WO2020166712A1 - Solution macromoléculaire aqueuse pour la cryoconservation et corps solidifié contenant un échantillon biologique - Google Patents

Solution macromoléculaire aqueuse pour la cryoconservation et corps solidifié contenant un échantillon biologique Download PDF

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WO2020166712A1
WO2020166712A1 PCT/JP2020/005870 JP2020005870W WO2020166712A1 WO 2020166712 A1 WO2020166712 A1 WO 2020166712A1 JP 2020005870 W JP2020005870 W JP 2020005870W WO 2020166712 A1 WO2020166712 A1 WO 2020166712A1
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water
polymer
biological sample
solution
cells
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Japanese (ja)
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田畑　泰彦
克秀 水野
行哉 駒田
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イビデン株式会社
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Priority to JP2020572347A priority Critical patent/JP7382355B2/ja
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Priority to JP2023172868A priority patent/JP2024001153A/ja

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/44—Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L101/00—Compositions of unspecified macromolecular compounds
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
    • C08L5/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/04—Preserving or maintaining viable microorganisms

Definitions

  • the present invention relates to a solidified body containing an aqueous polymer solution and a biological sample for cryopreservation of the biological sample.
  • regenerative medicine such as cell therapy is being actively performed not only in humans but also in the veterinary field.
  • the bone marrow-derived mesenchymal stem cells and adipose-derived mesenchymal stem cells collected from the living body are used in the above-mentioned regenerative medicine and regenerative medicine research after being collected in large quantities.
  • the surplus cells are cryopreserved and used appropriately.
  • the demand for stable supply of such cells is also increasing.
  • DMSO dimethylsulfoxide
  • glycerin glycerin
  • propylene glycol permeated into cells by cryopreservation. It is used by adding it to a buffer such as a culture medium as a cryoprotective reagent of the type (Patent Document 1). Of these, DMSO is most often used and has a good effect of protecting cells and organelles.
  • Non-Patent Document 1 Non-Patent Document 1
  • cryoprotectants instead of chemical substances, attempts are being made to use natural cryoprotectants as cryoprotective reagents.
  • a disaccharide, an oligosaccharide, or a high molecular polysaccharide is added to a buffer solution such as a culture medium as a non-penetrating cryoprotective reagent.
  • a method of retaining biological components in a crosslinked body that forms a hydrogel is under consideration.
  • a living body made of modified hyaluronic acid in which a side chain having a substituent that reacts with a hydroxyl group to form a crosslinked structure has been introduced into hyaluronic acid as a raw material having a weight average molecular weight of 5000 to 4,000,000 is used as a raw material.
  • Preservatives for the ingredients are described.
  • the modified hyaluronic acid reacts with the hydroxyl groups of a compound having a plurality of hydroxyl groups such as polyvinyl alcohol to form a crosslinked product of the modified hyaluronic acid, and by embedding a biological component in the agar-like hydrogel, a preservative Is used as.
  • the molecular weight of the hydrogel described in Patent Document 2 as an actual preservative is estimated to be several million or more.
  • biological components are stored in a refrigerator at about 4° C., and the storage period is about several days.
  • Patent Document 3 a cryopreservation composition containing a carboxylated polyamino acid in which an amino group of the polyamino acid is blocked by being carboxylated (or acetylated) with a carboxylic acid anhydride and an organic amphoteric agent is disclosed. Have been described. Further, in Patent Document 4, fructan is disclosed as an active ingredient of a cell preservation solution.
  • JP-A-63-216476 International Publication No. 2016/076317 Japanese Patent Publication No. 2018-533377 JP 2012-235728 A
  • ⁇ Intracellular penetration type cryoprotectants slow the formation rate of ice crystals formed in cells by promoting dehydration of cells and inhibit ice crystal formation.
  • DMSO easily penetrates into cells and is therefore effective for cryopreservation of cells having a complicated structure such as mammalian cells.
