Composition for preserving biomaterials

Abstract

The purpose of the present invention is to provide a biomaterial preserving composition capable of solving problems related to conventional biomaterial preserving liquids regarding not being sufficient for the survival or functional retention of cells in a biomaterial, and having many restrictions in preservation conditions such as the preservation temperature and the preservation treatment, resulting in a bottleneck for widespread use of cell therapy. Said purpose is achieved by having a thermo-reversible polymer included in a preserving composition.

Description

[Technical Field]

[0001] The present invention relates to a composition for preserving biomaterials, comprising a thermoreversible polymer. [Background technology]

[0002] In recent years, cell therapy has begun to be used to repair damaged tissues, etc. Cell therapy requires that a certain number of viable cells be maintained in biomaterials such as cells, tissues, or cell sheets, and that the biomaterials perform the desired function at the recipient site. Therefore, after biomaterials are collected, processed, etc., they must be preserved until transplantation, etc. Known preservation methods for such biomaterials include immersing the biomaterial in a preservation solution at low temperatures, continuously perfusing the biomaterial with a preservation solution, preserving the biomaterial in high-pressure gas, and preserving the biomaterial in an aerosol. However, none of these biomaterial preservation methods are sufficient to maintain the survival or function of cells in the biomaterial, and they also have many restrictions on preservation conditions such as storage temperature and preservation treatment, which have become a bottleneck in the widespread use of cell therapy (Patent Documents 1 and 2, Non-Patent Document 1). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Publication No. 2018-016654 [Patent Document 2] International Publication No. 2010 / 049996 [Non-patent literature]

[0004] [Non-Patent Document 1] Jamart et al., “Efficiency and limitation of Euro-Collins solution in kidney preservation”, J Surg Res. 1983 Mar;34(3):195-204 [Non-patent document 2] H. Yoshioka et al., “A Synthetic Hydrogel with Thermoreversible Gelation. I. Preparation and Rheological Properties”, Journal of Macromolecular Science, A31(1), 113-120 (1994) [Non-patent document 3] KRHolme.et al., “Chitosan derivatives bearing C10-alkyl glycoside branches: a temperature-induced gelling polysaccharide”, Macromolecules, 24, 3828-3833(1991) Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention aims to provide a composition for preserving biological materials. [Means for solving the problem]

[0006] While conducting intensive research to solve the above problems, the inventors discovered that the survival and / or function of cells in a biomaterial can be maintained by preserving the biomaterial with a thermoreversible polymer, and thus completed the present invention.

[0007] That is, the present invention relates to the following composition for preserving biological materials: (1) A composition for preserving a biomaterial, comprising a thermoreversible polymer. (2) A preservation composition according to (1) above, which is intended for storage at a constant or fluctuating temperature. (3) The preservation composition according to (2) above, wherein the constant or fluctuating temperature is a temperature at which cells do not substantially grow. (4) The preservation composition according to (3) above, wherein the temperature at which cells do not substantially grow is 4 to 30°C. (5) The preservation composition according to any one of (1) to (4), wherein the biological material is selected from the group consisting of cartilage tissue, oral mucosal tissue, corneal tissue, limbal tissue, dental pulp tissue, vascular tissue, gastrointestinal mucosal tissue, omentum tissue, skin tissue, and liver tissue. (6) The preservation composition according to any one of (1) to (4), wherein the biological material is a somatic cell, progenitor cell, or stem cell contained in a tissue selected from the group consisting of cartilage tissue, oral mucosal tissue, corneal tissue, limbal tissue, dental pulp tissue, vascular tissue, gastrointestinal mucosal tissue, omentum tissue, skin tissue, and liver tissue. (7) A preservative composition according to any one of (1) to (6), wherein the thermoreversible polymer is selected from the group consisting of polypropylene oxide, copolymers of propylene oxide and other alkylene oxides, poly N-substituted acrylamide derivatives, poly N-substituted methacrylamide derivatives, copolymers of N-substituted acrylamide derivatives and N-substituted methacrylamide derivatives, polyvinyl methyl ether, and partial acetylated polyvinyl alcohol, and is composed of multiple blocks having a cloud point and a hydrophilic block bonded together. (8) The preservative composition according to any one of (1) to (7) above, which can also be used for transportation. [Effects of the Invention]

[0008] The preservation composition of the present invention can maintain the viability and / or function of cells contained in biomaterials compared to conventional preservation solutions. Furthermore, since preservation does not necessarily require constant low-temperature conditions, biomaterials can be preserved over a wide range of temperature conditions. Furthermore, preservation using the preservative of the present invention requires minimal effort and cost, making it widely applicable to cell therapy, organ transplantation, and the like. [Brief explanation of the drawings]

[0009] [Figure 1] Figure 1 shows samples A and B for transporting cartilage tissue, which were prepared with a thermoreversible polymer (sample A) and PBS (sample B), respectively. [Figure 2]FIG. 2 shows the expression levels of COL2a1 expressed during culture after transport using a thermoreversible polymer (sample A) and ECS (sample B). [Figure 3] Figure 3 shows the proliferation of buccal mucosa cultured in planar culture (DMEM and Cnt-PR) and thermoreversible polymer (TGP) after transport with a thermoreversible polymer (Sample A) and ECS (Sample B). [Figure 4] FIG. 4 shows endothelial cells in the cornea when the cornea was transported with a thermoreversible polymer (Sample A) and an MK solution (Sample B), respectively. [Figure 5] FIG. 5 shows endothelial cells in the cornea when the cornea was transported with a thermoreversible polymer (Sample A) and Optisol GS solution (Sample B), respectively. [Figure 6] FIG. 6 shows the proliferation of corneal limbuses cultured after transport in a thermoreversible polymer (Sample A) and MK solution (Sample B).

[0010] [Figure 7] FIG. 7 shows the proliferation of intestinal tissue cultured after transportation in a thermoreversible polymer (Sample A) and DMEM (Sample B). [Figure 8] FIG. 8 shows the proliferation of vascular tissue cultured after transport with a thermoreversible polymer (Sample A) and M199 (Sample B). [Figure 9] FIG. 9 shows the proliferation of dental pulp tissue cultured after transportation in a thermoreversible polymer (Sample A) and DMEM (Sample B). [Figure 10] FIG. 10 shows HE staining images of dental pulp tissue cultured after transport in a thermoreversible polymer. [Figure 11] FIG. 11 shows the proliferation of skin tissue cultured after transportation in a thermoreversible polymer (Sample A) and HBSS (Sample B). [Figure 12] FIG. 12 shows the proliferation of liver tissue cultured after transportation in a thermoreversible polymer (Sample A) and DMEM (Sample B). DETAILED DESCRIPTION OF THE INVENTION

[0011] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All patents, applications, published applications and other publications referenced herein are incorporated herein by reference in their entirety.

[0012] The composition for preserving biomaterials of the present invention is characterized by containing a thermoreversible polymer.

[0013] (Biomaterials) In the present invention, the term "biomaterial" refers to cells or cell populations, cell cultures, structures, tissues, organs, etc. containing the same. The cells are preferably cells derived from a living organism, more preferably primary cells obtained from an individual organism. The cells may be primary cells grown by culturing for one or more generations. The cells may also be cells grown by culturing for multiple generations but whose growth has stopped due to certain biological or physical conditions. Cells include, but are not limited to, somatic cells (e.g., a "cell" may be a primary cell or an established cell line. Cells may be derived from, but are not limited to, cells from the central or peripheral nervous system, such as liver (e.g., hepatocytes, sinusoidal endothelial cells), pancreas (e.g., pancreatic islet β cells), lung, brain (e.g., nerve, glial, or ependymal cells) or spinal cord, kidney, eye (e.g., retinal cells, corneal endothelial cells), spleen, skin, thymus, testis, lung, diaphragm, heart (cardiac cells), muscle or psoas muscle, or intestine (e.g., endocrine cells), adipose tissue (white, brown, or beige adipocytes), muscle (e.g., fibroblasts), synovial cells, or cells from other tissues. These include cells such as follicles, chondrocytes, osteoclasts, epithelial cells, endothelial cells, salivary gland cells, inner ear neurons, or hematopoietic cells (e.g., blood cells or lymphocytes), or their precursor cells and stem cells (tissue stem cells such as epithelial stem cells, satellite cells, intestinal stem cells, endothelial stem cells, olfactory mucosa stem cells, hair follicle stem cells, mammary gland stem cells, neural stem cells, hematopoietic stem cells, cardiac stem cells, mesenchymal stem cells, embryonic stem cells, oocytes, blastomeres, inner cell mass cells, embryonic germ cells, embryoid body cells, morula-derived cells, teratoma (teratocarcinoma) cells, and pluripotent cells such as pluripotent partially differentiated embryonic stem cells derived from later stages of embryonic development, and iPS (induced pluripotent stem) cells), as well as sperm, eggs, fertilized eggs, and embryos.

