Cryopreserved preparations containing collagen hydrolysate

Abstract

The present invention relates to a cryopreservation preparation comprising collagen hydrolysate, preferably comprising dimethyl sulfoxide.The present invention further relates to a method for cryopreserving one or more biological materials, comprising providing one or more biological materials in a cryopreservation preparation comprising collagen hydrolysate and preferably dimethyl sulfoxide.The present invention also relates to the use of a cryopreservation preparation comprising collagen hydrolysate and preferably dimethyl sulfoxide for cryopreserving one or more biological materials selected from the group consisting of eukaryotic cells, prokaryotic cells, cell organelles, extracellular vesicles, organoids, tissues, and organs.The present invention also relates to the use of collagen hydrolysate in a cryopreservation preparation, preferably in combination with dimethyl sulfoxide.

Description

[Technical Field]

[0001] The present invention relates to the cryopreservation of biological materials such as cells and tissues, and to cryoprotectants for use therein. [Background technology]

[0002] ·Cryopreservation Cryopreservation is a process in which biological materials—most commonly single-cell suspensions, but also cellular tissues—are preserved by freezing. By freezing biological materials, the enzymatic and chemical activity of the cells is stopped. At any desired time, the biological material can be thawed, and cellular activity can resume. Cryopreservation confers an indefinite shelf life to biological materials, and the frozen state also allows biological materials to be easily transported between different laboratories. In particular, cell cryopreservation opens up a variety of new possibilities for medical therapy, where cells can be significantly expanded and then stored until their use is required. The relative ease and flexibility of cryopreservation make it an essential task in cell culture laboratories, whether for research or therapeutic purposes.

[0003] Cell stress and toxicity during cryopreservation Cryopreservation is most commonly performed at freezing temperatures on the order of -80°C or lower. A significant limitation of cryopreservation is the inherent toxicity of the freezing process to cells, which can lead to cell loss, cellular stress, and / or altered cellular responses. Cytotoxicity during cryopreservation is largely related to intracellular ice formation and disruption of intracellular osmotic balance (Bissoyi et al. Biopreserv Biobank. 2014 Feb;12(1):23-34). With this in mind, the success of cell recovery after cryopreservation is controlled, at least in part, by how cryopreservation is performed. A preferred method for cryopreserving cells is so-called "slow freezing." Slow freezing involves cooling cells at a controlled rate until the desired freezing temperature is reached. For example, a typical cooling rate of approximately 1°C / min is considered appropriate for many mammalian cells. The principle behind slow freezing is that it affects the external solute concentration, which dehydrates the cells to some extent and reduces the formation of intracellular ice crystals.

[0004] ·Cryoprotectant (CPA) Slow freezing is generally performed in combination with one or more cryoprotectants (i.e., cryoprotectants, CPAs). CPAs typically work by increasing intracellular solute concentrations, thereby helping to reduce freeze damage. Sulfoxides, glycols, and sugars form the most common CPAs, which are typically classified as either permeant or non-permeant CPAs. Permeant CPAs include dimethyl sulfoxide (DMSO), ethylene glycol, propylene glycol, and glycerol. Permeant CPAs are able to penetrate cell membranes. In cells, the structural ability to hydrogen bond with water helps reduce mechanical and osmotic damage. Non-permeant CPAs are typically larger molecules that exert their protective effect outside the cell. Non-permeable CPAs include polyethylene glycol, polyvinylpyrrolidone, methylcellulose, and sugars (e.g., sucrose, dextrose, and trehalose) (Whaley et al. Cryopreservation: An Overview of Principles and Cell-Specific Considerations. Cell Transplantation. January 2021).

[0005] Undesirable characteristics of a CPA CPAs also have well-known undesirable characteristics. These effects are best characterized for dimethyl sulfoxide (DMSO), which is also considered the most traditional CPA (e.g., the "gold standard CPA"). However, these undesirable characteristics are often found in all CPAs. Some of the most important undesirable characteristics are summarized below: 1) CPA is toxic to cells, which appears to be largely responsible for apoptosis through membrane pore formation (Notman et al. J. Am. Chem. Soc. 128, 13982-13983). 2) CPAs may interfere with various cellular processes and therefore alter the functionality of cells after cryopreservation. For example, changes in gene expression, cell proliferation, and differentiation have been reported after the use of CPAs (Tuncer et al. Scientific Reports volume 8, Article number: 14828, 2018). 3) CPAs can interfere with the therapeutic potential of cells after freezing. For example, stem cells cryopreserved with CPAs have been found to exhibit reduced engraftment after transplantation in patients (Mitrus et al. Randomized Controlled Trial Bone Marrow Transplant. 2018 Mar;53(3):274-280). 4) CPA can modulate the inflammatory response in cells. An altered inflammatory response can lead to unreliable test results and / or interfere with the therapeutic potential of cells (Li et al. Front Immunol. 2021 Nov 29;12:765667). 5) The use of CPA increases labor intensity. A washing step ideally ensures that all CPA is removed from the cell suspension. This increases the resources required in the cell culture lab. 6) Despite extensive washing steps, CPAs such as DMSO appear to persist in cell suspensions. DMSO contaminants cause direct side effects in recipients in clinical settings (e.g., hypertension, nausea, and vomiting). This limits their use in cell and tissue transplants and advanced therapeutic medicinal products (ATMPs). As a rule of thumb, it is typically considered that cell viability should be better than 80% (i.e., cell death less than 20%). For ATMPs in particular, it is further desirable that cryopreserved preparations be GMP-grade and preferably free of DMSO and / or serum. Currently available cryopreserved preparations do not meet this criterion, for example, because they often require serum and / or DMSO.

[0006] CPA is inappropriate for some cell types Another important limitation of traditional CPAs (e.g., DMSO, glycols, sugars) is that they are not compatible with all cell types. It is agreed among scientists worldwide that there is no general consensus on how to predict whether a particular cell type / cell line will be easy or difficult to freeze. It can be said that cell types may exhibit the following behavior after cryopreservation: 1. Cells are well frozen, with limited cell death (less than 20%) after freezing and thawing, and cells maintain their functionality. 2. Cells are well frozen and cell death after freezing and thawing is limited (less than 20%), but cells lose their specific functionality (quality). 3. Cells are frozen, but there is more than 20% cell death after thawing and / or there is loss of specific functionality (quality) of the cells. For simplicity, cells in category 1 will be referred to as "easily freezable," and cells in categories 2 and 3 will be referred to as "hardly freezable" and "non-freezable" cells, respectively.

[0007] To illustrate the mismatch between the maturity of cells and their behavior upon cryopreservation, several examples are given.

[0008] On the one hand, some differentiated and / or more mature cells, such as muscle cells and immune cells, are very easy to freeze. On the other hand, other types of differentiated cells, such as neural cells, are difficult to freeze because they lose their functionality after freezing. On the other hand, some pluripotent, less differentiated, and less mature cell phenotypes (e.g., fibroblasts, progenitor cells, mesenchymal stem cells, highly proliferative cells) are easy to freeze, but embryonic stem cells (ESCs) are more difficult to freeze (Li et al. Front. Cell Dev. Biol., 14 December 2021).

[0009] Because the current state of the art lacks a clear functional description of cells, the only way to describe their behavior during cryopreservation is to refer to them as "freezable," "hard to freeze," or "freeze-resistant." For example, human neuronal cells are typically classified as "freeze-resistant" in the CPA, which means that freezing neuronal cells results in extremely low cell recovery. In addition, recovered cells are affected in their cellular function. Based on the above, isolated neuronal cells must therefore be used in experiments immediately after isolation, which is impractical. Immortalized neuronal cell lines are also available for research purposes (Tremblay et al. J. Neurosci. Methods, 186, 2010, pp. 60-67); however, these cell lines do not exhibit the same behavior as primary neurons. Neuronal cell lines are therefore considered inappropriate for investigating neuron-specific functions such as synaptogenesis, activity, and plasticity (Ishizuka et al. Journal of Neuroscience Methods. Volume 333, 1 March 2020).

[0010] Other examples of difficult-to-freeze cells include many primary (isolated) and / or cardiac myocytes, skeletal muscle cells, differentiated hepatocytes, differentiated fibroblasts, monocytes, dendritic cells, beta cells, cardiomyocytes, pancreatic islets, primary human monocytes, osteocytes, and adipocytes. These cell types are available from the body in limited quantities and do not (or do not grow very often), so their numbers remain low. Therefore, further loss of these cells during cryopreservation is highly undesirable.

[0011] Reduces the toxicity of CPA In practice, proteins are commonly used to reduce the toxicity of CPAs. Serum or serum proteins, derived from humans or other animals, are examples of preferred protein sources. They can serve as universal growth supplements to support cell growth and differentiation. In the field of cryopreservation, serum or serum proteins are thought to reduce CPA-induced toxicity by stabilizing and protecting cell membranes against shear stress. However, even the use of high amounts of serum or serum proteins does not fully counteract the undesirable characteristics of CPAs. For example, a combination of 10% DMSO and 50–90% fetal bovine serum (FBS) is a preferred freezing composition used in the state of the art. Nevertheless, this preferred composition still leads to more than 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: -Serum supplements are generally considered inappropriate or undesirable for therapeutic use; -Serum supplements have batch-to-batch variations, which lead to inconsistent responses in cells -Endotoxin (lipopolysaccharide, LPS) levels are not controlled, which may hinder the reproducibility of results; -There is increasing ethical resistance to the use of FBS in cryopreservation (Jochems et al. Altern Lab Anim. Mar-Apr 2002;30(2):219-27). FBS is obtained from fetuses removed from pregnant cows during slaughter and is therefore considered animal-unfriendly.

[0012] In short, it can be concluded that the use of serum or other protein sources does not solve the problems most typically associated with CPA.

[0013] Thawing and thaw-related cellular stress and toxicity Even though toxicity can be minimized by adding cryoprotectants before or during the freezing process, the toxic effects can resume upon thawing and the accompanying temperature increase. Post-thaw toxicity is commonly overlooked, even though it may have the same damaging potential in all liquid phases of the cryopreservation cycle. Due to the fact that cells are already stressed by the freeze / thaw cycle, cells may be at least as sensitive to toxicity during thawing as when cooled, if not more so.

[0014] Despite advances in the field of cryopreservation of biological materials for research and therapeutic applications, conventional CPAs have several undesirable characteristics. There remains an unmet need for methods that reduce the reliance on conventional CPAs, most notably DMSO or other osmotic CPAs. In particular, there is a need for cryopreservation methods that: - Reduces the amount of conventional CPA needed during cryopreservation, thereby reducing the negative effects of CPA, such as cytotoxicity, altered cell functionality, and altered inflammatory responses. -Completely replaces conventional CPAs, thereby eliminating the negative effects of CPAs and making cryopreserved cells safer and more effective for therapeutic applications, especially cell and tissue transplantation (ATMP) -Allows for cryopreservation of cell types for which traditional CPA would be unsuccessful. These typically include slow-growing, well-differentiated cells, "hard to freeze" or "unfreezable" cells, such as neurons, cardiomyocytes, and / or other cell types. - Even if currently used CPAs are beneficial during freezing, they can still be toxic during thawing due to elevated temperatures. There is a need for a method or cryopreservation preparation that allows frozen cells to be transferred to room temperature, from a metabolically inactive state to an active state, without causing significant damage. It is an object of the present invention to provide a solution to one or more problems associated with cryopreservation and / or thawing of biological materials. Summary of the Invention

[0015] Surprisingly, the inventors have found that the use of a relatively high concentration of collagen hydrolysate (preferably 20% to 50% by weight) allows for a reduction in the concentration of the cryoprotectant dimethyl sulfoxide (i.e., DMSO) during cryopreservation. This strategy allows for a reduction in the amount of dimethyl sulfoxide, or even for DMSO not to be used at all, thus eliminating some of the known drawbacks of DMSO.