  • DMSO Various chemicals have cytotoxicity. It is considered that when the cryoprotective substance penetrates into cells and the intracellular concentration increases, the effect of toxicity also increases.
  • DMSO induces differentiation of HL-60 cells and P19CL6 cells (derived from mouse embryocarcinoma cells) (PNAS March 27, 2001 2001 98(7)3826-3831. and Biochem Biophys ResCommunity. 2004; Sep 24;322(3):759-65.), and it is also reported to affect the differentiation of ES cells (Cryobiology.2006 Oct;53(2):194-205.). Therefore, it is considered that the use of DMSO as a cryoprotective reagent is not suitable for cell preservation when it is necessary to maintain undifferentiated state or functionality in stem cells.
  • DMSO when a sample is stored for a long time using DMSO, it is indispensable to store the sample in liquid nitrogen or in an atmosphere that requires handling and management, which is considered to be an issue for the spread of regenerative medicine and regenerative medicine research.
  • cryoprotectants such as sugars are cell-friendly, but have a large molecular size and are difficult to be taken into cells. Therefore, it is considered that intracellular freezing cannot be sufficiently suppressed only by adding from outside the cells.
  • the tested preservation period is only a few days, and a better cryopreservation technique is required for preservation in units of several months. It is believed that there is.
  • a modifying group into hyaluronic acid, and in order to dissolve the gel for recovery of biological components after storage from the hydrogel.
  • the use of additives is also required. However, when such an additive is introduced into the cryopreservation method, it can be used for research tests, but it is difficult to use it for actual medical use due to the risk of contamination with impurities.
  • Patent Document 3 discloses a cryopreservation composition containing a carboxylated polyamino acid in which the amino group of the polyamino acid is modified with a carboxylic acid, an organic amphoteric agent, and optionally a polysaccharide, but the cells are cryopreserved. The survival rate was not sufficient when they were allowed to do so.
  • the fructan described in Patent Document 4 also showed cell death due to rupture and the like, and the cells could not be sufficiently frozen and stored.
  • a method using a non-penetrating cryoprotective reagent is also known, but such a preservative reagent alone does not have a sufficient effect on the cells, although the effect on cells is low. Therefore, a combination of an intracellular permeation type compound such as DMSO, glycerin and propylene glycol with an intracellular permeation type compound and a non-permeation type substance, in which the amount of the compound is reduced or replaced, is commercially available.
  • an intracellular permeation type compound such as DMSO, glycerin and propylene glycol
  • a non-permeation type substance in which the amount of the compound is reduced or replaced
  • the present invention has been made in view of the above problems, and provides a polymer aqueous solution for cryopreservation for appropriately preserving a biological sample.
  • biological samples can be obtained without basically using chemical substances such as dimethyl sulfoxide (DMSO), propylene glycol (PG), ethylene glycol (EG), and basically without adding serum or serum-derived protein.
  • Polymer solution for cryopreservation that enables stable storage at a temperature of -27°C or lower for a long time, a method for preventing or suppressing the growth of ice crystals in a biological sample using the same, and a solidified body containing the biological sample The purpose is to provide.
  • the present invention is a polymer aqueous solution containing a water-soluble polymer or a salt thereof in an aqueous solvent and used for cryopreservation of a biological sample, wherein the polymer aqueous solution has an intrinsic viscosity ( ⁇ ) of 0.20 dL/ g or more and 0.95 dL/g or less, and the orthogonal projection area of the biological sample in the state where the aqueous polymer solution is solidified at ⁇ 80° C. is the same as that of the biological sample in the culture solution (in the medium) before solidification.
  • ⁇ intrinsic viscosity
  • the calorific value of the exothermic peak in the temperature lowering process is 0 J/g, or 65% or less of the corresponding calorific value of the standard liquid consisting of water.
  • the DSC scanning was performed by (1) holding at 20° C. for 1 minute, lowering the temperature to ⁇ 80° C. at a temperature lowering rate of 5° C./min, and (2) holding at ⁇ 80° C. for 1 minute, then 10° C./min. The temperature is raised to 20° C. at a heating rate of min, and the heating is performed.