[0014] The cells also include cell populations isolated from tissues or organs collected from a living body. Cells that can be used in the form of a cell population include, but are not limited to, cell populations derived from epithelial tissues (e.g., oral mucosal epithelial cells, epithelial stem cells, etc.), cell populations derived from adipose tissues (adipocytes, mesenchymal stem cells, etc.), cell populations derived from cartilage tissues (synovial cells, chondrocytes, chondroprogenitor cells, mesenchymal stem cells), as well as cell populations obtained by culturing cultured cells. Furthermore, "biomaterials" may also include cell cultures or cell structures. Examples of cell cultures or cell structures include, but are not limited to, cell-polymer mixtures, cell sheets, cell aggregates, etc., and examples of cell structures include, but are not limited to, cell-polymer structures, structures combining cell sheets and tissues, etc.

[0015] The term "biomaterial" is not limited as long as it contains cells, and may be biological fluids including blood, bone marrow fluid, lymphatic fluid, etc., thymus tissue, thyroid tissue, skeletal muscle tissue, tracheal tissue, vascular tissue, lung tissue, liver tissue, gallbladder tissue, kidney tissue, ureteral tissue, appendix tissue, bladder tissue, urethral tissue, testicular tissue, uterine tissue, ovarian tissue, digestive tissue (such as stomach tissue, small intestine tissue, or large intestine tissue), heart tissue, esophageal tissue, diaphragm tissue, spleen tissue, pancreatic tissue, brain tissue (such as cerebral tissue and cerebellar tissue), spinal cord tissue, cartilage tissue, peripheral limb tissue, retinal tissue, skin tissue, oral mucosal tissue, corneal tissue, limbal tissue, dental pulp tissue, vascular tissue, digestive tract tissue, omentum tissue, skin tissue, liver tissue, amniotic membrane, and other tissues.

[0016] The "biomaterial" may also be an organ, including a salivary gland, palate, uvula, scrotum, teeth, pharynx, larynx, esophagus, liver, gallbladder, common bile duct, stomach, pancreas, pancreatic duct, small intestine (duodenum, jejunum, ileum), large intestine (transverse colon, ascending colon, cecum, descending colon, cecum, sigmoid colon, rectum), appendix, anus, heart, blood vessels, lymphatic vessels, lymph nodes, spleen, skin, thymus, nasal cavity, trachea, bronchi, lung, thorax, kidney, ureter, bladder, urethra, testis, uterus, ovaries, fallopian tubes, vas deferens, penis, vagina, eyeball, ear, brain, spinal cord, nerve fiber bundle, bone, cartilage, skeletal muscle, visceral muscle, tendon, and ligament. The biomaterial may be derived from any organism, including, but not limited to, humans, non-human primates, dogs, cats, pigs, horses, goats, sheep, rodents (e.g., mice, rats, hamsters, guinea pigs, etc.), rabbits, etc.

[0017] In the present invention, "preservation" refers to maintaining the viability and / or function of cells contained in a biomaterial to be preserved (hereinafter, the "biological material to be preserved" is referred to as the "target biomaterial"). The maintenance of cell viability can be confirmed by methods commonly used in the art for measuring the degree of cell viability, such as, but not limited to, measuring the number of viable cells contained in the target biomaterial or the respiratory activity of viable cells. It is preferable that the number of viable cells or respiratory activity contained in the target biomaterial be maintained by, but not limited to, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.1%, or 0.05% before and after preservation using the preservation composition of the present invention. The maintenance of function can be confirmed by methods commonly used in the art for measuring the function of biomaterials, such as, but not limited to, measuring the proliferation ability of cells contained in the target biomaterial, the maintenance of biomaterial morphology, the ability to secrete a specific component, or the expression level of a specific protein or gene. It is preferable that the same quality of function as that of the target biomaterial be maintained before and after preservation using the preservation composition of the present invention.

[0018] For example, if the target biomaterial is cartilage tissue immediately after collection, this may be confirmed by, but is not limited to, maintaining the expression of cartilage marker proteins, such as SOX9, COL2A1, COL9A1, COL9A2, COL9A3, COL11A1, COL11A2, ACAN, HAPLN1, COMP, or MATN3, or the genes encoding these proteins. Therefore, in the present invention, the term "preservation composition" refers to a composition used for preservation, i.e., a composition that has the above-described preservation effect when a biomaterial is immersed in the preservation composition. The term "preservation composition" encompasses both compositions that contain the target biomaterial and those that do not. As long as the survival and / or function of cells contained in the target biomaterial are maintained, preservation is not limited to static storage and includes any mode that may involve vibration, such as transportation. Therefore, in one aspect, the preservation composition of the present invention is a transportation composition.

[0019] (thermoreversible polymer) The thermoreversible polymer contained in the preservation composition of the present invention (Thermoreversible Gelation Polymer: also referred to as "TGP" herein) is a polymer that can thermoreversibly generate a crosslinked structure or network structure and, based on this structure, can thermoreversibly form a hydrogel that retains a separation liquid such as water inside it. The preservation composition of the present invention has the properties of this polymer. A hydrogel refers to a gel composed of a crosslinked or network structure made of a polymer and water supported or retained in the structure.

[0020] (Sol-Gel Transition Temperature) In the present invention, the definitions and measurements of the "sol state", "gel state", and "sol-gel transition temperature" are based on the definitions and methods described in Non-Patent Document 2 (H. Yoshioka et al., "A Synthetic Hydrogel with Thermoreversible Gelation. I. Preparation and Rheological Properties", Journal of Macromolecular Science, A31(1), 113-120 (1994)). That is, the dynamic elastic modulus of the sample at an observation frequency of 1 Hz is measured by gradually changing the temperature from the low temperature side to the high temperature side (1°C / 1 minute), and the temperature at which the storage elastic modulus (G', elastic term) of the sample exceeds the loss elastic modulus (G", viscous term) is defined as the sol-gel transition temperature. Generally, the state where G">G' is defined as the sol, and the state where G"<G' is defined as the gel. When measuring this sol-gel transition temperature, the following measurement conditions can be preferably used.

[0021] <Measurement Conditions for Dynamic and Loss Elastic Modulus> Measuring instrument (product name): Stress-controlled rheometer AR500, manufactured by TA Instruments Concentration of the sample solution (or separation liquid) (however, as the concentration of the "hydrogel-forming polymer having a sol-gel transition temperature"): 10 (wt)% Amount of the sample solution: approximately 0.8 g Shape and dimensions of the measuring cell: Acrylic parallel disk (diameter 4.0 cm), gap 600 μm Measurement frequency: 1Hz Applied stress: Within the linear region.

[0022] In the present invention, the sol-gel transition temperature of the thermoreversible polymer is preferably greater than 0°C and less than 37°C, and more preferably greater than 5°C and less than 35°C (particularly, greater than 10°C and less than 33°C). Because the preservation composition of the present invention can preserve the target biomaterial in either a sol or gel state, the sol-gel transition temperature is not limited and may be 10-35°C or 15-30°C. From the viewpoint of preventing agitation of the preservation medium due to vibration during storage or transportation by gelation and suppressing stress due to agitation, a sol-gel transition temperature of 17-25°C, at which gelation occurs at normal room temperature, is preferred, more preferably 19-23°C, and particularly preferably 19-21°C. TGPs having such suitable sol-gel transition temperatures can be easily selected from specific compounds described below using the screening method (sol-gel transition temperature measurement method) described above.

[0023] The TGP of the present invention is not particularly limited as long as it exhibits the above-mentioned thermoreversible sol-gel transition (i.e., has a sol-gel transition temperature). Specific examples of polymers whose aqueous solutions have a sol-gel transition temperature and reversibly exhibit a sol state at temperatures lower than the transition temperature include polyalkylene oxide block copolymers, such as block copolymers of polypropylene oxide and polyethylene oxide; etherified celluloses such as methyl cellulose and hydroxypropyl cellulose; and chitosan derivatives (KR Holme et al., "Chitosan derivatives bearing C10-alkyl glycoside branches: a temperature-induced gelling polysaccharide," Macromolecules, 24, 3828-3833 (1991) (Non-Patent Document 3)).

[0024] (Suitable Thermoreversible Polymers) The hydrogel-forming polymers that utilize hydrophobic bonds for crosslinking and that can be suitably used as the TGP of the present invention preferably comprise multiple blocks having a cloud point and hydrophilic blocks bonded together. The hydrophilic blocks are preferably present so that the hydrogel becomes water-soluble at temperatures below the sol-gel transition temperature, and the multiple blocks having a cloud point are present so that the hydrogel changes to a gel state at temperatures above the sol-gel transition temperature. In other words, a block having a cloud point dissolves in water at temperatures below the cloud point and becomes insoluble in water at temperatures above the cloud point. Therefore, at temperatures above the cloud point, the block serves as a crosslinking point consisting of hydrophobic bonds for forming a gel. In other words, the cloud point resulting from the hydrophobic bonds corresponds to the sol-gel transition temperature of the hydrogel. However, the cloud point and the sol-gel transition temperature do not necessarily have to coincide. This is because the cloud point of the "block having a cloud point" is generally influenced by the bond between the block and a hydrophilic block. Because of these properties, a target biomaterial can be immersed in the thermoreversible polymer preservation composition at low temperatures. Therefore, from the viewpoint of preventing damage to the target biomaterial, a thermoreversible polymer composed of multiple blocks having a cloud point and a hydrophilic block bonded together is preferred.