[0016] Suitable cryoprotectants generally contain 10% (v / v) or more dimethyl sulfoxide, which is often considered the "gold standard" for cryopreservation preparations. The present inventors have discovered 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 lead to the highest cell recovery rates. Cells cryopreserved using this combination also exhibited improved viability (e.g., higher proliferation capacity and functionality) compared to cells frozen in other cryopreservation preparations. Thus, the cryopreservation preparations of the present invention not only provide higher cell recovery compared to other cryopreservation preparations, but also provide higher viability of the recovered cells.

[0017] The collagen hydrolysate of the present invention, especially the described high concentration collagen hydrolysate, allows frozen cells to be transferred from a metabolically inactive state to an active state at room temperature without apparent injury. Thus, the collagen hydrolysate may exert beneficial effects on cells during the thawing process and / or reduce the undesirable effects of, for example, dimethyl sulfoxide.

[0018] At the preferred high concentrations, collagen hydrolysate has a strong cryoprotective effect. For example, collagen hydrolysate can function as a cryoprotectant. Additionally or alternatively, collagen hydrolysate can further enhance the cryoprotective effect of dimethyl sulfoxide, reduce the toxic effects of dimethyl sulfoxide, and / or preserve the genotype and phenotype of cells. Collagen hydrolysate can eliminate the need for serum and serum-derived proteins, which are typically included to reduce the toxicity of dimethyl sulfoxide. The cryopreserved preparations of the present invention are particularly suitable for the preservation of biological materials intended for human transplantation, such as advanced therapeutic medicinal products (ATMPs). The cryopreserved preparations of the present invention are particularly suitable for the cryopreservation of cells that typically cannot be cryopreserved with dimethyl sulfoxide alone and therefore can be considered difficult-to-freeze or unfreezable cells.

[0019] Overall, the best results were obtained when the amount of collagen hydrolysate was above 20% by weight, especially 30% or 40% by weight.

[0020] This finding by the inventors is surprising because it contradicts the existing idea that large amounts of collagen hydrolysate should be avoided during cryopreservation: for example, the state of the art recommends using collagen hydrolysate at 15% by weight or less, especially in combination with dimethyl sulfoxide or other permeating cryoprotectants, so as not to impair cell viability after cryopreservation.

[0021] In one aspect, the present invention relates to a cryopreserved preparation comprising 20 to 70% by weight of collagen hydrolysate, calculated relative to the weight of the cryopreserved preparation.

[0022] In one aspect, the present invention relates to the use of the cryopreservation preparations disclosed in the present disclosure for cryopreservation of one or more biological materials selected from the group consisting of eukaryotic cells, prokaryotic cells, cellular organelles, extracellular vesicles, organoids, tissues, and organs.

[0023] In one aspect, the invention relates to the use of collagen hydrolysate in a cryopreserved preparation, wherein the collagen hydrolysate is used in an amount of 20-70% by weight calculated on the weight of the cryopreserved preparation.

[0024] In certain aspects, the present invention relates to a method for cryopreserving a biological material, comprising providing the biological material in a cryopreservation preparation disclosed in the present disclosure, and reducing the temperature of the cryopreservation preparation below the freezing point of the cryopreservation preparation.

[0025] In one aspect, the present invention relates to the use of collagen hydrolysate to reduce the amount of dimethyl sulfoxide in a cryopreserved preparation, wherein the collagen hydrolysate is provided in an amount of 20 to 70% by weight calculated on the weight of the cryopreserved preparation.

[0026] In one aspect, the present invention relates to the use of collagen hydrolysate to reduce toxicity and / or improve viability in cells during thawing after cryopreservation, wherein the collagen hydrolysate is provided in the cryopreserved preparation in an amount of 20-70% by weight calculated on the weight of the cryopreserved preparation. [Brief explanation of the drawings]

[0027] Text description of the illustration image022.gif. [Figure 1] Cell death in fibroblast 3T3 cells after cryopreservation using cryopreservation preparations based on 25% (v / v) collagen hydrolysate, combined with 6.7% (v / v) DMSO and containing 1250 EU / g LPS ("low LPS", P5000) or 13350 EU / g LPS ("high LPS", P2000). [Figure 2] Cell death in fibroblast 3T3 cells in cryopreserved preparations containing 8% (v / v) DMSO with or without 20,000 EU / ml LPS ("LPS" group). Cell death was determined without or with a centrifugal wash step at 300 × g for 3 minutes ("Spin" group). [Figure 3]Cell death (%) in neuropil cells after freezing in 10% (v / v) DMSO ("control") or in 6.7% (v / v) DMSO supplemented with 30% (w / v) collagen hydrolysate ("+CH"). [Figure 4] Neuronal cell death after cryopreservation in various cryopreservation preparations: 30% HC=30 (w / v) collagen hydrolysate with an average molecular weight of approximately 5000 Da ("P5000"). [Figure 5] Staining of glial and neuronal markers in fresh (left panel) and frozen (middle and right panels) neuropil cells. Neuropil cells were frozen in 10% (v / v) DMSO (middle panel) or 6.7% (v / v) DMSO supplemented with 30% (w / v) collagen hydrolysate (CH, "P5000"). Staining shows combined staining for GFAP (glial marker) and MAP2 (neuronal marker). [Figure 6] Cell viability determined by staining. Fibroblast 3T3 cells were cryopreserved in freezing medium containing 0 to 13.3% (v / v) DMSO without (upper panel) or with (lower panel) hydrolyzed gelatin ("HG", 20% (w / v)). Cells were thawed and cultured for 3 days. They were then stained for viability. [Figure 7] Cell viability determined by counting. Fibroblast 3T3 cells were cryopreserved in freezing medium containing 0-20% (v / v) DMSO without (upper panel) or with (lower panel) hydrolyzed gelatin ("HG", 20% (w / v)). Cells were thawed and cultured for 3 days. The number of viable cells was counted after 3 days. [Figure 8] Cell viability (% relative to control) of 3D organoids determined using the WST-1 assay 1 hour after thawing. DETAILED DESCRIPTION OF THE INVENTION

[0028] The cryopreserved preparation of the present invention relates to a composition containing a collagen hydrolysate, preferably a composition containing the collagen hydrolysate in an amount of 20 to 70% by weight calculated relative to the weight of the cryopreserved preparation.

[0029] The present invention further relates to the use of one or more of the various embodiments of the cryopreserved preparations disclosed in this disclosure.

[0030] The present invention further relates to methods for cryopreserving biological materials using one or more of the various embodiments of the cryopreservation preparations disclosed in this disclosure.

[0031] In the context of the present invention, the term "collagen" refers to an amino acid sequence comprising a repeating (Gly-X-Y) sequence, preferably comprising 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 20 or more, 50 or more, 100 or more, 200 or more, 300 or more, or 400 or more sequences comprising the sequence Gly-X-Y, where X and Y are amino acid residues independently selected from each other, and X and / or Y are more preferably proline. "Collagen" preferably has a sequence found in native collagen in one or more animal species. Additionally or alternatively, "collagen" can refer to the full-length sequence or a fragment or subunit thereof of (native) collagen, preferably one or more of types I to XXVII collagen, more preferably one or 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" can refer to the alpha-1(I) chain, the alpha-2(I) chain, the alpha-1(II) chain, or the alpha-1(III) chain, or fragments thereof. The term "collagen" encompasses the triple helix structure formed by the three subunits present in native collagen.

[0032] In the context of the present invention, the term "collagen hydrolysate" refers to a mixture of short chains of amino acids (dipeptides, tripeptides, oligopeptides, polypeptides) resulting from (partial) hydrolysis, e.g., enzymatic hydrolysis, of natural (full-length) collagen. The degree of hydrolysis has an impact on the average molecular weight (expressed in Da) of the final product. The term "collagen hydrolysate" can be used interchangeably and synonymously with the terms "hydrolyzed collagen" or "collagen peptides." In the context of the present invention, "collagen hydrolysate" includes within its category collagen that has been subjected to hydrolysis or partial hydrolysis.

[0033] In some embodiments, the hydrolysis is alkaline hydrolysis. In some embodiments, the hydrolysis is acid hydrolysis. In some embodiments, the hydrolysis is enzymatic hydrolysis. The collagen hydrolysate may be one or more of an enzymatically hydrolyzed collagen hydrolysate, an alkaline hydrolyzed collagen hydrolysate, and an acid hydrolyzed collagen hydrolysate. Additionally or alternatively, the collagen hydrolysate may be obtained by one or a combination of the following processes: alkaline hydrolysis, acid hydrolysis, and enzymatic hydrolysis.

[0034] "Collagen hydrolysate" in the context of the present invention may be produced from collagen-containing materials in a one-step process or via an intermediate gelatin step (i.e., thus obtaining "hydrolyzed gelatin"). Therefore, the term "collagen hydrolysate" encompasses hydrolyzed gelatin. As used in this disclosure, the term "gelatin" refers to the (irreversible) form of collagen obtained by partial hydrolysis of collagen. Depending on the process used, two types of gelatin are generally obtained: type A (acid hydrolysis) and type B (alkaline hydrolysis). In the context of the present invention, "gelatin" can refer to type A gelatin or type B gelatin, or a combination thereof. Depending on the physical and chemical method of partial hydrolysis, the molecular weight of the peptides can fall within a wide range (e.g., 10-95 kDa). Partial hydrolysis gives gelatin the ability to retain water and its gelling ability, which typically distinguishes it from "collagen peptides" or "hydrolyzed collagen," i.e., products obtained by complete hydrolysis of collagen. In the context of the present invention, the term "hydrolyzed gelatin" refers to a product obtained by hydrolysis of gelatin, leading to a lower molecular weight of gelatin. The hydrolysis reaction to obtain hydrolyzed gelatin involves the breaking of one or more peptide bonds along with the addition of one mole of water. Hydrolyzed gelatin is distinguished from gelatin, inter alia, by its gelling ability; that is, hydrolyzed gelatin has reduced or no gelling ability, while gelatin has gelling ability. Gelatin can be hydrolyzed by acid (hydrogen ions), thereby obtaining "acid hydrolyzed gelatin." Gelatin can also be hydrolyzed by alkali (hydroxyl ions), thereby obtaining "alkali hydrolyzed gelatin." Gelatin may be hydrolyzed by one or more enzymes (e.g., pepsin, trypsin), thus obtaining "enzymatically hydrolyzed gelatin." The terms "hydrolyzed" and "hydrolysed" may be used interchangeably in the context of the present invention.

[0035] In a preferred embodiment, the collagen hydrolysate is hydrolyzed gelatin.

[0036] Collagen or collagen hydrolysate in the context of the present invention may include proteins or peptides that have been artificially synthesized, for example, recombinantly or chemically synthesized, including where the protein or peptide is encoded by recombinant DNA that is expressed by a synthetic system. Expression systems for recombinant peptides may be cells such as bacterial cells (e.g., Escherichia coli, Bacillus subtilis species), yeast cells (e.g., Saccharomyces cerevisiae, Pichia pastoris, or Ogataea angusta (Hansenula polymorpha), Candida bodini), fungal cells (e.g., Aspergillus oryzae, Aspergillus niger, Trichoderma reesei), mammalian cells (e.g., CHO cells, HeLa cells, HEK293 cells, NS0, Sp2 / 0), insect cells, and plant cells (e.g., tobacco, cereal, legume, fruit, vegetable).

[0037] In various embodiments of the present invention, the collagen hydrolysate may be produced by enzymatic or partial enzymatic hydrolysis of collagen, wherein the enzyme used for this purpose may be one or more selected from the group consisting of serine proteases, alkaline proteases, neutral proteases, flavor proteases, complex proteases, thiol proteases, bromelain, metalloproteases, proteases, carboxypeptidases, pepsin, chymotrypsin, trypsin, cathepsin K, chymotrypsin, papain, and subtilisin.