  • the endothermic peak measured by DSC is not confirmed in the DSC curve in the temperature rising process obtained under the measurement conditions (1) and (2) using a differential scanning calorimeter (DSC).
  • DSC differential scanning calorimeter
  • the orthogonal projection area of the biological sample in the state where the aqueous polymer solution is solidified at ⁇ 80° C. is 1/3.th of the orthogonal projection area of the biological sample in the culture solution (in the medium) before solidification. It is desirable that the size is 5 or more.
  • the intrinsic viscosity ( ⁇ ) of the aqueous polymer solution is preferably 0.32 dL/g or more and 0.65 dL/g or less.
  • the temperature at which the endothermic peak is confirmed is preferably ⁇ 1.3° C. or higher and 0.83° C. or lower.
  • the endothermic peak is not confirmed means that “the endothermic peak is not confirmed” unless the endothermic peak is confirmed in the range of -30°C to +5°C. This is because if water containing a water-soluble polymer exhibits an endothermic peak, it is in this range.
  • the orthographic area of the biological sample and the brightness of the Munsell color system of the area occupied by the biological sample and the matrix area do not depend on the temperature after freeze-solidification, but When the lightness of the Munsell color system in the area occupied by the sample and the matrix area has temperature dependence, the orthogonal projection area and the lightness of the Munsell color system are measured at ⁇ 80° C., respectively.
  • a metal salt, a halogen salt or a sulfate salt is desirable.
  • the metal salt is preferably an alkali metal salt or an alkaline earth metal salt.
  • the alkali metal or alkaline earth metal sodium, potassium, calcium or the like is selected.
  • the halogen chlorine, bromine or the like can be used.
  • the polymer aqueous solution for cryopreservation of the biological sample of the present invention does not contain dimethyl sulfoxide, which is an intracellular permeation type cryoprotectant. Further, it is desirable that the aqueous polymer solution for cryopreservation of the biological sample of the present invention does not contain a cytotoxic cryoprotective agent such as ethylene glycol. These are harmful to the cells after thawing.
  • the aqueous polymer solution of the present invention has a DSC curve in the temperature decreasing process obtained under the measurement conditions of DSC (1) to (2) above, wherein the exothermic amount of the exothermic peak in the temperature decreasing process is 0 J/g, or water. It is preferably 40% or less of the corresponding calorific value of the standard liquid consisting of
  • the polymer aqueous solution is preferably a polymer aqueous solution for cryopreservation of a biological sample containing a water-soluble polymer or a salt thereof in a content of 0.1 w/v% or more and 20 w/v% or less.
  • aqueous polymer solution for cryopreservation of a biological sample in which the water-soluble polymer is a water-soluble polymer containing a sugar residue is preferable.
  • the polymer solution for cryopreservation of the biological sample in which the biological sample is cells, tissues, or a tissue-like substance that is a membrane or an aggregate is preferable.
  • the polymer sample is preferably an aqueous polymer solution for cryopreservation of the biological sample, which is a mesenchymal stem cell, a blood cell, an endothelial cell, or a tissue for transplantation.
  • the above-mentioned biological sample is preferably a polymer aqueous solution for cryopreservation of the biological sample which is a sperm, an egg, or a fertilized egg.
  • the polymer aqueous solution for cryopreservation of biological samples of the present invention is preferably used for vitrification preservation of biological samples.
  • the present invention also provides a method for preventing or suppressing the growth of ice crystals in a biological sample during cryopreservation of the biological sample by contacting the biological sample with an aqueous polymer solution for cryopreservation of the biological sample of the present invention.
  • the present invention also provides a solidified body containing a biological sample and a matrix containing an aqueous solvent and a water-soluble polymer or a salt thereof, wherein the biological sample is compared with a solidified culture solution (in a medium) before solidification.
