Method for preserving or transporting stem cells without freezing

Abstract

To provide a method for preserving stem cells while maintaining a high survival rate at low temperatures without freezing them.SOLUTION: The present disclosure relates to a method for preserving stem cells, the method comprising: 1) preparing a storage solution containing a gelling agent in a non-gelling state at a temperature higher than the gelling temperature and having a pH of about 5.0 to about 8.0; 2) lowering the temperature to the gelling temperature of the gelling agent after placing the stem cells in the storage solution; and 3) storing the storage solution containing the stem cells at the gel maintenance temperature of the gelling agent. In some embodiments, the gelling agent can contain collagen, denatured collagen, collagen-like peptide, gelatin, or any combination thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to techniques for storing or transporting stem cells. [Background technology]

[0002] Currently, several regenerative medicine products using stem cells are being developed and partially approved in Japan, bringing new hope for diseases for which there was no cure (severe burns, spinal cord injury, head trauma, cerebral infarction, cerebral hemorrhage, Parkinson's disease, etc.), and great development is expected in the future. However, many of these products are transported in a frozen state at ultra-low temperatures (-170°C). This presents the following problems: (1) a decrease in cell quality (cell viability, trophic factor secretion ability), and (2) transportation costs (cost of liquid nitrogen and purchase and maintenance of ultra-low temperature transportation equipment). On the other hand, when cells are transported at room temperature, there are problems such as the depletion of oxygen and nutrients in the transport liquid as cell metabolism proceeds normally, and physical damage due to vibrations as the cells are not completely fixed, resulting in an extremely low survival rate over long periods of time (more than one day). Although transporting cells at low temperatures suppresses metabolism, this does not improve the resistance to vibration. A method is needed to transport cells at low cost that can withstand transport conditions such as vibration while maintaining cell viability. Summary of the Invention [Means for solving the problem]

[0003] As a result of extensive research and development, the present inventors have discovered a method for preserving stem cells with a high viability. Specifically, the present inventors have found that preserving stem cells with a gelling agent maintains a high viability and the ability to secrete trophic factors without freezing. Therefore, the preservation method disclosed herein allows therapeutic stem cells to be transported at low temperatures (e.g., 4°C) without freezing.

[0004] Thus, the present disclosure provides: (Item 1) 1. A method for preserving stem cells, the method comprising: 1) preparing a storage solution containing a gelling agent at a temperature higher than the gelation temperature and having a pH of about 5.0 to about 8.0; 2) placing the stem cells in the preservation solution and then lowering the temperature to the gelling temperature of the gelling agent; 3) storing the preservation solution containing the stem cells at a gel-maintaining temperature of the gelling agent; A method comprising: (Item 1A) 1. A method for preserving stem cells, the method comprising: 1) preparing a storage solution containing a gelling agent in a non-gel state at a temperature higher than the gelation temperature and having a pH of about 5.0 to about 8.0; 2) placing the stem cells in the preservation solution and then lowering the temperature to the gelling temperature of the gelling agent; 3) storing the preservation solution containing the stem cells at a gel-maintaining temperature of the gelling agent; A method comprising: (Item 2) The method according to the preceding item, wherein the gelling agent comprises a peptide. (Item 3) 2. The method of claim 1, wherein the gelling agent comprises collagen, denatured collagen, collagen-like peptides, gelatin, or any combination thereof. (Item 4) The gelling agent comprises a collagen-like peptide, and the collagen-like peptide is Gly-X -Y, wherein X and Y are independently the same or different amino acids. (Item 5) The method according to any of the preceding items, wherein the gelling agent is contained in the preservation solution at a concentration of about 1% (w / w) or more. (Item 6) The method according to any of the preceding items, wherein the gelling agent is contained in the preservative solution at a concentration of about 1% (w / w) to about 10% (w / w). (Item 7) The method according to any of the preceding items, wherein the gelling agent is contained in the preservative solution at a concentration of about 2.5% (w / w). (Item 8) The method according to any one of the preceding items, wherein the preservation solution is a cell culture medium, a physiological saline solution, or an electrolyte solution. (Item 9) Item 10. The method according to any one of the preceding items, wherein the pH of the preservation solution is about 6.0 to about 7.5. Item 11. The method according to any one of the preceding items, wherein the pH of the preservation solution is about 6.4 to about 7.4. The method according to any of the preceding items, wherein the gel maintenance temperature is about 0°C to about 37°C. (Item 12) The method according to any of the preceding items, wherein the gel maintenance temperature is about 0°C to about 24°C. (Item 13) The method according to any of the preceding items, wherein the stem cells are mesenchymal stem cells. (Item 14) The method according to any of the preceding items, wherein the mesenchymal stem cells are derived from bone marrow, adipose tissue, placental tissue, synovial tissue, umbilical cord tissue (e.g., umbilical cord blood), dental pulp, or amniotic membrane, or are mesenchymal stem cells differentiated from ES cells or iPS cells. (Item 15) The method according to any one of the preceding items, wherein the mesenchymal stem cells are bone marrow-derived mesenchymal stem cells. (Item 16) The method according to any one of the preceding items, wherein the stem cells are cells used in cell infusion therapy and drug discovery research, and are cells obtained from a subject undergoing the cell infusion therapy. (Item 17) 2. The method according to any of the preceding items, further comprising a step of non-gelling treatment. (Item 18) 2. The method of claim 1, wherein the non-gelling step comprises increasing the temperature to a temperature at or above the melting point of the gelling agent. (Item 19) The method according to any of the preceding items, further comprising the step of recovering the stem cells. (Item 20) The method according to any of the preceding items, wherein the step of recovering the stem cells comprises diluting the preservation solution 2 to 20 times and separating the stem cells from the preservation solution. (Item 21) The stem cells are about 1×10 6 ~Approx. 1×10 7 The method according to any one of the preceding items, wherein the cells are stored at a cell density of 1000 cells / ml. (Item 22) A kit for preserving stem cells, the kit comprising: (1) a preservative solution containing a gelling agent; (2) a buffer solution for adjusting the pH of the preservative solution to about 5.0 to about 8.0; wherein the stem cells are stored at a gel-maintaining temperature. (Item 23) A composition for preserving stem cells, comprising a gelling agent. (Item 24) The composition according to any one of the preceding items, wherein the composition is used at a pH of about 5.0 to about 8.0 when preserving stem cells. (Item 25) The composition described in any of the above items, wherein the composition is used to store stem cells at the gelling temperature of the gelling agent. (Item 26) A formulation comprising a gelling agent and stem cells, and having a pH of about 5.0 to about 8.0. (Item 29) The formulation according to any of the preceding items, which is a ready-to-use formulation. (Item 30) 1. A method for preserving tissue or organs, the method comprising: 1) preparing a storage solution containing a gelling agent at a temperature higher than the gelation temperature and having a pH of about 5.0 to about 8.0; 2) placing the tissue or organ in the preservation solution and then lowering the temperature to the gelling temperature of the gelling agent; 3) storing the preservation solution containing the tissue or organ at a gel-maintaining temperature of the gelling agent. (Item 30A) 1. A method for preserving tissue or organs, the method comprising: 1) preparing a storage solution containing a gelling agent in a non-gel state at a temperature higher than the gelation temperature and having a pH of about 5.0 to about 8.0; 2) placing the tissue or organ in the preservation solution and then lowering the temperature to the gelling temperature of the gelling agent; 3) storing the preservation solution containing the tissue or organ at a gel-maintaining temperature of the gelling agent. (Item 31) A kit for preserving tissue or organs, the kit comprising: (1) a preservative solution containing a gelling agent; (2) a buffer solution for adjusting the pH of the preservative solution to about 5.0 to about 8.0; wherein the tissue or organ is preserved at a gel-maintaining temperature. (Item 32) A composition for preserving tissue or organs, comprising a gelling agent. (Item 33) The composition according to any one of the preceding items, wherein the composition is used at a pH of about 5.0 to about 8.0 when preserving tissues or organs. (Item 34) 1. A method for delivering stem cells, the method comprising: 1) preparing a storage solution containing a gelling agent at a temperature higher than the gelation temperature and having a pH of about 5.0 to about 8.0; 2) placing the stem cells in the preservation solution and then lowering the temperature to the gelling temperature of the gelling agent; 3) transporting the preservation solution containing the stem cells at a gel-maintaining temperature of the gelling agent; A method comprising: (Item 35) A gelling agent for preserving stem cells, which is used at a pH of about 5.0 to about 8.0 and at a gel-maintaining temperature when preserving the stem cells. (Item 36) A gelling agent for preserving tissues or organs, wherein the gelling agent is used at a pH of about 5.0 to about 8.0 and at a temperature at which the gelling agent maintains its gel when preserving the tissue or organ.