[0025] The hydrogel used in the present invention utilizes the property that hydrophobic bonds not only become stronger with increasing temperature, but also that this change is reversible with temperature. From the viewpoint of forming multiple crosslinking points within one molecule, forming a highly stable gel, and thereby improving the shelf life of the target biomaterial, it is preferable for TGP to have multiple "blocks with a cloud point." On the other hand, as described above, the hydrophilic block in the TGP functions to convert the TGP to water-soluble at temperatures below the sol-gel transition temperature, and to form a hydrogel state while preventing the hydrophobic bonding strength from increasing excessively at temperatures above the transition temperature, which would cause the hydrogel to aggregate and precipitate. Furthermore, it is desirable for the TGP used in the present invention to be degraded and absorbed in vivo. That is, it is preferable for the TGP of the present invention to be degraded in vivo by hydrolysis or enzymatic reaction, resulting in a low molecular weight substance that is harmless to the body and is absorbed and excreted. When the TGP of the present invention is composed of multiple blocks having a cloud point and a hydrophilic block bonded together, it is preferable that at least one of the blocks having a cloud point and the hydrophilic block, and preferably both of them, be degraded and absorbed in vivo.

[0026] (Multiple blocks with cloud points) The block having a cloud point is preferably a polymer block exhibiting a negative solubility-temperature coefficient in water, and more specifically, a polymer selected from the group consisting of polypropylene oxide, copolymers of propylene oxide with other alkylene oxide-substituted acrylamide derivatives and N-substituted acrylamides, poly(N-substituted acrylamide derivatives), poly(N-substituted methacrylamide derivatives), copolymers of N-substituted methacrylamide derivatives, poly(vinyl methyl ether), and poly(vinyl alcohol) partially acetylated polymers can be preferably used. From the viewpoint of forming a gel with excellent stability, thereby increasing the preservation of the target biomaterial, copolymers of poly(N-substituted acrylamide derivatives), poly(N-substituted methacrylamide derivatives), and N-substituted methacrylamide derivatives are preferred.

[0027] To make a cloud point block biodegradable and absorbable in vivo, it is effective to use a polypeptide consisting of hydrophobic and hydrophilic amino acids as the cloud point block. Alternatively, a polyester-type biodegradable polymer such as polylactic acid or polyglycolic acid can be used as the cloud point block biodegradable and absorbable in vivo. The cloud point of the polymer (block having a cloud point) is preferably higher than 4° C. and lower than 40° C., since this allows the sol-gel transition temperature of the polymer used in the present invention (a compound in which multiple blocks having a cloud point and a hydrophilic block are bonded) to be higher than 0° C. and lower than 37° C. Here, the cloud point can be measured, for example, by cooling an aqueous solution of about 1% by mass of the polymer (block having a cloud point) to form a transparent, homogeneous solution, and then gradually increasing the temperature (at a rate of about 1° C. / min) until the solution first becomes cloudy.

[0028] Specific examples of poly N-substituted acrylamide derivatives and poly N-substituted methacrylamide derivatives that can be used in the present invention are listed below. Poly-N-acryloylpiperidine; Poly-Nn-propylmethacrylamide; Poly-N-isopropylacrylamide; Poly-N,N-diethylacrylamide; Poly-N-isopropylmethacrylamide; Poly-N-cyclopropylacrylamide; Poly-N-acryloylpyrrolidine; Poly-N,N-ethylmethylacrylamide; Poly-N-cyclopropylmethacrylamide; Poly-N-ethylacrylamide. The above polymers may be homopolymers or copolymers of the monomers constituting the above polymers with other monomers. The other monomers constituting such copolymers may be either hydrophilic or hydrophobic monomers. Generally, copolymerization with a hydrophilic monomer increases the cloud point of the product, whereas copolymerization with a hydrophobic monomer decreases the cloud point of the product. Therefore, by selecting the monomers to be copolymerized, a polymer having a desired cloud point (e.g., a cloud point higher than 4°C and lower than 40°C) can be obtained.

[0029] (hydrophilic monomer) Examples of the hydrophilic monomer include, but are not limited to, N-vinylpyrrolidone, vinylpyridine, acrylamide, methacrylamide, N-methylacrylamide, hydroxyethyl methacrylate, hydroxyethyl acrylate, hydroxymethyl methacrylate, hydroxymethyl acrylate, acrylic acid, methacrylic acid and salts thereof having an acidic group, vinyl sulfonic acid, styrene sulfonic acid, etc., and N,N-dimethylaminoethyl methacrylate, N,N-diethylaminoethyl methcrete, N,N-dimethylaminopropyl acrylamide and salts thereof having a basic group.

[0030] (hydrophobic monomer) On the other hand, examples of the hydrophobic monomer include acrylate derivatives and methacrylate derivatives such as ethyl acrylate, methyl methacrylate, and glycidyl methacrylate, N-substituted alkyl methacrylamide derivatives such as Nn-butyl methacrylamide, vinyl chloride, acrylonitrile, styrene, and vinyl acetate, but are not limited to these.

[0031] (hydrophilic block) On the other hand, specific examples of the hydrophilic block to be bonded to the block having the above-mentioned cloud point include methyl cellulose, dextran, polyethylene oxide, polyvinyl alcohol, poly N-vinylpyrrolidone, polyvinylpyridine, polyacrylamide, polymethacrylamide, poly N-methylacrylamide, polyhydroxymethyl acrylate, polyacrylic acid, polymethacrylic acid, polyvinyl sulfonic acid, polystyrene sulfonic acid, and salts thereof; poly N,N-dimethylaminoethyl methacrylate, poly N,N-diethylaminoethyl methacrylate, poly N,N-dimethylaminopropylacrylamide, and salts thereof. Furthermore, it is desirable that the hydrophilic block be decomposed, metabolized and excreted in the body, and hydrophilic biopolymers such as proteins, such as albumin and gelatin, and polysaccharides, such as hyaluronic acid, heparin, chitin and chitosan, are preferably used.

[0032] The method for bonding the block having a cloud point and the hydrophilic block is not particularly limited. For example, this can be achieved by introducing a polymerizable functional group (e.g., an acryloyl group) into one of the blocks and copolymerizing the monomer that provides the other block. The bonded product of the block having a cloud point and the hydrophilic block can also be obtained by block copolymerization of a monomer that provides the block having a cloud point and a monomer that provides the hydrophilic block. The bond between the block having a cloud point and the hydrophilic block can also be achieved by introducing reactive functional groups (e.g., hydroxyl, amino, carboxyl, isocyanate, etc.) into both blocks in advance and then bonding them by chemical reaction. In this case, multiple reactive functional groups are usually introduced into the hydrophilic block. Furthermore, polypropylene oxide having a cloud point and a hydrophilic block can be bonded, for example, by anionic or cationic polymerization, in which propylene oxide and a monomer (e.g., ethylene oxide) constituting the "other hydrophilic block" are sequentially polymerized repeatedly to obtain a block copolymer in which polypropylene oxide and a "hydrophilic block" (e.g., polyethylene oxide) are bonded. Such a block copolymer can also be obtained by introducing a polymerizable group (e.g., an acryloyl group) into the terminal of polypropylene oxide and then copolymerizing the monomer constituting the hydrophilic block. Furthermore, the polymer used in the present invention can also be obtained by introducing into the hydrophilic block a functional group capable of bonding with the functional group (e.g., hydroxyl group) at the terminal of polypropylene oxide and then reacting the two. The TGP used in the present invention can also be obtained by linking a material such as Pluronic® F-127 (trade name, manufactured by Asahi Denka Kogyo Co., Ltd.), in which polyethylene glycol is bonded to both ends of polypropylene glycol.

[0033] In the embodiment of the polymer of the present invention containing this cloud point block, the "cloud point block" present in the molecule is water-soluble together with the hydrophilic block, so that the polymer completely dissolves in water and exhibits a sol state at temperatures below the cloud point. However, when the temperature of an aqueous solution of this polymer is heated to a temperature above the cloud point, the "cloud point block" present in the molecule becomes hydrophobic and associates with other molecules through hydrophobic interactions. On the other hand, since the hydrophilic block remains water-soluble even at this temperature (when heated to a temperature above the cloud point), the polymer of the present invention forms a hydrogel in water with a three-dimensional network structure in which the hydrophobic associations between the cloud point blocks serve as crosslinking points. When the temperature of this hydrogel is again cooled to a temperature below the cloud point of the "cloud point block" present in the molecule, the cloud point block becomes water-soluble, the crosslinking points due to hydrophobic associations are released, the hydrogel structure disappears, and the TGP of the present invention becomes a completely aqueous solution again. Thus, in a preferred embodiment, the sol-gel transition of the polymer of the present invention is based on the reversible change in hydrophilicity and hydrophobicity of the cloud point-containing block present in the molecule at the cloud point, and is therefore completely reversible in response to temperature changes. In one embodiment of the present invention, such hydrophobic interactions allow association between distinct molecules, preventing the gel from being dissolved by tissue fluids from the target biomaterial, thereby maintaining a constant environment for the target biomaterial. The delicate hydrophilic-hydrophobic balance of TGP in water described above may contribute to the stability of the target biomaterial during storage.