  • a solidified culture solution in a medium
  • the difference between the lightness in the color system and the lightness in the Munsell color system of the area occupied by the matrix is 3 or less, and the measurement conditions of (1) and (2) below using the differential scanning calorimeter (DSC) of the matrix
  • DSC differential scanning calorimeter
  • the endothermic amount of the endothermic peak in the temperature raising process is 0 J/g or 65% or less of the corresponding endothermic amount of the reference liquid made of water.
  • the DSC scanning was performed by (1) holding at 20° C. for 1 minute, lowering the temperature to ⁇ 80° C. at a temperature lowering rate of 5° C./min, and (2) holding at ⁇ 80° C. for 1 minute, then 10° C./min.
  • the temperature is raised to 20° C. at a heating rate of min, and the heating is performed.
  • the intrinsic viscosity ( ⁇ ) of the polymer aqueous solution containing the above aqueous solvent and the water-soluble polymer or its salt before freeze solidification is 0.20 dL/g or more and 0.95 dL/g or less.
  • the endothermic peak is not confirmed, or the temperature at which the endothermic peak is confirmed exceeds -1.4°C.
  • a solidified body having a temperature of 1° C. or lower is desirable.
  • no endothermic peak is confirmed here means that "the endothermic peak is not confirmed” unless the endothermic peak is confirmed in the range of -30°C to +5°C. This is because if water containing a water-soluble polymer exhibits an endothermic peak, it is in this range.
  • a metal salt, a halogen salt or a sulfate salt is desirable.
  • the metal salt is preferably an alkali metal salt or an alkaline earth metal salt.
  • the alkali metal or alkaline earth metal sodium, potassium, calcium or the like is selected.
  • the halogen chlorine, bromine or the like can be used.
  • the solidified product of the present invention does not contain dimethyl sulfoxide, which is an intracellular permeation type cryoprotectant for biological samples. Further, it is preferable that the solidified product of the present invention does not contain a cryoprotective agent such as ethylene glycol that is cytotoxic to a biological sample. These are harmful to the biological sample after thawing.
  • a cryoprotective agent such as ethylene glycol that is cytotoxic to a biological sample.
  • the solidified product of the present invention is preferably a solidified frozen product (frozen solidified product).
  • the solidified product of the present invention is preferably a solidified product containing a water-soluble polymer or a salt thereof in an amount of 0.1 w/v% or more and 20 w/v% or less.
  • the endothermic peak endothermic amount in the temperature rising process is 0 J/g, or It is preferably 40% or less of the corresponding endothermic amount of the reference liquid made of water.
  • the solidified body in which the biological sample contained in the solidified body is cells and the cells are mammalian cells is preferable.
  • the biological sample contained in the solidified body is cells, and the cells are mammalian mesenchymal stem cells, mammalian blood cells, or mammalian endothelial cells.
  • the solidified product of the present invention can be preferably thawed and used as a solution for biological sample administration. Since the solidified body of the present invention does not contain cell-penetrating and toxic compounds such as DMSO and ethylene glycol, and does not contain serum or serum-derived protein, it can be directly used as a biological sample administration solution after thawing. It is possible to add a protein that is not contaminated with bacteria or viruses. It is also possible to use compounds that are permeable to cells and toxic at low concentrations that do not impair cell function.
  • the amount of HGF produced by the biological sample contained in the solution for administration of the biological sample in which the solidified body of the present invention is melted is such that the biological sample in the solidified body containing DMSO instead of the water-soluble polymer or its salt is melted. It is preferably suppressed as compared with the amount of HGF produced later.
  • the amount of IL-10 produced by a biological sample contained in a solution for administration of a biological sample in which the solidified substance of the present invention is melted is DMSO instead of the water-soluble polymer or a salt thereof. Is preferably increased compared to the amount of IL-10 produced after thawing.
  • the polymer aqueous solution for cryopreservation of the biological sample of the present invention and the solidified body thereof do not contain a compound having cell permeability and cytotoxicity such as dimethyl sulfoxide or ethylene glycol. This is because these are toxic to cells after thawing.