[0005] It is contemplated that the present disclosure may provide one or more of the above-described features in combinations other than those explicitly stated. Still further embodiments and advantages of the present disclosure will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary. [Brief explanation of the drawings]

[0006] [Figure 1] FIG. 1 shows a graph of cell viability after 72 hours of storage and 72 hours after reseeding in the presence or absence of RCP. [Figure 2] FIG. 2 shows a graph of cell viability after 72 hours of storage and 72 hours after reseeding in the presence of different concentrations of RCP. [Figure 3] FIG. 3 shows a graph of cell viability after 72 hours of storage and 72 hours after reseeding in different preservation solutions containing RCP. [Figure 4] FIG. 4 shows a graph of cell viability after 72 hours of storage and 72 hours after reseeding at different temperatures. [Figure 5] FIG. 5 shows a graph of cell viability after 72 hours of storage and 72 hours after reseeding for different storage days. [Figure 6] FIG. 6 shows a graph of cell viability after 72 hours of storage and 72 hours of reseeding in preservation solutions containing different gelling agents. [Figure 7] FIG. 7 shows a graph of cell viability after 72 hours of storage and 72 hours of reseeding at different pH levels. [Figure 8] FIG. 8 shows a graph of cell viability after shipping and static storage of the cells. [Figure 9] FIG. 9 shows a graph of cell viability when the cells after storage were diluted 1-fold and 5-fold with the storage solution. DETAILED DESCRIPTION OF THE INVENTION

[0007] The present disclosure will be described below. Throughout this specification, singular expressions should be understood to include the plural concept unless otherwise specified. Therefore, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include the plural concept unless otherwise specified. Furthermore, it should be understood that terms used in this specification are used in the sense commonly used in the relevant field unless otherwise specified. Therefore, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In case of conflict, the present specification (including definitions) will prevail.

[0008] (definition) As used herein, "about" means ±10% of the value that follows.

[0009] As used herein, "stem cells" refer to immature cells that have the ability to self-renew and differentiate and proliferate. Stem cells include subpopulations such as pluripotent stem cells (pluripotent stem cell ce11), multipotent stem cells (multipotent stem cell ce11), and unipotent stem cells (unipotent stem cell ce11), depending on their differentiation potential. Pluripotent stem cells refer to cells that cannot become an individual by themselves but have the ability to differentiate into all tissues and cells that make up an organism. Multipotent stem cells refer to cells that have the ability to differentiate into multiple types of tissues and cells, but not all types. Unipotent stem cells refer to cells that have the ability to differentiate into specific tissues or cells.

[0010] As used herein, "mesenchymal stem cells," also known as mesenchymal stromal cells, refer to stem cells capable of differentiating into mesenchymal cells such as osteoblasts, adipocytes, muscle cells, and chondrocytes. Mesenchymal stem cells may include mesenchymal stem cells derived from bone marrow, adipose tissue, placental tissue, synovial tissue, umbilical cord tissue (e.g., umbilical cord blood), dental pulp, or amniotic membrane, or mesenchymal stem cells differentiated from ES cells or iPS cells.

[0011] As used herein, "tissue" refers to any tissue type, including any kind of cell type and combinations thereof, such as ovarian tissue, testicular tissue, umbilical cord tissue, placental tissue, connective tissue, cardiac tissue, corneal tissue, tissue derived from muscle, cartilage or bone, endocrine tissue, and neural tissue.

[0012] As used herein, the term "organ" refers to the lung, liver, kidney, heart, ovary, pancreas, and umbilical cord. The organ may be a human organ or a non-human animal organ. The non-human animal may be a rodent, including a mouse and a rat; an ungulate, including a pig, a goat, and a sheep; a non-human primate, including a chimpanzee; or another non-human mammal, or an animal other than a mammal.

[0013] As used herein, "storage" refers to storing cells in a container for a certain period of time for any purpose (e.g., cell infusion therapy, tissue or organ transplantation, or transportation therefor), and refers to maintaining the cells in a container while maintaining the function of the cells, tissue, or organ, without the purpose of proliferating the cells. Storage differs from "culturing," which aims to proliferate the cells. Furthermore, cell storage does not refer to transferring cells into a container such as a syringe and temporarily storing them just before administration, nor does it refer to temporarily storing cells in a container for preparation just before administration.

[0014] As used herein, the term "storage solution" refers to a solution for maintaining cells for a certain period of time, and refers to a solution having physiological osmotic pressure and electrolytes to the extent that cells can survive.

