for use. The relative ease and flexibility of cryopreservation make it an indispensable operation in cell culture laboratories, whether for research purposes or for therapeutic applications.

[0004] Cell Stress and Toxicity in Cryopreservation

[0005] Cryopreservation is most commonly at cryogenic temperatures, such as around -80 °C or lower. An important limitation of cryopreservation is the inherent toxicity of the freezing process to cells, such as causing cell loss, cell stress, and / or changes in cell response. The cytotoxicity in cryopreservation is largely related to the formation of intracellular ice and osmotic imbalances in cells (Bissoyi et al. Biopreserv Biobank. 2014 Feb;12(1):23 - 34). Taking this into account, the success of recovering cells after cryopreservation can be at least partially affected by the method of implementing cryopreservation. The preferred method for cryopreserving cells is by so-called "slow freezing". Slow freezing involves cooling the cells at a controlled rate until the desired cryogenic temperature is reached. For example, for many mammalian cells, a typical cooling rate of about 1 °C / minute is considered suitable. The principle behind slow freezing is that it affects the external solute concentration and thus dehydrates the cells to some extent and reduces the formation of intracellular ice crystals.

[0006] Cryoprotective Agents (CPA)

[0007] Slow freezing is usually carried out in combination with one or more cryoprotective agents (i.e., cryoprotective agent, CPA). CPAs typically act by increasing the solute concentration in cells, thereby helping to reduce freezing damage. Sulfoxides, glycols, and sugars form the most common CPAs and are generally classified as either permeating CPAs or non-permeating CPAs. Permeating CPAs include dimethyl sulfoxide (DMSO), ethylene glycol, propylene glycol, and glycerol. Permeating CPAs can penetrate cell membranes. In cells, the ability of this structure to form hydrogen bonds with water helps to reduce mechanical and osmotic damage. Non-permeating CPAs are typically large molecules and exert their protective effects extracellularly. Non-permeating CPAs include polyethylene glycol, polyvinylpyrrolidone, methylcellulose, and sugars (such as sucrose, dextran, and trehalose) (Whaley et al. Cryopreservation: An Overview of Principles and Cell-Specific Considerations. Cell Transplantation. January 2021).

[0008] Undesirable characteristics of CPAs

[0009] CPAs also have well-known undesirable characteristics. These effects have been best characterized for dimethyl sulfoxide (DMSO), which is also considered the most conventional CPA (e.g., also referred to as the "gold standard CPA"), but these undesirable characteristics can generally be seen in all CPAs. Some of the most relevant undesirable characteristics are summarized below:

[0010] 1) CPAs are toxic to cells, which has been shown to be mainly associated with apoptosis due to plasma membrane pore formation (Notman et al. J. Am. Chem. Soc. 128, 13982–13983).

[0011] 2) CPAs interfere with multiple cellular processes and can therefore alter cell function after cryopreservation. For example, changes in gene expression, cell proliferation, and differentiation have been reported after the use of CPAs ( et al. Scientific Reports Volume 8, Article number: 14828, 2018).

[0012] 3) CPAs can impede the therapeutic potential of cells after cryopreservation. For example, it has been found that stem cells cryopreserved with CPAs show reduced engraftment in patients after transplantation (Mitrus et al. Randomized Controlled Trial Bone Marrow Transplant. 2018 Mar;53(3):274-280).

[0013] 4) CPAs can modulate the inflammatory response in cells. Altered inflammatory responses can lead to unreliable test results and / or impede their therapeutic potential (Li et al. Front Immunol. 2021 Nov 29;12:765667).

[0014] 5) The use of CPAs increases the labor intensity. The washing step ideally ensures the removal of all CPAs from the cell suspension. This increases the resources required in the cell culture laboratory.

[0015] 6) Despite numerous washing steps, CPAs such as DMSO have been shown to persist in the cell suspension. DMSO contaminants cause direct side effects in recipients in a clinical setting (e.g., causing hypertension, nausea, and vomiting). This limits their use in cell and tissue transplantation and advanced therapy medicinal products (ATMPs).

[0016] It is generally considered that, as a rule of thumb, cell viability should be higher than 80% (i.e., cell death is lower than 20%). Especially for ATMPs, it is also desirable that the cryopreservation formulation is of GMP grade and preferably free of DMSO and / or serum. Currently available cryopreservation formulations do not meet this standard, for example because they generally require serum and / or DMSO.

[0017] CPAs are not suitable for several cell types

[0018] Another important limitation of conventional CPAs (e.g., DMSO, ethylene glycol, sugars) is that they are not compatible with all cell types. Among scientists worldwide, there is no general consensus on how to predict whether a particular cell type / cell line is easy or difficult to cryopreserve. It can be said that cell types can exhibit the following behaviors after cryopreservation:

[0019] 1. Cells cryopreserve well, and there is limited cell death (lower than 20%) after freeze-thawing, while the cells maintain their function;

[0020] 2. Cells cryopreserve well, and there is limited cell death (lower than 20%) after freeze-thawing, but the cells have lost their specific function (quality);

[0021] 3. The cells are frozen, but after thawing, more than 20% of the cells die and / or lose their specific functions (quality).

[0022] For simplicity, the cells under point 1 are referred to as "easily freezable", and the cells under points 2 and 3 are referred to as "difficult to freeze" or "non-freezable" cells, respectively.

[0023] To show the differences between the maturity of cells and their behavior in cryopreservation, some examples are given.

[0024] On the one hand, some differentiated and / or more mature cells such as muscle cells and immune cells are easily frozen. While other types of differentiated cells such as nerve cells are difficult to freeze because these cells lose their functions after freezing. On the other hand, some pluripotent and less differentiated and less mature cell phenotypes (e.g., fibroblasts, progenitor cells, mesenchymal stem cells, highly proliferating cells) are easily frozen, while embryonic stem cells (ESC) are more difficult to freeze (Li et al. Front. Cell Dev. Biol., 14 December 2021).

[0025] Due to the lack of clear functional descriptions of cells in the prior art, the only way to describe the performance of these cells in cryopreservation is to describe them as "easily freezable", "difficult to freeze" or "non-freezable" cells. For example, (human) neuronal cells are generally classified as "non-freezable" in the use of CPA, meaning that the freezing of neuronal cells results in extremely poor recovery of the cells. In addition, the recovered cells are affected in terms of their cell functions. Based on the above, isolated neuronal cells must therefore be used immediately for experiments after isolation, which is not practical. Immortalized neuronal cell lines can also be used for research purposes (Tremblay et al. J. Neurosci. Methods, 186, 2010, pp. 60 - 67), however, these cell lines do not show the same behavior as primary neuronal cells. Therefore, it is considered that neuronal cell lines are not suitable for studying the specific functions of neuronal cells, such as synapse formation, activity and plasticity (Ishizuka et al. Journal of Neuroscience Methods. Vol. 333, 1 March 2020).

[0026] Other examples of cells that are difficult to freeze include many primary (isolated cells) and / or cardiomyocytes, skeletal muscle cells, terminally differentiated hepatocytes, terminally differentiated fibroblasts, monocytes, dendritic cells, beta cells, cardiomyocytes, islets of Langerhans, primary human monocytes, osteocytes, and adipocytes. These cell types can only be obtained in limited quantities from the body and remain scarce due to their non-(or almost non-) proliferative nature. Therefore, further loss of these cells during cryopreservation is highly undesirable.

[0027] Reducing the toxicity of CPA

[0028] In practice, proteins are commonly used to reduce the toxicity of CPA. Serum or serum proteins from human or other animal sources are examples of preferred protein sources. They can serve as general growth supplements to support cell growth and differentiation. In the field of cryopreservation, it is believed that serum or serum proteins reduce the toxicity of CPA by stabilizing and protecting cell membranes against shear stress. However, even using large amounts of serum or serum proteins is not sufficient to counteract the undesirable characteristics of CPA. For example, a combination of 10% DMSO and 50% to 90% fetal bovine serum (FBS) is a preferred cryopreservation composition used in the prior art. However, this preferred composition still results in at least 20% cell death, even for cells that are considered relatively easy to freeze (Yong et al. Scientific Reports Volume 5, Article number: 9596, 2015). Other limitations include the following:

[0029] - Generally, it is considered that serum supplements are not suitable or desirable for therapeutic applications;

[0030] - There are batch-to-batch differences in serum supplements, resulting in inconsistent responses in cells;

[0031] - The endotoxin (lipopolysaccharide, LPS) level is uncontrolled, and it can hinder result reproducibility;

[0032] - The ethical objections to the use of FBS in cryopreservation are growing (Jochems et al. Altern Lab Anim. March - April 2002; 30(2):219 - 27). At slaughter, FBS is obtained from the fetuses of pregnant cows and is therefore considered animal-unfriendly.

[0033] In summary, it can be concluded that using serum or other protein sources does not solve the problems most typically associated with CPA.

[0034] Thawing and cell stress and toxicity associated with thawing

[0035] However, even if toxicity is minimized by adding cryoprotectants before and during the freezing process, toxic effects may resume with thawing and the associated increase in temperature. Toxicity after thawing may have the same damaging potential in all liquid phases of the cryopreservation cycle but is often overlooked. The sensitivity of cells to toxicity during thawing can be at least as high, if not higher, than during cooling due to the fact that they have been stressed by the freeze / thaw cycle.

[0036] Despite developments in the field of cryopreservation of biological materials for research and therapeutic applications, conventional CPAs have several undesirable features. There is an unmet need for methods to reduce reliance on conventional CPAs (most notably DMSO or other permeating CPAs). Specifically, there is a need for cryopreservation methods that:

[0037] - Reduce the amount of conventional CPA required during cryopreservation, thereby reducing the negative effects of the CPA, such as cytotoxicity, changes in cell function, and changes in the inflammatory response;

[0038] - Completely replace conventional CPAs, thereby eliminating the negative effects of the CPA and further making cryopreserved cells safer and more effective in therapeutic applications, particularly in advanced therapy medicinal products (ATMPs);

[0039] - Allow cryopreservation of cell types that are considered unsuccessful with conventional CPAs. These typically include slowly proliferating, terminally differentiated cells, "difficult-to-freeze" cells, or "non-freezable" cells, such as neuronal cells, cardiomyocytes, and / or other cell types;

[0040] - Even if currently used CPAs are beneficial during freezing, due to the increase in temperature, they may be toxic during thawing. Methods or cryopreservation formulations are needed that allow bringing cryopreserved cells to room temperature from a metabolically inactive state to an active state without causing much damage.

[0041] One object of the present invention is to provide a solution to one or more problems associated with the cryopreservation and / or thawing of biological materials. Summary of the Invention

[0042] Unexpectedly, the inventors have found that using a relatively high concentration of hydrolyzed collagen (preferably 20 to 50% by weight) allows reducing the concentration of the cryoprotectant dimethyl sulfoxide (i.e., DMSO) in cryopreservation. This strategy allows reducing the amount of dimethyl sulfoxide or even no longer using dimethyl sulfoxide at all, and thus it can eliminate several known drawbacks of dimethyl sulfoxide.

[0043] Suitable cryopreservatives typically contain 10% (v / v) or more of dimethyl sulfoxide and are generally considered the "gold standard" in cryopreservation formulations. The inventors have found that a combination of a relatively high concentration of collagen hydrolysate (preferably 20 to 50% by weight) and a relatively low concentration of dimethyl sulfoxide (preferably 0.5 to 8.5% by weight) can result in the highest cell recovery. Compared to freezing in other cryopreservation formulations, cells cryopreserved by this combination also show improved viability (e.g., higher proliferation ability and function). Thus, compared to other cryopreservation formulations, the cryopreservation formulation of the present invention not only provides higher cell recovery but also provides higher viability of the recovered cells.