  • the method for cryopreserving a biological sample using the aqueous polymer solution of the present invention comprises the step of preserving a biological sample by holding the aqueous polymer solution containing the biological sample at a temperature of ⁇ 27° C. or lower.
  • the method is preferred.
  • cryopreservation temperature in the cryopreservation method of a biological sample using the aqueous polymer solution of the present invention is not limited as long as it is -27°C or lower, but the upper limit is preferably -70°C or lower, It is preferably ⁇ 80° C. or lower.
  • the lower limit is preferably ⁇ 196° C. or higher, preferably ⁇ 150° C. or higher.
  • a preferred method is a cryopreservation method of a biological sample in which the step of cooling and solidifying the aqueous polymer solution containing the biological sample is performed by a slow freezing method at a cooling rate of 10°C/or less. Desirably, the step of cooling and solidifying the polymer aqueous solution containing the biological sample is performed by a slow freezing method at a cooling rate of 1° C./min or less.
  • the method of cryopreserving a biological sample in which the biological sample is cells, tissues, or a tissue-like substance that is a membrane or an aggregate is preferable.
  • the method for cryopreserving a biological sample in which the biological sample is a sperm, an egg, or a fertilized egg is preferable.
  • the “intrinsic viscosity ( ⁇ )” of the aqueous polymer solution containing the water-soluble polymer or salt used in the present invention is obtained by the following method and calculation formula.
  • a predetermined amount of NaCl is dissolved in ion-exchanged water at 30° C. to prepare a 0.2 M NaCl solution (standard solution).
  • the solid content obtained by removing the solvent from the solution is used as the sample of the water-soluble polymer or its salt.
  • the sample of the mixture is used as it is. Even a mixture of water-soluble polymer and impurities is used as a sample as it is.
  • the viscosities of the standard solution and the stock solution are measured, and the relative viscosity of the stock solution with respect to the standard solution is adjusted to be 2.0 to 2.4.
  • the stock solution at 30° C. is diluted to 5/4, 5/3, 5/2 times with the standard solution at 30° C., respectively.
  • Dividing the viscosities of the undiluted solution and the diluted solution by the viscosity of the standard solution is taken as the relative viscosity ( ⁇ r ) and the reduced viscosity is derived based on the following equation.
  • ⁇ sp water-soluble or reduced viscosity of salt thereof [mL/g]
  • ⁇ r relative viscosity of water-soluble polymer or salt thereof [ ⁇ ]
  • C concentration of water-soluble polymer or salt thereof [g /ML].
  • aqueous solvent used for dissolving the water-soluble polymer or salt thereof in the present invention examples include water, physiological solution, or sodium ion or potassium ion so that the osmotic pressure of body fluid or cell fluid is almost the same.
  • An isotonic aqueous solution whose salt concentration, sugar concentration and the like are adjusted by calcium ion or the like is preferable.
  • physiological saline phosphate buffered saline (PBS) which is a physiological saline having a buffering effect
  • Dulbecco's phosphate buffered saline Tris buffered saline ( Buffered Saline (TBS), HEPES buffered saline
  • balanced salt solution such as Hanks balanced salt solution, Ringer's solution, Ringer's lactate, Ringer's acetate, Ringer's bicarbonate, or D-MEM, E-MEM, ⁇ MEM, RPMI- 1640 medium
  • basal medium for animal cell culture such as Ham's F-12, Ham's F-10, M-199, general culture medium for various cells or tissues, commercially available medium, etc. it can.
  • a biological sample such as a cell is cryoprotected by highly toxic DMSO or ethylene glycol.
  • the biological sample can be cryopreserved while maintaining the viability and properties of the biological sample without using any substance.
  • the water-soluble polymer or its salt contained as a solute in the polymer aqueous solution of the present invention which is a cryoprotective agent for the polymer aqueous solution of the present invention, is not permeable to cell membranes, and therefore, when the biological sample such as cells is vitrified, It does not permeate into the sample and swell the living tissue, but rather promotes water removal from the cells.