[0015] As used herein, the term "gelling agent" refers to an agent for gelling a preservation solution. The gelling agent used herein advantageously has the property of being in a non-gel state when contained in a preservation solution under normal circumstances (for example, at a temperature higher than the gelling temperature (e.g., 37°C when the gelling temperature is 24°C or lower) and gelling when heated to the gelling temperature.

[0016] As used herein, the term "gelation temperature" refers to the temperature at which a material changes from a non-gelled state to a gelled state.

[0017] As used herein, the term "gel maintenance temperature" refers to the temperature at which the gel remains intact after gelation without melting.

[0018] As used herein, "non-gelling" refers to the release of the gel state by heating, diluting with a solvent, or a combination of these. The state in which the gel state has been released is also referred to as a "non-gelled state." "De-gelling temperature" refers to the temperature at which the gelled state changes to a non-gelled state. The de-gelling temperature is usually higher than the gelling temperature.

[0019] In this specification, "collagen" refers to a protein derived from animals, in which three polypeptide chains made of amino acids form a triple helix structure. There are 28 types of collagen confirmed in humans, and there are others. When collagen is heated, it is thermally denatured, and the polypeptide chains are dissolved to form gelatin. There are two types of collagen: "denatured" and "non-denatured." In this specification, when simply referring to "collagen," unless otherwise specified, it refers to "non-denatured" collagen, but it also includes both "denatured" and "non-denatured" depending on the context. "Denatured" refers to collagen that has been thermally denatured. "Non-denatured" collagen is extracted without the addition of heat, and is therefore recognized as collagen by the body, absorbed intact, and used as such. Collagen is a type of gelling agent.

[0020] As used herein, "denatured collagen" refers to collagen that has been denatured by heat, chemical denaturation (e.g., denaturation by collagenase), or denaturation by mechanical damage, and also refers to collagen that partially maintains its collagen structure. Denatured collagen does not include collagen that has been completely denatured and converted into gelatin. Denatured collagen is a type of gelling agent.

[0021] As used herein, "gelatin" refers to collagen that has been completely heat-denatured and no longer has the triple helix structure characteristic of collagen. Gelatin is a type of gelling agent.

[0022] As used herein, the term "collagen-like peptide" refers to a chemically synthesized peptide designed to mimic the unique triple helix structure that characterizes collagen molecules. Collagen-like peptides are a type of gelling agent.

[0023] As used herein, "RCP" is an abbreviation for recombinant collagen-like peptide, and refers to a recombinantly expressed collagen-like peptide. It has the same structure as collagen-like peptides and is recombinantly expressed. RCP is a type of gelling agent.

[0024] As used herein, the term "cell infusion therapy" refers to a treatment method in which cells (eg, stem cells) are infused to treat a disease (eg, cerebral infarction).

[0025] As used herein, the term "ready-to-use preparation" refers to a preparation that can be used as is after storage without further culturing of therapeutic cells.

[0026] (Preferred embodiment) Although the following description of preferred embodiments is given, it should be understood that these embodiments are merely examples of the present disclosure and that the scope of the present disclosure is not limited to such preferred embodiments. It should also be understood that those skilled in the art can easily make modifications, changes, etc. within the scope of the present disclosure by referring to the following preferred examples. Those skilled in the art can combine any of these embodiments as appropriate.

[0027] (Stem cell preservation method) In one aspect, the present disclosure provides a method for preserving stem cells, the method comprising the steps of: 1) preparing a preservation solution containing a gelling agent in a non-gelled state at a temperature higher than the gelling temperature, the pH of the solution being about 5.0 to about 8.0; 2) placing the stem cells in the preservation solution and then lowering the temperature to the gelling temperature of the gelling agent; and 3) preserving the preservation solution containing the stem cells at the gel-maintaining temperature of the gelling agent. The preservation method of the present disclosure enables preservation of stem cells while maintaining a high viability and the ability to secrete trophic factors. It has been found that vibration during preservation does not reduce viability, making the preservation method of the present disclosure applicable to transportation where vibration may occur.

[0028] In some embodiments, storing may involve transportation.

[0029] In some embodiments, the gelling agent is any substance that causes the preservation solution to gel, such as peptides, collagen, denatured collagen, collagen-like peptides, gelatin, fibrin, silicone, glycosaminoglycans, VitroGel TM 3D, VitroGel TM 3D-RGB (TheWell BIOSIENCE), BD Matrigel TM Matrix (BD Bioscience), carboxyvinyl polymers, acrylic copolymers (e.g., acrylate / alkyl acrylate copolymers), polyacrylamides, polysaccharides, natural gums, metal salts of fatty acids, hydrophobic silica, polyethylene, crosslinked acrylic acid polymers (e.g., carbomer, carboxypolyalkylene, Carbopol®), polyethylene oxide, polyoxyethylene-polyoxypropylene copolymer, polyvinyl alcohol, cellulosic polymers (e.g., hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and methylcellulose), gums (e.g., tragacanth gum and xanthan gum), sodium alginate, gellan gum, agar, carrageenan, pectin, furcellaran, alginic acid or a salt thereof, glucomannan, tara gum, locust bean gum, tamarind, pullulan, guar gum, starch phosphate, polyacrylates, gum arabic, curdlan, gum ghatti, Aeromonas gum, tamarind seed polysaccharide, and the like.

[0030] In certain embodiments, the gelling agent can be at least one selected from the group consisting of collagen, denatured collagen, collagen-like peptides, and gelatin.

[0031] In certain embodiments, the gelling agent can be a collagen-like peptide. Collagen-like peptides are commercially available, for example, those provided by Nitta Gelatin (beMATRIX® Collagen) or Fujifilm (recombinant peptide (RCP)). The collagen-like peptide contains repeats of a sequence represented by Gly-XY, where X and Y may each independently be the same amino acid or different amino acids.

[0032] In further embodiments, the gelling agent may be a recombinant collagen-like peptide (RCP). In certain embodiments, the RCP may consist of or comprise the amino acid sequence set forth in SEQ ID NO: 1, or an amino acid sequence having at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity thereto. The RCP may be prepared as appropriate according to WO 2018 / 159797 or purchased (Fujifilm).

[0033] The molecular weight of the recombinant collagen-like peptide is not particularly limited, but is preferably 2,000 to 100,000 (2 kDa (kilodaltons) to 100 kDa), more preferably 2,500 to 95,000 (2.5 kDa to 95 kDa), even more preferably 5,000 to 90,000 (5 kDa to 90 kDa), and most preferably 10,000 to 90,000 (10 kDa to 90 kDa).