[0044] The collagen hydrolysate of the present invention, especially at the aforementioned high concentrations, allows cryopreserved cells to be brought to room temperature from a metabolically inactive state to an active state with significantly less damage. Thus, the collagen hydrolysate can exert a beneficial effect on the cells and / or reduce, for example, the undesirable effects of dimethyl sulfoxide during thawing.

[0045] At a preferred high concentration, the collagen hydrolysate has a strong cryoprotective effect. For example, the collagen hydrolysate can act as a cryoprotectant. As a supplement or alternative, the collagen hydrolysate can also enhance the cryoprotective effect of dimethyl sulfoxide, reduce the toxic effects of dimethyl sulfoxide, and / or maintain the genotype and phenotype of the cells. The collagen hydrolysate can eliminate the need for serum and serum-derived proteins, which are typically included to reduce the toxicity of dimethyl sulfoxide. The cryopreservation formulation of the present invention is particularly suitable for preserving biomaterials intended for human transplantation, such as advanced therapy medicinal products (ATMP). The cryopreservation formulation of the present invention is particularly suitable for cryopreserving cells that generally cannot be cryopreserved alone with dimethyl sulfoxide and are thus considered difficult or impossible to freeze.

[0046] In summary, the best results are obtained when the amount of collagen hydrolysate exceeds 20% by weight, especially when it is 30% or 40% by weight.

[0047] The current findings of the inventors are unexpected because they contradict the existing view that large amounts of collagen hydrolysate should be avoided in cryopreservation. For example, the prior art indicates using no more than 15% by weight of collagen hydrolysate, especially in combination with dimethyl sulfoxide or other permeating cryoprotectants, so as not to compromise the viability of cells after cryopreservation.

[0048] In one aspect, the present invention relates to a cryopreservation formulation comprising 20 to 70% by weight of collagen hydrolysate based on the weight of the cryopreservation formulation.

[0049] In one aspect, the present invention relates to the use of a cryopreservation preparation as disclosed herein for cryopreserving one or more biological materials selected from eukaryotic cells, prokaryotic cells, organelles, extracellular vesicles, organoids, tissues, and organs.

[0050] In one aspect, the present invention relates to the use of a collagen hydrolysate in a cryopreservation preparation, wherein the collagen hydrolysate is used in an amount of 20 to 70% by weight based on the weight of the cryopreservation preparation.

[0051] In one aspect, the present invention relates to a method for cryopreserving a biological material, the method comprising the steps of: providing the biological material in a cryopreservation preparation as disclosed herein, and lowering the temperature of the cryopreservation preparation to below its freezing point.

[0052] In one aspect, the present invention relates to the use of a collagen hydrolysate for reducing the amount of dimethyl sulfoxide in a cryopreservation preparation, wherein the collagen hydrolysate is provided in an amount of 20 to 70% by weight based on the weight of the cryopreservation preparation.

[0053] In one aspect, the present invention relates to the use of a collagen hydrolysate for reducing toxicity and / or enhancing cell viability during thawing after cryopreservation, wherein the collagen hydrolysate is provided in an amount of 20 to 70% by weight based on the weight of the cryopreservation preparation. Detailed Description

[0054] The cryopreservation preparation of the present invention relates to a composition comprising a collagen hydrolysate, preferably in an amount of 20 to 70% by weight based on the weight of the cryopreservation preparation.

[0055] The present invention also relates to the use of one or more of the embodiments of the cryopreservation preparation described in the present disclosure.

[0056] The present invention also relates to a method for cryopreserving a biological material using one or more of the embodiments of the cryopreservation preparation described in the present disclosure.

[0057] In the context of the present invention, the term "collagen" means an amino acid sequence comprising repeating (Gly-X-Y) sequences, preferably comprising at least 2, 3, 4, 5, 10, 20, 50, 100, 200, 300 or 400 sequences containing the sequence Gly-X-Y, where X and Y are amino acid residues independently selected from each other, but X and / or Y are more preferably proline. "Collagen" preferably has the sequence of natural collagen present in one or more animal species. As a supplement or alternative, "collagen" may mean the full-length sequence of (natural) collagen or a fragment or subunit thereof, said (natural) collagen preferably being one or more of types I to XXVII collagen, more preferably one or even more of types I, II, III, V or X collagen, even more preferably one or more of types I, II or III collagen. For example, the term "collagen" may refer to the α-1(I), α-2(I), α-1(II) or α-1(III) chains, or fragments thereof. The term "collagen" encompasses the triple helix structure formed by three subunits as present in natural collagen.

[0058] In the context of the present invention, the term "collagen hydrolysate" means a mixture of short-chain amino acids (dipeptides, tripeptides, oligopeptides, polypeptides) derived from the (partial) hydrolysis (e.g., by enzymatic hydrolysis) of natural (full-length) collagen. The degree of hydrolysis has an impact on the average molecular weight of the final product (expressed in Daltons, Da). The term "collagen hydrolysate" may be used interchangeably and synonymously with the terms "hydrolyzed collagen" or "collagen peptide". In the context of the present invention, "collagen hydrolysate" encompasses collagen that has undergone hydrolysis or partial hydrolysis.

[0059] In one embodiment, the hydrolysis is alkaline hydrolysis. In one embodiment, the hydrolysis is acid hydrolysis. In one embodiment, the hydrolysis is enzymatic hydrolysis. The collagen hydrolysate can be one or more of an enzyme-hydrolyzed, alkaline-hydrolyzed and acid-hydrolyzed collagen hydrolysate. As a supplement or alternative, the collagen hydrolysate can be obtained by one or a combination of the following methods: alkaline hydrolysis, acid hydrolysis and enzymatic hydrolysis.

[0060] In the context of the present invention, "collagen hydrolysate" can be produced from a collagen-containing material in a one-step process or via an intermediate gelatin stage (i.e., "hydrolyzed gelatin" is thus obtained). Thus, the term "collagen hydrolysate" encompasses hydrolyzed gelatin (i.e., hydrolyzed gelatin). The term "gelatin" as used herein means the (irreversible) form of collagen obtained by partial hydrolysis of collagen. Depending on the method used, two types of gelatin are generally obtained, namely type A (acid hydrolysis) and type B (alkaline hydrolysis). In the context of the present invention, "gelatin" can refer to type A or type B gelatin, or a combination thereof. Depending on the physical and chemical methods of partial hydrolysis, the molecular weight of the peptides can fall within a wide range (e.g., 10 to 95 kDa). Partial hydrolysis provides gelatin with water retention capacity and its gelling ability, which is generally distinguished from "collagen peptides" or "hydrolyzed collagen" (i.e., the product obtained by complete hydrolysis of collagen). In the context of the present invention, the term "hydrolyzed gelatin" means the product obtained by hydrolysis of gelatin and which results in a lower molecular weight of gelatin. As for the hydrolysis reaction to obtain hydrolyzed gelatin, it involves breaking one or more peptide linkages with the addition of 1 molecule of water. In terms of gelling ability, hydrolyzed gelatin is different from other gelatins, i.e., hydrolyzed gelatin has a reduced / no gelling ability, while gelatin has a gelling ability. Gelatin can be hydrolyzed by an acid (hydrogen ions), i.e., to obtain "acid-hydrolyzed gelatin". Gelatin can be hydrolyzed by a base (hydroxide ions), i.e., to obtain "alkaline-hydrolyzed gelatin". Gelatin can be hydrolyzed by one or more enzymes (e.g., pepsin, trypsin), i.e., to obtain "enzymatically hydrolyzed gelatin". In the context of the present invention, the terms "hydrolyzed" and "hydrolysed" can be used interchangeably.

[0061] In a preferred embodiment, the collagen hydrolysate is hydrolyzed gelatin.

[0062] In the context of the present invention, the collagen or collagen hydrolysate may include synthetic (e.g., recombinant or by chemical synthesis) proteins or peptides. Recombinant synthesis encompasses proteins or peptides encoded by recombinant DNA expressed in an expression system. The expression system for recombinant peptides may be a cell, such as a bacterial cell (e.g., Escherichia coli, Bacillus subtilis species), a yeast cell [e.g., Saccharomyces cerevisiae, Pichia pastoris or Ogataea angusta (Hansenula polymorpha), Candida bodini], a fungal cell (e.g., Aspergillus oryzae, Aspergillus niger, Trichoderma reesei), a mammalian cell (e.g., CHO cell, HeLa cell, HEK293 cell, NS0, Sp2 / 0), an insect cell, and a plant cell (e.g., tobacco, cereals, legumes, fruits, vegetables).

[0063] In several embodiments of the present invention, the collagen hydrolysate may be produced by enzymatic hydrolysis or partial enzymatic hydrolysis of collagen, and the enzyme used for this purpose may be one or more selected from the following: serine protease, alkaline protease, neutral protease, flavor protease, compound protease, sulfhydryl protease, bromelain, metalloprotease, protease, carboxypeptidase, pepsin, chymotrypsin, trypsin, cathepsin K, chymotrypsin, papain, and subtilisin.

[0064] In one embodiment, the hydrolysis (preferably enzymatic hydrolysis) is carried out at a pH of 5 to 8, preferably pH 6 to 7. In one embodiment, the hydrolysis (preferably enzymatic hydrolysis) is carried out at a temperature of 55 to 70 °C, preferably 60 to 65 °C. In one embodiment, the hydrolysis (preferably enzymatic hydrolysis) is carried out for 3 to 8 hours, preferably 4 to 7 hours, more preferably 5 to 6 hours.

[0065] In the context of the present invention, the collagen hydrolysate may be derived from one or more types of collagen selected from type I to type XXVII collagen, preferably one or more of type I, type II, type III, type V, or type X collagen, more preferably one or more of type I, type II, or type III collagen. As a supplement or alternative, the collagen hydrolysate as disclosed herein may be a mixture of two or more types of collagen, preferably two or more of type I, type II, and type III collagen.

[0066] The collagen disclosed herein can be derived from any one or more animals or animal species, such as cows (species), pigs (species), chickens, and fish (species). In one embodiment, the collagen is derived from cows (cow). In one embodiment, the collagen is derived from pigs. In one embodiment, the collagen is derived from fish. In one embodiment, the collagen is derived from chickens. In multiple embodiments, the collagen is a mixture of collagens from different sources (e.g., collagens derived from multiple animal species and / or collagens derived from different tissues). For example, the collagen disclosed herein can be a mixture of two or more collagens selected from the following: fish collagen, pig collagen, chicken collagen, and cow collagen.

[0067] In the context of the present invention, the collagen can be derived from one or more tissues selected from the following: skin, scales, antlers, protrusions (e.g., humps), horns, head, brain, neck, ear, eye, nose, tongue, lip, mouth, esophagus, trachea, sternum, larynx, bronchus, limb, foot, toe, palm, claw, bone, cartilage, bone marrow, joint, membrane, hind leg (hind), ligament, tendon, rib, diaphragm, muscle, skeletal muscle, smooth muscle, intestine, blood vessel, bladder, stomach, aorta, heart, liver, kidney, chest, lung, spleen, pancreas, egg, sperm, testis, ovary, nerve, gallbladder, and abdomen. The term "skin" as disclosed herein encompasses "hide", which means the outer covering of a large animal such as from cows (species) or any other large animal. The terms "skin" and "hide" can be used interchangeably herein and can refer to the outer covering of an animal, regardless of size.

[0068] In a preferred embodiment, the collagen taught herein is derived from skin and / or skin connective tissue. In a preferred embodiment, the collagen taught herein is derived from cartilage. In a preferred embodiment, the collagen taught herein is derived from bone. In a preferred embodiment, the collagen taught herein is derived from the sternum. The collagen disclosed herein can be a mixture of collagens derived from two or more tissues and / or two or more animals. In one embodiment, the collagen disclosed herein is a mixture of two or more collagens selected from the group comprising: skin collagen, cartilage collagen, sternum cartilage collagen, and bone collagen.