[0034] (Gel solubility) As described above, the hydrogel-forming polymer of the present invention, which contains at least a polymer having a sol-gel transition temperature in an aqueous solution, is substantially water-insoluble at a temperature (d°C) higher than the sol-gel transition temperature, and is reversibly water-soluble at a temperature (e°C) lower than the sol-gel transition temperature. The high temperature (d°C) is preferably at least 1°C higher than the sol-gel transition temperature, and more preferably at least 2°C (particularly at least 5°C) higher. Furthermore, the term "substantially water-insoluble" preferably means that the amount of the polymer that dissolves in 100 ml (liters) of water at the temperature (d°C) is 5.0 g or less (more preferably 0.5 g or less, particularly 0.1 g or less).

[0035] On the other hand, the above-mentioned low temperature (e°C) is preferably a temperature lower than the sol-gel transition temperature by 1°C or more (in absolute terms), and more preferably by 2°C or more (particularly by 5°C or more). Furthermore, the term "water-soluble" means that the amount of the polymer dissolved in 100 ml (liters) of water at the above-mentioned temperature (e°C) is preferably 0.5 g or more (even more preferably 1.0 g or more). Furthermore, the term "reversibly water-soluble" means that an aqueous solution of the TGP exhibits the above-mentioned water-solubility at temperatures lower than the sol-gel transition temperature even after it has been gelled (at a temperature higher than the sol-gel transition temperature).

[0036] The polymer preferably exhibits a viscosity of 10 to 3,000 centipoise (more preferably 50 to 1,000 centipoise) in a 10% aqueous solution at 5° C. Such a viscosity is preferably measured under the following measurement conditions, for example. Viscometer: Stress-controlled rheometer (model: AR500, manufactured by TA Instruments) Rotor diameter: 60mm Rotor shape: parallel plate

[0037] The aqueous solution of TGP of the present invention is gelled at a temperature higher than the sol-gel transition temperature, and the gel does not substantially dissolve even when immersed in a large amount of water. The above-mentioned properties of the hydrogel formed by TGP can be confirmed, for example, as follows. Specifically, 0.15 g of TGP was dissolved in 1.35 g of distilled water at a temperature below the sol-gel transition temperature (e.g., under ice cooling) to prepare a 10 wt% aqueous solution. The solution was poured into a 35 mm diameter plastic dish and heated to 37°C to form a gel approximately 1.5 mm thick. The weight (f grams) of the entire dish containing the gel was then measured. The entire dish containing the gel was then placed in 250 ml (liters) of water at 37°C for 10 hours, after which the weight (g grams) of the entire dish containing the gel was measured to evaluate whether the gel had dissolved from the gel surface. In this case, for the hydrogel-forming polymer of the present invention, the weight loss rate of the gel, i.e., (f g) / f, is preferably 5.0% or less, and more preferably 1.0% or less (particularly 0.1% or less).

[0038] The aqueous solution of TGP of the present invention is gelled at a temperature higher than the sol-gel transition temperature, and the gel does not dissolve even when immersed in a large amount of water (about 0.1 to 100 times the volume of the gel) for a long period of time. Such properties of the polymer used in the present invention are achieved, for example, by the presence of two or more (plural) blocks having a cloud point in the polymer. In contrast, the inventors have found that when a similar gel is prepared using the aforementioned Pluronic (registered trademark) F-127, which is made of polypropylene oxide with polyethylene oxide bonded to both ends, the gel completely dissolves in water after being left to stand for several hours. In order to keep the cytotoxicity in the non-gelled state as low as possible, it is preferable to use TGP that can gel at a concentration relative to water, i.e., {(polymer) / (polymer+water)}×100(%), of 20% or less (preferably 15% or less, and particularly 10% or less). The molecular weight of the TGP used in the present invention is preferably 30,000 to 30,000,000, more preferably 100,000 to 10,000,000, and even more preferably 500,000 to 5,000,000.

[0039] (TGP concentration in the preservative composition, etc.) The TGP in the preservation composition of the present invention may be dissolved in any medium. The TGP concentration in the preservation composition may be any concentration, including, but not limited to, 1-40%, 3-30%, 5-20%, 7-15%, 8-12%, or 9-11%, as long as it maintains the viability of cells contained in the target biomaterial. The TGP concentration is preferably 7-15%, 8-12%, or 9-11%, from the viewpoint of providing a viscosity sufficient to allow the target biomaterial to float without contacting the bottom of the storage container in a sol state. The TGP concentration of 9-11% is preferred, from the viewpoint of providing a crosslinked or network structure having a pressure sufficient to allow the target biomaterial to gel and maintain a pressure equivalent to that of the target biomaterial in vivo. The TGP contained in the preservation composition of the present invention may be dissolved in any medium. The medium is not particularly limited as long as it can maintain cell viability, but typically, physiological saline, various physiological buffer solutions (e.g., PBS, HBSS, etc.), various basal media for cell culture, preservation solutions, transport solutions, etc. can be used. The composition of physiological saline and various physiological buffer solutions may be appropriately modified depending on the target biomaterial and other preservation conditions.

[0040] Basal media include, but are not limited to, DMEM, MEM, F12, DME, RPMI1640, MCDB (MCDB102, 104, 105 (M199), 107, 120, 131, 153, 199, etc.), L15, SkBM, RITC80-7, CnT-PR, etc. Many of these basal media are commercially available, and their compositions are publicly known. Basal media may be used with their standard composition (e.g., as commercially available), or their composition may be modified appropriately depending on the cell type and cell conditions. Preservation solutions include, but are not limited to, EPII solution, MK solution, and Optisol GS solution. Tissue or organ transport solutions include, but are not limited to, choline solution, Eurocholine solution, UW solution, HTK solution, Celsior solution, Polysol, Dsol, etc. The physiological saline, basal medium, storage solution, and transport solution used as the medium of the present invention are not limited to those with known compositions, and include those in which one or more components have been added, removed, increased, or decreased in amount.

[0041] In addition to the above, the medium may contain one or more additives such as serum, growth factors (e.g., EGF, insulin, etc.), steroid components, and selenium components. In one embodiment of the present invention, the medium for dissolving TGP does not contain serum. In one embodiment of the present invention, the medium for dissolving TGP may contain serum. The serum may be xenogeneic or allogeneic serum. Allogeneic serum is preferred, and among allogeneic serum, autologous serum is particularly preferred. The concentration of serum is not limited, and may be 1% or more, 3% or more, 5% or more, 10% or more, or 20% or more in the medium. Preferably, it is 10%.

[0042] The preservative composition of the present invention may further contain any additional component as long as it does not inhibit the preservative effect of the preservative composition. The additional component may include, for example, an acceptable carrier, any component that enhances the viability of the cell culture (vitamins, amino acids, etc.), antibiotics, and preservatives. Any known additional component can be used as such an additional component, and those skilled in the art are familiar with these additional components. Components that can enhance the effect of the preservative composition of the present invention are preferred.

[0043] In the present invention, the state of TGP may be either a sol state or a gel state as long as the target biomaterial can be preserved. Storage in a gel state is preferred from the viewpoint of suppressing agitation during transportation. Storage in a sol state can be typically achieved by immersing the target biomaterial in TGP sol at a low temperature, and then not raising the temperature of the sol. Storage in a gel state can be typically achieved by immersing the target biomaterial in TGP sol, and then raising the temperature of the sol above the sol-gel transition temperature to create a gel state. In one embodiment of the present invention, cells to be preserved, etc., may be immersed in TGP sol to form a gel, and then a medium may be added on top of the TGP. This allows nutrients to be supplied from the medium to the TGP, making it preferable for long-term preservation. The medium added on top of the gelled TGP may be the same as or different from the medium in which TGP is dissolved. Preferably, the medium added on top of TGP is the same as the medium in which TGP is dissolved, except that it does not contain serum.