[0034] The recombinant collagen-like peptide preferably has a repeat of the Gly-XY sequence characteristic of collagen. Here, the multiple Gly-XYs may be the same or different. In Gly-XY, Gly represents glycine, and X and Y represent any amino acid (preferably any amino acid other than glycine). The Gly-XY sequence characteristic of collagen is a highly specific substructure in the amino acid composition and sequence of gelatin and collagen compared to other proteins. In this substructure, glycine accounts for approximately one-third of the total, with one repeat cluster occurring every three amino acids in the amino acid sequence. Glycine is the simplest amino acid, exerting minimal constraints on the molecular chain configuration and contributing significantly to the regeneration of the helix structure during gelation. The amino acids represented by X and Y are rich in imino acids (proline and oxyproline), preferably accounting for 10% to 45% of the total. Preferably, 80% or more, more preferably 95% or more, and most preferably 99% or more of the amino acids in the recombinant collagen-like peptide sequence are Gly-XY repeat structures.

[0035] In general gelatin, charged and uncharged polar amino acids exist in a 1:1 ratio. Here, polar amino acids specifically refer to cysteine, aspartic acid, glutamic acid, histidine, lysine, asparagine, glutamine, serine, threonine, tyrosine, and arginine, and uncharged polar amino acids refer to cysteine, asparagine, glutamine, serine, threonine, and tyrosine. In the recombinant collagen-like peptide used in the present disclosure, the proportion of polar amino acids among all constituent amino acids is 10 to 40%, preferably 20 to 30%. Furthermore, the proportion of uncharged amino acids among the polar amino acids is preferably 5% or more but less than 20%, more preferably 5% or more but less than 10%. Furthermore, it is preferable that the sequence does not contain any one, preferably two or more, of the amino acids serine, threonine, asparagine, tyrosine, and cysteine.

[0036] In general, the minimum amino acid sequence that functions as a cell adhesion signal in polypeptides is known (for example, Nagai Publishing Co., Ltd., "Pathophysiology," Vol. 9, No. 7 (1990), p. 527). The recombinant collagen-like peptide used in the present disclosure may have two or more of these cell adhesion signals in one molecule. Specific sequences, expressed by single-letter amino acid code, are preferred, in view of the wide variety of cell types that can be adhered to them (RGD, LDV, REDV, YIGSR, PDSGR, RYVVLPR, LGTIPG, RNIAEIIKDI, IKVAV, LRE, DGEA, and HAV sequences (SEQ ID NOS: 2 to 9). The RGD, YIGSR, PDSGR, LGTIPG, IKVAV, and HAV sequences are more preferred, with the RGD sequence being particularly preferred. Of the RGD sequences, the ERGD sequence is preferred.

[0037] The arrangement of RGD sequences in the recombinant collagen-like peptides used in the present disclosure is preferably such that the number of amino acids between RGDs is not uniform and is between 0 and 100, preferably between 25 and 60.

[0038] The content of this minimum amino acid sequence in one protein molecule is preferably 3 to 50, more preferably 4 to 30, particularly preferably 5 to 20, and most preferably 12.

[0039] In the recombinant collagen-like peptides used in the present disclosure, the ratio of RGD motifs to the total number of amino acids is preferably at least 0.4%. When the recombinant gelatin contains 350 or more amino acids, it is preferred that each stretch of 350 amino acids contains at least one RGD motif. The ratio of RGD motifs to the total number of amino acids is more preferably at least 0.6%, even more preferably at least 0.8%, even more preferably at least 1.0%, particularly preferably at least 1.2%, and most preferably at least 1.5%. The number of RGD motifs in the recombinant peptide is preferably at least 4, more preferably 6, even more preferably 8, and particularly preferably 12 to 16 per 250 amino acids. A ratio of 0.4% of RGD motifs corresponds to at least one RGD sequence per 250 amino acids. Since the number of RGD motifs is an integer, to meet the 0.4% characteristic, a gelatin consisting of 251 amino acids must contain at least two RGD sequences. Preferably, the recombinant gelatin of the present invention contains at least two RGD sequences per 250 amino acids, more preferably at least three RGD sequences per 250 amino acids, and even more preferably at least four RGD sequences per 250 amino acids. In a further embodiment, the recombinant gelatin of the present invention contains at least four RGD motifs, preferably six, more preferably eight, and even more preferably 12 to 16 RGD motifs.

[0040] The recombinant collagen-like peptide may be partially hydrolyzed.

[0041] More preferably, the polypeptide used in the present disclosure is represented by Formula 2 below: Gly-Ala-Pro-[(Gly-XY)63 ]3-Gly In the formula, 63 X's each independently represent any amino acid, and 63 Y's each independently represent any amino acid. Note that 63 Gly-X-Y's may be the same or different.

[0042] The repeating unit preferably comprises multiple naturally occurring collagen sequence units linked together. The naturally occurring collagen referred to here may be any naturally occurring collagen, but is preferably type I, type II, type III, type IV, or type V collagen. More preferably, it is type I, type II, or type III collagen. In another embodiment, the collagen is preferably derived from human, bovine, porcine, mouse, or rat, more preferably human.

[0043] The isoelectric point of the recombinant collagen-like peptide used in the present disclosure is preferably 5 to 10, more preferably 6 to 10, and even more preferably 7 to 9.5. The isoelectric point of recombinant gelatin can be measured by measuring the pH after passing a 1% by mass peptide solution through a mixed crystal column of cation and anion exchange resins, as described in isoelectric focusing (see Maxey, CR (1976; Photogr. Gelatin 2, Editor Cox, PJ Academic, London, Engl.).

[0044] Preferably, the recombinant collagen-like peptide is not deaminated.

[0045] Preferably, the recombinant collagen-like peptides are telopeptide-free.

[0046] Preferably, the recombinant collagen-like peptide is a substantially pure polypeptide prepared from a nucleic acid encoding the amino acid sequence.

[0047] The recombinant collagen-like peptide is particularly preferably (1) the amino acid sequence set forth in SEQ ID NO: 1; or (2) An amino acid sequence having 80% or more (preferably 90% or more, more preferably 95% or more, particularly preferably 98% or more) sequence identity with the amino acid sequence set forth in SEQ ID NO: 1 and having bioaffinity: It has.

[0048] As used herein, sequence identity refers to a value calculated using the following formula: % sequence identity = [(number of identical residues) / (length of alignment)] × 100 The sequence identity between two amino acid sequences can be determined by any method known to those skilled in the art, such as using the BLAST (Basic Local Alignment Search Tool) program (J. Mol. Biol. 215:403-410, 1990).

[0049] The recombinant collagen-like peptide may have an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 1, and which has bioaffinity.

[0050] In the "amino acid sequence in which one or several amino acids are deleted, substituted or added," "one or several" preferably means 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and particularly preferably 1 to 3.