[0069] In the context of the present invention, the term "cryopreservation preparation" means a preparation suitable for and / or intended for cryopreservation of cells. The preparation is preferably a liquid preparation, or at least a preparation in which biological substances (including cells) can be suspended. The term "cryopreservation" as used herein means cooling, preferably freezing, biological substances, preferably with the aim of allowing storage of the biological substances and / or preservation of the biological substances for future use. In the context of the present invention, most commonly and preferably, cryopreservation is achieved at a temperature of about -10 to -30 °C, more preferably at about -70 to -90 °C (e.g., using solid carbon dioxide) or about -180 to -220 °C (e.g., using liquid nitrogen). In the context of the present invention, the term "cryopreservation" can be used interchangeably with the terms "cryoprotection" or "cryobank". The term "cryopreservation" as used herein encompasses cooling a substance above its freezing point, e.g., to avoid the formation of ice crystals. The term "cryopreservation" encompasses slow freezing. "Slow freezing" means a gradual decrease in temperature, most commonly at an average rate of 0.2 to 5 °C per minute (e.g., 0.5 to 2 °C) until the final storage temperature is reached. This means that the biological substance is cooled over the course of several hours (e.g., to reach a temperature of about -196 °C). In the context of the present invention, "slow freezing" can be used interchangeably with the terms "controlled-rate freezing" or "slow programmable freezing (SPF)". The term "cryopreservation" as used herein encompasses "vitrification" (also known as rapid freezing), which involves rapid cooling with the aim of preventing the formation of ice crystals and thus helping to prevent cryopreservation damage. The processes of "slow freezing" and "vitrification" are well known in the art, e.g., as described in the review article by Son et al. (Expert Rev Med Devices 2009 Jan; 6(1): 1-7).

[0070] In the context of the present invention, the term "cryopreservation" preferably encompasses the following steps: incorporating a biological substance into a cryopreservation preparation, freezing, and thawing the cells after freezing. In the context of the present invention, a preparation that results in an improvement in cells during thawing is generally considered to improve cryopreservation. The present invention further relates to the thawing of biological substances after freezing. In particular, the inventors have found that an appropriate concentration of collagen hydrolysate allows frozen cells to be brought to room temperature from a metabolically inactive state to an active state without significantly causing damage. Thus, collagen hydrolysate can play a beneficial role during thawing and / or reduce the negative (toxic) effects of dimethyl sulfoxide during thawing.

[0071] In one embodiment, the use of a collagen hydrolysate and / or a cryopreservation formulation as disclosed herein relates to use for reducing toxicity and / or enhancing cell viability during thawing after cryopreservation, wherein the collagen hydrolysate is preferably provided in the cryopreservation formulation in an amount of 20 to 70% by weight based on the weight of the cryopreservation formulation. In a preferred embodiment, cytotoxicity is reduced and / or cell viability is enhanced by reducing one or more toxic effects of non-permeating and / or permeating cryoprotectants in cells during thawing. In a preferred embodiment, cytotoxicity is reduced and / or cell viability is enhanced by reducing one or more toxic effects of a permeating cryoprotectant in cells during thawing, wherein the permeating cryoprotectant is dimethyl sulfoxide.

[0072] The method of the present invention involves providing a biological material in a cryopreservation formulation as disclosed herein and reducing the temperature of the cryopreservation formulation to below the freezing point of the cryopreservation formulation. The freezing point may depend on the composition of the liquid cryopreservation formulation and is defined as the temperature at which the liquid cryopreservation formulation becomes solid at normal atmospheric pressure. As a supplement or alternative, considering that the cryopreservation formulation may be aqueous, the freezing point is preferably 0 °C or close to 0 °C (e.g., -10, -9, -8, -7, -6, -5, -4, -3, -2, -1, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 °C).

[0073] In one embodiment, the cryopreservation formulation containing the biological material is directly frozen, which means that the freezing rate is not controlled, thus excluding the use of programmed and / or slow freezing.

[0074] The cryopreservation formulation may comprise at least 5% by weight, or 10% by weight, or 15% by weight, or 20% by weight, or 22.5% by weight, or 25% by weight, or 27.5% by weight, or 30% by weight, or 32.5% by weight, or 35% by weight, or 37.5% by weight, or 40% by weight, or 45% by weight, or 50% by weight of collagen hydrolysate based on the weight of the cryopreservation formulation. As a supplement or alternative, the cryopreservation formulation may comprise no more than 80% by weight, or 75% by weight, or 70% by weight, or 65% by weight, or 50% by weight, or 45% by weight, or 40% by weight, or 37.5% by weight, or 35% by weight, or 32.5% by weight, or 30% by weight, or 27.5% by weight, or 25% by weight, or 22.5% by weight, or 20% by weight of collagen hydrolysate based on the weight of the cryopreservation formulation. In a preferred embodiment, the cryopreservation formulation comprises 20 to 70% by weight, preferably 20 to 50% by weight, more preferably 25 to 40% by weight, even more preferably 27.5 to 35% by weight of collagen hydrolysate based on the weight of the cryopreservation formulation.

[0075] In one embodiment, the cryopreservation formulation further comprises dimethyl sulfoxide.

[0076] The cryopreservation formulation may comprise at least 0.01 wt%, or 0.05 wt%, or 0.1 wt%, or 0.2 wt%, or 0.5 wt%, or 0.6 wt%, or 0.7 wt%, or 0.8 wt%, or 0.9 wt%, or 1 wt%, or 1.25 wt%, or 1.5 wt%, or 1.75 wt%, or 2 wt%, or 2.25 wt%, or 2.5 wt%, or 2.75 wt%, or 3 wt%, or 3.25 wt%, or 4 wt%, or 4.25 wt%, or 4.5 wt%, or 4.75 wt%, or 5 wt%, or 5.25 wt%, or 5.25 wt%, or 5.5 wt%, or 5.75 wt%, or 6.0 wt%, or 6.25 wt%, or 6.5 wt%, or 7 wt%, or 7.5 wt%, or 8 wt%, or 8.5 wt%, or 9 wt%, or 9.5 wt%, or 10 wt%, or 11 wt%, or 12 wt%, or 13 wt%, or 14 wt%, or 15 wt% of dimethyl sulfoxide, based on the weight of the cryopreservation formulation. As a supplement or alternative, the cryopreservation formulation may comprise no more than 20%, or 17.5%, or 15%, or 12.5, or 12%, or 11%, or 10.9%, or 10.8%, or 10.7%, or 10.6%, or 10.5%, or 10.4%, or 10.3%, or 10.2%, or 10.1%, or 10%, or 9.5%, or 9%, or 8.5%, or 8%, or 7.5%, or 7%, or 6.5%, or 6.25%, or 6%, or 5.75%, or 5.5%, or 5.25%, or 5%, or 4.75%, or 4.5%, or 4.25%, or 4%, or 3.75%, or 3.5%, or 3.25%, or 3%, or 2.75%, or 2.5% of dimethyl sulfoxide, based on the weight of the cryopreservation formulation.

[0077] For example, a cryopreservation preparation for use as described herein may comprise at least 0.01% v / v, or 0.05% v / v, or 0.1% v / v, or 0.2% v / v, or 0.5% v / v, or 0.6% v / v, or 0.7% v / v, or 0.8% v / v, or 0.9% v / v, or 1% v / v, or 1.25% v / v, or 1.5% v / v, or 1.75% v / v, or 2% v / v, or 2.25% v / v, or 2.5% v / v, or 2.75% v / v, or 3% v / v, or 3.25% v / v, or 4% v / v, or 4.25% v / v, or 4.5% v / v, or 4.75% v / v, or 5% v / v, or 5.25% v / v, or 5.25% v / v, or 5.5% v / v, or 5.75% v / v, or 6.0% v / v, or 6.25% v / v, or 6.5% v / v, or 7% v / v, or 7.5% v / v, or 8% v / v, or 8.5% v / v, or 9% v / v, or 9.5% v / v, or 10% v / v, or 11% v / v, or 12% v / v, or 13% v / v, or 14% v / v, or 15% v / v of dimethyl sulfoxide, based on the total volume of the cryopreservation preparation. As a supplement or alternative, the cryopreservation preparation may comprise no more than 20% v / v, or 17.5% v / v, or 15% v / v, or 12.5 v / v, or 12% v / v, or 11% v / v, or 10.9% v / v, or 10.8% v / v, or 10.7% v / v, or 10.6% v / v, or 10.5% v / v, or 10.4% v / v, or 10.3% v / v, or 10.2% v / v, or 10.1% v / v, or 10% v / v, or 9.5% v / v, or 9% v / v, or 8.5% v / v, or 8% v / v, or 7.5% v / v, or 7% v / v, or 6.5% v / v, or 6.25% v / v, or 6% v / v, or 5.75% v / v, or 5.5% v / v, or 5.25% v / v, or 5% v / v, or 4.75% v / v, or 4.5% v / v, or 4.25% v / v, or 4% v / v, or 3.75% v / v, or 3.5% v / v, or 3.25% v / v, or 3% v / v, or 2.75% v / v, or 2.5% v / v of dimethyl sulfoxide, based on the total volume of the cryopreservation preparation.

[0078] In a preferred embodiment, a cryopreservation formulation for use, for example, as described herein contains more than 20% by weight, preferably more than 22.5% by weight, and even more preferably more than 25% by weight of hydrolyzed collagen, based on the weight of the cryopreservation formulation. In a preferred embodiment, a cryopreservation formulation for use, for example, as described herein contains less than 50% by weight, preferably less than 45% by weight, and even more preferably less than 40% by weight of hydrolyzed collagen, based on the weight of the cryopreservation formulation. In a preferred embodiment, a cryopreservation formulation for use, for example, as described herein contains less than 10% v / v, preferably less than 9.5% v / v, and even more preferably less than 9.0% v / v of dimethyl sulfoxide, based on the total volume of the cryopreservation formulation.

[0079] In a preferred embodiment, the cryopreservation formulation contains at least 0.1% by weight of dimethyl sulfoxide, based on the weight of the cryopreservation formulation. In a preferred embodiment, the cryopreservation formulation contains less than 10.9% by weight of dimethyl sulfoxide, based on the weight of the cryopreservation formulation.

[0080] In a preferred embodiment, the cryopreservation formulation contains 0.2 to 10.5% by weight, preferably 0.5 to 8.5% by weight, more preferably 1 to 7% by weight, even more preferably 2 to 6% by weight, and most preferably 3 to 5% of dimethyl sulfoxide, based on the weight of the cryopreservation formulation.

[0081] Those skilled in the art know how to calculate based on the known density of DMSO (1.1 g / cm 3 ) and the density of the aqueous medium in which DMSO is present (usually a density of about 1.0 g / cm 3)Convert between weight % and volume % amounts of DMSO. For example, a 10% (v / v) DMSO concentration calculated based on the volume of an aqueous formulation is equal to 10.9 wt% DMSO calculated based on the total weight of the formulation. The same conversion applies for example to DMSO concentrations of 0.5% v / v (i.e., 0.55 wt%), 1.0% v / v (i.e., 1.10 wt%), 1.5% v / v (i.e., 1.65 wt%), 2.0% v / v (i.e., 2.2 wt%), 2.5% v / v (i.e., 2.74 wt%), 3.0% v / v (i.e., 3.29 wt%), 3.5% v / v (i.e., 3.84 wt%), 4.0% v / v (i.e., 4.38 wt%), 4.5 v / v (i.e., 4.93 wt%), 5.0% v / v (i.e., 5.24 wt%), 5.5% v / v (i.e., 6.02 wt%), 6.0% v / v (i.e., 6.56 wt%), 6.5% v / v (i.e., 7.10 wt%), 7.0% v / v (i.e., 7.65 wt%), 7.5% v / v (i.e., 8.16 wt%), 8.0% v / v (i.e., 8.73 wt%), 8.5% v / v (i.e., 9.27 wt%), 9.0% v / v (i.e., 9.81 wt%), 9.5% v / v (i.e., 10.35 wt%), 10% v / v (i.e., 10.90 wt%), 12.5% v / v (i.e., 13.58 wt%), 15% v / v (i.e., 16.26 wt%).