  • aqueous polymer solution of the present invention by adjusting the molecular species and the degree of polymerization (molecular weight) of the polymer and/or by adding a low molecular weight or monomolecular compound to the aqueous solution of the water-soluble polymer, The intrinsic viscosity ( ⁇ ) of the aqueous solution of the molecule and the temperature and the amount of heat of the endothermic peak of melting during the heating process are adjusted.
  • the intrinsic viscosity ( ⁇ ) of the aqueous polymer solution is in the range of 0.20 dL/g or more and 0.95 dL/g or less
  • the calorific value of the exothermic peak measured by DSC is 0 J/g or standard water
  • the polymer aqueous solution for cryopreservation of the biological sample of the present invention is an intracellular non-penetrating cryopreservation liquid, and the polymer aqueous solution and its frozen solidified product are intracellular permeation type such as DMSO and ethylene glycol. Contains no chemicals. Therefore, the damage to the biological sample, the cytotoxicity, and the effect on the cell properties as reported for DMSO can be suppressed to a low level. That is, the properties of the biological sample in the biological sample during and after cryopreservation can be maintained.
  • the polymer aqueous solution for cryopreservation of the biological sample of the present invention and the frozen solidified product thereof are cryopreserved without preserving the survival rate and properties of the biological sample without using serum and/or serum-derived protein. Therefore, the biological sample is not contaminated with bacteria or viruses.
  • a frozen biological sample is used with a cytotoxic chemical substance such as DMSO or ethylene glycol, or a protein such as serum. It can be stored stably at a temperature of ⁇ 27° C. or lower, that is, in a deep freezer for a long period of time. It is possible to add a protein that is not contaminated with bacteria or viruses. It is also possible to use compounds that are permeable to cells and toxic at low concentrations that do not impair cell function.
  • cryopreservation temperature is not limited as long as it is ⁇ 27° C. or lower, but the upper limit is desirably ⁇ 70° C. or lower, preferably ⁇ 80° C. or lower.
  • the lower limit is preferably ⁇ 196° C. or higher, preferably ⁇ 150° C. or higher.
  • FIG. 3 is a graph showing the amount of IL-10 produced in primary human mesenchymal stem cells after cryopreservation using the test sample solution of the present invention.
  • FIG. 3 is a diagram showing an intracellular vitrification state using the test sample solution of Example 1. It is a figure which shows the brightness difference of an intracellular vitrification state.
  • FIG. 3 is a diagram in which the difference in brightness between intracellular vitrification states was quantified according to the Munsell brightness (0 to 10), and the difference in brightness between the solvent and the cells was determined.
  • a polymer aqueous solution for cryopreservation of a biological sample of the present invention contains a water-soluble polymer or a salt thereof, and is 0.20 dL/g or more and 0.95 dL/g or less. It has an intrinsic viscosity ( ⁇ ) in the range of.
  • the “intrinsic viscosity ( ⁇ )” of an aqueous solution containing a water-soluble polymer or a salt thereof used in the present invention means a value calculated from the following method and calculation formula.
  • Intrinsic viscosity measurement (1) A predetermined amount of NaCl is dissolved in ion-exchanged water at 30° C. to prepare a 0.2 M NaCl solution (standard solution). (2) Dissolve a sample of water-soluble polymer or its salt in a standard solution at 30°C to prepare a stock solution. When a sample of the water-soluble polymer or its salt is obtained as a solution, the solid content obtained by removing the solvent from the solution is used as the sample of the water-soluble polymer or its salt. In the case of a mixed sample containing a plurality of water-soluble polymers, the sample of the mixture is used as it is. Even if the water-soluble polymer contains impurities, use it as it is.
  • each water-soluble polymer is separated and fractionated, and then the solvent is removed from each solution to obtain a sample of each water-soluble polymer.