[0051] Recombinant collagen-like peptides can be produced by genetic recombination techniques known to those skilled in the art, for example, by methods described in EP1014176A2, U.S. Patent No. 6,992,172, International Publication No. WO2004 / 85473, International Publication No. WO2008 / 103041, etc. Specifically, a gene encoding the amino acid sequence of a given recombinant collagen-like peptide is obtained, incorporated into an expression vector to prepare a recombinant expression vector, and this is then introduced into a suitable host to produce a transformant. The resulting transformant is cultured in an appropriate medium to produce the recombinant collagen-like peptide, and the recombinant collagen-like peptide used in the present invention can be prepared by recovering the recombinant collagen-like peptide produced from the culture.

[0052] In some embodiments, the gelling agent is present in an amount of about 1% (w / w) or more, for example, about 1% (w / w) to about 10% (w / w), about 2% (w / w) to about 10% (w / w), about 1% (w / w) to about 9% (w / w), about 2% (w / w) to about 9% (w / w), about 1% (w / w) to about 8% (w / w), about 2% (w / w) to about 8% (w / w), about 1% (w / w) to about 7% (w / w), or about 2% (w / w). In some embodiments, the gelling agent may be contained in the preservative solution at a concentration of about 2.5% (w / w). In further embodiments, the gelling agent may be included in the preservative solution at a concentration of 2.5±1.5% (w / w), 2.5±1% (w / w), 2.5±0.5% (w / w).

[0053] In some embodiments, the preservation solution may be a cell culture medium, physiological saline, or an electrolyte solution. In some embodiments, the pH of the preservation solution may be about 5.0 to about 8.0, about 6.0 to about 8.0, about 6.0 to about 7.5, about 6.1 to about 7.4, about 6.2 to about 7.4, about 6.3 to about 7.4, about 6.4 to about 7.4, about 6.4 to about 7.3, about 6.4 to about 7.2, about 6.4 to about 7.1, about 6.4 to about 7.0, about 6.4 to about 6.9, about 6.4 to about 6.8, about 6.4 to about 6.7, about 6.4 to about 6.6, or about 6.4 to about 6.5. In certain embodiments, the pH of the preservation solution may be about 6.4 to about 7.4.

[0054] In some embodiments, the preservation solution may be a cell culture medium. The preservation solution may or may not contain growth factors. In the disclosed method, cells are preserved at low temperatures, so cell proliferation does not occur, regardless of the presence or absence of growth factors. Preservation solutions include, but are not limited to, MEMα, physiological saline, PBS, DMEM, CELSIOR® COLD STORAGE SOLUTION (WATERS MEDICAL SYSTEMS), ETK® Organ and Tissue Preservation Solution (ETK Solution) (Otsuka Pharmaceutical Co., Ltd.), and Belzer UW® Cold Preservation Solution (UW Solution) (Asteral Pharmaceutical Co., Ltd.).

[0055] In some embodiments, the gel maintenance temperature can be about 0°C to about 37°C, about 0°C to about 30°C, about 0°C to about 24°C, about 0°C to about 20°C, about 0°C to about 15°C, or about 0°C to about 10°C. In further embodiments, the gel maintenance temperature can be about 4°C. In certain embodiments, the gel maintenance temperature can be 4±2.0°C, 4±1.5°C, 4±1.0°C, or 4±0.5°C.

[0056] In some embodiments, the gelling temperature and the gel-maintenance temperature may be the same or different.

[0057] In some embodiments, the stored cells may be mesenchymal stem cells of any origin (e.g., from bone marrow, adipose tissue, placental tissue, synovial tissue, umbilical cord tissue (e.g., umbilical cord blood), dental pulp, or amniotic membrane), or mesenchymal stem cells differentiated from ES cells or iPS cells. In certain embodiments, the mesenchymal stem cells may be bone marrow-derived mesenchymal stem cells. In further embodiments, the stem cells may be cells used in cell infusion therapy and drug discovery research. In further embodiments, the stem cells may be cells obtained from a subject undergoing cell infusion therapy.

[0058] The preservation method of the present disclosure may further include a step of subjecting the preservation solution to a non-gelling treatment. The non-gelling treatment step includes raising the temperature of the preservation solution to a temperature at or above the melting point of the gelling agent. The melting point of the gelling agent may vary depending on the concentration, pH, etc. of the gelling agent, but may be 37°C or lower. The temperature during the non-gelling treatment is at or above the melting point of the gelling agent and can be determined appropriately. In certain embodiments, the gelling agent is a recombinant collagen-like peptide, and the temperature of the preservation solution during the non-gelling treatment step may be about 10°C to about 30°C, about 15°C to 25°C, or about 20°C.

[0059] The preservation method of the present disclosure may further include a step of recovering stem cells. In some embodiments, the step of recovering stem cells may include diluting the post-storage preservation solution 2-20 times, for example, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 11-fold, about 12-fold, about 13-fold, about 14-fold, about 15-fold, about 16-fold, about 17-fold, about 18-fold, about 19-fold, or about 20-fold. In preferred embodiments, the post-storage preservation solution may be diluted by about 5-fold or more, up to about 20-fold. In some embodiments, the step of recovering stem cells may include separating the stem cells from the preservation solution (e.g., by centrifugation). While not wishing to be bound by theory, if the preservation solution is heated to or above its melting point to render it non-gelled, the viscosity of the preservation solution increases, resulting in a low cell recovery rate. Therefore, diluting the preservation solution is preferable; typically, diluting it by 5-fold or more increases the cell recovery rate. The solution for dilution can be a preservative solution that does not contain a gelling agent.

[0060] In some embodiments, the stem cells are about 1×10 5 ~Approx. 1×10 8 / ml, preferably about 5 × 10 5 ~Approx. 5×10 6 / ml, more preferably about 1 × 10 6 ~Approx. 1×10 7 The cells can be stored at a cell density of 1000 cells / ml.

[0061] (Stem cell preservative) In another aspect, the present disclosure provides a composition (e.g., a preservation solution) for preserving stem cells, comprising a gelling agent. The composition (e.g., a preservation solution) of the present disclosure can be used at a pH of about 5.0 to about 8.0 when preserving stem cells. The composition (e.g., a preservation solution) of the present disclosure can be used when preserving stem cells, so that it is stored at the gel-maintaining temperature of the gelling agent. The composition of the present disclosure can have one or more of the features of the above embodiments.

[0062] (Stem Cell Preservation Kit) In a further aspect, the present disclosure provides a kit for preserving stem cells, the kit comprising (1) a preservation solution containing a gelling agent, and (2) a buffer for adjusting the pH of the preservation solution to about 5.0 to about 8.0, wherein the stem cells are preserved at a gel-maintaining temperature. The kit of the present disclosure may have one or more of the features of the above embodiments.