[0082] In one embodiment, the present invention relates to the use of the collagen hydrolysate disclosed herein in the cryopreservation of biological materials, wherein the biological materials are preferably selected from eukaryotic cells, prokaryotic cells, organelles, extracellular vesicles, organoids, tissues and organs. In a preferred embodiment, the collagen hydrolysate in the use described herein is present in an amount of 10 to 70 wt%, preferably 20 to 60 wt%, more preferably more than 20 wt% based on the total weight of the cryopreservation formulation. In a preferred embodiment, the dimethyl sulfoxide hydrolysate in the use described herein is present in an amount of less than 10% (v / v), preferably less than 9.5% (v / v), more preferably less than 9% (v / v) based on the total volume of the cryopreservation formulation. In a preferred embodiment, the cryopreservation formulation in the use described herein is (substantially) free of dimethyl sulfoxide, or contains no more than 0.1% (v / v), or no more than 0.01% (v / v), or no more than 0.001% (v / v), or no more than 0.0001% (v / v) of dimethyl sulfoxide based on the total volume of the cryopreservation formulation. In a preferred embodiment, the collagen hydrolysate in the use described herein is (substantially) free of one or more of the following: additional cryoprotectants, serum and serum proteins, more preferably free of additional permeating cryoprotectants.

[0083] In one embodiment, the method of the present invention utilizes a cryopreservation preparation that contains an amount of hydrolyzed collagen of 10 to 70% by weight, preferably 20 to 60% by weight, more preferably more than 20% by weight, based on the total weight of the cryopreservation preparation. In a preferred embodiment, the method of the present invention utilizes a cryopreservation preparation that contains an amount of dimethyl sulfoxide of less than 10% (v / v), preferably less than 9.5% (v / v), more preferably less than 9% (v / v), based on the total volume of the cryopreservation preparation. In a preferred embodiment, the method of the present invention utilizes a cryopreservation preparation that is (substantially) free of dimethyl sulfoxide, or contains no more than 0.1% (v / v), or no more than 0.01% (v / v), or no more than 0.001% (v / v), or no more than 0.0001% (v / v) of dimethyl sulfoxide, based on the total volume of the cryopreservation preparation. In a preferred embodiment, the method of the present invention utilizes a cryopreservation preparation that is (substantially) free of one or more of the following: additional cryoprotectants, serum, and serum proteins, and more preferably is free of additional permeating cryoprotectants.

[0084] In one embodiment, in the absence of one or more of additional cryoprotectants, serum, and serum proteins, and more preferably in the absence of DMSO, the method and / or use of the present invention utilizes hydrolyzed collagen as a cryoprotectant.

[0085] In embodiments where hydrolyzed collagen is used in the absence of one or more of additional cryoprotectants, serum, and serum proteins, hydrolyzed collagen is preferably hydrolyzed gelatin, as this has been shown to result in the highest viability after cryopreservation, although other forms of hydrolyzed collagen may also be suitable for the present invention.

[0086] In embodiments where hydrolyzed collagen is used in the absence of one or more of additional cryoprotectants, serum, and serum proteins, hydrolyzed collagen preferably has a molecular weight of 3500 to 7500 Da, preferably 4000 to 6000 Da, more preferably 4500 to 5500 Da, as this has been shown to result in the highest viability after cryopreservation, although other forms of hydrolyzed collagen may also be suitable for the present invention.

[0087] In embodiments where the collagen hydrolysate is used in the absence of one or more of additional cryoprotectants, serum, and serum proteins, the amount of the collagen hydrolysate is preferably more than 20 to 50% by weight, preferably 22.5 to 45% by weight, more preferably 25 to 40% by weight, even more preferably 27.5 to 35% by weight, based on the total weight of the formulation, because this has been shown to result in the highest viability after cryopreservation, but other forms of collagen hydrolysate may also be suitable for the present invention.

[0088] In one embodiment, the use of the present invention relates to the use of a collagen hydrolysate for reducing the amount of dimethyl sulfoxide in a cryopreservation formulation, wherein the collagen hydrolysate is preferably provided in an amount of 20 to 70% by weight based on the weight of the cryopreservation formulation. For example, the amount of dimethyl sulfoxide can be reduced due to the collagen hydrolysate while still achieving at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99% cell recovery.

[0089] In one embodiment, in the cryopreservation formulation of the present invention, cryopreservation of the biological material (preferably cells) results in similar or higher cell recovery (and / or lower cell death) and / or cell viability compared to the same cryopreservation formulation without the collagen hydrolysate. For example, a combination of 25% by weight of the collagen hydrolysate and 5% by weight of DMSO can result in similar or higher cell recovery (and / or lower cell death) and / or cell viability compared to 5% by weight of DMSO without the collagen hydrolysate.

[0090] In one embodiment, the use of a collagen hydrolysate in the cryopreservation of biological material (preferably cells) allows for the use of a lower amount of DMSO while resulting in similar or higher cell recovery (and / or lower cell death) and / or cell viability. For example, a combination of 25% by weight of the collagen hydrolysate and 5% by weight of DMSO can result in similar or higher cell recovery (and / or lower cell death) and / or cell viability compared to the use of 10% by weight of DMSO without the collagen hydrolysate. For example, "a lower amount of DMSO" can be a reduction in the amount of DMSO of at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 99% and / or a reduction to any of the preferred DMSO concentrations disclosed herein.

[0091] In the context of the present invention, "cell recovery" and / or "cell death" are preferably determined after slow freezing of the cells and the percentage of live and dead cells is determined after thawing. The person skilled in the art knows that slow freezing and thawing can depend on the type of cells and the protocol can be adjusted accordingly. Cell recovery after thawing can be determined by counting the live cells and comparing them to the number of frozen cells, for example using a stain that selectively stains dead cells, such as trypan blue. The % cell death can be determined by comparing the number of live cells and frozen cells. As a supplement or alternative, live and dead cells can be determined using a commercial assay, such as the Live Dead assay, which uses a mixture of two fluorescent dyes that differentially label live and dead cells and measures live and dead cells using flow cytometry or fluorescence microscopy.

[0092] In the context of the present invention, the term "live cell" means a cell with an intact cell membrane, for example defined by the inability of a (DNA-binding) cell membrane-impermeable dye to enter the cell. In the context of the present invention, the term "dead cell" means a cell that is not a live cell.

[0093] In the context of the present invention, "cell viability" is preferably determined based on one or more of cell metabolic activity, cell proliferation, and (adenosine triphosphate) ATP concentration, i.e., higher cell metabolic activity, higher cell proliferation, and / or higher ATP concentration can be indicators of higher cell viability.

[0094] For example, cell metabolic activity can be measured using an assay based on a tetrazolium salt (such as MTT, XTT) or Alamar Blue. For example, cell proliferation can be measured using an assay directed against a cell proliferation marker or a cell cycle regulatory marker (such as Ki-67, proliferating cell nuclear antigen (PCNA), topoisomerase IIB, or phosphorylated histone H3). For example, ATP can be detected using the bioluminescent luciferase and its substrate (luciferin).

[0095] In one embodiment, the use of the present invention relates to the use of a collagen hydrolysate for reducing the amount of dimethyl sulfoxide in a cryopreservation preparation, wherein the collagen hydrolysate is preferably provided in an amount of 20 to 70% by weight based on the weight of the cryopreservation preparation.

[0096] The average molecular weight of the collagen hydrolysate can be in the range of 500 Da to 25,000 Da, such as 1000 Da to 15000 Da or 2000 Da to 10000 Da. The average molecular weight of the collagen hydrolysate can be at least 500 Da, or 600 Da, or 700 Da or 800 Da, or 900 Da, or 1000 Da or 1100 Da, or 1200 Da, or 1300 Da, or 1400 Da, or 1500 Da, or 1750 Da, or 2000 Da, or 2250 Da, or 2500 Da, or 2750 Da, or 3000 Da, or 3250 Da, or 3500 Da, or 4000 Da, or 4500 Da, or 5000 Da, or 5500 Da. As a supplement or alternative, the average molecular weight of the collagen hydrolysate can be not more than 10000 Da, or 9500 Da, or 9000 Da, or 8750 Da, or 8500 Da, or 8250 Da, or 8000 Da, or 7750 Da, or 7500 Da, or 7250 Da, or 7000 Da, or 6750 Da, or 6500 Da, or 6250 Da, or 6000 Da, or 5750 Da, or 5500 Da, or 5250 Da, or 5000 Da, or 4500 Da, or 4000 Da. In a preferred embodiment, the average molecular weight of the collagen hydrolysate is 500 to 10000 Da, preferably 1000 to 8000 Da, more preferably 2000 to 7000 Da, even more preferably 3000 to 6000 Da.

[0097] In the context of the present invention, the average molecular weight is preferably the weight-average molecular weight. The preferred method for measuring the (average) molecular weight and / or molecular weight distribution of the collagen hydrolysate or gelatin is by high performance size exclusion chromatography (HPSEC). The following protocol is a preferred HPSEC protocol:

[0098] Use an Agilent HPLC with a TSKgel SWXL guard column and a G2000SWXL column (Tosoh Bioscience), 1260 Infinity series (G1316A, G1329B, G1311C, G1315D). Analyze using WinGPC software (PSS). The eluent is 100 mM phosphate buffer at pH 5.3. Elute the sample from the column (e.g., 0.5 mL / min, isocratic) and monitor with a UV detector (e.g., 214 nm, analysis time: 40 minutes / injection + 180 minutes equilibration). Calibrate using narrow calibration standards (low FILK).

[0099] The inventors have found that in the context of the present invention, it would be beneficial if no LPS (endotoxin) or a minimal amount of LPS (endotoxin) is present, especially when performing a centrifugation washing step. The absence of LPS or the presence of low LPS makes the centrifugation washing step more redundant. In addition, it has been found that the presence of LPS during cryopreservation can induce an undesirable toxic and / or pro-inflammatory response in the cells. As a supplement or alternative, the use of collagen hydrolysate can also provide protection against the undesirable effects of LPS in the cryopreservation formulation.

[0100] The endotoxin level of the collagen hydrolysate is preferably not more than 10,000 EU (i.e., endotoxin units) / g of collagen hydrolysate or / mL of cryopreservation formulation, preferably not more than 1,000 EU (e.g., not more than 500, 400, 300, or 200 EU), more preferably not more than 100 EU (e.g., not more than 90, 80, 70, 60, 50, 40, or 30 or 20 EU), even more preferably not more than 10 EU (e.g., not more than 9, 8, 7, 6, 5, 4, 3, 2, 1 EU), and most preferably not more than 1 EU, all per g of collagen hydrolysate or / mL of cryopreservation formulation. As a supplement or alternative, the endotoxin level of the collagen hydrolysate can be at least 1 EU / g of collagen hydrolysate or / mL of cryopreservation formulation, preferably at least 10 EU, e.g., at least 20 EU (e.g., at least 30, 40, 50, 60, 70, 80, 90, 100, or 150 EU), more preferably at least 100 EU (e.g., at least 200, 300, 400, 500, 600, 700, 800, 900, 1,000, or 1,500 EU), even more preferably at least 1,000 EU (e.g., at least 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000 EU), and most preferably at least 10,000 EU, all per g of collagen hydrolysate or / mL of cryopreservation formulation.