[0044] (storage temperature) The temperature at which the target biomaterial is stored is not limited as long as it maintains the viability of the cells in the target biomaterial, and may be, for example, 1°C to 42°C, 4°C to 38°C, 6°C to 35°C, 10°C to 35°C, 12°C to 30°C, 15°C to 30°C, 20°C to 30°C, 22°C to 28°C, 23°C to 27°C, or 24°C to 26°C, with 15°C to 30°C being preferred and 20°C to 30°C being more preferred. While storage at a constant temperature is preferred to prevent damage to the target biomaterial due to temperature changes, the composition can also be used as a composition for storing the target biomaterial at fluctuating temperatures. Thus, in one embodiment of the present invention, the storage temperature may be constant or fluctuating. The temperature range to be varied is not limited as long as it maintains cell viability in the target biomaterial, and may typically be within the ranges of 1°C to 42°C, 4°C to 38°C, 6°C to 35°C, 10°C to 35°C, 12°C to 30°C, 15°C to 30°C, 20°C to 30°C, 22°C to 28°C, 23°C to 27°C, or 24°C to 26°C, with a variation of 15°C to 30°C or 20°C to 30°C being preferred, and a variation of 20°C to 30°C being more preferred. In one embodiment of the present invention, the temperature to be varied may be the ambient temperature, and the aforementioned temperature range may be varied diurnally.

[0045] In one embodiment of the present invention, the storage temperature may be a temperature at which cells or the like to be preserved do not substantially grow. The term "temperature at which cells do not substantially grow" refers to a temperature at which cell growth slows or stops in the art, and is a temperature at which the growth or respiration rate of the target biomaterial is at least 1 / 3, 1 / 5, 1 / 10, 1 / 20, or 1 / 100 of the growth or respiration rate at 37°C. The temperature may be, but is not limited to, 30°C or lower, 27°C or lower, 25°C or lower, 23°C or lower, 20°C or lower, 17°C or lower, 15°C or lower, 12°C or lower, 10°C or lower, 7°C or lower, 6°C or lower, or 4°C or lower. Specifically, the temperature may be, for example, 4 to 30°C, 6 to 30°C, 8 to 30°C, 10 to 30°C, 12 to 30°C, 14 to 30°C, 16 to 30°C, 18 to 30°C, or 20 to 30°C, with 16 to 30°C being preferred and 20 to 30°C being particularly preferred. In one embodiment of the present invention, the storage temperature may be constant or may vary at a temperature at which the cells or the like to be stored do not substantially grow.

[0046] (Storage time) The storage time is not limited as long as the target biomaterial can be preserved, and may be, for example, an upper limit of 1 hour or more, 3 hours or more, 5 hours or more, 12 hours or more, 18 hours, 24 hours or more, 2 days or more, 4 days or more, 8 days or more, 12 days or more, 16 days or more, 20 days or more, 30 days or more, 40 days or more, 50 days or more, or 60 days or more; and, for example, a lower limit of 45 days or less, 35 days or less, 25 days or less, 14 days or less, 10 days or less, 6 days or less, 3 days or less, 20 hours or less, 16 hours or less, 14 hours or less, 10 hours or less, 8 hours or less, 6 hours or less, 5 hours or less, 4 hours or less, 3 hours or less, 2 hours or less, or 1 hour or less. The storage time may be any combination of these upper and lower limits, and may include, but is not limited to, ranges of 3 hours to 60 days, 6 hours to 50 days, 8 hours to 40 days, 10 hours to 25 days, 12 hours to 20 days, 18 hours to 15 days, 24 hours to 10 days, 36 hours to 8 days, etc. Long-term storage is not preferred because the target biomaterial will be damaged by long-term storage.

[0047] The preservation composition of the present invention has excellent preservation properties for biological materials and can therefore be suitably used, without limitation, as a preservation composition or transportation composition for biological materials used in fields such as medicine and medical experiments, particularly cell therapy and organ transplantation. [Example]

[0048] Manufacturing Example 1 42.0 g of N-isopropylacrylamide and 4.0 g of n-butyl methacrylate were dissolved in 592 g of ethanol. An aqueous solution of 11.5 g of polyethylene glycol dimethacrylate (PDE6000, manufactured by Nippon Oil & Fats Co., Ltd.) dissolved in 65.1 g of water was added and heated to 70°C under a nitrogen stream. While maintaining the temperature at 70°C under a nitrogen stream, 0.4 mL of N,N,N',N'-tetramethylethylenediamine (TEMED) and 4 mL of 10% aqueous ammonium persulfate (APS) were added and the reaction was stirred for 30 minutes. Further additions of 0.4 mL of TEMED and 4 mL of 10% aqueous APS were made four times at 30-minute intervals to complete the polymerization reaction. The reaction solution was cooled to below 5°C, diluted with 5 L of cooled distilled water at 5°C, and concentrated to 2 L at 5°C using an ultrafiltration membrane with a molecular weight cutoff of 100,000.

[0049] The concentrated solution was diluted with 4 L of chilled distilled water, and the above-described ultrafiltration concentration procedure was repeated. The above dilution and ultrafiltration concentration procedures were repeated five more times to remove materials with molecular weights of 100,000 or less. The material that was not filtered by ultrafiltration (i.e., the material remaining in the ultrafiltration membrane) was collected and freeze-dried to obtain 40 g of a hydrogel-forming polymer of the present invention ("hydrogel-forming polymer"-6) with a molecular weight of 100,000 or more. 1 g of the hydrogel-forming polymer of the present invention ("hydrogel-forming polymer"-6) obtained above was dissolved in 9 g of distilled water under ice cooling to obtain a 10 wt% aqueous solution. The storage modulus of this aqueous solution was measured using a stress-controlled rheometer (AR500, manufactured by TA Instruments) at an applied frequency of 1 Hz, and was found to be 43 Pa at 10°C, 680 Pa at 25°C, and 1310 Pa at 37°C. This temperature-dependent change in storage modulus was observed repeatedly and reversibly. The sol-gel transition temperature was approximately 20°C.

[0050] Example 1: Transport of cartilage tissue A portion of the cartilage tissue (approximately 10 x 5 mm, 3 mm thick, age: 35 years) excised during artificial joint replacement surgery was collected and immersed in PBS containing antibiotics (gentamicin (50 μg / ml), amphotericin (0.25 μg / ml), penicillin (100 Units / ml) / streptomycin (100 μg / ml)) for 30 minutes. The cartilage tissue was cut into approximately 3 mm pieces using a scalpel. 3 The tissue was then minced into small pieces to form tissue fragments. Next, 1 g of TGP prepared as described in the Preparation Example was dissolved in 9 ml of DMEM at 4°C to prepare a 10% TGP solution. The tissue fragments were added to the TGP solution and pipetted to uniformly disperse the tissue fragments. The flask was then left at room temperature to gel, after which 7–8 ml of 10% serum-containing DMEM medium (Thermo Fisher Scientific, DMEM, high glucose, Cat. No. 11965-084) was added, and the tissue fragments were cultured in a 5% CO₂ incubator (ESPEC BNA-111). The medium was changed weekly and the culture was continued for 42 days. After 42 days, 4°C PBS was added to the TGP gel containing the tissue fragments, and the TGP gel was dissolved by pipetting. The tissue fragments were transferred to a 50 ml test tube. 20 ml of 4°C PBS was added to the test tube, followed by centrifugation and washing. This process was repeated twice. The obtained tissue pieces were divided into two, and each was weighed and then added to two 10 ml test tubes (test tubes A and B).

[0051] Sample A: 1 g of TGP prepared in the manufacturing example was dissolved in 9 ml of DMEM at 4°C to prepare a 10% TGP solution, and 10 ml of this solution was added to test tube A containing the tissue pieces. The solution was allowed to gel at 30°C for 1 hour, and then transported for 3 hours at ambient temperatures ranging from 5 to 42°C. The weight of the tissue pieces was 0.23 g (n=4). Sample B: 10 ml of PBS (phosphate buffer solution) was added to test tube B containing the tissue pieces, and the tissue pieces were transported at 4° C. for 3 hours. The weight of the tissue pieces was 0.16 g (n=4). Photographs of samples A and B containing cartilage tissue fragments are shown in Figure 1.

[0052] After washing the tissue pieces from samples A and B with 4°C PBS, each was treated with Tripsin-EDTA solution (0.25%) at 37°C for 30 minutes, followed by digestion with collagenase II solution (1 mg / ml) at 37°C for 19 hours. After filtering through a 100 μm cell strainer (PLS, Cat No: 43-50100-03), the tissue was centrifuged (150 rpm, 5 minutes) and resuspended in 2 ml of PBS. A 100 μl aliquot was taken from each test tube, and 400 μL of 0.4% trypan blue solution was added. The tissue was then counted using a cell counting chamber. The results are shown in Table 1. [Table 1]

[0053] When cartilage tissue was transported in TGP, the viable cell count and relative viable cell count were maintained at a higher level than when it was transported in PBS. These results indicated that TGP is suitable for transporting cartilage tissue.

[0054] Example 1-2: Transport of cartilage tissue Cartilage tissue fragments were prepared in the same manner as in Example 1-1, except that cartilage tissue from a different specimen was used, and added to test tubes A and B.