[0038] In one embodiment, the hydrolysis, preferably the enzymatic hydrolysis, is carried out at a pH of 5 to 8, preferably 6 to 7. In one embodiment, the hydrolysis, preferably the enzymatic hydrolysis, is carried out at a temperature of 55 to 70° C., preferably 60 to 65° C. In one embodiment, the hydrolysis, preferably the enzymatic hydrolysis, is carried out for 3 to 8 hours, preferably 4 to 7 hours, more preferably 5 to 6 hours.

[0039] Collagen hydrolysates in the context of the present invention may be derived from one or more collagens selected from collagen types I to XXVII, preferably one or more of collagen types I, II, III, V, or X, more preferably one or more of collagen types I, II, or III. Additionally or alternatively, the collagen hydrolysates disclosed in the present disclosure may be derived from two or more collagens, preferably one or two or more of collagen types I, II, and III.

[0040] The collagen disclosed in the present disclosure may be derived from any one or more animals or animal species, such as bovine (species), porcine (species), chicken, and fish (species). In some embodiments, the collagen is derived from cows. In some embodiments, the collagen is derived from pigs. In some embodiments, the collagen is derived from fish. In some embodiments, the collagen is derived from chickens. In various embodiments, the collagen is a mixture of collagens from different sources, such as collagens from multiple animal species and / or collagens from different tissues. For example, the collagen disclosed in the present disclosure may be a mixture of two or more collagens selected from the group consisting of fish collagen, porcine collagen, chicken collagen, and bovine collagen.

[0041] Collagen in the context of the present invention may be derived from one or more tissues selected from the group consisting of skin, scales, antlers, protrusions (e.g., humps), horns, head, brain, neck, ears, eyes, nose, tongue, lips, mouth, esophagus, trachea, sternum, pharynx, bronchi, limbs, feet, toes, palms, claws, bone, cartilage, bone marrow, joints, membranes, hind, ligaments, tendons, ribs, diaphragm, muscle, skeletal muscle, smooth muscle, intestine, blood vessels, bladder, stomach, aorta, heart, liver, kidneys, chest, lungs, spleen, pancreas, eggs, sperm, testes, ovaries, nerves, gallbladder, and connective tissue from the belly. As used in this disclosure, the term "skin" also includes "hide," which means the outer covering of a large animal, such as a bovine (species) or any other large animal. The terms "skin" and "leather" can be used interchangeably in this disclosure and can refer to the outer covering of an animal, regardless of size.

[0042] In preferred embodiments, the collagen taught in this disclosure is derived from skin and / or dermal connective tissue. In certain preferred embodiments, the collagen taught in this disclosure is derived from cartilage. In certain preferred embodiments, the collagen taught in this disclosure is derived from bone. In certain preferred embodiments, the collagen taught in this disclosure is derived from sternum. The collagen disclosed in this disclosure may be a mixture of collagens derived from two or more tissues and / or two or more animals. In certain embodiments, the collagen disclosed in this disclosure is a mixture of two or more collagens selected from the group consisting of skin collagen, cartilage collagen, sternum collagen, and bone collagen.

[0043] In the context of the present invention, the term "cryopreservation" refers to a preparation suitable for and / or intended for use in the cryopreservation of cells. The preparation is preferably a liquid preparation, or at least a preparation capable of suspending biological material, including cells. As used in the present disclosure, the term "cryopreservation" refers to cooling, preferably freezing, of biological material, preferably for the purpose of preserving and / or saving the biological material for future use. Most typically, and preferably in the context of the present invention, cryopreservation achieves temperatures on the order of -10°C to -30°C, more preferably on the order of -70°C to -90°C (e.g., using solid carbon dioxide) or -180°C to -220°C (e.g., using liquid nitrogen). The term "cryopreservation" can be used interchangeably with the terms "cryoprotection" or "cryobanking" in the context of the present invention. As used in the present disclosure, the term "cryoprotection" also encompasses cooling above the freezing point, e.g., to avoid ice crystal formation. The term "cryoprotection" also encompasses slow freezing. "Slow freezing" refers to a gradual decrease in temperature, most typically at an average rate of 0.2-5°C (e.g., 0.5-2°C) per minute until the final storage temperature is reached. This means that the biological material is cooled over several hours (e.g., to reach a temperature around -196°C). "Slow freezing," in the context of the present invention, can be used interchangeably with "controlled-rate freezing" or "slow programmable freezing (SPF)." As used in this disclosure, the term "cryopreservation" also encompasses "vitrification" (also called flash freezing), which involves rapid cooling to prevent ice crystal formation and thereby help prevent cryopreservation damage.The processes of "slow freezing" and "vitrification" are well known in the art and are described, for example, in the review by Son et al. (Expert Rev Med Devices 2009 Jan;6(1):1-7).

[0044] In the context of the present invention, the term "cryopreservation" preferably includes the steps of incorporating biological material into a cryopreservation preparation, freezing, and thawing the cells after freezing. In the context of the present invention, a preparation that leads to improved cell survival during thawing is considered to improve cryopreservation in general. The present invention further relates to the thawing of biological material after freezing. In particular, the inventors have found that a collagen hydrolysate at an appropriate concentration allows frozen cells to be transferred from a metabolically inactive state to an active state at room temperature without causing any appreciable damage. Thus, collagen hydrolysate may have a beneficial effect during thawing and / or may reduce the negative (toxic) effects of dimethyl sulfoxide during thawing.

[0045] In some embodiments, the collagen hydrolysate and / or cryopreserved preparation disclosed herein is used to reduce toxicity and / or increase viability in cells during thawing after cryopreservation, wherein the collagen hydrolysate is preferably provided in the cryopreserved preparation in an amount of 20-70% by weight, calculated based on the weight of the cryopreserved preparation. In some preferred embodiments, reducing toxicity and / or increasing viability in cells is achieved by reducing one or more toxic effects of non-permeating and / or permeating cryoprotectants on cells during thawing. In some preferred embodiments, reducing toxicity and / or increasing viability in cells is achieved by reducing one or more toxic effects of permeating cryoprotectants, such as dimethyl sulfoxide, on cells during thawing.

[0046] The method of the present invention involves providing a biological material in a cryopreservation preparation disclosed herein and lowering the temperature of the cryopreservation preparation to below the freezing point of the cryopreservation preparation. The freezing point may depend on the composition of the liquid cryopreservation preparation and is defined as the temperature at which the liquid cryopreservation preparation becomes solid at normal atmospheric pressure. Additionally or alternatively, given that the cryopreservation preparation may be aqueous, the freezing point is preferably at or near 0°C (e.g., -10°C, -9°C, -8°C, -7°C, -6°C, -5°C, -4°C, -3°C, -2°C, -1°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, or 10°C).

[0047] In some embodiments, the cryopreserved preparation with the biological material is directly frozen, which means that the freezing rate is not controlled, thus precluding the use of programmable and / or slow freezing.

[0048] The cryopreserved preparation may comprise 5% or more, or 10% or more, or 15% or more, or 20% or more, or 22.5% or more, or 25% or more, or 27.5% or more, or 30% or more, or 32.5% or more, or 35% or more, or 37.5% or more, or 40% or more, or 45% or more, or 50% or more by weight of collagen hydrolysate, calculated on the weight of the cryopreserved preparation. Additionally or alternatively, the cryopreserved preparation may comprise 80% by weight or less, or 75% by weight or less, or 70% by weight or less, or 65% by weight or less, or 50% by weight or less, or 45% by weight or less, or 40% by weight or less, or 37.5% by weight or less, or 35% by weight or less, or 32.5% by weight or less, or 30% by weight or less, or 27.5% by weight or less, or 25% by weight or less, or 22.5% by weight or less, or 20% by weight or less of collagen hydrolysate, calculated relative to the weight of the cryopreserved preparation. In a preferred embodiment, the cryopreserved preparation may comprise 20-70% by weight, preferably 20-50% by weight, more preferably 25-40% by weight, and even more preferably 27.5-35% by weight of collagen hydrolysate, calculated relative to the weight of the cryopreserved preparation.

[0049] In some embodiments, the cryopreserved preparation further comprises dimethyl sulfoxide.

[0050] Cryopreserved preparations may contain at least 0.01% by weight, or at least 0.05% by weight, or at least 0.1% by weight, or at least 0.2% by weight, or at least 0.5% by weight, or at least 0.6% by weight, or at least 0.7% by weight, or at least 0.8% by weight, or at least 0.9% by weight, or at least 1% by weight, or at least 1.25% by weight, or at least 1.5% by weight, or at least 1.75% by weight, or at least 2% by weight, or at least 2.25% by weight, or at least 2.5% by weight, or at least 2.75% by weight, or at least 3% by weight, or at least 3.25% by weight, or at least 4% by weight, or at least 4.25% by weight or more, or 4.5% by weight or more, or 4.75% by weight or more, or 5% by weight or more, or 5.25% by weight or more, or 5.25% by weight or more, or 5.5% by weight or more, or 5.75% by weight or more, or 6.0% by weight or more, or 6.25% by weight or more, or 6.5% by weight or more, or 7% by weight or more, or 7.5% by weight or more, or 8% by weight or more, or 8.5% by weight or more, or 9% by weight or more, or 9.5% by weight or more, or 10% by weight or more, or 11% by weight or more, or 12% by weight or more, or 13% by weight or more, or 14% by weight or more, or 15% by weight or more of dimethyl sulfoxide. Additionally or alternatively, the cryopreserved preparation has no more than 20%, or no more than 17.5%, or no more than 15%, or no more than 12.5%, or no more than 12%, or no more than 11%, or no more than 10.9%, or no more than 10.8%, or no more than 10.7%, or no more than 10.6%, or no more than 10.5%, or no more than 10.4%, or no more than 10.3%, or no more than 10.2%, or no more than 10.1%, or no more than 10%, or no more than 9.5%, or no more than 9%. , or 8.5% or less, or 8% or less, or 7.5% or less, or 7% or less, or 6.5% or less, or 6.25% or less, or 6% or less, or 5.75% or less, or 5.5% or less, or 5.25% or less, or 5% or less, or 4.75% or less, or 4.5% or less, or 4.25% or less, or 4% or less, or 3.75% or less, or 3.5% or less, or 3.25% or less, or 3% or less, or 2.75% or less, or 2.5% or less dimethyl sulfoxide.