[0101] In a preferred embodiment, the endotoxin level of the collagen hydrolysate is not more than 10,000 EU / g based on the weight of the collagen hydrolysate, preferably not more than 1,000 EU / g, more preferably not more than 100 EU / g, and most preferably not more than 10 EU / g.

[0102] In a preferred embodiment, the endotoxin level of the cryopreservation formulation is less than 2,500 EU / ml based on the volume of the cryopreservation formulation, preferably less than 250 EU / ml, more preferably less than 25 EU / ml, even more preferably less than 2.5 EU / ml, and most preferably less than 1 EU / ml. For example, the endotoxin level of the cryopreservation formulation based on the volume of the cryopreservation formulation can be 0.5 to 2,500 EU / ml, or 1 to 1,000 EU / ml, or 5 to 500 EU / ml or 10 to 250 EU / ml.

[0103] In one embodiment, the collagen hydrolysate is endotoxin-free. In one embodiment, the cryopreservation formulation is endotoxin-free. The term "endotoxin-free" may mean the absence of endotoxins and / or may mean "substantially free of endotoxins".

[0104] The term "free of" may have the same meaning as "substantially free of". The term "substantially free of" encompasses that the amount of a substance or compound is not measurable and / or is below certain thresholds according to standard techniques in the art, preferably below 0.1 wt%, more preferably below 0.01 wt%, even more preferably below 0.001 wt%, and most preferably below 0.0001 wt%. In the context of the present invention, "substantially free of" may mean an endotoxin level below 10, preferably below 1, more preferably below 0.1, even more preferably below 0.01, and most preferably below 0.001 (both in EU / g or EU / ml). The wording "free of" or "substantially free of" encompasses that a substance or compound is completely absent (e.g., 0 wt%). In the context of the present invention, the terms "absence", "free of", and "substantially free of" may be used interchangeably.

[0105] In the context of the present invention, the widely used and preferred method for measuring endotoxin levels (e.g., for determining EU / g or EU / ml) is the Limulus Amebocyte Lysate (LAL) test or the recombinant Factor C (rFC) test, which are familiar to those skilled in the art.

[0106] In one embodiment, the cryopreservation formulation is free of one or more of the following: additional cryoprotectants, serum, and serum proteins.

[0107] In the context of the present invention, the term "cryoprotectant" means any substance or compound used for the cryopreservation of biological materials, and which prevents (i.e., at least reduces) cryo-damage in biological materials, for example by reducing ice crystal formation. In the context of the present invention, the terms "cryoprotectant", "cryopreservative" and their variations, as well as "antifreeze agent" are used interchangeably. When applied outside of cryobiology, cryoprotectants are commonly referred to as "antifreeze agents". In the context of the present invention, a substance or compound is considered to be a cryoprotectant if a solution containing the substance or compound vitrifies after cooling (e.g., at -80 °C or in liquid nitrogen). In the context of the present invention, "vitrification" means the formation of solid water with an irregular, amorphous structure. Vitrification is preferably determined by preparing a solution containing the substance or solution and cooling it at -80 °C or in liquid nitrogen for 30 minutes. If the cooled solution becomes transparent (as opposed to milky), for example if the transparency is equal to or higher than that observed for a reference (well-known) cryoprotectant (e.g., a permeating or non-permeating cryoprotectant as disclosed herein), the cooled solution is considered to be vitrified. For example, the transparency of a solution containing 20% (w / v) hydrolyzed collagen can be compared to that of dimethyl sulfoxide (10% v / v), ethylene glycol (1 M), propylene glycol (1 M) or glycerol (20% v / v) as a reference. A dose-response curve can be obtained to determine optimal vitrification (for hydrolyzed collagen).

[0108] In the context of the present invention, cryoprotectants encompass both permeating and non-permeating cryoprotectants. The term "permeating cryoprotectant" means a cryoprotectant that has the ability to penetrate into cells and thereby provide intracellular protection against cryo-damage. Permeating cryoprotectants are able to cross biological membranes. The term "non-permeating cryoprotectant" means a cryoprotectant that (largely) does not have the ability to penetrate into cells and / or (largely) does not require penetration into cells to mediate cryoprotection. Non-permeating cryoprotectants generally induce vitrification by the same mechanism as permeating cryoprotectants, but extracellularly. In the context of the present invention, the cell permeability of a compound or substance is preferably defined by an increase in cell volume after exposing cells to a solution containing the compound or substance. Thus, a substance or compound can be classified, for example, as a permeating or non-permeating cryoprotectant or cryopreservative. The rate of penetration into cells can be determined by measuring cell volume at specific time intervals after exposing the cells to the solution. A preferred protocol is as described by Eto et al (Cryobiology. 2014 Feb;68(1):147-51). Next, examples are provided to determine the possible cell permeability of hydrolyzed collagen:

[0109] Cells (such as the 3T3 fibroblast cell line) are exposed to a solution containing 20% w / v of collagen hydrolysate at 25 ± 0.5 °C. A solution containing permeating dimethyl sulfoxide (10% v / v), ethylene glycol (1 M), propylene glycol (1 M), or glycerol (20% v / v) is used as a reference, and the cell diameter is measured (control) after 30, 60, 120, 180, 240, and 300 seconds. The volume is calculated using the formula V = S 3 / 2 (S: relative cross-sectional area; V: relative volume, S = πab; a: major axis radius; b: minor axis radius) and the volume ratio at each time point is calculated using the control volume. If the average relative volume of the collagen hydrolysate solution matches at least the average volume of the reference (for one or more measured time points), the collagen hydrolysate is considered to permeate the cells.

[0110] Cells exposed to the collagen hydrolysate solution may initially contract and then recover (once the collagen hydrolysate enters the cells). Therefore, it is preferred to calculate the volume change starting from the lowest V value.

[0111] It has been found that collagen hydrolysate can also enhance the cryoprotective effect of dimethyl sulfoxide, reduce the toxic effect of dimethyl sulfoxide, and / or maintain the genotype and phenotype of the cells (e.g., by DNA sequencing, methylation, cell surface marker expression, cell differentiation assays, or other functional tests).

[0112] In one embodiment, the collagen hydrolysate is a cryoprotectant. In one embodiment, the collagen hydrolysate is a permeating cryoprotectant. In one embodiment, the collagen hydrolysate is a non-permeating cryoprotectant. In one embodiment, the collagen hydrolysate is not a cryoprotectant. In one embodiment, the collagen hydrolysate enhances the effect and / or reduces the toxicity of an (additional) cryoprotectant, which is preferably a permeating cryoprotectant, more preferably dimethyl sulfoxide.

[0113] Without being bound by theory, the inventors have found that collagen hydrolysate can have one or more mechanisms (in combination with DMSO) for improving cell recovery and viability after cryopreservation, such as providing cell binding sites, water binding / absorption, affecting osmotic pressure, affecting intracellular water content, and ice crystal formation. Therefore, in the context of the present invention, collagen hydrolysate can provide one or more of the various mechanisms of action.

[0114] In a preferred embodiment, the cryopreservation formulation comprises one or more of the following: additional cryoprotectants, serum, and serum proteins.

[0115] In one embodiment, the permeating cryoprotectant is one or more selected from the group consisting of dimethyl sulfoxide, ethylene glycol, propylene glycol, and glycerol.

[0116] If dimethyl sulfoxide is present in the formulation, the term "additional cryoprotectant" refers to any cryoprotectant other than dimethyl sulfoxide. If dimethyl sulfoxide is not present in the formulation, the term "additional cryoprotectant" can refer to any cryoprotectant, including dimethyl sulfoxide.

[0117] In one embodiment, the non-permeating cryoprotectant is one or more selected from the following: polyethylene glycol, sugars (such as sucrose, dextran, raffinose, trehalose, lactose), polyvinylpyrrolidone, and methylcellulose.

[0118] It has been found that the addition of collagen hydrolysate can eliminate the need for serum and serum-derived proteins that are commonly used in the art to reduce the toxicity of cryoprotectants. Without being bound by theory, collagen hydrolysate can protect cell membranes from shear stress and / or counteract the undesirable characteristics of dimethyl sulfoxide or other cryoprotectants. Additionally or alternatively, replacing serum and serum-derived proteins can reduce batch-to-batch variability, inconsistent cell responses, and usage limitations in terms of ethical or therapeutic considerations.

[0119] The term "serum" as disclosed herein encompasses human and animal sera, including but not limited to fetal bovine serum (FBS), fetal calf serum (FCS), or newborn bovine serum (NBS), newborn calf serum. The term "serum" as disclosed herein encompasses heat-inactivated serum. In the context of the present invention, the term "serum protein" encompasses human serum proteins and animal serum proteins, preferably human serum albumin (HSA) or bovine serum albumin (BSA).

[0120] In the present invention, salts may or may not be present in the cryopreservation formulation.

[0121] In one embodiment, the cryopreservation formulation is (substantially) free of salts.

[0122] In one embodiment, the cryopreservation formulation is (substantially) free of addition salts.

[0123] In one embodiment, the cryopreservation formulation is of non-animal origin, meaning it does not contain components of animal origin, such as when collagen (hydrolysate) or gelatin (hydrolysate) is obtained recombinantly or synthetically.

[0124] In one embodiment, the cryopreservation formulation comprises one or more of a permeating and / or non-permeating cryoprotectant in an amount of 0.1 to 50% by weight, preferably 0.2 to 25% by weight, more preferably 0.5 to 10% by weight, even more preferably 1 to 5% by weight, and most preferably 2 to 4% by weight, based on the weight of the cryopreservation formulation. As a supplement or alternative, in one embodiment, the cryopreservation formulation comprises one or more of a permeating and / or non-permeating cryoprotectant in an amount of 0.1 to 50% by weight, preferably 0.2 to 25% by weight, more preferably 0.5 to 10% by weight, even more preferably 1 to 5% by weight, and most preferably 2 to 4% by weight, based on the weight of the cryopreservation formulation.

[0125] In one embodiment, the cryopreservation formulation comprises one or more permeating diols in an amount of 0.1 to 20% by weight, preferably 0.2 to 10% by weight, more preferably 0.5 to 5% by weight, even more preferably 1 to 2.5% by weight, based on the weight of the cryopreservation formulation. "Permeating diols" as used herein are preferably ethylene glycol and / or propylene glycol.

[0126] In one embodiment, the cryopreservation formulation comprises one or more sugars in an amount of 0.1 to 20% by weight, preferably 0.2 to 10% by weight, more preferably 0.5 to 5% by weight, even more preferably 1 to 2.5% by weight, based on the weight of the cryopreservation formulation.

[0127] In the context of the present invention, "sugar" is preferably one or more selected from the following: sucrose, dextrose, raffinose, trehalose, and lactose.

[0128] In the context of the present invention, the cryopreservation formulation is suitable for cryopreserving one or more biological materials, such as one or more selected from the following: cells (eukaryotic or prokaryotic), multicellular eukaryotes (such as (nematode) worms), organoids (such as intestinal organoids, lung organoids, embryonic organoids, kidney organoids, epithelial organoids), microbial cultures (such as fungi, bacteria), blood (such as cord blood), semen, thrombosome, tissues (such as living or non-living tissues, such as tumors and histological cross-sections), ovarian tissue, skin tissue, musculoskeletal tissues (such as bone, cartilage, tendon), testicular tissue, sperm, oocytes, embryos, organs (such as liver, heart, kidney, skin, lung, pancreas, intestine), cell vesicles (such as extracellular vesicles, including exosomes), organoids, plant materials (such as plant seeds, shoot tips, dormant buds, bryophytes).