[0063] (Cell preparations) In a further aspect, the present disclosure may provide a formulation comprising a gelling agent and stem cells, and having a pH of about 5.0 to about 8.0. The formulation may be used to preserve stem cells by storing the formulation at a gel-maintaining temperature of the gelling agent. The formulation may be gelled. The formulation of the present disclosure may be a ready-to-use formulation.

[0064] (Tissue or organ preservation) In a further aspect, the present disclosure provides a method for preserving a tissue or organ, the method comprising the steps of: 1) preparing a preservation solution containing a gelling agent in a non-gelled state at a temperature higher than the gelling temperature, the preservation solution having a pH of about 5.0 to about 8.0; 2) placing the tissue or organ in the preservation solution and then lowering the temperature to the gelling temperature of the gelling agent; and 3) storing the preservation solution containing the tissue or organ at the gel-maintaining temperature of the gelling agent.

[0065] In yet another aspect, the present disclosure provides a kit for preserving tissues or organs, the kit comprising (1) a preservation solution containing a gelling agent and (2) a buffer for adjusting the pH of the preservation solution to about 5.0 to about 8.0, and the tissue or organ is preserved at a gel-maintaining temperature. Tissues and organs are aggregates of cells, and since cell preservation can be achieved by the techniques of the present disclosure as described herein, tissues and organs that are aggregates can also be preserved by applying the techniques of the present disclosure under similar conditions. It will be understood that those skilled in the art will be able to adjust the optimal conditions as appropriate, taking into account the characteristics of each tissue and / or organ, in light of the disclosure herein.

[0066] In yet another aspect, the present disclosure provides a composition for preserving tissues or organs, comprising a gelling agent. For tissue or organ preservation, a tissue or organ preservation solution may be used. Examples of tissue or organ preservation solutions include, but are not limited to, ETK® Organ Tissue Preservation Solution (ETK Solution) (Otsuka Pharmaceutical Co., Ltd.), Belzer UW® Cold Preservation Solution (UW Solution) (Asteral Pharmaceutical Co., Ltd.), and Celsior Solution. A gelling agent may be dissolved in the preservation solution and used.

[0067] In some embodiments, the ETK solution may contain sodium gluconate, potassium dihydrogen phosphate, dipotassium phosphate, hydroxyethyl starch, trehalose hydrate, and potassium hydroxide. In a specific embodiment, the ETK solution may contain 21.814 g of sodium gluconate, 0.885 g of potassium dihydrogen phosphate, 3.222 g of dipotassium phosphate, 30.0 g of hydroxyethyl starch, 45.3 g of trehalose hydrate, and an appropriate amount of potassium hydroxide per 1000 mL.

[0068] In some embodiments, the UW solution comprises pentafraction, lactobionic acid (lactobionic acid), ton), potassium dihydrogen phosphate, magnesium sulfate heptahydrate, raffinose pentahydrate In a specific embodiment, the UW solution may contain pentafraction 50 g / L, lactobionic acid (as lactone) 35.83 g / L, potassium dihydrogen phosphate 3.4 g / L, magnesium sulfate heptahydrate 1.23 g / L, raffinose pentahydrate 17.83 g / L, adenosine 1.34 g / L, allopurinol 0.136 g / L, total glutathione 0.922 g / L, potassium hydroxide 5.61 g / L, sodium hydroxide / hydrochloric acid (to adjust pH to 7.4), and an appropriate amount of water for injection.

[0069] In some embodiments, the Celsior solution may contain mannitol, lactobionic acid, glutamic acid, histidine, calcium chloride, potassium chloride, magnesium chloride, sodium hydroxide, reduced glutathione, and water for injection. In certain embodiments, the Celsior solution may contain mannitol 10.930 g / L, lactobionic acid 28.664 g / L, glutamic acid 2.942 g / L, histidine 4.650 g / L, calcium chloride 0.037 g / L, potassium chloride 1.118 g / L, magnesium chloride 2.642 g / L, sodium hydroxide 4.000 g / L, reduced glutathione 0.921 g / L, and an appropriate amount of water for injection.

[0070] One or more of the embodiments described above with respect to cell preservation may be employed as embodiments in the tissue or organ preservation aspect, where appropriate.

[0071] (Transportation method) In a further aspect, the present disclosure provides a method for transporting stem cells, the method comprising the steps of: 1) preparing a preservation solution containing a gelling agent in a non-gelled state at a temperature higher than the gelling temperature, the pH of which is about 5.0 to about 8.0; 2) placing the stem cells in the preservation solution and then lowering the temperature to the gelling temperature of the gelling agent; and 3) transporting the preservation solution containing the stem cells at the gel-maintaining temperature of the gelling agent. This method allows for transport without freezing, while preserving cell viability and capable of withstanding transport conditions such as vibration. Transport may include land transport, sea transport, air transport, or a combination thereof.

[0072] (cell) In a further aspect, the present disclosure provides a composition for treating or preventing a disease, disorder, or condition in a subject, comprising stem cells preserved or delivered according to the above-described method. In some embodiments, the condition may be treatable with stem cell-based regenerative medicine, such as, but not limited to, severe burns, spinal cord injury, head trauma, cerebral infarction, cerebral hemorrhage, and Parkinson's disease.

[0073] (syringe formulation) In a further aspect, the present disclosure provides a syringe preparation that can be administered to a subject without the need for further processing after storing or transporting the stem cells by the above-described method, comprising a suspension of stem cells and a syringe.

[0074] (Treatment method) In a further aspect, the present disclosure provides a method of treating or preventing a disease, disorder, or condition in a subject, the method comprising storing or transporting stem cells according to the above method, and administering the stored or transported stem cells to the subject in need thereof.

[0075] (use) In a further aspect, the present disclosure provides the use of stem cells preserved or transported according to the above method in the manufacture of a medicament (e.g., the above syringe formulation) for treating or preventing a disease, disorder, or condition in a subject.

[0076] The present disclosure has been described above by showing preferred embodiments for ease of understanding. The present disclosure will be described below based on examples. However, the above description and the following examples are provided for illustrative purposes only and are not intended to limit the present disclosure. Therefore, the scope of the present disclosure is not limited to the embodiments or examples specifically described herein, but is limited only by the scope of the claims. [Example]

[0077] The present disclosure will be described more specifically below based on examples.