[0051] Cryopreserved preparations, such as those for the uses described in this disclosure, have a soluble ... , or 0.9% (v / v) or more, or 1% (v / v) or more, or 1.25% (v / v) or more, or 1.5% (v / v) or more, or 1.75% (v / v) or more, or 2% (v / v) or more, or 2.25% (v / v) or more, or 2.5% (v / v) or more, or 2.75% (v / v) or more, or 3% (v / v) or more, or 3.25% (v / v) or more, or 4% (v / v) or more, or 4.25% (v / v) or more, or 4.5% (v / v) or more, or 4.75% (v / v) or more, or 5% (v / v) or more, or 5.25% (v / v) or more, or 5.25% (v / v) or more, or 5.5% (v / v) or more, or 5.75% (v / v) or more, or 6.0% (v / v) or more, or 6.25% (v / v) or more, or 6.5% (v / v) or more, or 7% (v / v ) or more, or 7.5% (v / v) or more, or 8% (v / v) or more, or 8.5% (v / v) or more, or 9% (v / v) or more, or 9.5% (v / v) or more, or 10% (v / v) or more, or 11% (v / v) or more, or 12% (v / v) or more, or 13% (v / v) or more, or 14% (v / v) or more, or 15% (v / v) or more of dimethyl sulfoxide.Additionally or alternatively, the cryopreserved preparation may contain no more than 20% (v / v), or no more than 17.5% (v / v), or no more than 15% (v / v), or no more than 12.5% ​​(v / v), calculated on the total volume of the cryopreserved preparation. v / v, or 12% (v / v) or less, or 11% (v / v) or less, or 10.9% (v / v) or less, or 10.8% (v / v) or less, or 10.7% (v / v) or less, or 10.6% (v / v) or less, or 10.5% (v / v) or less, or 10.4% (v / v) or less, or 10.3% (v / v) or less, or 10.2% (v / v) or less, or 10.1% (v / v) or less, or 10% (v / v) or less, or 9.5% (v / v) or less, or 9% (v / v) or less, or 8.5% (v / v) or less, or 8% (v / v) or less, or 7.5% (v / v) or less, or 7% (v / v) or less, or 6.5% (v / v) or less, or 6.25% (v / v) or less, or 6% (v / v) or less, or 5.75% (v / v) or less, or 5.5% (v / v) or less, or 5.25% (v / v) or less, or 5% (v / v) or less, or 4.75% (v / v) or less, or 4.5% (v / v) or less, or 4.25% (v / v) or less, or 4% (v / v) or less, or 3.75% (v / v) or less, or 3.5% (v / v) or less, or 3.25% (v / v) or less, or 3% (v / v) or less, or 2.75% (v / v) or less, or 2.5% (v / v) or less of dimethyl sulfoxide.

[0052] In a preferred embodiment, the cryopreserved preparation, e.g., a cryopreserved preparation for the uses described herein, comprises more than 20% by weight, preferably more than 22.5% by weight, and even more preferably more than 25% by weight, of collagen hydrolysate, calculated relative to the weight of the cryopreserved preparation. In a preferred embodiment, the cryopreserved preparation, e.g., a cryopreserved preparation for the uses described herein, comprises less than 50% by weight, preferably less than 45% by weight, and even more preferably less than 40% by weight, of collagen hydrolysate, calculated relative to the weight of the cryopreserved preparation. In a preferred embodiment, the cryopreserved preparation, e.g., a cryopreserved preparation for the uses described herein, comprises 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, calculated relative to the total volume of the cryopreserved preparation.

[0053] In a preferred embodiment, the cryopreserved preparation contains 0.1% or more by weight of dimethyl sulfoxide, calculated relative to the weight of the cryopreserved preparation. In a preferred embodiment, the cryopreserved preparation contains less than 10.9% by weight of dimethyl sulfoxide, calculated relative to the weight of the cryopreserved preparation. In a preferred embodiment, the cryopreserved preparation 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% by weight of dimethyl sulfoxide, calculated relative to the weight of the cryopreserved preparation.

[0054] Those skilled in the art will appreciate that DMSO has a known density (1.1 g / cm 3 ) and the density of the aqueous medium in which DMSO resides (typical density is approximately 1.0 g / cm 3), we know how to convert between % by weight and % by volume amounts of DMSO. For example, a 10% (v / v) DMSO concentration calculated relative to the volume of an aqueous preparation is equivalent to 10.9% DMSO by weight calculated relative to the total weight of the preparation. Similar conversions are, for example, 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., 7.02 wt%), 7.0% (v / v) (i.e., 8.02 wt%), 8.0% (v / v) (i.e., 9.02 wt%), 9.0% (v / v) (i.e., 10.02 wt%), 10.0% (v / v) (i.e., 11.02 wt%), 11.0% (v / v) (i.e., 12.02 wt%), 12.0% (v / v) (i.e., 13.02 wt%), 13.0% (v / v) (i.e., 14.02 wt%), 14.0% (v / v) (i.e., 15. This also applies to the following DMSO concentrations: (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%), and 15% (v / v) (i.e., 16.26 wt%).

[0055] In one embodiment, the present invention relates to the use of the collagen hydrolysate disclosed herein in the cryopreservation of biological materials, preferably selected from the group consisting of eukaryotic cells, prokaryotic cells, cell 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.%, calculated based on the total weight of the cryopreservation preparation. 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), calculated based on the total volume of the cryopreservation preparation. In a preferred embodiment, the cryopreserved preparation for use according to the present disclosure is (essentially) free of dimethyl sulfoxide or contains 0.1% (v / v) or less, or 0.01% (v / v) or less, or 0.001% (v / v) or less, or 0.0001% (v / v) or less, of dimethyl sulfoxide relative to the total volume of the cryopreserved preparation. In a preferred embodiment, the collagen hydrolysate for use according to the present disclosure is (essentially) free of one or more of additional cryoprotectants, serum, and serum proteins, more preferably free of additional penetrating cryoprotectants.

[0056] In one embodiment, the method of the present invention uses a cryopreserved preparation containing collagen hydrolysate in an amount of 10 to 70% by weight, preferably 20 to 60% by weight, and more preferably more than 20% by weight, calculated based on the total weight of the cryopreserved preparation. In a preferred embodiment, the method of the present invention uses a cryopreserved preparation containing dimethyl sulfoxide in an amount of less than 10% (v / v), preferably less than 9.5% (v / v), and more preferably less than 9% (v / v), calculated based on the total volume of the cryopreserved preparation. In a preferred embodiment, the method of the present invention uses a cryopreserved preparation that is (essentially) dimethyl sulfoxide-free or contains 0.1% (v / v) or less, or 0.01% (v / v) or less, or 0.001% (v / v) or less, or 0.0001% (v / v) or less, of dimethyl sulfoxide, based on the total volume of the cryopreserved preparation. In a preferred embodiment, the methods of the present invention use cryopreserved preparations that are (essentially) free of one or more of additional cryoprotectants, serum, and serum proteins, and more preferably free of additional permeating cryoprotectants.

[0057] In one embodiment, the methods and / or uses of the invention use collagen hydrolysate as a cryoprotectant in the absence of one or more of further cryoprotectants, serum, and serum proteins, more preferably in the absence of DMSO.

[0058] In certain embodiments in which collagen hydrolysate is used in the absence of additional cryoprotectants, serum, and / or serum proteins, the collagen hydrolysate is preferably hydrolyzed gelatin, as this appears to result in the highest viability after cryopreservation, although other types of collagen hydrolysates may also be suitable for the present invention.

[0059] In embodiments in which collagen hydrolysate is used in the absence of additional cryoprotectants, serum, and / or serum proteins, the collagen hydrolysate preferably has a molecular weight of 3500-7500 Da, preferably 4000-6000 Da, and more preferably 4500-5500 Da, as this appears to result in the highest viability after cryopreservation, although other types of collagen hydrolysates may also be suitable for the present invention.

[0060] In embodiments where collagen hydrolysate is used in the absence of one or more of an additional cryoprotectant, serum, and serum proteins, the amount of collagen hydrolysate, calculated on the total weight of the preparation, is preferably greater than 20% to 50% by weight, preferably 22.5 to 45% by weight, more preferably 25 to 40% by weight, and even more preferably 27.5 to 35% by weight, since this appears to result in the highest viability after cryopreservation, although other types of collagen hydrolysate may also be suitable for the present invention.

[0061] In one embodiment, the use of the present invention relates to the use of a collagen hydrolysate to reduce the amount of dimethyl sulfoxide in a cryopreserved preparation, wherein the collagen hydrolysate is preferably provided in an amount of 20-70% by weight, calculated relative to the weight of the cryopreserved preparation. For example, the amount of dimethyl sulfoxide can be reduced thanks to the collagen hydrolysate, while still achieving a cell recovery of 75% or more, or 80% or more, or 85% or more, or 90% or more, or 95% or more, or 99% or more.

[0062] In certain embodiments, cryopreservation of biological material, preferably cells, in the cryopreservation preparations of the present invention leads to similar or higher cell recovery (and / or lower cell death) and / or cell viability compared to a similar cryopreservation preparation without collagen hydrolysate. For example, a combination of 25 wt% collagen hydrolysate and 5 wt% DMSO may lead to similar or higher cell recovery (and / or lower cell death) and / or cell viability compared to 5 wt% DMSO without collagen hydrolysate.

[0063] In certain embodiments, the use of collagen hydrolysate in the cryopreservation of biological materials, preferably cells, allows for the use of smaller amounts of DMSO while still achieving similar or higher cell recovery (and / or lower cell death) and / or cell viability. For example, a combination of 25% by weight collagen hydrolysate and 5% by weight DMSO may result in similar or higher cell recovery (and / or lower cell death) and / or cell viability compared to the use of 10% by weight DMSO without collagen hydrolysate. "Less DMSO" can be, for example, a reduction in the amount of DMSO of 10% or more, or 20% or more, or 30% or more, or 40% or more, or 50% or more, or 60% or more, or 70% or more, or 80% or more, or 90% or more, or 95% or more, or 99% or more, and / or a reduction to any of the preferred DMSO concentrations described herein.

[0064] "Cell recovery" and / or "cell death" in the context of the present invention are preferably determined after slow freezing of cells and measuring the percentage of live and dead cells after thawing. Those skilled in the art know that slow freezing and thawing may depend on the cell type and can adapt protocols accordingly. Post-thaw cell recovery can be determined by counting live cells and comparing it to the number of frozen cells, for example, using a stain that selectively stains dead cells, such as trypan blue. The percentage of cell death can be determined by comparing the number of live cells to the number of frozen cells. Additionally or alternatively, live and dead cells can be determined using a commercial assay that uses a mixture of two fluorescent dyes that differentially label live and dead cells and then measuring live and dead cells using flow cytometry or fluorescence microscopy, such as a live-dead assay.

[0065] In the context of the present invention, the term "viable cell" refers to a cell with an intact cell membrane, as defined, for example, 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" refers to a cell that is not viable.

[0066] In the context of the present invention, "cell viability" is preferably determined by one or more of cell metabolic activity, cell proliferation, and (adenosine triphosphate) ATP concentration. That is, higher cell metabolic activity, higher cell proliferation, and / or higher ATP concentration can be indicators of higher cell viability. Cell metabolic activity can be measured, for example, using an assay based on tetrazolium salts (e.g., MTT, XTT) or Alamar Blue. Cell proliferation can be measured, for example, using an assay for cell proliferation markers or cell cycle control markers, such as Ki-67, proliferating cell nuclear antigen (PCNA), topoisomerase IIB, or phosphorylated histone H3. ATP can be detected, for example, using bioluminescent luciferase and its substrate, luciferin.

[0067] In one embodiment, the use of the present invention relates to the use of a collagen hydrolysate to reduce the amount of dimethyl sulfoxide in a cryopreserved preparation, wherein the collagen hydrolysate is preferably provided in an amount of 20-70% by weight calculated on the weight of the cryopreserved preparation.

[0068] The collagen hydrolysate may have an average molecular weight in the range of 500 Da to 25,000 Da, for example 1000 Da to 15000 Da or 2000 Da to 10000 Da. The collagen hydrolysate may have an average molecular weight of 500 Da or more, or 600 Da or more, or 700 Da or more, or 800 Da or more, or 900 Da or more, or 1000 Da or more, or 1100 Da or more, or 1200 Da or more, or 1300 Da or more, or 1400 Da or more, or 1500 Da or more, or 1750 Da or more, or 2000 Da or more, or 2250 Da or more, or 2500 Da or more, or 2750 Da or more, or 3000 Da or more, or 3250 Da or more, or 3500 Da or more, or 4000 Da or more, or 4500 Da or more, or 5000 Da or more, or 5500 Da or more. Additionally or alternatively, the collagen hydrolysate may have an average molecular weight of 10,000 Da or less, or 9,500 Da or less, or 9,000 Da or less, or 8,750 Da or less, or 8,500 Da or less, or 8,250 Da or less, or 8,000 Da or less, or 7,750 Da or less, or 7,500 Da or less, or 7,250 Da or less, or 7,000 Da or less, or 6,750 Da or less, or 6,500 Da or less, or 6,250 Da or less, or 6,000 Da or less, or 5,750 Da or less, or 5,500 Da or less, or 5,250 Da or less, or 5,000 Da or less, or 4,500 Da or less, or 4,000 Da or less. In a preferred embodiment, the collagen hydrolysate has an average molecular weight of 500 to 10,000 Da, preferably 1,000 to 8,000 Da, more preferably 2,000 to 7,000 Da, and even more preferably 3,000 to 6,000 Da.