[0055] Sample A: 1 g of TGP prepared in the manufacturing example was dissolved in 9 ml of DMEM at 4°C to prepare a 10% TGP solution. 10 ml of this solution was added to test tube A containing the tissue fragments and allowed to gel at 30°C for 1 hour. The tissue fragments were kept at approximately 20°C and transported for 3 hours using a Bio Box (Sugiyamagen Co., Ltd., Cat. No. SBE-10W) and Thermo Storage 20 (Sugiyamagen Co., Ltd., Cat. No. TP-20-350). The weight of the tissue fragments placed in Sample A was 0.21 g. Sample B: 10 ml of Corning Glucose Solution (Euro-Collins), Cat No. 99-408-CM (hereafter referred to as "ECS") was added to test tube B containing the cheek tissue fragment, and the tissue fragment was transported at 4°C for 3 hours. The weight of the tissue fragment placed in Sample B was 0.19 g.

[0056] Each tissue piece is cut into 1mm pieces using a scalpel. 2 The tissue was then cut into small pieces as follows: The tissue fragments were treated with Tripsin-EDTA solution (0.25%) at 37°C for 30 minutes, then digested with collagenase II solution (1 mg / ml) at 37°C for 12-16 hours. After washing with DMEM, the tissue was filtered through a 100 μm filter and centrifuged (1800 rpm, 10 minutes). The precipitate was diluted with 10% serum-containing DMEM, placed in a T25 flask, and cultured in a 5% CO2 incubator. The culture medium was changed every three days and cultured for two weeks. After two weeks, the culture supernatant was discarded and the cells were dispersed in Tripsin-EDTA (0.25%).

[0057] One gram of TGP prepared as described in the Preparation Example was dissolved in DMEM at 10°C to prepare a 10% TGP solution. Cells derived from cartilage tissue fragments were then dispersed in the solution and dispensed into a 6-well plate. After allowing the solution to gel at room temperature, 7-8 ml of medium containing 10% serum-containing DMEM containing antibiotics (gentamicin (50 μg / ml), amphotericin (0.25 μg / ml), penicillin (100 units / ml), streptomycin (100 μg / ml), and L-ascorbic acid (5 mg / ml)) was added and the cells were cultured in a 5% CO₂ incubator (ESPEC BNA-111). The culture medium was changed weekly for 4-16 weeks.

[0058] The resulting chondrocyte cultures were harvested on day 42. mRNA was isolated from the harvested cultures using the RNeasy Mini Kit (Qiagen). cDNA was synthesized by reverse transcription using 1 μg of the total RNA as a template with Superscript III reverse transcriptase (Invitrogen). Real-time PCR analysis was performed using TB Green Premix Ex Taq II (Takara, Cat. No. RR820S / A / B) on a Thermal Cycler Dice Real Time System II (Takara, Cat. No. TP900). The primer sequences used are shown below. SOX9: Fwd 5'-ggagatgaaatctgttctgggaatg-3' (SEQ ID NO: 1) SOX9:Rvs 5'-ttgaaggttaactgctggtgttctg-3' (SEQ ID NO: 2) COL2A1:Fwd 5'-ccagttgggagtaatgcaagga-3' (SEQ ID NO: 3) COL2A1:Rvs 5'-acaccaggttcaccaggttca-3' (SEQ ID NO: 4)

[0059] Real-time PCR showed that SOX9 was expressed at similar levels in samples A and B. On the other hand, sample B (ECS transport) did not express COL2A1 on day 42, but sample A (TGP transport) expressed COL2A1 on day 42. The results for COL2A1 are shown in Figure 2.

[0060] When transported using TGP, SOX9 and COL2A1, known as markers of healthy cartilage tissue, were found to be expressed even after 42 days of culture. Combined with the results of Example 1-1, transport using TGP not only had a favorable effect on the number of viable cells but also on the properties of the subsequent cultured tissue compared to transport using PBS or ECS. These results demonstrate that TGP is suitable for transporting cartilage tissue.

[0061] Example 2-1: Transport of oral tissue Oral mucosal tissue (3 mm) was extracted from the oral cavity of humans (age 54 (#1080), age 21 (#1081), age 17 (#1082), age 36 (#1083)) using the same procedure as in Example 1. 3 Four tissue pieces were collected from each individual and washed. Each tissue piece was cut into equal pieces and placed in two 10 ml test tubes (samples A and B).

[0062] [Transportation of tissue] Sample A: 1 g of TGP prepared in the manufacturing example was dissolved in 9 ml of DMEM at 4°C to prepare a 10% TGP solution, and 10 ml of this was added to test tube A containing the tissue pieces and allowed to gel at 30°C for 1 hour. The solution was then transported for 4 hours, kept at approximately 20°C, using a Biobox (Sugiyamagen Co., Ltd., Cat. No. SBE-10W) and Thermostorage 20 (Sugiyamagen Co., Ltd., Cat. No. TP-20-350). Sample B: 10 ml of PBS was added to test tube B containing the cheek tissue fragment and transported at 4°C for 4 hours.

[0063] [Measurement of viable cell count] After washing the tissue pieces from samples A and B with 4°C PBS, 0.05 mg of each was weighed and treated with Tripsin-EDTA solution (0.25%) at 37°C for 30 minutes, followed by digestion with Collagenase II solution (1 mg / ml) at 37°C for 2 hours. After filtering through a 100 μm cell strainer, the tissue was centrifuged (150 rpm, 5 minutes) and resuspended in 2 ml of PBS. A 100 μl aliquot was taken from each sample, and 400 μL of 0.4% trypan blue solution was added. The tissue was then counted using a cell counting chamber. The results are shown in Table 2. [Table 2]

[0064] [result] Many viable cells were confirmed in all epithelial tissues transported using TGP, indicating that preservation in TGP is suitable for transporting epithelial tissues.

[0065] Example 2-2: Transport of oral tissue A portion of healthy cheek tissue was collected from the oral cavity of a human (age 34) (#1084) and transported using the same procedures as in Example 2, except that ECS (Corning Glucose Solution (Euro-Collins), Product Number 99-408-CM) was used instead of PBS.

[0066] [Confirmation of proliferation] Each tissue fragment from Samples A and B was washed with 4°C PBS. Then, each tissue fragment from Samples A and B was seeded in 10% TGP, prepared by dissolving 1 g of TGP prepared in the manufacturing example in 9 ml of culture medium. After gelation at 30°C for 1 hour, culture medium was added and the tissue was cultured in a 5% carbon dioxide incubator (Sample A-TGP, Sample B-TGP). The proliferation of the tissue during culture was confirmed using an inverted microscope.

[0067] Some tissue pieces of samples A and B were cut into 25cm pieces. 2 The tissue fragments from sample B were added to a 25cm flask containing 10 ml of Cnt-PR and cultured (sample A-DMEM, sample B-DMEM). 2 The cells were added to a flask and cultured (Sample B-Cnt-PR). Inverted microscope images of Sample A-DMEM and Sample A-TGP after 7 and 17 days of culture are shown on the left side of Figure 3. Sample B-DMEM, Sample B-CnT-PR, and Sample B-TGP are shown on the right side of Figure 3. All images are at 10x magnification except for Sample A-TGP, which is at 40x magnification, and Sample B-TGP on Day 17, which is at 40x magnification.

[0068] When tissue explants from sample A were cultured in DMEM and TGP, cell proliferation was confirmed at a relatively early stage (sample A-DMEM, -TGP). As shown in Figure 3, in sample A-DMEM, a large number of cells proliferated from the tissue explants after 17 days of culture. In sample A-TGP, cell proliferation was confirmed the earliest among all samples. In sample A-TGP, an extremely large number of cells proliferated from the tissue explants after 17 days of culture.

[0069] When tissue explants from sample B were cultured in DMEM, Cnt-PR, and TGP (samples B-DMEM, -Cnt-PR, and -TGP), the proliferation rate was slow in all cases, but was relatively fast when cultured in TGP. As shown in Figure 3, in sample B-DMEM, a small amount of cells proliferated from the tissue explants, but fibroblasts began to proliferate from the tissue explants after 7 days of culture, and the bottom of the culture dish was covered with fibroblasts after 17 days of culture. In addition, in sample B-Cnt-PR, a small amount of cells proliferated from the tissue explants after 17 days of culture. In sample B - TGP, many cells proliferated from the tissue explants after 17 days of culture. Regardless of the culture medium, epithelial cell proliferation was superior in sample A, indicating that the use of TGP for transport causes less damage to the tissue than ECS.

[0070] At day 21 of incubation, tissue fragments from Sample A-TGP, Sample A-DMEM, Sample B-TGP, and Sample B-DMEM were each washed with 4°C PBS. After washing the tissue fragments from Sample A and B with 4°C PBS, each was treated with Tripsin-EDTA solution (0.25%) at 37°C for 30 minutes, followed by digestion with collagenase II solution (1 mg / ml) at 37°C for 5 hours. After filtering through a 100 μm cell strainer, the cells were centrifuged (150 rpm, 5 minutes) and resuspended in 2 ml of PBS. A 100 μl aliquot was taken from each sample, and 400 μL of 0.4% trypan blue solution was added. The cells were counted using a cell counting chamber. The results are shown in Table 3.