  • the substance of the water-soluble polymer is identified by HPLC, LC-MS, LC-IR or the like, and the viscosity average molecular weight is calculated as described later.
  • each component is separated and fractionated, and each water-soluble polymer is identified by HPLC, LC-MS, LC-IR, etc.
  • the polymer aqueous solution of the present invention is a cryopreservation liquid for appropriately preserving a biological sample when the biological sample is cryopreserved.
  • the intrinsic viscosity ( ⁇ ) of the polymer aqueous solution of the present invention is set to the range of 0.20 dL/g or more and 0.95 dL/g or less, the polymer aqueous solution of the present invention is formed by polymer chains in the cooling process.
  • the aqueous solvent molecules can be well trapped in the matrix. As a result, it is possible to limit the molecular motion of water in the aqueous solvent during cooling and solidify and/or freeze the water in a vitrified state without crystallizing.
  • the freezing method called the vitrification method increases the salt concentration in the residual solution by removing the solute (cryoprotectant to prevent frost damage) from the crystals when the solution freezes, and It is a method of dehydrating the inside of the cell and vitrifying the inside of the cell by generating an osmotic pressure difference, and is applied to cells having a particularly low survival rate after thawing.
  • the concentration of the solute (cryoprotectant) is increased and the cooling rate is increased in order to make the water more vitrified.
  • the osmotic pressure difference damage to cells also increases, and that recrystallization during lysis causes cells to be damaged.
  • the inside of the biological sample when used as a cryopreservation solution for biological samples such as cells, the inside of the biological sample is dehydrated by the action of the polymer chains of the water-soluble polymer dissolved in the aqueous polymer solution. Since the inside of the biological sample is vitrified, the formation of ice crystals in the biological sample is suppressed or prevented, and further, the osmotic shock in the biological sample during freezing, which is a problem in the conventional vitrification method, is weakened. be able to. Therefore, it is not necessary to contain a chemical substance such as dimethyl sulfoxide (DMSO) or ethylene glycol having cytotoxicity, and the polymer aqueous solution of the present invention does not contain DMSO and/or ethylene glycol.
  • DMSO dimethyl sulfoxide
  • ethylene glycol having cytotoxicity
  • the water molecules of the solvent are present in the polymer network matrix during freezing, and free water or bound water (a state in which the water molecules are entangled and trapped in the polymer) It is thought that it exists in the state of. Further, it is considered that the water molecules that have been eliminated from the inside of cells due to the osmotic pressure difference are replaced with macromolecules around the biological sample such as cells. In order to realize this state, it is necessary to adjust the intrinsic viscosity of the polymer aqueous solution to 0.20 to 0.95 dL/g.
  • the intrinsic viscosity of the aqueous polymer solution is too small, the polymer diffuses, and water molecules cannot be trapped and caught in the water-soluble polymer network. On the other hand, if the intrinsic viscosity of the aqueous polymer solution is too high, the substitution with water molecules in the vicinity of the biological sample does not proceed. In addition, handling of the biological sample may be deteriorated when the biological sample is included in the aqueous polymer solution for cryopreserving the biological sample.
  • the intrinsic viscosity of the polymer aqueous solution By setting the intrinsic viscosity of the polymer aqueous solution to be in the range of 0.20 dL/g or more and 0.95 dL/g or less, when the polymer aqueous solution of the present invention is cooled, it is a glass state that is amorphous in a frozen state. Is stabilized. Therefore, the cells are not easily damaged by cooling and freezing, and the cells can be stably and efficiently cryopreserved. Cell destruction due to the formation of ice crystals is also unlikely to occur. Therefore, the cell survival rate in the biological sample after thawing the frozen biological sample of the present invention is high.
  • the intrinsic viscosity ( ⁇ ) of the aqueous polymer solution is about 0.32 dL/g or more and about 0.65 dL/g or less.