[0069] Average molecular weight in the context of the present invention is preferably weight average molecular weight. A preferred way to measure the (average) molecular weight and / or molecular weight distribution of collagen hydrolysates or gelatins is by High Performance Size Exclusion Chromatography (HPSEC). The following protocol is a preferred HPSEC protocol: An Agilent HPLC 1260 Infinity series (G1316A, G1329B, G1311C, G1315D) with a TSKgel SWXL precolumn and a G2000SWXL column (Tosoh Bioscience) was used. Analysis was performed using WinGPC software (PSS). The eluent was 100 mM phosphate buffer, pH 5.3. The sample was eluted from the column (e.g., 0.5 mL / min, isocratically) and monitored by UV detection (e.g., 214 nm, analysis time 40 min per injection + 180 min equilibration). Calibration was performed using a Narrow Calibration Standard (Low FILK).

[0070] The inventors have found that the absence or minimal presence of LPS (endotoxin) is beneficial in the context of the present invention, particularly when a centrifugal wash step is performed. The absence or minimal presence of LPS (endotoxin) makes the centrifugal wash step even redundant. It has further been found that the presence of LPS during cryopreservation can induce undesirable toxic and / or pro-inflammatory responses in cells. The use of collagen hydrolysate may additionally or alternatively provide protection from the undesirable effects of LPS in cryopreservation preparations.

[0071] The collagen hydrolysate preferably has an endotoxin level of 10,000 EU (i.e., endotoxin units) or less per gram of collagen hydrolysate or per mL of cryopreserved preparation, and preferably has an endotoxin level of 1,000 EU or less (e.g., 500 EU or less, 400 EU or less, 300 EU or less, or 200 EU or less), more preferably 100 EU or less (e.g., 90 EU or less, 80 EU or less, 70 EU or less, 60 EU or less, 50 EU or less, 40 EU or less, 30 EU or less, or 20 EU or less), even more preferably 10 EU or less (e.g., 9 EU or less, 8 EU or less, 7 EU or less, 6 EU or less, 5 EU or less, 4 EU or less, 3 EU or less, 2 EU or less, 1 EU or less), and most preferably 1 EU or less, both per gram of collagen hydrolysate or per mL of cryopreserved preparation. Additionally or alternatively, the collagen hydrolysate may have an endotoxin level of 1 EU or more per gram of collagen hydrolysate or per mL of cryopreserved preparation, and the endotoxin level is preferably 10 EU or more, such as 20 EU or more (e.g., 30 EU or more, 40 EU or more, 50 EU or more, 60 EU or more, 70 EU or more, 80 EU or more, 90 EU or more, 100 EU or more, or 150 EU or more, either per gram of collagen hydrolysate or per mL of cryopreserved preparation. or more), more preferably 100 EU or more (e.g., 200 EU or more, 300 EU or more, 400 EU or more, 500 EU or more, 600 EU or more, 700 EU or more, 800 EU or more, 900 EU or more, 1000 EU or more, or 1500 EU or more), even more preferably 1000 EU or more (e.g., 3000 EU or more, 4000 EU or more, 5000 EU or more, 6000 EU or more, 7000 EU or more, 8000, 9000 EU or more, or 1000 EU or more), and most preferably 10000 EU or more.

[0072] In a preferred embodiment, the collagen hydrolysate has an endotoxin level of 10,000 EU / g or less, preferably 1,000 EU / g or less, more preferably 100 EU / g or less, and most preferably 10 EU / g or less, calculated on the weight of the collagen hydrolysate.

[0073] In a preferred embodiment, the cryopreserved preparation has an endotoxin level, calculated relative to the volume of the cryopreserved preparation, of less than 2500 EU / ml, 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 cryopreserved preparation may have an endotoxin level, calculated relative to the volume of the cryopreserved preparation, of 0.5 to 2500 EU / ml, or 1 to 1000 EU / ml, or 5 to 500 EU / ml, or 10 to 250 EU / ml.

[0074] In some embodiments, the collagen hydrolysate is endotoxin-free. In some embodiments, the cryopreserved preparation is endotoxin-free. The term "endotoxin-free" can mean the absence of endotoxin and / or can mean "essentially endotoxin-free."

[0075] The term "free" may mean the same as "essentially free." The term "essentially free" encompasses an amount of a substance or compound that is not measurable by standard techniques in the art and / or is below a certain threshold, preferably less than 0.1% by weight, more preferably less than 0.01% by weight, even more preferably less than 0.001% by weight, and most preferably less than 0.0001% by weight. In the context of the present invention, "essentially free" may also mean an endotoxin level of less than 10, preferably less than 1, more preferably less than 0.1, even more preferably less than 0.01, and most preferably less than 0.001 (all in EU / g or EU / ml). The terms "free" or "essentially free" encompass the complete absence of a substance or compound (e.g., 0% by weight). The terms "absent," "free," and "essentially free" may be used interchangeably in the context of the present invention.

[0076] In the context of the present invention, well-known and preferred methods for measuring endotoxin levels (e.g., determining EU / g or EU / ml) are the Limulus Amebocyte Extract (LAL) test or the recombinant Factor C (rFC) test, which will be familiar to those skilled in the art.

[0077] In certain embodiments, the cryopreserved preparation is free of one or more of additional cryoprotectants, serum, and serum proteins.

[0078] In the context of the present invention, the term "cryoprotectant" refers to any substance or compound used in the cryopreservation of biological materials that provides protection against freeze damage (i.e., at least reduces freeze damage) in the biological material, for example, by reducing ice crystal formation. The terms "cryoprotectant," "cryopreservative," "cryopreservative," and "antifreeze (agent)" may be used interchangeably in the context of the present invention. Cryoprotectants are typically referred to as "cryoprotectants" when applied outside of cryobiology. 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). "Vitrification" in the context of the present invention refers to the formation of solid water with an irregular, amorphous structure. Vitrification is preferably determined by preparing a solution containing the substance or compound and cooling it for 30 minutes at -80°C or in liquid nitrogen. The cooled solution is considered vitrified if it becomes clear (and not milky), e.g., the same as or greater than the clarity observed for a reference (known) cryoprotectant, such as a permeating or non-permeating cryoprotectant disclosed in this disclosure. For example, the clarity of a solution containing 20% ​​(v / v) collagen hydrolysate may be compared to 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 generated to determine optimal vitrification (for collagen hydrolysate).

[0079] Cryoprotectants in the context of the present invention include both permeating and non-permeating cryoprotectants. The term "permeating cryoprotectant" refers to a cryoprotectant that has the ability to penetrate into cells, thereby providing intracellular protection against freeze damage. Permeating cryoprotectants can cross biological membranes. The term "non-permeating cryoprotectant" refers to a cryoprotectant that does not (mostly) have the ability to penetrate into cells and / or does not (mostly) require intracellular penetration to mediate its cryoprotective effect. Non-permeating cryoprotectants generally induce vitrification via 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 the increase in cell volume after exposing cells to a solution containing the compound or substance. Thus, the substance or compound can be classified, for example, as a permeating or non-permeating cryoprotectant or cryopreservative. The rate of cell penetration can be determined by measuring cell volume at specific time intervals after exposing the cells to the solution. A preferred protocol is that described by Eto et al. (Cryobiology. 2014 Feb;68(1):147-51). An example is now given for determining the cell permeability that a collagen hydrolysate may have.

[0080] Cells (e.g., 3T3 fibroblast cell line) are exposed to a solution containing 20% ​​(v / v) collagen hydrolysate at 25±0.5°C. Solutions containing permeant dimethyl sulfoxide (10% (v / v)), ethylene glycol (1 M), propylene glycol (1 M), or glycerol (20% (v / v)) are used as references, and cell diameters are measured after 30, 60, 120, 180, 240, and 300 seconds (control). The volume is calculated as V = S. 3 / 2(where S is the relative cross-sectional area; V is the relative volume, S=πab, a is the major radius, and b is the minor radius), and the ratio of the volume at each time point to the control volume is calculated. If the average relative volume for the collagen hydrolysate solution at least matches the average volume for the reference (for one or more of the measured time points), the collagen hydrolysate is considered to have penetrated the cells. Cells exposed to the collagen hydrolysate solution may initially shrink and then recover (once the collagen hydrolysate has entered the cells). Therefore, the volume change is preferably calculated starting from the lowest V value.

[0081] It has been found that collagen hydrolysates can further enhance the cryoprotective effect of dimethyl sulfoxide, reduce the toxic effects of dimethyl sulfoxide, and / or preserve the genotype and phenotype of cells (e.g., by DNA sequencing assays, methylation assays, cell surface marker expression assays, cell differentiation assays, or other functional tests).

[0082] In some embodiments, the collagen hydrolysate is a cryoprotectant. In some embodiments, the collagen hydrolysate is a penetrating cryoprotectant. In some embodiments, the collagen hydrolysate is a non-penetrating cryoprotectant. In some embodiments, the collagen hydrolysate is not a cryoprotectant. In some embodiments, the collagen hydrolysate improves the efficacy of and / or reduces the toxicity of a (further) cryoprotectant, preferably a penetrating cryoprotectant, more preferably dimethyl sulfoxide.

[0083] Without wishing to be bound by theory, the inventors have found that collagen hydrolysate (in combination with DMSO) may have one or more mechanisms in improving cell recovery and viability after cryopreservation, including, for example, providing cell binding sites, water binding / absorption, affecting osmotic pressure, affecting intracellular water content, and ice crystal formation. Thus, collagen hydrolysate may exert one or more multiple modes of actions in the context of the present invention.

[0084] In preferred embodiments, the cryopreserved preparation comprises one or more of an additional cryoprotectant, serum, and serum proteins.

[0085] In certain embodiments, the permeating cryoprotectant is one or more selected from the group consisting of dimethyl sulfoxide, ethylene glycol, propylene glycol, and glycerol.

[0086] If dimethyl sulfoxide is present in the preparation, the term "additional cryoprotectant" refers to any cryoprotectant other than dimethyl sulfoxide. If dimethyl sulfoxide is not present in the preparation, the term "additional cryoprotectant" can refer to any cryoprotectant, including dimethyl sulfoxide.

[0087] In certain embodiments, the non-permeable cryoprotectant is one or more selected from the group consisting of polyethylene glycol, sugars (e.g., sucrose, dextrose, raffinose, trehalose, lactose), polyvinylpyrrolidone, and methylcellulose.

[0088] It has been discovered that the addition of collagen hydrolysate can eliminate the need for serum and serum-derived proteins, which are commonly used in the art to reduce the toxicity of cryoprotectants. Without wishing to be bound by theory, collagen hydrolysate may protect cell membranes against shear stress and / or counteract the undesirable properties of dimethyl sulfoxide or other cryoprotectants. Replacing serum and serum-derived proteins may additionally or alternatively reduce batch-to-batch variability, inconsistent cell responses, and resistance to use due to ethical or therapeutic considerations.

[0089] The term "serum" disclosed in the present disclosure encompasses human and animal serum, including, but not limited to, fetal bovine serum (FBS), fetal calf serum (FCS), newborn bovine serum (NBS), and newborn calf serum. The term "serum" disclosed in the present disclosure encompasses heat-inactivated serum. The term "serum protein" in the context of the present invention encompasses human serum proteins and animal serum proteins, preferably human serum albumin (HSA) or bovine serum albumin (BSA).