[0071] In the table, E:N indicates the ratio of epithelial cells to non-epithelial cells. [Table 3]

[0072] Sample A (TGP) transported and then cultured in TGP showed the highest cell count. Sample B (ECS) plate cultured after transportation showed the lowest cell count. Furthermore, even Sample B, which showed a low viable cell count after transportation in Example 2, showed a sufficient viable cell count when cultured with TGP. This indicates that TGP enhances the proliferation ability of cells in epithelial tissue, and in particular, restores the proliferation ability of epithelial tissue in which the viable cell count has decreased.

[0073] Furthermore, it was found that the proportion of non-epithelial cells was higher in all TGP cultures. While epithelial cells have a polygonal, cobblestone-like morphology, non-epithelial cells have a round morphology characteristic of epithelial stem cells, and the proliferated non-epithelial cells are considered to be epithelial stem cells. Therefore, it was found that culturing epithelial tissue using TGP also proliferates epithelial stem cells in epithelial tissue.

[0074] Example 3-1: Corneal transport (MK solution) Eyeballs were collected from human cadavers and immersed in a sterilized 0.5% solution of I-PVP (iodine-polyvinylpyrrolidone) for 2 minutes. Two corneas were collected according to standard procedures and immersed in PBS containing antibiotics (gentamicin (50 μg / ml), amphotericin (0.25 μg / ml), penicillin (100 Units / ml) / streptomycin (100 μg / ml)) for 30 minutes. Next, 1 g of TGP prepared in the manufacturing example was added to 9 ml of MK solution (M199 containing 5% dextran 40 (Thermo Fisher Scientific)). A 10% TGP solution was prepared by dissolving TGP in a corneal gel (no. 11150-067) at 4°C. One of the two corneas was transferred to a corneal storage container, and the TGP solution was added so that the entire cornea was submerged. The solution was allowed to gel at 30°C for 1 hour, after which 10 ml of MK solution was added. The sample was then transported at temperatures ranging from 5 to 42°C for 96 hours (Sample A). The other cornea was transferred to a corneal storage container, and 10 ml of MK solution was added (Sample B). The sample was then transported at 4°C for 96 hours. The corneas of Samples A and B were observed before storage and after 96 hours of transport using a specular microscope (Keratoanalyzer EKA-10, Konan Medical) and software (KSS-EB10, Konan Medical). The results are shown in Figure 4. The top two photographs show the corneal tissue before storage (0 hours), and the bottom two photographs show the corneal tissue after 96 hours of transport.

[0075] After 96 hours of storage, it was clear that Sample A contained many high-quality endothelial cells, which is a characteristic of corneal quality. On the other hand, no viable endothelial cells were confirmed in Sample B after 96 hours. Therefore, the corneas were better preserved in Sample A compared to Sample B. This demonstrates that TGP is suitable for transporting corneas.

[0076] Example 3-2: Corneal transport (Optisol GS solution) Two corneas were collected from a different specimen using the same procedure as in Example 3-1. Samples A and B were prepared and transported using the same procedure as in Example 3-1, except that OptiSol-GS Corneal Storage Media (Box of 12) (Bausch & Lomb 50006-OPT) was used instead of MK solution. The corneas of Samples A and B were observed using a specular microscope (KeratoAnalyzer EKA-10, Konan Medical) and software (KSS-EB10, Konan Medical) before storage and after 96 hours of transport. The results are shown in Figure 5. The top two photographs show the corneal tissue before storage (0 hours), and the bottom two photographs show the corneal tissue after 96 hours of transport. After 96 hours of storage, Sample A clearly exhibited the presence of numerous high-quality endothelial cells, a characteristic of corneal quality. On the other hand, no viable endothelial cells were observed in Sample B on the fourth day. Therefore, the corneas of Sample A were better preserved than those of Sample B. Therefore, it was revealed that TGP is suitable for transporting corneas.

[0077] Example 4-1: Transport of the limbus Samples A and B were prepared and transported using the same procedure as in Example 3-1, except that limbal tissue fragments were collected from a different specimen instead of the cornea, and a 10 ml test tube was used instead of the corneal storage container. The limbal tissue fragments of transported samples A and B were washed with PBS at 4°C. A 10% TGP solution was prepared by dissolving DMEM at 4°C, and 25 cm samples containing partial tissue fragments of samples A and B were placed in a 10 ml test tube. 210 ml of the medium was added to the flask and pipetted to disperse the tissue fragments evenly. 7-8 ml of 10% serum-containing DMEM was added, and the tissue was cultured in a 5% CO₂ incubator for 14 days. The proliferation of the tissue during culture was observed under an inverted microscope (10x magnification). The results are shown in Figure 6.

[0078] In sample B, cell proliferation from the graft was confirmed on day 7, whereas in sample A, good cell proliferation from the graft was confirmed on day 2. This indicates that transport in TGP causes less damage to the corneal limbus than transport in MK medium. Therefore, it has become clear that TGP is suitable for transporting the corneal limbus.

[0079] Example 5: Intestinal Tissue Transport 3mm of colonic tissue from a human who had undergone intestinal resection due to Hirschsprung's disease 3 The tissue was collected, cut into small pieces, and immersed in PBS containing antibiotics (gentamicin (50 μg / ml), amphotericin (0.25 μg / ml), penicillin (100 Units / ml) / streptomycin (100 μg / ml)) for 30 minutes. Each tissue piece was washed twice by centrifugation in PBS at 4°C. Each tissue piece was cut into equal pieces and placed in two 10 ml test tubes (test tubes A and B).

[0080] Sample A: 1 g of TGP prepared in the manufacturing example was dissolved in 9 ml of DMEM / F12 at 4°C to prepare a 10% TGP solution, and 10 ml of this was added to test tube A containing tissue fragments. After gelling at 30°C for 1 hour, the solution was transported for 2 hours at ambient temperatures ranging from 5 to 42°C. Sample B: 10 ml of PBS was added to test tube B containing tissue fragments, and the solution was transported for 2 hours at 4°C.

[0081] After washing the tissue pieces of samples A and B with PBS at 4°C, each was treated with Tripsin-EDTA solution (0.25%) at 37°C for 30 minutes, and then digested with collagenase II solution (1 mg / ml) at 37°C for 19 hours. After filtering through a 100 μm cell strainer, the pieces were centrifuged (150 rpm, 5 minutes) and resuspended in a solution of TGP (Mebiol 25 cm) dissolved in 9 ml of DMEM / F12 at 4°C. 2 The cells were uniformly dispersed in a 25cm flask. 2 After adding the mixture to a flask and allowing it to gel at 30°C for 1 hour, 7-8 ml of DMEM / F12 was added and the mixture was cultured in a 5% CO₂ incubator for 2 weeks (Sample A-TGP, Sample B-TGP). Cell proliferation during culture was confirmed using an inverted microscope (10x magnification), and then the number of viable cells was measured using trypan blue. The results are shown in Figure 7. In Sample A, enteric neural stem cells proliferated well as neurosphere-like bodies, but proliferation was poor in Sample B. The cell count after culture was approximately 20-30 times higher in Sample A than in Sample B. This demonstrates that TGP is suitable for transporting intestinal tissue.

[0082] Example 6: Vascular Tissue Transport Saphenous vein tissue was collected from human cadavers and minced into tissue fragments. The tissue fragments were immersed in PBS containing antibiotics (gentamicin (50 μg / ml), amphotericin (0.25 μg / ml), penicillin (100 units / ml) / streptomycin (100 μg / ml)) for 30 minutes. Each tissue fragment was washed twice by centrifugation in PBS at 4°C. Each tissue fragment was cut into equal pieces and placed in two 10 ml test tubes (test tubes A and B).

[0083] Sample A: 1 g of TGP prepared in the manufacturing example was dissolved in 9 ml of M199 at 4°C to prepare a 10% TGP solution, and 10 ml of this was added to test tube A containing the tissue fragments.The solution was allowed to gel at 30°C for 1 hour and then transported for 24 hours at a temperature (ambient temperature) that could vary between 5 and 42°C. Sample B: 10 ml of HBSS was added to test tube B containing the tissue fragments, and the mixture was transported at 4°C for 2 hours.

[0084] After washing the tissue pieces of samples A and B with PBS at 4°C, each was treated with Tripsin-EDTA solution (0.25%) at 37°C for 30 minutes, and then digested with collagenase II solution (1 mg / ml) at 37°C for 19 hours. After filtering through a 100 μm cell strainer and centrifuging (150 rpm, 5 minutes), the tissue pieces were resuspended in a solution of TGP (Mebiol 25 cm) dissolved in 9 ml of M199 at 4°C. 2 The cells were uniformly dispersed in a 25cm flask. 2 After adding the cells to the flask and allowing them to gel at 30°C for 1 hour, 7-8 ml of 10% serum-containing M199 was added and the cells were cultured in a 5% CO₂ incubator for 1 week (Sample A-TGP, Sample B-TGP). Cell proliferation during culture was confirmed using an inverted microscope (10x magnification), and then the number of viable cells was measured using trypan blue. The observation results are shown in Figure 8.