[0129] The biological material is preferably mammalian, more preferably human.

[0130] In a preferred embodiment, the use of the present invention relates to the cryopreservation of one or more biomaterials selected from: eukaryotic cells, prokaryotic cells, organelles, extracellular vesicles (EVs), organoids, tissues, and organs.

[0131] 3D organoids closely mimic the in vivo situation and contain proliferative stem and progenitor cells as well as differentiated cell types, including absorptive intestinal epithelial cells, secretory mucus-producing cells, Paneth cells, or enteroendocrine cells. The cell types are located in different proliferative (crypt) or differentiated (villus) zones throughout the organoid structure. 3D organoids represent a structurally closed system characterized by the formation of a continuous epithelium with in vivo-like characteristics. Additionally, molecular features comparable to native tissues classify primary 3D organoid cultures as important in vitro models for more precise drug screening in the future pharmaceutical industry.

[0132] Those skilled in the art are aware of different types of organoids and methods for obtaining them (e.g., organoids as described at least in the review article by Kim et al., Nature Reviews Molecular Cell Biology, Volume 21, pages 571 - 584 (2020)).

[0133] If the biomaterial is a cell, the cells can be cryopreserved, for example, at the following concentrations based on the volume of the cryopreservation formulation: 1×10 3 to 1×10 9 cells / ml, preferably 1×10 4 to 1×10 8 cells / ml, more preferably 1×10 5 to 1×10 7 cells / ml. If the biomaterial is an extracellular vesicle (i.e., an EV), the EVs can be cryopreserved, for example, at the following concentrations based on the volume of the cryopreservation formulation: 1×10 5 to 1×10 11 EVs / ml, preferably 1×10 6 to 1×10 10 EVs / ml, more preferably 1×10 7 to 1×10 9 EVs / ml. If the biomaterial is an organoid, the organoids can be cryopreserved, for example, at the following concentrations based on the volume of the cryopreservation formulation: 10 to 10,000 organoids / ml, preferably 50 to 1000 organoids / ml, more preferably 100 to 500 organoids / ml.

[0134] In one embodiment, in the context of the present invention, the cells are cells for advanced therapy medicinal products (ATMP) and / or one or more selected from the following: hematopoietic cells (such as erythroid cells, hematopoietic stem cells), myeloid cells (such as granulocytes, megakaryocytes, macrophages), immune cells (such as neutrophils, natural killer cells, T lymphocytes, B lymphocytes, monocytes, dendritic cells), peripheral blood mononuclear cells (PBMC), stem cells (such as hematopoietic stem cells, induced pluripotent stem cells (iPSC), mesenchymal stem cells (MSC), embryonic stem cells), amniotic epithelial cells, hepatocytes, and hepatic stellate cells.

[0135] In one embodiment, in the context of the present invention, the cells are chimeric antigen receptor (CAR) T lymphocytes. In one embodiment, in the context of the present invention, the cells are CD3+ and / or CD45+ T lymphocytes. In one embodiment, in the context of the present invention, the cells are CD45+ and / or CD34+ stem cells.

[0136] The cryopreservation preparation of the present invention is particularly suitable for cryopreserving one or more of "difficult-to-freeze cells", "non-freezable cells", terminally differentiated cells, and slowly growing cells, which generally cannot achieve the desired low cell death and / or high cell viability using 10% (v / v) (10.9 wt%) DMSO, but can generally be achieved by the combination of collagen hydrolysate and DMSO.

[0137] In one embodiment, in the context of the present invention, the cells are "easy-to-freeze cells". As used herein, "easy-to-freeze cells" refers to such cell types that, after cryopreservation in a preparation based on permeating and / or non-permeating cryoprotectants rather than collagen hydrolysate as disclosed herein, show less than 20% cell death (under optimal conditions) and maintain viability (e.g., the viability reduction does not exceed 20%). Preferably, "easy-to-freeze cells" as used herein refers to such cell types that show less than 20% cell death and maintain viability (e.g., the viability reduction does not exceed 20%) after cryopreservation in 10% (v / v) DMSO.

[0138] In one embodiment, in the context of the present invention, the cells are "difficult-to-freeze cells". As used herein, "difficult-to-freeze cells" refers to a cell type that, after cryopreservation in a formulation based on permeating and / or non-permeating cryoprotectants as disclosed herein rather than collagen hydrolysate, shows less than 20% cell death (under optimal conditions) but shows a decrease in viability (e.g., a decrease in viability of at least 20%). Preferably, as used herein, "difficult-to-freeze cells" refers to a cell type that shows less than 20% cell death but shows a decrease in viability (e.g., a decrease in viability of at least 20%) after cryopreservation in 10% (v / v) DMSO.

[0139] In one embodiment, in the context of the present invention, the cells are "non-freezable cells". As used herein, "non-freezable cells" refers to a cell type that shows at least 20% cell death (under optimal conditions) after cryopreservation in a formulation based on permeating and / or non-permeating cryoprotectants as disclosed herein rather than collagen hydrolysate. Preferably, as used herein, "non-freezable cells" refers to a cell type that shows at least 20% cell death after cryopreservation in 10% (v / v) DMSO.

[0140] In one embodiment, the cells are primary cells, meaning cells freshly isolated from a living tissue (preferably human tissue).

[0141] In one embodiment, the cells are terminally differentiated cells, preferably selected from one or more of the following: neuronal cells, neuropil, cardiomyocytes, skeletal muscle cells, terminally differentiated fibroblasts, terminally differentiated hepatocytes, monocytes, dendritic cells, beta cells, islets of Langerhans, primary human hepatocytes, primary human monocytes, chondrocytes, osteocytes, and adipocytes. As used herein, the term "terminally differentiated cells" refers to cells that rarely (i.e., no more than once every 48 hours, preferably no more than once every 7 days) or do not proliferate at all, especially in vitro. As used herein, the term "terminally differentiated cells" preferably refers to cells that have undergone cell cycle arrest. In the context of the present invention, the proliferative capacity preferably relates to the ability to proliferate in vitro. Generally speaking, terminally differentiated cells do not transform into other cell types and have differentiated to perform specific functions.

[0142] In one embodiment, the biomaterial as disclosed herein is (intended) for transplantation into a host, preferably into a human. In one embodiment, the biomaterial is an advanced therapy medicinal product (ATMP), such as one or more of a gene therapy medicinal product, a somatic cell therapy medicinal product, and a tissue engineered product. For ATMP products, it is generally considered, as a rule of thumb, that cell viability should be higher than 80% (i.e., cell death is lower than 20%), and furthermore, the cryopreservation formulation should be GMP grade and preferably free of DMSO and / or (animal) serum. Currently available cryopreservation formulations do not meet this standard, for example because they generally require serum (protein) and / or DMSO. However, the cryopreservation formulation of the present invention can meet this standard.

[0143] In one embodiment, the cryopreservation formulation is GMP (Good Manufacturing Practice) grade and / or can be manufactured under GMP conditions.

[0144] In one embodiment, the cryopreservation formulation can additionally be used as a cell carrier, such as scaffolds and / or hydrogels known in the art, including bioinks suitable for 3D printing. The cryopreservation formulation for this purpose contains additional crosslinkable polymers that allow the formation of a hydrogel (e.g., as discussed by Chaudhary et al. Beni-Suef Univ J Basic Appl Sci 11, 3 2022). For example, the cryopreservation formulation can contain a photoinitiator to facilitate crosslinking before and / or after cryopreservation, such as to obtain a 3D construct (Tan et al. Micromachines (Basel). 2022 Jul;13(7):1038).

[0145] In one embodiment, the cryopreservation formulation of the present invention is suitable for the long-term preservation of biomaterials, such as cells, more preferably mammalian cells, even more preferably human cells. In one embodiment, the cryopreservation formulation of the present invention is used for cryopreservation for at least 1 year (e.g., 1 year to 20 years), preferably at least 5 years, more preferably at least 10 years.

[0146] When using the cryopreservation preparation of the present invention, it unexpectedly shows that if a centrifugation washing step is not included after thawing, cell recovery, viability and / or functionality can be improved. In one embodiment, the biological material is not subjected to a centrifugation washing step after cryopreservation and / or after freeze-thawing, but is directly used for, for example, in vitro culture and / or in vivo implantation, without excluding the possibility of diluting the cryopreservation preparation in a selected additional (liquid) preparation. In the context of the present invention, the terms "after cryopreservation" and / or "after freeze-thawing" preferably mean a time period of 15 minutes, preferably 30 minutes, more preferably 60 minutes, even more preferably 1 day, and most preferably 1 week after thawing the cells. In the context of the present invention, the term "centrifugation washing" can be any method of aggregating and / or precipitating at least part of the biological material (such as cells) by applying centrifugal force. Centrifugation washing allows the removal of the supernatant (cryopreservation preparation), and then the biological material can be resuspended in a different aqueous medium or another carrier substance. The term "centrifugation washing" encompasses centrifugation using a commercial (laboratory) centrifuge, for example, at 50 to 10,000×g, or 100 to 5000×g, or 200 to 1000×g, or 300 to 500×g.

[0147] In the context of the present invention, the terms "comprising / including" or "comprises / includes" and variations thereof are used in their non-limiting sense to mean including the item(s) following the word, but not excluding items not specifically mentioned.

[0148] Unless the context clearly requires the presence of one and only one element, the mention of an element by a noun without a quantifier does not exclude the possibility of there being more than one element. Thus, a noun without a quantifier generally means "at least one / species".

[0149] In the context of the present invention, a level is "increased" or "decreased" when it is at least 1% (such as 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%) higher or lower, respectively, than the corresponding level in a control or reference. As a supplement or alternative, a level in a sample can be considered increased or decreased when it is statistically significantly higher or lower, respectively, than the level in a control or reference (including an earlier time point), regardless of the magnitude of the change. In the context of the present invention, the term "to reduce" can be used interchangeably with the term "to decrease". Description of the Drawings

[0150] Figure 1. Cell death in fibroblast 3T3 cells after cryopreservation with a cryopreservation preparation based on a combination of 25% (w / v) collagen hydrolysate (containing 1250 EU / g LPS (“low LPS”, P5000) or 13350 EU / g LPS (“high LPS”, P2000)) and 6.7% (v / v) DMSO.

[0151] Figure 2 . Cell death in fibroblast 3T3 cells in a cryopreservation preparation containing 8% (v / v) DMSO and with or without 20000 EU / ml LPS (“LPS” group). Cell death was determined in the absence of a centrifugation washing step or in the presence of a centrifugation washing step (for 3 minutes at 300×g force) (“spin” group).

[0152] Figure 3 . Cell death (%) in neuroglial cells after cryopreservation in 10% (v / v) DMSO (“control”) or 6.7% (v / v) DMSO supplemented with 30% (w / v) collagen hydrolysate (“+CH”).

[0153] Figure 4 . Neuronal cell death after cryopreservation in various cryopreservation preparations. 30% HC = collagen hydrolysate with an average molecular weight of approximately 5000 Da at 30 w / v (“P5000”).

[0154] Figure 5 . Staining of glial markers and neuronal markers in fresh neuroglial cells (left panel) and cryopreserved neuroglial cells (middle and right panels). The neuroglial cells were cryopreserved in 10% (v / v) DMSO (middle panel) or a combination of 6.7% (v / v) DMSO and 30% (w / v) collagen hydrolysate (CH, “P5000”). The staining shows combined staining of GFAP (glial cell marker) and MAP2 (neuronal marker).