[0090] In the present invention, salt may or may not be present in the cryopreserved preparation.

[0091] In certain embodiments, the cryopreserved preparation is (essentially) salt-free. In some embodiments, the cryopreserved preparation is (essentially) free of added salt.

[0092] In some embodiments, the cryopreserved preparations are animal-free, meaning that they do not contain any animal-derived components, for example, collagen (hydrolysate) or gelatin (hydrolysate) are animal-free if they are recombinantly or synthetically derived.

[0093] In some embodiments, the cryopreserved preparation comprises one or more permeating and / or non-permeating cryoprotectants 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, calculated relative to the weight of the cryopreserved preparation. Additionally or alternatively, in some embodiments, the cryopreserved preparation comprises one or more permeating and / or non-permeating cryoprotectants 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, calculated relative to the weight of the cryopreserved preparation.

[0094] In certain embodiments, the cryopreserved preparation comprises one or more penetrating glycols in an amount of 0.1-20% by weight, preferably 0.2-10% by weight, more preferably 0.5-5% by weight, and even more preferably 1-2.5% by weight, calculated on the weight of the cryopreserved preparation. As used in this disclosure, "penetrating glycol" is preferably ethylene glycol and / or propylene glycol.

[0095] In one embodiment, the cryopreserved preparation 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, and even more preferably 1 to 2.5% by weight, calculated on the weight of the cryopreserved preparation.

[0096] "Sugar" in the context of the present invention is preferably one or more selected from the group consisting of sucrose, dextrose, raffinose, trehalose, and lactose.

[0097] In the context of the present invention, cryopreservation preparation is suitable for cryopreservation of one or more biological materials.Such biological materials include one or more selected from the group consisting of cell (eukaryotic or prokaryotic), multicellular eukaryote (for example, nematode worms), organoid (for example, intestinal organoid, lung organoid, embryonic organoid, kidney organoid, epithelial organoid), microbial culture (for example, fungus, bacteria), blood (for example, umbilical cord blood), semen, thrombosome, tissue (for example, living tissue or non-living tissue, such as tumor and histological section), ovarian tissue, skin tissue, musculoskeletal tissue (for example, bone, cartilage, tendon), testicular tissue, sperm, egg, embryo, organ (for example, liver, heart, kidney, skin, lung, pancreas, intestine), cellular vesicle (for example, extracellular vesicle, including exosome), organoid, plant material (for example, plant seed, shoot tip, dormant bud, bryophyte).

[0098] The biological material is preferably mammalian, more preferably human.

[0099] In a preferred embodiment, the use of the present invention relates to the cryopreservation of one or more biological materials selected from the group consisting of eukaryotic cells, prokaryotic cells, cellular organelles, extracellular vesicles (EVs), organoids, tissues, and organs.

[0100] 3D organoids closely mimic the in vivo situation, containing proliferative stem and progenitor cells and differentiated cell types, including absorptive enterocytes, secretory mucus-producing cells, Paneth cells, or enteroendocrine cells. These cell types are localized in distinct proliferative (crypt) or differentiated (villus) zones within the overall organoid architecture. 3D organoids represent a structurally closed system characterized by the formation of a consistent epithelium with in vivo-like characteristics. Additionally, due to molecular signatures comparable to native tissue, primary 3D organoid cultures qualify as important in vitro models for future, more precise drug screening in the pharmaceutical industry.

[0101] Those skilled in the art are aware of various types of organoids and methods for obtaining them (e.g., at least those described in the review by Kim et al., Nature Reviews Molecular Cell Biology volume 21, pages 571-584 (2020)).

[0102] If the biological material is a cell, the cell may be, for example, 1 x 10 cells calculated relative to the volume of the cryopreserved preparation. 3 ~1×10 9 cells / ml, preferably 1 x 10 4 ~1×10 8 cells / ml, more preferably 1 x 10 5 ~1×10 7 If the biological material is extracellular vesicles (EVs), the EVs can be cryopreserved at a concentration of, for example, 1 x 10 cells / ml, calculated relative to the volume of the cryopreserved preparation. 5 ~1×10 11 EVs / ml, preferably 1 x 10 6 ~1×10 10 EVs / ml, more preferably 1 x 10 7 ~1×10 9 If the biological material is an organoid, the organoid can be cryopreserved at a concentration of 10 to 10,000 organoids / ml, preferably 50 to 1,000 organoids / ml, more preferably 100 to 500 organoids / ml, calculated based on the volume of the cryopreserved preparation.

[0103] In certain embodiments, the cell in the context of the present invention is a cell used in an advanced therapy medicinal product (ATMP) and / or is one or more selected from the group consisting of hematopoietic cells (e.g., erythroid cells, hematopoietic stem cells), myeloid cells (e.g., granulocytes, megakaryocytes, macrophages), immune cells (e.g., neutrophils, natural killer cells, T lymphocytes, B lymphocytes, monocytes, dendritic cells), peripheral blood mononuclear cells (PBMCs), stem cells (e.g., hematopoietic stem cells, induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs), embryonic stem cells), amniotic epithelial cells, hepatocytes, and hepatic stellate cells.

[0104] In one embodiment, the cells in the context of the present invention are chimeric antigen receptor (CAR) T lymphocytes. In one embodiment, the cells in the context of the present invention are CD3+ and / or CD45+ T lymphocytes. In one embodiment, the cells in the context of the present invention are CD45+ and / or CD34+ stem cells.

[0105] The cryopreserved preparations of the present invention are particularly suitable for cryopreserving one or more of "hard-to-freeze cells," "unfreezable cells," well-differentiated cells, and slow-growing cells, for which 10% (v / v) (10.9% by weight) DMSO typically does not achieve the desired low cell death and / or high cell viability, whereas a combination of collagen hydrolysate and DMSO typically does.

[0106] In one embodiment, the cells in the context of the present invention are "freezeable cells." As used in this disclosure, "freezeable cells" refers to a cell type that exhibits less than 20% cell death (under optimal conditions) and maintains viability (e.g., no more than a 20% loss in viability) after cryopreservation in a formulation based on a permeating cryoprotectant and / or a non-permeating cryoprotectant disclosed in this disclosure other than collagen hydrolysate. Preferably, as used in this disclosure, "freezeable cells" refers to a cell type that exhibits less than 20% cell death and maintains viability (e.g., no more than a 20% loss in viability) after cryopreservation in 10% (v / v) DMSO.

[0107] In one embodiment, the cells in the context of the present invention are "hard-to-freeze cells." As used in the present disclosure, "hard-to-freeze cells" refers to cell types that exhibit less than 20% cell death (under optimal conditions) but reduced viability (e.g., a loss of viability of 20% or more) after cryopreservation in a preparation based on a permeating cryoprotectant and / or a non-permeating cryoprotectant disclosed in the present disclosure other than collagen hydrolysate. Preferably, as used in the present disclosure, "hard-to-freeze cells" refers to cell types that exhibit less than 20% cell death but reduced viability (e.g., a loss of viability of 20% or more) after cryopreservation in 10% (v / v) DMSO.

[0108] In one embodiment, the cells in the context of the present invention are "freezable cells." As used in this disclosure, "freezable cells" refers to cell types that exhibit 20% or more cell death (under optimal conditions) after cryopreservation in a formulation based on a permeating cryoprotectant and / or a non-permeating cryoprotectant disclosed herein other than collagen hydrolysate. Preferably, as used in this disclosure, "freezable cells" refers to cell types that exhibit 20% or more cell death after cryopreservation in 10% (v / v) DMSO.

[0109] In one embodiment, the cells are primary cells, which means cells that have been (freshly) isolated from living tissue, preferably human tissue.

[0110] In one embodiment, the cells are well-differentiated cells, preferably one or more selected from the group consisting of neurons, neuropil, cardiomyocytes, skeletal muscle cells, well-differentiated fibroblasts, well-differentiated hepatocytes, monocytes, dendritic cells, beta cells, pancreatic islets, primary human hepatocytes, primary human monocytes, chondrocytes, osteocytes, and adipocytes. As used herein, the term "well-differentiated cells" refers to cells that proliferate little (i.e., less than once per 48 hours, preferably less than once per 7 days) or not at all, particularly in vitro. As used herein, the term "well-differentiated cells" preferably refers to cells that have undergone cell cycle arrest. The ability to proliferate in the context of the present invention preferably relates to the ability to proliferate in vitro. Typically, well-differentiated cells do not transform into other types of cells and are differentiated to perform a specific function.

[0111] In one embodiment, the biological material disclosed in the present disclosure is a biological material intended for transplantation into a host, preferably a human. In one embodiment, the biological material is an advanced therapeutic 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 typically considered a rule of thumb that cell survival should be better than 80% (i.e., cell death should be less than 20%), and further, the cryopreserved preparation should be GMP-grade and preferably free of DMSO and / or (animal) serum. Currently available cryopreserved preparations do not meet this criterion, for example, because they often require serum (proteins) and / or DMSO. However, the cryopreserved preparations of the present invention may meet this criterion.

[0112] In certain embodiments, the cryopreserved preparation is GMP (Good manufacturing practice)-grade and / or can be manufactured under GMP conditions.

[0113] In some embodiments, the cryopreserved preparation may additionally serve as a scaffold and / or cell carrier, as known in the art, such as a hydrogel, including a bioink suitable for 3D printing. Such cryopreserved preparations may contain additional crosslinkable polymers to allow for the formation of a hydrogel (e.g., as discussed in Chaudhary et al. Beni-Suef Univ J Basic Appl Sci 11, 3 2022). For example, the cryopreserved preparation may contain a photoinitiator to promote crosslinking before and / or after cryopreservation, thereby obtaining, for example, a 3D construct (Tan et al. Micromachines (Basel). 2022 Jul; 13(7): 1038).

[0114] In one embodiment, the cryopreserved preparations of the present invention are suitable for long-term storage of biological materials, such as cells, preferably mammalian cells, and even more preferably human cells. In one embodiment, the cryopreserved preparations of the present invention are used for cryopreservation for 1 year or more (e.g., 1 to 20 years), preferably 5 years or more, and more preferably 10 years or more.

[0115] When the cryopreserved preparations of the present invention are used, it appears that cell recovery, viability, and / or functionality can be surprisingly improved if a post-thaw centrifugal wash step is not included. In certain embodiments, the biological material is not subjected to a post-cryopreservation and / or post-freeze-thaw centrifugal wash step but is used directly, e.g., for in vitro culture and / or in vivo implantation. However, this does not exclude the possibility of diluting the cryopreserved preparation in a selected additional (liquid) preparation. In the context of the present invention, the terms "post-cryopreservation" and / or "post-freeze-thaw" preferably refer to a 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 "centrifugal wash" may refer to any method that aggregates and / or pellets at least a portion of the biological material (e.g., cells) by applying centrifugal force. Centrifugal wash allows the supernatant of the cryopreserved preparation to be removed, after which the biological material can be resuspended in a different aqueous medium or another carrier material. The term "centrifugal washing" includes 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.

[0116] The terms "comprising" or "to comprise" and their conjugations are used in the context of the present invention in their open-ended sense to mean that the items followed by the words are included but not to exclude items not specifically mentioned.

[0117] The recitation of an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of that element is present, unless the context clearly requires that one and only one of that element is present. The indefinite article "a" or "an" thus normally means "at least one."