[0085] Good proliferation was observed in sample A, and the cells were relatively large and well-shaped. In sample B, proliferation was poor and the cells were small. This demonstrated that TGP is suitable for transport through vascular tissue.

[0086] Example 7: Transport of dental pulp tissue The same procedure as in Example 6 was used, except that instead of saphenous vein tissue from human cadavers, dental pulp tissue from 20 avulsed deciduous incisors, molars, and canines obtained from 15 healthy humans was used.

[0087] Sample A: 1 g of TGP prepared in the manufacturing example was dissolved in 9 ml of DMEM at 4°C to prepare a 10% TGP solution, and 10 ml of this was added to test tube A containing the tissue fragments. The solution was allowed to gel at 30°C for 1 hour, and then transported at temperatures (ambient temperature) that could vary between 5 and 42°C for 24, 48, and 96 hours. Sample B: 10 ml of PBS was added to test tube B containing the tissue fragments and transported at 4°C for 2 hours.

[0088] The tissue pieces of samples A and B were washed with PBS at 4°C, centrifuged (150 rpm, 5 min), and then added to 9 ml of DMEM at 4°C in a TGP solution (Mebiol 25 cm 2 The dental pulp tissue was dispersed evenly in a 25cm flask. 2 The cells were added to a flask and allowed to gel at 20°C for 1 hour. After that, 7-8 ml of 10% serum-containing DMEM was added and the cells were cultured in a 5% CO₂ incubator for 2 weeks. Cell proliferation during culture was monitored using an inverted microscope (10x magnification). The results are shown in Figure 9. In addition, H&E staining of the dental pulp tissues of Samples A and B after 21 days of culture was performed. The H&E staining of the dental pulp tissue of Sample A is shown in Figure 10.

[0089] Observation under an inverted microscope revealed that cell proliferation from the cultured tissue was better in Sample A than in Sample B. Furthermore, H&E of Sample A on the 21st day confirmed healthy dental pulp tissue, whereas no stained dental pulp tissue was observed in Sample B. This demonstrated that TGP is suitable for transporting dental pulp tissue.

[0090] Example 8: Preservation of foreskin tissue 1 cm of foreskin tissue from a circumcised human penis 2 The tissue was collected and cut into small pieces. The pieces were immersed in PBS containing antibiotics (gentamicin (50 μg / ml), amphotericin (0.25 μg / ml), penicillin (100 Units / ml) / streptomycin (100 μg / ml)) for 30 minutes. Each piece was washed twice by centrifugation in PBS at 4°C. Each piece was cut into equal pieces and placed in two 10 ml test tubes (test tubes A and B).

[0091] Sample A: 1 g of TGP prepared in the manufacturing example was dissolved in 9 ml of DMEM at 4°C to prepare a 10% TGP solution, and 10 ml of this was added to test tube A containing the tissue fragments.The solution was allowed to gel at 30°C for 1 hour, and then left to stand for 24 hours at a temperature (ambient temperature) that could vary between 5 and 42°C. Sample B: 10 ml of Hank's balanced salt solution (HBSS) was added to test tube B containing the tissue pieces, and the tissue pieces were left to stand at 4°C for 24 hours.

[0092] After washing the tissue pieces of samples A and B with PBS at 4°C, each was treated with Tripsin-EDTA solution (0.25%) at 37°C for 30 minutes, and then digested with collagenase II solution (1 mg / ml) at 37°C for 19 hours. After filtering through a 100 μm cell strainer, the pieces were centrifuged (150 rpm, 5 minutes) and resuspended in a solution of TGP (Mebiol 25 cm) dissolved in 9 ml of DMEM at 4°C. 2 The cells were uniformly dispersed in a 25cm flask. 2 The cells were added to a flask and allowed to gel at 30°C for 1 hour. Then, 7-8 ml of 10% serum-containing DMEM was added and the cells were cultured in a 5% CO₂ incubator for 2 weeks. Cell proliferation during culture was monitored using an inverted microscope (10x magnification), and the number of viable cells was then measured using trypan blue. The microscopic images are shown in Figure 11. Compared to sample B, sample A had cells growing out of the tissue, and by day 7 had 5 to 8 times more cells than sample B. This demonstrated that TGP is suitable for preserving skin tissue.

[0093] Example 9: Transport and culture of omental tissue Instead of foreskin tissue, 2-3 cm of omental tissue was taken from a human cadaver. 2 The samples were prepared in the same manner as in Example 8, except that M199 was used instead of DMEM, and transported under the following conditions. Sample A: Transported for 12 hours at temperatures (ambient temperature) that could vary between 5 and 42°C. Sample B: Transported at 4°C for 12 hours.

[0094] After washing the tissue pieces of samples A and B with 4°C PBS, each was treated with Tripsin-EDTA solution (0.25%) at 37°C for 30 minutes, and then digested with collagenase II solution (1 mg / ml) at 37°C for 19 hours. After filtering through a 100 μm cell strainer, the tissue was centrifuged (150 rpm, 5 minutes). 9 ml of M199 was added to TGP solution (Mebiol 25 cm 2 The dispersed cells of sample A were added to a flask containing TGP solution prepared by adding the cells to a 25 cm 2 After adding the cells to a flask and allowing them to gel at 30°C for 1 hour, 7-8 ml of 10% serum-containing M199 was added and the cells were cultured in a 5% CO2 incubator for 10 days. The dispersed cells of sample B were added to 10 ml of M199 and cultured in a 5% CO2 incubator for 2 weeks. Cell proliferation during culture was confirmed using an inverted microscope (10x magnification) on the 10th day. The results are shown in Figure 12. Compared to sample B, cell growth was better in sample A, with approximately 15 to 20 times the cell number observed. This demonstrated that TGP is suitable for transporting omental tissue.

[0095] Example 10: Delivery of human fetal liver cells Instead of omental tissue, 2-3 cm of liver tissue was extracted from a human fetus. 2 Samples A and B were prepared in triplicate using the same procedures as in Example 9, except that six pieces of tissue were used and DMEM / HAM F-12 was used instead of M199. Samples A and B were prepared and transported under the following conditions: Sample A: Transported for 4 to 8 hours at temperatures (ambient temperature) that could vary between 5 and 42°C. Sample B: Transported for 4 to 8 hours at temperatures (ambient temperature) that could vary between 5 and 42°C.

[0096] After washing the tissue pieces from samples A and B with 4°C PBS, each was treated with Tripsin-EDTA solution (0.25%) at 37°C for 30 minutes, followed by digestion with collagenase II solution (1 mg / ml) at 37°C for 19 hours. After filtering through a 100 μm cell strainer, the tissue was centrifuged (150 rpm, 5 minutes) and resuspended in 2 ml of PBS. A 100 μl aliquot was taken from each test tube, and 400 μL of 0.4% trypan blue solution was added. The cells were counted using a cell counting chamber. The results are shown in Table 4. [Table 4]

[0097] A higher number of viable cells was observed when transported in TGP compared to PBS. This indicates that TGP can be transported without damaging liver tissue, and is therefore suitable for transporting liver tissue. The results of Examples 1 to 10 demonstrate that TGP is suitable for the storage and transportation of various biomaterials.

Claims

1. 1. A composition for transporting and preserving biological materials, comprising a thermoreversible polymer, the thermoreversible polymer is composed of a plurality of blocks having a cloud point and a hydrophilic block bonded together, the blocks being selected from the group consisting of polypropylene oxide, a copolymer of propylene oxide and another alkylene oxide, a poly(N-substituted acrylamide derivative), a poly(N-substituted methacrylamide derivative), a copolymer of an N-substituted acrylamide derivative and an N-substituted methacrylamide derivative, polyvinyl methyl ether, and a partially acetylated polyvinyl alcohol; The molecular weight of the thermoreversible polymer is 100,000 or more and 10,000,000 or less, For transport at a temperature in the range of 5 to 42°C and for storage at a temperature in the range of 1 to 42°C; The transport and storage composition.

2. 10. The transport and storage composition of claim 1, for transport and storage at constant or fluctuating temperatures.

3. 3. The transport and storage composition of claim 2, wherein the constant or fluctuating temperature is a temperature at which cells do not substantially grow.

4. 4. The composition for transportation and storage according to claim 3, wherein the temperature at which cells do not substantially grow is 6 to 30°C.

5. The composition for transportation and storage according to any one of claims 1 to 4, wherein the biological material is selected from the group consisting of cartilage tissue, oral mucosal tissue, corneal tissue, limbal tissue, dental pulp tissue, vascular tissue, gastrointestinal mucosal tissue, omentum tissue, skin tissue, and liver tissue.

6. The composition for transportation and storage according to any one of claims 1 to 4, wherein the biological material is a somatic cell, progenitor cell, or stem cell contained in a tissue selected from the group consisting of cartilage tissue, oral mucosal tissue, corneal tissue, limbal tissue, dental pulp tissue, vascular tissue, gastrointestinal mucosal tissue, omentum tissue, skin tissue, and liver tissue.

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