[0078] (Measurement method) In the following examples, the cell count was measured twice, once after storage and once after reseeding. The cell viability after storage was calculated by the formula "cell viability after storage = cell number after storage ÷ cell number before storage". The cell viability after reseeding was calculated by seeding the pre-storage cells into a flask after storage, adding a culture medium (MEMα + PL (platelet lysate)), and culturing for 72 hours, and then calculating the formula "cell viability after reseeding = cell number after reseeding ÷ cell number before storage".

[0079] Cell counts were measured using a Luna Automated Cell counter (https: / / logosbio.com / automated-cell-counters / brightfield / luna) to fluorescently count live and dead cells.

[0080] (Example 1: Even after 72 hours of shaking at 4°C in the presence of RCP protein, cells remain viable at or above the level of frozen storage) (material and method) ·material The following commercially available reagents or cells were used:

[0081] RCP (recombinant collagen-like peptide): Reagent manufactured by Fujifilm Corporation (https: / / www.fujifilm.com / jp / ja / business / materials / rm / rcp) Human bone marrow stem cells, PL (platelet lysate): collected from healthy volunteers at Hokkaido University, approved by the ethics committee, and consent forms obtained MEMα: Catalog number: Invitrogen, 32571-036, USA MEMα PL solution: PL is made in-house from platelet solution collected from healthy volunteers Cryopreservation solution Bambanker (registered trademark) Catalog number: CS-02-001 (Nippon Genetics) ·method Five hundred thousand human bone marrow stem cells isolated and cultured from bone marrow fluid obtained from healthy volunteers were (1) diluted in MEMα and adjusted to approximately pH 7.0, (2) dissolved in MEMα PL solution (final concentration 2.5%) and adjusted to approximately pH 7.0, and (3) diluted in Bambanker® (Nippon Genetics) cryopreservation solution. (1) and (2) were then stored in a 4°C refrigerator with shaking for 72 hours using a vortex machine (Heathrow Scientific HS120318, 3000 rpm). (3) was stored stationary in a laboratory freezer at -80°C.

[0082] (result) The results are shown in Figure 1. In the absence of RCP, the survival rate of cells exposed to vibration at 4°C was nearly 0%, and no proliferation was observed even after reseeding. On the other hand, cells in the preservation solution gelled with RCP had a survival rate of 95% and were able to proliferate by nearly 1.6 times after reseeding, which was equivalent to the results of cryopreservation.

[0083] (Example 2: Examination of RCP concentration in preservative solution) (material and method) Five hundred thousand human bone marrow stem cells isolated and cultured from bone marrow fluid obtained from healthy volunteers were dissolved in RCP solutions at concentrations of 0, 0.5, 1.0, 2.5, 5.0, and 10.0% and adjusted to a pH of approximately 7.0. The solutions were stored in a 4°C refrigerator with shaking in a vortex machine (Heathrow Scientific HS120318, 3000 rpm) for 72 hours. After 72 hours, the cells were liquefied at 20°C and the cell count was determined. Liquefaction was performed as follows.

[0084] The gel was returned to liquid by placing it in an incubator at 20°C. It was then diluted by adding five times the volume of medium (MEMα) and centrifuged to pellet the cells, which were collected at the bottom of the centrifuge tube. The entire supernatant was aspirated to remove the RCP component of the gel, and a small amount (1 ml) of medium (MEMα) was added and stirred before counting the number of cells. If the gel was left to liquefy at 20°C and then centrifuged to pellet the cells, some cells would remain in the supernatant, resulting in a poor recovery rate.

[0085] (result) The results are shown in Figure 2. The survival rate after storage increased depending on the RCP concentration, but the survival rate after reseeding was highest at 2.5%.

[0086] (Example 3: Study of media for dissolving RCP) (material and method) To preserve 500,000 human bone marrow stem cells isolated and cultured from bone marrow aspirates obtained from healthy volunteers, a 2.5% RCP solution was diluted with MEMαPL, normal saline, Belzer UW cold storage solution (organ preservation solution, Astellas Pharma), Hypothermosol-FRS (low-temperature cell preservation reagent, Sigma-Aldrich), phosphate-buffered saline (PBS; commercially available), or 5% glucose solution (commercially available). The pH was adjusted to approximately 7.0 and the cells were stored in a 4°C refrigerator with shaking in a vortex machine for 72 hours. After 72 hours, the cells were liquefied at 20°C and counted.

[0087] (result) The results are shown in Figure 3. Neither solvent appears to have a negative effect on viability after storage. PBS and glucose resulted in low viability after seeding. Therefore, depending on the type of preservation solution, it may be advisable not to reseed after storage. In any case, a high viability was maintained regardless of the preservation solution used, so reseeding is not necessary as long as the desired cell number is achieved.

[0088] (Example 4: Examination of storage temperature) (material and method) Five hundred thousand human bone marrow stem cells isolated and cultured from bone marrow aspirates obtained from healthy volunteers were stored in MEMαPL (pH 7.0) containing 2.5% RCP at -20°C, 4°C, 24°C, or 37°C for 72 hours. After 72 hours, the cells were liquefied at 20°C and the cell count was determined. All cells were then seeded into flasks and cultured in MEMαPL for 72 hours to confirm their proliferation potential.

[0089] (result) The results are shown in Figure 4. At 4°C, cell viability remained above 100% after 72 hours of storage and 72 hours after reseeding. At -20°C, cell viability was approximately 70% after 72 hours of storage, but almost no cells survived 72 hours after reseeding. This suggests that although the cells were viable immediately after storage, they were damaged in some way. This suggests that storage without freezing is preferable in the storage method disclosed herein. Furthermore, when stored at 24°C, cell viability after 72 hours of storage was low at approximately 20%, but cell viability after reseeding was approximately 100%. At 37°C, cell viability after 72 hours of storage and 72 hours after reseeding was below 50%. From the above, it was found that the storage temperature should be a temperature that does not freeze (e.g., 0°C or higher) and is preferably below 37°C (e.g., 24°C or lower), with 4°C being optimal.

[0090] (Example 5: Examination of storage period) (material and method) 500,000 human bone marrow stem cells isolated and cultured from bone marrow aspirates obtained from healthy volunteers were stored in MEMαPL (pH 7.0) containing 2.5% RCP for 1, 3, 5, 7, or 14 days at 4°C with shaking. After the specified number of days, the cells were liquefied at 20°C and counted.

[0091] (result) The results are shown in Figure 5. The survival rate after storage was roughly the same for all days, but the survival rate after reseeding was particularly good from 1 to 3 days, and then decreased with time, but was still high by 14 days. However, the survival rate remains around 80%.