[0118] In the context of the present invention, a level is "increased" or "decreased" if the level is 1% or more, e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%, higher or lower, respectively, than the corresponding level in a control or reference. Additionally or alternatively, a level in a sample may be considered to be increased or decreased if it is statistically significantly higher or lower, respectively, compared to the control or reference (including at an earlier time point), regardless of the magnitude of the change. The term "decreased" may be used interchangeably with the term "reduced" in the context of the present invention. [Example]

[0119] Example 1 ·method Table 1 gives an overview of the starting materials used in the examples (all obtained from Rousselot BV, Belgium).

[0120] Table 1. Summary of starting materials used. MW = molecular weight; LPS = lipopolysaccharide; EU = endotoxin units; IEP = isoelectric point. [Table 1]

[0121] The 3T3 cell line (fibroblast culture) was used in the experiment. Cells were frozen in cryovials by slow cooling at a cooling rate of -1°C / min. Then, the cells were stored at -80°C overnight for 18 hours or more. The freezing solution was prepared by supplementing DMEM (Dulbecco's Modified Eagle's Medium) with DMSO (varying between 0 and 13% (v / v)) and / or collagen hydrolysate (varying between 0 and 40% (w / v)).

[0122] For thawing, cells in cryovials were thawed in a water bath at 37°C until only small pieces of ice remained, after which the cryovials were kept on ice. The cell suspension was transferred to a tube, excess medium (which had been brought to room temperature) was carefully added, and the suspension was mixed. The cells were centrifuged at 300 x g for 3 minutes, after which the supernatant was discarded and the cells were resuspended in warm (approximately 37°C) cell culture medium at the desired concentration. The % cell death after thawing was determined by counting viable cells and comparing it to the number of frozen cells.

[0123] ·result Table 2 shows the cell death of fibroblast 3T3 cells after cryopreservation using cryopreservation preparations based on 0-40% (w / v) collagen hydrolysate ("P5000") and 6.7% (v / v) DMSO. It can be seen that higher concentrations of collagen hydrolysate (e.g., 30% or 40% (w / v)) in combination with 6.7% (v / v) DMSO reduce cell death to less than 10%, which is lower than the cell death achieved with 20% (w / v) collagen hydrolysate and 6.7% (v / v) DMSO.

[0124] Table 2. Cell death in fibroblast 3T3 cells after cryopreservation using cryopreservation preparations based on different concentrations of collagen hydrolysate (CH, "P5000") and 6.7% (v / v) DMSO. [Table 2]

[0125] Table 3 shows the percent cell death in fibroblast 3T3 cells after cryopreservation using various concentrations of DMSO (0%, 0.8%, 1.7%, 3.3%, and 6.7% (v / v)), alone or in combination with 20% (w / v) collagen hydrolysate ("P5000"). It was found that 20% collagen hydrolysate can reduce cell death when used as a stand-alone cryoprotectant. Furthermore, the combination of collagen hydrolysate and DMSO was found to reduce cell death to less than 20%, even at DMSO concentrations as low as 0.8%. Compared to 10% DMSO (often considered a "gold standard"), the addition of collagen hydrolysate allowed for a reduction in the amount of DMSO concentration required by more than 90%. It was also found that cell death could be reduced to 2-5% when collagen hydrolysate was used in combination with 0.8-6.7% DMSO. Such low toxicity could not be achieved when DMSO was used alone.

[0126] Table 3. Cell death (%) in fibroblast 3T3 cells after cryopreservation in various 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"). [Table 3]

[0127] Table 4 shows cell death in fibroblast 3T3 cells after cryopreservation using cryopreservation preparations based on collagen hydrolysate with a molecular weight of approximately 2000 Da ("P2000") or approximately 5000 Da ("P5000") in combination with 6.7% DMSO. It can be seen that in both cases, low cell death occurs in the presence of 20%, 30%, or 40% (all w / v) collagen hydrolysate, whether it is the 2000 Da or the 5000 Da collagen hydrolysate.

[0128] Table 4. Cell death in fibroblast 3T3 cells after cryopreservation using cryopreservation preparations based on collagen hydrolysate (CH) with a molecular weight of approximately 2000 Da ("P2000") or approximately 5000 Da ("P5000") in combination with 6.7% DMSO. [Table 4]

[0129] To examine the effect of the centrifugal washing step, after freeze-thawing, cells were resuspended in fresh DMEM medium with or without centrifugation (300 × g force for 3 minutes), and the percentage of cell death was then determined as described above.

[0130] Table 5 shows cell death in fibroblast 3T3 cells in cryopreserved preparations containing various concentrations of collagen hydrolysate (P5000) and DMSO. Cell death was determined without or with a centrifugal wash step (300 × g force for 3 minutes). Cell death was found to be lowest in the presence of 24% (v / v) collagen hydrolysate and less than 8% DMSO.

[0131] Table 5. Cell death in fibroblast 3T3 cells in cryopreserved preparations containing various concentrations of collagen hydrolysate (CH, "P5000") and DMSO. Cell death was determined with or without a centrifugal wash step.

[0132] [Table 5]

[0133] Overall, a combination of no DMSO or low amounts of DMSO (e.g., less than 10%) and 20% or more collagen hydrolysate was found to be most suitable for cryopreservation. Collagen hydrolysates with an average molecular weight of approximately 2000 Da and those with an average molecular weight of approximately 5000 Da performed similarly.

[0134] When "P5000" and "H5000" were tested at 0%, 10%, 20%, 30%, or 40% (w / v) without DMSO, an inverse concentration-dependent relationship was observed between the amount of collagen hydrolysate and the percentage of cell death. The lowest percentage of cell death was observed with 30% or 40% (w / v) collagen hydrolysate. Cells also exhibited more informative genotypes and phenotypes (by DNA sequencing assays, methylation assays, cell surface marker expression assays, cell differentiation assays, or other functional tests) in the absence of DMSO.

[0135] It was found that decreasing the amount of DMSO resulted in smaller genotypic and phenotypic changes in the cells, therefore, it is believed that collagen hydrolysate may reduce the negative effects of DMSO (Tuncer et al. Scientific Reports volume 8, Article number: 14828, 2018).

[0136] Overall, the best results were obtained when the amount of collagen hydrolysate was above 20%, but especially when the amount of collagen hydrolysate was 30% or 40%.

[0137] Example 2 ·method A comparison was made between hydrolyzed gelatins with different LPS contents, "P5000" (1250 EU / g LPS) and "P2000" (13350 EU / g LPS). Cells were frozen and thawed according to the method in Example 1, and the percentage of cell death was determined.

[0138] To further investigate the effect of LPS during the washing centrifugation, freezing solutions were compared with and without spiking with 20,000 EU / ml LPS. The freezing solutions were based on DMEM according to Example 1 and contained 24% (w / v) hydrolyzed gelatin ("P5000") and 8% (v / v) DMSO. Freezing and thawing were performed according to Example 1.

[0139] After freezing and thawing, the cells were resuspended in fresh DMEM medium with or without centrifugation, and the percentage of cell death was determined as described above.

[0140] LPS levels were determined using the endozyme recombinant factor C method (see supplier Hygloss; FDA approved method).

[0141] ·result Figure 1: Cell death in fibroblast 3T3 cells after cryopreservation using cryopreservation preparations based on 25% (v / v) collagen hydrolysate containing 1250 EU / g LPS ("low LPS", P5000) or 13350 EU / g LPS ("high LPS", P2000) combined with 6.7% (v / v) DMSO. The use of collagen hydrolysate with low LPS content was found to reduce cell death by 75% compared to high LPS conditions.

[0142] Figure 2 shows cell death in fibroblast 3T3 cells in cryopreserved preparations containing 8% (v / v) DMSO spiked with or without 20,000 EU / ml LPS ("LPS" group). Cell death was determined without or with a centrifugal wash step ("Spin" group). In the presence of 8% DMSO, centrifugal wash was found not to increase cell death. However, in the presence of 20,000 EU / ml, centrifugal wash increased cell death.

[0143] Similarly, for cryopreserved preparations containing 24% (w / v) collagen hydrolysate ("P5000"), spiked with or without 20,000 EU / ml LPS, the centrifugal wash step was found to lead to increased cell death, especially in the presence of LPS. The effects of centrifugation and LPS on increased cell death were observed for cryopreserved preparations containing 24% (w / v) collagen hydrolysate ("P5000"), both with and without 8% (v / v) DMSO.

[0144] It has further been found that the presence of LPS during cryopreservation can induce undesirable toxic and / or pro-inflammatory responses.

[0145] Overall, it was further found that cryopreservation was further improved in the absence or minimal presence of LPS, especially when centrifugal washes were performed. No or low amounts of LPS can make centrifugal washes redundant.

[0146] Overall, the best results were obtained when the amount of collagen hydrolysate was approximately 20% (w / v) or more, especially when the amount of collagen hydrolysate was 30% (w / v) or 40% (w / v).

[0147] Example 3 ·method Experiments were performed using a population of neuropil cells (including neurons) according to the methodology described in Example 1. Neuropil cells were used as received from the supplier. The ratio of neuropil cells used was approximately 1:3 astrocytes:neurons. Cells were stained for GFAP (glial marker) and MAP2 (neuronal marker).

[0148] ·result Figure 3 shows cell death (%) in neuropil cells after freezing in 10% (v / v) DMSO ("control") or in 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 seen when 10% (v / v) DMSO alone was used.

[0149] Figure 4 shows cell death in neuropil cells after cryopreservation in various cryopreservation preparations. The combination of 30% (w / v) collagen hydrolysate ("P5000") with DMSO reduces cell death compared to DMSO alone. Notably, cell death in neuropil cells was nearly 70% in 6.7% (v / v) DMSO, demonstrating the significant improvement achieved by adding collagen hydrolysate.

[0150] Figure 5 shows staining for glial and neuronal markers in fresh neuropil cells (i.e., not cryopreserved; left panel) and frozen and thawed neuropil cells (middle and right panels). As can be seen, the left and right panels show a mixture of neurons and astrocytes with dendrites at the expected density. In the middle panel, the density is lower, and therefore no activity is expected. When analyzing cells, dendritic density and formation (processes / outgrowth) were examined. Neuropil cells frozen in a preparation containing 6.7% (v / v) DMSO in combination with 30% (w / v) collagen hydrolysate ("P5000") were found to exhibit an improved phenotype compared to cells frozen in a preparation with 10% (v / v) DMSO.

[0151] It was further found that 20-40% (w / v) collagen hydrolysate (with or without additional DMSO) was the optimal concentration range.

[0152] Overall, for cells that exhibit high cell death and / or low viability in 10% (v / v) DMSO (and are therefore considered "difficult to freeze" or "unfreezable"), such as neuronal cells, freezing media based on more than 20% (w / v) collagen hydrolysate are found to be more efficient than freezing media based solely on DMSO (at 10% (v / v)). The lowest cell death and highest viability are seen when more than 20% (w / v) collagen hydrolysate is combined with 5% (v / v) or other low amounts of DMSO.

[0153] Similar results as for neurons were obtained for non-differentiated cells such as ESCs: ESCs showed undesirably high cell death with 10% (v / v) DMSO alone, but this was improved to less than 5% in combination with 30% (w / v) collagen hydrolysate.

[0154] 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 DMSO concentrations of 10% (v / v) or higher did not result in satisfactory cell recovery and viability in ESCs and neuronal cells. However, the use of collagen hydrolysate at levels above 20% (w / v) can achieve at least 80% cell recovery or better for certain cells. This is especially true when the DMSO concentration is low (e.g., 5% or other low concentrations).

[0155] Other cells tested include human primary cardiomyocytes, pancreatic islets, primary human hepatocytes, and primary human monocytes. For these cell types, freezing in 10% (v / v) DMSO results in less than 20% or even less than 10% cell recovery. Cell recovery and viability are improved with a combination of more than 20% (particularly 30 or 40% by weight) collagen hydrolysate in combination with 1-5% (v / v) DMSO.