[0155] Figure 6 . Cell viability was determined by staining. Fibroblast 3T3 cells were cryopreserved in a cryopreservation medium without (upper panel) or with (lower panel) hydrolyzed gelatin (“HG”, 20% w / v) and 0 to 13.3% (v / v) DMSO. The cells were thawed and cultured for 3 days. Then the live cells were stained.

[0156] Figure 7.Cell viability was determined by counting. Fibroblast 3T3 cells were cryopreserved in cryopreservation medium without (upper figure) or with (lower figure) hydrolyzed gelatin (“HG”, 20% w / v) and 0 to 20% (v / v) DMSO. The cells were thawed and cultured for 3 days. The number of live cells was counted after 3 days.

[0157] Figure 8 .Cell viability of 3D organoids determined by WST-1 assay 1 hour after thawing (% relative to control).

[0158] Examples

[0159] Example 1

[0160] Methods

[0161] Table 1 provides an overview of the starting materials used in the examples (all from Rousselot B.V., Belgium).

[0162] Table 1. Overview of starting materials used. MW = molecular weight; LPS = lipopolysaccharide, EU = endotoxin unit; IEP = isoelectric point.

[0163]

[0164] The 3T3 cell line (fibroblast culture) was used in the experiments. The cells were cryopreserved in cryovials by slow freezing using a cooling rate of -1°C / minute. The cells were then stored at -80°C for at least 18 hours overnight. The cryopreservation solution was prepared by supplementing DMEM (Dulbecco's Modified Eagle Medium) medium with DMSO (varying between 0 and 13% v / v) and / or collagen hydrolysate (varying between 0 and 40% w / v).

[0165] For thawing, the cells in the cryovial were thawed in a water bath at 37°C until only a small piece of ice remained, and then the cryovial was kept on ice. The cell suspension was transferred to a tube and an excess of medium (to room temperature) was carefully added and the suspension was mixed. The cells were centrifuged at 300×g for 3 minutes, after which the supernatant was discarded and the cells were resuspended in warm (about 37°C) cell culture medium at the desired concentration.

[0166] The % of cell death after thawing was determined by counting the live cells and comparing them with the number of frozen cells.

[0167] Results

[0168] Table 2 shows cell death in fibroblast 3T3 cells after cryopreservation with cryopreservation formulations based on 0 to 40% (w / v) collagen hydrolysate (“P5000”) and 6.7% (v / v) DMSO. It was found that the combination of relatively high concentrations of collagen hydrolysate (e.g., 30% or 40% w / v) with 6.7% (v / v) DMSO reduced cell death to below 10%. This was lower than the cell death achieved with 20% (w / v) collagen hydrolysate and 6.7% (v / v) DMSO.

[0169] Table 2. Cell death in fibroblast 3T3 cells after cryopreservation with cryopreservation formulations based on different concentrations of collagen hydrolysate (CH, “P5000”) and 6.7% (v / v) DMSO.

[0170] CH Concentration (w / v) DMSO Concentration (v / v) Cell Death 0％ 6.7％ >20％ 20％ 6.7％ 2.3％ 30％ 6.7％ 2.9％ 40％ 6.7％ 6％ 20％ 10％ 56％

[0171] Table 3 shows cell death (%) in fibroblast 3T3 cells after cryopreservation with different concentrations of DMSO (0%, 0.8%, 1.7%, 3.3%, 6.7% v / v), alone or in combination with 20% (w / v) collagen hydrolysate (“P5000”). It was found that 20% collagen hydrolysate could reduce cell death when used as an independent cryopreservative. In addition, for the combination of collagen hydrolysate and DMSO, even when the DMSO concentration was as low as 0.8%, cell death was reduced to below 20%. Compared with 10% DMSO (commonly considered the “gold standard”), the addition of collagen hydrolysate allowed a >90% reduction in the amount of DMSO concentration required. It was also found that when collagen hydrolysate was used in combination with 0.8% to 6.7% DMSO, cell death could be reduced to 2% to 5%. Such low toxicity could not be achieved when DMSO was used alone.

[0172] Table 3. Cell death (%) in fibroblast 3T3 cells after cryopreservation in different concentrations of DMSO (0%, 0.8%, 1.7%, 3.3%, 6.7% v / v) alone or in combination with 20% (w / v) collagen hydrolysate (CH, “P5000”).

[0173] DMSO Concentration Cell Death in DMSO Only Cell Death in DMSO + 20% CH 0％ 75.8％ 48％ 0.8％ 65.2％ 18.8％ 1.7％ 48.2％ 4.1％ 3.3％ 23.1％ 3.3％ 6.7％ >20％ 2.3％ 10％ 20％ No Data

[0174] Table 4 shows cell death in fibroblast 3T3 cells after cryopreservation with cryopreservation formulations combining collagen hydrolysates with a molecular weight of approximately 2000 Da (“P2000”) or approximately 5000 Da (“P5000”) with 6.7% DMSO. It was found that both collagen hydrolysates with a molecular weight of 2000 Da or 5000 Da resulted in low cell death in the presence of 20%, 30%, or 40% (all w / v) collagen hydrolysate.

[0175] Table 4. Cell death in fibroblast 3T3 cells after cryopreservation with cryopreservation formulations combining collagen hydrolysate (CH) with a molecular weight of approximately 2000 Da (“P2000”) or approximately 5000 Da (“P5000”) with 6.7% DMSO.

[0176] CH Concentration (w / v) CH Approximately 2000 Da CH Approximately 5000 Da 0％ 15.5％ 15.5％ 20％ 8.2％ 2.3％ 30％ 2.3％ 2.9％ 40％ 6.0％ 6.0％

[0177] To study the effect of the centrifugation washing step, cells were centrifuged (for 3 minutes at 300×g force) or not centrifuged and resuspended in fresh DMEM medium after freeze-thawing. Then the % cell death was determined as described above.

[0178] Table 5 shows cell death in fibroblast 3T3 cells in cryopreservation formulations containing different concentrations of collagen hydrolysate (“P5000”) and DMSO. Cell death was determined in the absence or presence of a centrifugation washing step (for 3 minutes at 300×g force). It was found that cell death was lowest in the presence of 24% (w / v) collagen hydrolysate and less than 8% DMSO.

[0179] Table 5. Cell death in fibroblast 3T3 cells in cryopreservation formulations containing different concentrations of collagen hydrolysate (CH, “P5000”) and DMSO. Cell death was determined in the absence or presence of a centrifugation washing step.

[0180] Cryopreservative Composition Cell Death of Uncentrifuged and Washed Cell Death of Centrifuged and Washed 0% CH + 8% DMSO 15.5％ 20％ 24% CH + 0% DMSO 1％ 19％ 24% CH + 2.7% DMSO 1％ 4％ 24% CH + 5.3% DMSO 2％ 4％

[0181] Overall, it was found that combinations of 20% or more collagen hydrolysate with no DMSO or low DMSO (e.g., <10%) were most suitable for cryopreservation. Collagen hydrolysates with an average molecular weight of approximately 2000 Da and approximately 5000 Da behaved similarly.

[0182] When “P5000” and “H5000” were tested at 0%, 10%, 20%, 30% or 40% (w / v) in the absence of DMSO, an inverse concentration-dependent relationship was found between the amount of collagen hydrolysate and the % cell death. The % cell death was lowest when using 30% or 40% (w / v) of collagen hydrolysate. In the absence of DMSO, the cells also showed more beneficial genotypes and phenotypes (by DNA sequencing, methylation, cell surface marker expression, cell differentiation assays or other functional tests).

[0183] A decrease in the amount of DMSO was found to result in less change in cell genotype and phenotype, and thus it was considered that collagen hydrolysate could reduce the negative effects of DMSO ( et al. Scientific Reports Volume 8, Article number: 14828, 2018).

[0184] Overall, when the amount of collagen hydrolysate exceeded 20%, especially when it was 30% or 40%, the best results were obtained.

[0185] Example 2

[0186] Methods

[0187] A comparison was made between “P5000” (1250 EU / g LPS) and “P2000” (13350 EU / g LPS) hydrolyzed gelatin, which had different LPS contents. The cells were frozen and thawed according to the method in Example 1, and the % cell death was determined.

[0188] To further investigate the role of LPS during washing and centrifugation, frozen solutions with or without 20000 EU / ml LPS were compared. The frozen solution was based on DMEM according to Example 1 and contained 24% (w / v) hydrolyzed gelatin (“P5000”) and 8% (v / v) DMSO. Freezing and thawing were carried out according to Example 1.

[0189] After freezing and thawing, the cells were centrifuged or not and resuspended in fresh DMEM medium. Then the % cell death was determined as described above.

[0190] The LPS level was determined using the Endozyme recombinant factor-C method. (Reference: Supplier Hygloss; FDA-approved method).

[0191] Results

[0192] Figure 1Cell death in fibroblast 3T3 cells after cryopreservation with a cryopreservation preparation based on a combination of 25% (w / v) collagen hydrolysate (containing 1250 EU / g LPS (“low LPS”, P5000) or 13350 EU / g LPS (“high LPS”, P2000)) and 6.7% (v / v) DMSO was examined. It was found that compared with the high LPS condition, the use of collagen hydrolysate with a low LPS content reduced cell death by 75%.

[0193] Figure 2 Cell death in fibroblast 3T3 cells in a cryopreservation preparation containing 8% (v / v) DMSO, with or without 20000 EU / ml LPS added (“LPS” group) is shown. Cell death was determined in the absence or presence of a centrifugation washing step (“spin” group). It was found that in the presence of 8% DMSO, centrifugation washing did not increase cell death. However, in the presence of 20000 EU / ml, centrifugation washing increased cell death.

[0194] Similarly, for cryopreservation preparations containing 24% (w / v) collagen hydrolysate (“P5000”), with or without 20000 EU / ml LPS added, the centrifugation washing step led to higher cell death, especially in the presence of LPS. For cryopreservation preparations containing 24% (w / v) collagen hydrolysate (“P5000”) and with or without 8% (v / v) DMSO, the effects of centrifugation and LPS on increasing cell death were observed.

[0195] In addition, it was found that the presence of LPS during cryopreservation could induce unwanted toxicity and / or pro-inflammatory responses.

[0196] Overall, it was found that cryopreservation was further improved when LPS was absent or present in the lowest amount, especially when centrifugation washing was performed. The absence of LPS or low LPS made the centrifugation washing redundant.

[0197] Overall, when the amount of collagen hydrolysate was about 20% (w / v) or more than 20% (w / v), especially when it was 30% (w / v) or 40% (w / v), the best results were obtained.

[0198] Example 3

[0199] Method

[0200] According to the method described in Example 1, experiments were conducted using a neurofelt cell population (containing neuronal cells). The neurofelt cells were obtained from a supplier. The neurofelt cells used were approximately 1:3 astrocytes:neurons. The cells were stained with GFAP (a glial cell marker) and MAP2 (a neuronal marker).

[0201] Results

[0202] Figure 3 Shows the cell death (%) in neurofelt cells after freezing in 10% (v / v) DMSO (“control”) or 6.7% (v / v) DMSO supplemented with 30% (w / v) collagen hydrolysate (“+CH”). It was found that the combination of DMSO and collagen hydrolysate led to a strong reduction in cell death, which was not observed when only 10% (v / v) DMSO was used.

[0203] Figure 4 Shows the cell death in neurofelt cells after cryopreservation in various cryopreservation formulations. The combination of 30% (w / v) collagen hydrolysate (“P5000”) and DMSO reduced cell death compared to DMSO alone. Notably, in neurofelt cells, cell death was almost 70% in 6.7% (v / v) DMSO, indicating a large improvement achieved by adding collagen hydrolysate.

[0204] Figure 5 Shows the staining of glial and neuronal markers in fresh neurofelt cells (i.e., not cryopreserved, left panel) and cryopreserved and thawed neurofelt cells (middle and right panels).