[0092] (Example 6: Examination of types of gelling agents) (material and method) To preserve 500,000 human bone marrow stem cells isolated and cultured from bone marrow fluid obtained from healthy volunteers, 2.5%, 5%, and 10% B-Matrix Gelatin (Nitta Gelatin, LS-250), Cook Gelatin (Morinaga & Co., Ltd.), Jellice Gelatin Powder (Maruha Nichiro), and RCP were dissolved in MEMαPL and adjusted to approximately pH 7.0. B-Matrix and RCP were stored at 4°C with shaking, while Cook Gelatin and Jellice Gelatin Powder were stored stationary, after which the cell count was measured.

[0093] (result) The results are shown in Figure 6. Cell preservation was possible with all gelatins: B-Matrix Gelatin, Cook Gelatin, and Gellice. Therefore, it is important that the preservation solution gels.

[0094] (Example 7: Study of pH during storage) (material and method) To preserve 500,000 human bone marrow stem cells isolated and cultured from bone marrow fluid obtained from healthy volunteers, a preservation solution was used, consisting of MEMαPL with 2.5% RCP, Cook's gelatin, and rice gelatin powder. The pH was adjusted to 4.4-8.4, and the cells were stored at 4°C with or without shaking, after which the cell count was measured.

[0095] (result) The results are shown in Figure 7. Almost no cells survived without pH adjustment (approximately 9). Furthermore, high survival rates were observed at pH 5.4 to 8.4, but almost no cells survived at pH 4.4. After seeding, the survival rates were high at pH 6.4 and 7.4.

[0096] Example 8: Tissue and organ preservation (material and method) ·material Tissue and organ preservation solution ETK (registered trademark) (ETK solution): Otsuka Pharmaceutical Co., Ltd., or Belzer UW (registered trademark) Cold Preservation Solution (UW Solution): Asteral Pharmaceutical Co., Ltd. RCP (recombinant collagen-like peptide): Reagent manufactured by Fujifilm Corporation (https: / / www.fujifilm.com / jp / ja / business / materials / rm / rcp) ·method Dissolve RCP (final concentration 2-10%) in ETK or UW solution and adjust the pH to around 7. Place tissue (e.g., corneal tissue, skin tissue, etc.) or organ (e.g., lung, kidney, liver, pancreas, etc.) in a container containing ETK or UW solution, seal aseptically, and cool the container containing the tissue or organ.

[0097] Example 9: Cell Delivery In this example, the animals were actually transported by airplane, and the survival rate after transportation was measured.

[0098] (material and method) ·material MEMα+2.5%RCP 50ml Falcon tube or 100ml sterile bottle bone marrow stem cells Standard temperature-controlled transport package TACPack0208F (Tamai Kasei Co., Ltd.)

[0099] ·method RCP 2.5% (final concentration) was dissolved in MEMα, adjusted to pH 6.05, and used as a storage solution. 6 The cells were placed in a container (50 ml Falcon tube) containing a storage solution at a concentration of 1 / ml, and the inside was kept under 5% CO2 (20% O2, 75% N2) atmosphere. After replacing the air, the container was aseptically sealed and cooled to 4°C. The container was placed horizontally in a standard constant temperature transport package TACPack0208F (Tamai Kasei Co., Ltd.) and transported while maintaining the temperature at 4°C. The cells were shipped from Sapporo and arrived in Wakayama two days later. After being stored at 4°C for one day, the cells were shipped again from Wakayama three days later and arrived in Sapporo five days later. This transport involved land and air transport. As a control, cells were stored stationary.

[0100] (result) The results are shown in Figure 8. As shown, the transported cells exhibited cell viability equivalent to that of cells preserved stationary. Furthermore, it was confirmed that the preserved cells differentiated normally into fat, bone, and cartilage. Therefore, the preservation solution of the present invention enables the transportation of cells without freezing, while maintaining cell viability and allowing them to withstand transportation conditions such as vibration.

[0101] Example 10: Change in viability due to dilution of preservative solution upon cell recovery In this example, it was confirmed whether the cell viability would change if the preservation solution was diluted when the cells were collected.

[0102] (material and method) ·material MEMα+2.5%RCP 50ml Falcon tube or Froze Bag® (Nipro) Human bone marrow stem cells

[0103] ·method RCP 2.5% (final concentration) was dissolved in MEMα, adjusted to pH 7.4-8.0, and used as a storage solution. 6 The cells were placed in a container (50 ml Falcon tube or Froze Bag (registered trademark)) containing a storage solution at a concentration of cells / ml, aseptically sealed, and the container was cooled to 4°C and stored at 4°C for 72 hours. Thawing was carried out at 37°C for 10 minutes to 3 hours.

[0104] After storage, the cells were diluted with MEMα (1x or 5x dilution), centrifuged to pellet the cells, and collected at the bottom of the centrifuge tube. The supernatant was aspirated to remove the RCP, a component of the gel. A small amount (1 ml) of medium (MEMα) was added and the cells were counted while stirring.

[0105] (result) The results are shown in Figure 9. As shown, diluting the preservative solution 5-fold when recovering the cells increased cell viability compared to diluting it 1-fold. While not wishing to be bound by theory, the increase in cell viability with a 5-fold dilution is thought to be due to the viscosity of the gelling agent. Even with centrifugation, it is thought that a highly viscous liquid will not be able to adequately precipitate the cells, resulting in a lower recovery rate. Diluting the solution 5-fold or more reduces the viscosity, making it possible to precipitate and recover almost all cells using the standard centrifugation method (1000G).

[0106] While the present disclosure has been illustrated using preferred embodiments thereof, it is understood that the scope of the present disclosure is to be construed solely in terms of the claims. It is understood that the contents thereof are to be incorporated by reference into this specification as if written.

[0107] This application claims the benefit of priority from Japanese Patent Application No. 2021-126706, filed on August 2, 2021, the entire contents of which are incorporated herein by reference. [Industrial Applicability]

[0108] The present invention provides a preservation method that can maintain cells with a high viability. The preserved cells can be used in cell injection therapy, and therefore can be used in fields such as pharmaceuticals. [Sequence List Free Text]

[0109] SEQ ID NO: 1: Amino acid sequence of recombinant collagen-like peptide (RCP) SEQ ID NO: 2: Cell adhesion signal sequence (REDV) SEQ ID NO: 3: Cell adhesion signal sequence (YIGSR) SEQ ID NO: 4: Cell adhesion signal sequence (PDSGR) SEQ ID NO: 5: Cell adhesion signal sequence (RYVVLPR) SEQ ID NO: 6: Cell adhesion signal sequence (LGTIPG) SEQ ID NO: 7: Cell adhesion signal sequence (RNIAEIIKDI) SEQ ID NO: 8: Cell adhesion signal sequence (IKVAV) SEQ ID NO: 9: Cell adhesion signal sequence (DGEA)

Claims

[Claim 1] The invention described in the specification.

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