[0156] Overall, the best results were obtained when the amount of collagen hydrolysate combined with low concentrations (e.g., 1-5% (v / v)) of DMSO exceeded 20% (v / v), especially 30% (v / v) or 40% (v / v).

[0157] Example 4 ·method Fibroblasts were cryopreserved according to Example 1. After thawing, the cells were cultured in tissue culture plates. After 3 days, cell viability was determined by staining live cells and then counting them. ·result Figures 6 and 7 show the viability of fibroblast 3T3 cells cryopreserved in various freezing media compositions and cultured for 3 days, then stained for live cells.

[0158] Compared with cells cryopreserved in DMSO alone, greater cell proliferation was observed over time for cells cryopreserved in hydrolyzed gelatin combined with DMSO. This indicates that collagen hydrolysate not only reduces cell death but also improves cell viability of recovered cells. Furthermore, cell viability was found to be highest when DMSO concentrations were less than 10% (v / v), particularly for the 3.3% and 6.7% (v / v) DMSO groups. Viability was also determined after 1 or 2 days, and again showed highest cell viability when DMSO concentrations were less than 10% (v / v).

[0159] Example 5 ·method 3T3 cell lines (fibroblast cultures) were cryopreserved according to Example 1 in freezing media containing 2.5% or 10% (v / v) DMSO with or without 30% (w / w) hydrolyzed gelatin. A colorimetric MTT assay was performed at 96 hours to assess the metabolic activity of the cells. The MTT assay is based on the metabolic reduction of the yellow substance MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) to a blue formazan product by mitochondrial dehydrogenases. Only viable cells can catalyze this reaction.

[0160] ·result Table 6 shows the metabolic activity in fibroblast 3T3 cells after cryopreservation in various freezing media. The highest metabolic activity is observed when cells are cryopreserved in a combination of hydrolyzed gelatin and a relatively low concentration of DMSO.

[0161] Table 6. Metabolic activity in fibroblast 3T3 cells after cryopreservation in frozen preparations containing various concentrations of DMSO with or without 30% by weight hydrolyzed gelatin (collagen hydrolysate, CH). [Table 6]

[0162] Overall, it appears that using a relatively low amount of DMSO is beneficial. Similar effects of DMSO concentration were observed for other concentrations of collagen hydrolysate.

[0163] Example 6 ·method Primary mouse cortical cells (E18) were cryopreserved according to the freezing method described in Example 1, except that the freezing period was increased to 4 weeks. Different freezing media were compared (n=2 per group) as described in Table 7. 1. Cell metabolic activity was assessed by a colorimetric MTT assay. The MTT assay is based on the metabolic reduction of the yellow substance MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) to a blue formazan product by mitochondrial dehydrogenase. Only viable cells were able to catalyze this reaction. 2. Cytotoxicity was assessed in the supernatants of primary cortical neurons using a lactate dehydrogenase (LDH) assay. LDH is a stable cytosolic enzyme present in all cells and is rapidly released into the cell culture supernatant upon cell membrane damage. LDH activity in the cell culture supernatant was determined by a coupled enzymatic reaction in which the tetrazolium salt INT was reduced to formazan. 3. To assess neuronal activity, primary cortical neurons were transduced with Neuroblast™ lentivirus on DIV2 and neuronal activity was monitored over 72 hours (e.g., DIV10-12) using live-cell imaging of RFP oscillations at 120 seconds per read on an IncuCyte™ instrument. The instrument automatically assessed the number of active cells, average burst rate, and average correlation.

[0164] Table 7. Various cryopreservation preparations based on DMSO and / or hydrolyzed gelatin (collagen hydrolysate, CH) were tested for their effect on viability and metabolic activity in primary mouse cortical cells after cryopreservation. [Table 7]

[0165] ·result It has been found that 10% (v / v) DMSO alone leads to low viability and metabolic activity. Overall, high viability and metabolic activity is observed when DMSO is combined with collagen hydrolysate, preferably at a concentration of less than 10% (v / v). Similar experiments were performed on cortical rat cells (neuropil), where GFAP / MAP2 staining was also performed as described in Example 3.

[0166] Example 7 Example 7 shows the viability of intestinal organoids frozen in hydrolyzed gelatin X-Pure HGP LS (Rousselot BV, Belgium) or Peptan P5000 LD (Rousselot BV, Belgium) compared to freezing media based on FCS and 10% DMSO.

[0167] Figure 8 shows the cell viability of 3D intestinal organoids determined by WST-1 assay 1 hour after thawing (% relative to control).

[0168] Higher organoid viability after 1 hour of thawing can be seen when frozen in X-Pure HGP LS and Peptan P5000 LD compared to freezing media based on FCS + 10% DMSO. The present disclosure also includes the following aspects. <1> Contains collagen hydrolysate and dimethyl sulfoxide, The amount of collagen hydrolysate is 10 to 70% by weight calculated based on the weight of the frozen preparation; The amount of dimethyl sulfoxide is less than 10% (v / v) calculated relative to the volume of the cryopreserved preparation; Cryopreserved preparations. <2> The content of dimethyl sulfoxide is 8.5% or less, preferably 7% (v / v) or less. <1> A cryopreserved preparation according to claim 1. <3> <1> containing dimethyl sulfoxide at 1% (v / v) or more, preferably 2% (v / v) or more; or <2> A cryopreserved preparation according to claim 1. <4> Contains 20 to 50% by weight of collagen hydrolysate, <1> ~ <3> 10. The cryopreserved preparation according to any one of the preceding claims. <5> Contains more than 20% and less than 45% by weight of collagen hydrolysate, <1> ~ <4> 10. The cryopreserved preparation according to any one of the preceding claims. <6> The collagen hydrolysate is hydrolyzed gelatin. <1> ~ <5> 10. The cryopreserved preparation according to any one of the preceding claims. <7> The collagen hydrolysate has an average molecular weight of 500 to 10,000 Da. <1> ~ <6> 10. The cryopreserved preparation according to any one of the preceding claims. <8> the collagen hydrolysate has an endotoxin level of less than 10,000 EU / g calculated on the weight of the collagen hydrolysate, preferably less than 1,000 EU / g, more preferably less than 100 EU / g; <1> ~ <7> 10. The cryopreserved preparation according to any one of the preceding claims. <9> The collagen hydrolysate is endotoxin-free. <8> A cryopreserved preparation according to claim 1. <10> The cryopreserved preparation , cryopreserved preparations and an endotoxin level of less than 2500 EU / ml calculated on the volume of the solution, the endotoxin level being preferably less than 250 EU / ml, more preferably less than 25 EU / ml. <1> ~ <9> 10. The cryopreserved preparation according to any one of the preceding claims. <11> The cryopreserved preparation is endotoxin-free. <10> A cryopreserved preparation according to claim 1. <12> The cryopreserved preparation is free of one or more of additional cryoprotectants, serum, and serum proteins. <1> ~ <11> 10. The cryopreserved preparation according to any one of the preceding claims. <13> further comprising one or more of a cryoprotectant, serum, and serum proteins; <1> ~ <11> 10. The cryopreserved preparation according to any one of the preceding claims. <14> The further cryoprotectant is a penetrating cryoprotectant and / or a non-penetrating cryoprotectant. <12> or <13> A cryopreserved preparation according to claim 1. <15> The permeable cryoprotectant is a permeable glycol, preferably ethylene glycol, propylene glycol, or a combination thereof; <14> A cryopreserved preparation according to claim 1. <16> The non-permeable cryoprotectant is one or more selected from the group consisting of polyethylene glycol, glycerol, polyvinylpyrrolidone, methylcellulose, sugar, or combinations thereof; <14> A cryopreserved preparation according to claim 1. <17> <1> ~ <16> and reducing the temperature of the cryopreserved preparation to below the freezing point of said cryopreserved preparation. A method for cryopreserving biological materials, comprising: <18> providing the biological material in a cryopreserved preparation comprising collagen hydrolysate and reducing the temperature of the cryopreserved preparation to below the freezing point of said cryopreserved preparation; The amount of collagen hydrolysate is 10 to 70% by weight calculated based on the weight of the cryopreserved preparation; the amount of dimethyl sulfoxide is less than 10% (v / v) calculated relative to the volume of the cryopreserved preparation; Methods for cryopreservation of biological materials. <19> The biological material is selected from the group consisting of eukaryotic cells, prokaryotic cells, cell organelles, extracellular vesicles, organoids, tissues, and organs; <17> or <18> The method described below. <20> The collagen hydrolysate <1> ~ <16> 10. Use of collagen hydrolysate in cryopreservation of biological material, provided in a cryopreservation preparation according to any one of the preceding claims. <21> Collagen hydrolysate is provided in a cryopreserved preparation, The amount of collagen hydrolysate is 10 to 70% by weight calculated based on the weight of the cryopreserved preparation; the amount of dimethyl sulfoxide is less than 10% (v / v) calculated relative to the volume of the cryopreserved preparation; Use of collagen hydrolysates in the cryopreservation of biological materials. <22> The biological material is selected from the group consisting of eukaryotic cells, prokaryotic cells, cell organelles, extracellular vesicles, organoids, tissues, and organs; <20> or <21> Use as described in.

Claims

1. Contains collagen hydrolysate and dimethyl sulfoxide, the amount of collagen hydrolysate is 10-70% by weight calculated relative to the weight of the cryopreserved preparation; The amount of dimethyl sulfoxide is less than 1-8.5% (v / v) calculated on the volume of the cryopreserved preparation; Cryopreservative preparations for cryopreservation of biological materials.

2. 2. The cryopreserved preparation of claim 1, wherein the amount of dimethyl sulfoxide is 2-7% (v / v) calculated relative to the volume of the cryopreserved preparation.

3. 10. The cryopreserved preparation of claim 1, comprising 20-50% by weight of collagen hydrolysate.

4. 10. The cryopreserved preparation of claim 1, comprising more than 20% and less than 45% by weight of collagen hydrolysate.

5. 2. The cryopreserved preparation of claim 1, wherein the collagen hydrolysate is hydrolyzed gelatin.

6. 2. The cryopreserved preparation of claim 1, wherein the collagen hydrolysate has an average molecular weight of 500 to 10,000 Da.

7. 2. The cryopreserved preparation of claim 1, wherein the collagen hydrolysate has an endotoxin level of less than 10,000 EU / g calculated on the weight of the collagen hydrolysate.

8. 2. The cryopreserved preparation of claim 1, wherein the cryopreserved preparation has an endotoxin level of less than 2500 EU / ml calculated on the volume of the cryopreserved preparation.

9. 10. The cryopreserved preparation of claim 1, wherein the cryopreserved preparation is free of one or more of additional cryoprotectants, serum, and serum proteins.

10. 10. The cryopreserved preparation of claim 1, further comprising one or more of a cryoprotectant, serum, and serum proteins.

11. 11. The cryopreserved preparation of claim 9 or claim 10, wherein the further cryoprotectant is a permeating cryoprotectant and / or a non-permeating cryoprotectant.

12. 12. The cryopreserved preparation of claim 11, wherein the permeating cryoprotectant is a permeating glycol.

13. 12. The cryopreserved preparation of claim 11, wherein the non-permeable cryoprotectant is one or more selected from the group consisting of polyethylene glycol, glycerol, polyvinylpyrrolidone, methylcellulose, sugars, or combinations thereof.

14. 11. A method for cryopreserving a biological material, comprising the steps of providing the biological material in a cryopreservation preparation according to any one of claims 1 to 10, and reducing the temperature of the cryopreservation preparation to below the freezing point of the cryopreservation preparation.

15. 15. The method of claim 14, wherein the biological material is selected from the group consisting of eukaryotic cells, prokaryotic cells, cell organelles, extracellular vesicles, organoids, tissues, and organs.

Images