[0205] As shown, the left and right panels show a mixture of neurons and astrocytes with dendrites of the expected density. In the middle panel, the density is lower and thus no activity is expected. When analyzing the cells, the density and formation of dendrites (processes / outgrowths) were studied. It was found that neurofelt cells cryopreserved in a formulation containing a combination of 6.7% (v / v) DMSO and 30% (w / v) collagen hydrolysate (“P5000”) showed an improved phenotype compared to cells cryopreserved in a 10% (v / v) DMSO formulation.

[0206] It was also found that 20% to 40% (w / v) collagen hydrolysate is the optimal concentration range (with or without additional DMSO).

[0207] Generally, it was found that for cells showing high cell death and / or low viability in 10% (v / v) DMSO (and thus considered "difficult to freeze" or "non-freezable" cells), such as neuronal cells, cryopreservation media based on more than 20% (w / v) collagen hydrolysate were more effective than cryopreservation media based solely on (10% v / v) DMSO. The lowest cell death and the highest viability were observed when more than 20% by weight of collagen hydrolysate was combined with 5% (v / v) or other low amounts of DMSO.

[0208] For undifferentiated cells, such as ESCs, similar results were obtained. With only 10% (v / v) DMSO, ESCs showed undesirably high cell death, however, cell death improved to less than 5% when combined with 30% (w / v) collagen hydrolysate.

[0209] Comparisons were also made between 3T3 fibroblasts, HEK293 cells, CaLu-3 cells, mesenchymal stem cells (MSCs), embryonic stem cells (ESCs) and neuronal cells. It was found that 10% (v / v) or more DMSO did not result in satisfactory cell recovery and viability in ESCs and neuronal cells. However, at least 80% cell recovery was achieved using more than 20% (w / v) collagen hydrolysate or better for specific cells, especially when using low concentrations of DMSO (such as 5% or other low concentrations).

[0210] Other cells tested included human primary cardiomyocytes, islets, human primary hepatocytes and human primary monocytes. For these cell types, cell recovery upon freezing in 10% (v / v) DMSO was less than 20% or even less than 10%. Cell recovery and viability were improved when combined with more than 20% by weight (especially 30 or 40% by weight) of collagen hydrolysate and with 1% to 5% (v / v) DMSO.

[0211] Generally, the best results were obtained when the amount of collagen hydrolysate exceeded 20% (v / v), especially when it was 30% (v / v) or 40% (v / v), in combination with low concentrations (such as 1% to 5% v / v) of DMSO.

[0212] Example 4

[0213] Method

[0214] Cryopreserve fibroblasts according to Example 1. After thawing, culture the cells in a tissue culture plate. After 3 days, determine cell viability by staining the live cells and then counting them.

[0215] Results

[0216] Figure 6 and 7 showed the viability of fibroblast 3T3 cells cryopreserved and cultured for three days in different cryogenic medium compositions. The live cells were then stained.

[0217] Over time, relatively higher cell expansion was observed for cells that had been cryopreserved in hydrolyzed gelatin in combination with DMSO compared to cells cryopreserved in DMSO alone. This indicates that collagen hydrolysate not only reduces cell death but also enhances the viability of the recovered cells. In addition, it was found that cell viability was highest when the DMSO concentration was below 10% (v / v), and was particularly high for the 3.3% and 6.7% (v / v) DMSO groups. Viability was also determined after one or two days, and the highest cell viability was also shown when the DMSO concentration was below 10% (v / v).

[0218] Example 5

[0219] Method

[0220] According to Example 1, the 3T3 cell line (fibroblast culture) was cryopreserved in a cryogenic medium containing 2.5% or 10% (v / v) DMSO, with or without 30 wt% hydrolyzed gelatin.

[0221] For cell metabolic activity, a colorimetric MTT assay was performed at 96 hours. The MTT assay is based on the metabolic reduction of MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide, a substance with a yellow color) to blue formazan product by mitochondrial dehydrogenase. Only live cells are able to catalyze this reaction.

[0222] Results

[0223] Table 6 shows the metabolic activity of fibroblast 3T3 cells after cryopreservation in different cryogenic media. The highest metabolic activity was observed when the cells were cryopreserved in a combination of hydrolyzed gelatin and a relatively low concentration of DMSO.

[0224] Table 6. Metabolic activity in fibroblast 3T3 cells after cryopreservation in cryopreservation formulations with different concentrations of DMSO and with or without 30 wt% hydrolyzed gelatin (collagen hydrolysate, CH).

[0225] Cryopreservative Composition Metabolic Activity 10% DMSO Average 2.5% DMSO Low 10% DMSO + 30% CH High 2.5% DMSO + 30% CH Very High

[0226] Overall, it was found that using a relatively low amount of DMSO was beneficial. Similar effects of DMSO concentration were observed for other concentrations of collagen hydrolysate.

[0227] Example 6

[0228] Method

[0229] Mouse primary cortical cells (E18) were cryopreserved according to the cryopreservation method described in Example 1, but the cryopreservation period was increased to 4 weeks. Different cryopreservation media were compared as described in Table 7 (n = 2 per group).

[0230] 1. For cell metabolic activity, a colorimetric MTT assay was performed. The MTT assay is based on the metabolic reduction of MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide, a yellow substance) to blue formazan product by mitochondrial dehydrogenase. Only living cells can catalyze this reaction.

[0231] 2. Lactate dehydrogenase (LDH) assay was used to evaluate cytotoxicity in the supernatant of primary cortical neurons. LDH is a stable cytoplasmic enzyme present in all cells and is rapidly released into the cell culture supernatant when the plasma membrane is damaged. The LDH activity in the cell culture supernatant was determined by a coupled enzymatic reaction in which the tetrazolium salt INT is reduced to formazan .

[0232] 3. To evaluate neuronal activity, primary cortical neurons were transduced with Neuroburst TM lentivirus at DIV2, and live cell imaging on an IncuCyteTM device was used to monitor the evaluation of neuronal activity for 72 hours (e.g., DIV10 to 12), with RFP oscillations read every 120 seconds. The IncuCyteTM device automatically evaluated the number of active cells, the average burst rate, and the average correlation.

[0233] Table 7. The effects of different cryopreservation formulations based on DMSO and / or hydrolyzed gelatin (collagen hydrolysate, CH) on the viability and metabolic activity of mouse primary cortical cells after cryopreservation were tested.

[0234]

[0235] Results

[0236] It was found that 10% (v / v) DMSO alone led to poor viability and metabolic activity. When DMSO was combined with collagen hydrolysate, generally high viability and metabolic activity were observed, and the concentration of DMSO was preferably less than 10% (v / v).

[0237] Similar experiments were performed on cortical rat cells (neuropil), in which GFAP / MAP2 staining was also performed as described in Example 3.

[0238] Example 7

[0239] Example 7 shows the viability of intestinal organoids cryopreserved in hydrolyzed gelatin X-PureHGP LS (Rousselot B.V., Belgium) or Peptan P5000 LD (Rousselot B.V., Belgium) compared to a cryomedia based on FCS and 10% DMSO.

[0240] Figure 8 The cell viability (% relative to control) of 3D intestinal organoids determined by WST-1 assay 1 hour after thawing is shown.

[0241] It was found that the viability of organoids cryopreserved in X-Pure HGP LS and Peptan P5000 LD was higher 1 hour after thawing compared to a cryomedia based on FCS + 10% DMSO.

Claims

1. A cryopreservation preparation comprising a collagen hydrolysate and dimethyl sulfoxide, wherein the amount of the collagen hydrolysate is 10 to 70% by weight based on the weight of the cryopreservation preparation, wherein the amount of dimethyl sulfoxide is less than 10% (v / v) based on the volume of the cryopreservation preparation.

2. The cryopreservation preparation according to claim 1, comprising not more than 8.5%, preferably not more than 7% (v / v) of dimethyl sulfoxide.

3. The cryopreservation preparation according to claim 1 or 2, comprising at least 1% (v / v), preferably at least 2% (v / v) of dimethyl sulfoxide.

4. The cryopreservation preparation according to any one of the preceding claims, comprising 20 to 50% by weight of a collagen hydrolysate.

5. The cryopreservation preparation according to any one of the preceding claims, comprising more than 20% by weight and less than 45% by weight of a collagen hydrolysate.

6. The cryopreservation preparation according to any one of the preceding claims, wherein the collagen hydrolysate is hydrolyzed gelatin.

7. The cryopreservation preparation according to any one of the preceding claims, wherein the average molecular weight of the collagen hydrolysate is 500 to 10,000 Da.

8. The cryopreservation preparation according to any one of the preceding claims, wherein the endotoxin level of the collagen hydrolysate is less than 10,000 EU / g, preferably less than 1,000 EU / g, more preferably less than 100 EU / g based on the weight of the collagen hydrolysate.

9. The cryopreservation preparation according to claim 8, wherein the collagen hydrolysate is endotoxin-free.

10. The cryopreservation preparation according to any one of the preceding claims, wherein the endotoxin level of the cryopreservation preparation is less than 2,500 EU / ml, preferably less than 250 EU / ml, more preferably less than 25 EU / ml based on the volume of the cryopreservation preparation.

11. The cryopreservation preparation according to claim 10, wherein the cryopreservation preparation is endotoxin-free.

12. The cryopreservation preparation according to any one of claims 1 to 11, wherein the cryopreservation preparation does not contain one or more of the following: additional cryoprotectants, serum, and serum proteins.

13. The cryopreservation preparation according to any one of claims 1 to 11, comprising one or more of the following: additional cryoprotectants, serum, and serum proteins.

14. The cryopreservation preparation according to claim 12 or 13, wherein the additional cryoprotectant is a permeating and / or non-permeating cryoprotectant.

15. The cryopreservation preparation according to claim 14, wherein the permeating cryoprotectant is a permeating diol, preferably ethylene glycol, propylene glycol, or a combination thereof.

16. The cryopreservation preparation according to claim 14, wherein the non-permeating cryoprotectant is one or more selected from the following: polyethylene glycol, glycerol, polyvinylpyrrolidone, methylcellulose, sugar, or a combination thereof.

17. A method for cryopreserving biological substances, the method comprising the following steps: Provide the biological substance in a cryopreservation preparation as defined in any one of claims 1 to 16, and lower the temperature of the cryopreservation preparation to below the freezing point of the cryopreservation preparation.

18. A method for cryopreserving a biological substance, the method comprising the steps of: providing the biological substance in a cryopreservation preparation comprising a collagen hydrolyzate, and lowering the temperature of the cryopreservation preparation to below the freezing point of the cryopreservation preparation, wherein the amount of the collagen hydrolyzate is 10 to 70% by weight based on the weight of the cryopreservation preparation, wherein the amount of dimethyl sulfoxide is less than 10% (v / v) based on the volume of the cryopreservation preparation.

19. The method according to claim 17 or 18, wherein the biological substance is selected from eukaryotic cells, prokaryotic cells, organelles, extracellular vesicles, organoids, tissues and organs.

20. Use of a collagen hydrolyzate in the cryopreservation of a biological substance, wherein the collagen hydrolyzate is provided in a cryopreservation preparation as defined in any one of claims 1 to 16.

21. Use of a collagen hydrolyzate in the cryopreservation of a biological substance, wherein the collagen hydrolyzate is provided in a cryopreservation preparation, wherein the amount of the collagen hydrolyzate is 10 to 70% by weight based on the weight of the cryopreservation preparation, wherein the amount of dimethyl sulfoxide is less than 10% (v / v) based on the volume of the cryopreservation preparation.

22. The use according to claim 20 or 21, wherein the biological substance is selected from eukaryotic cells, prokaryotic cells, organelles, extracellular vesicles, organoids, tissues and organs.

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