  • the intrinsic viscosity of the aqueous polymer solution can be adjusted by selecting the molecular species of the water-soluble polymer and the degree of polymerization (viscosity average molecular weight).
  • the water-soluble polymer in the present invention a polymer containing a monomer having a hydrophilic group as a repeating unit can be used.
  • the hydrophilic group is, for example, a hydroxyl group and a carboxylic acid group or a salt thereof.
  • the water-soluble polymer may contain, as a repeating unit, a nitrogen-containing monomer having an optionally substituted amino group or an optionally substituted amide group.
  • the structure of the water-soluble polymer has a hydroxyl group at the equatorial position. This is because it is considered that the water of the solvent can be better trapped in the matrix formed of polymer chains when frozen.
  • the monomer having a hydrophilic group is, for example, a sugar residue.
  • the polymer in the present invention may be a water-soluble polymer containing a polymer in which a sugar residue is linked by a glycoside bond as a repeating unit and a derivative thereof.
  • the sugar residue may be a monosaccharide or a monosaccharide in which a hydroxyl group and/or a hydroxymethyl group of the monosaccharide is substituted, for example, a hydroxyl group and/or a hydroxymethyl group may be a carboxyl group, an amino group or an N-acetyl group. Examples thereof include, but are not limited to, monosaccharides substituted with at least one substituent selected from the group consisting of an amino group, a sulfooxy group, a methoxycarbonyl group and a carboxymethyl group.
  • Examples of monosaccharides include triose, tetrose, pentose, hexose and heptose.
  • Examples of pentoses include ribose, arabinose, xylose, lyxose, xylulose, ribulose, deoxyribose and the like.
  • Examples of the hexose include glucose, mannose, galactose, fructose, sorbose, tagatose, fucose, fuculose and rhamnose.
  • Urine may be uronic acid as a monosaccharide substituted with a carboxyl group.
  • uronic acid include glucuronic acid, iduronic acid, mannuronic acid and galacturonic acid.
  • monosaccharides substituted with an amino group include amino sugars.
  • amino sugars include glucosamine, galactosamine, mannosamine and muramic acid.
  • monosaccharide substituted with an N-acetylamino group include N-acetylglucosamine, N-acetylmannosamine, N-acetylgalactosamine and N-acetylmuramic acid.
  • Examples of the monosaccharide substituted with a sulfoxy group include galactose-3-sulfate.
  • Examples of monosaccharides having a plurality of substituents include N-acetylglucosamine-4-sulfate, iduronic acid-2-sulfate, glucuronic acid-2-sulfate, N-acetylgalactosamine-4-sulfate, neuraminic acid and N. -Acetylneuraminic acid and the like.
  • the water-soluble polymer in the present invention is a polymer containing the above-mentioned monosaccharide as a repeating unit.
  • the water-soluble polymer in the present invention may be a polymer containing an optionally substituted pentose, hexose or uronic acid or a combination thereof as a repeating unit.
  • the water-soluble polymer in the present invention may include a 6-membered ring structure in its main chain. This is because a polysaccharide or the like having a six-membered ring structure in its main chain may easily form a polymer network structure around cells via hydrogen bonds.
  • the water-soluble polymer in the present invention may be an alternating copolymer of a monomer having a hydrophilic group and a nitrogen-containing monomer.
  • the nitrogen-containing monomer may be, for example, an amino sugar.
  • the water-soluble polymer in the present invention can be glycosaminoglycan. It may also be a sulfated polysaccharide in which one or more hydroxyl groups have been replaced by sulfoxy groups.
  • Polymers that can be used in the present invention include, but are not limited to, hyaluronic acid, dextran, pullulan, and chondroitin sulfate.
  • the water-soluble polymer or its salt used in the present invention may be of natural origin or may be chemically synthesized.
  • a commercially available water-soluble polymer or its salt may be used as it is.
  • a naturally occurring polymer compound with a higher molecular weight or a commercially available polymer compound is used to obtain a cleavage product by subjecting it to hydrolysis, enzyme treatment, subcritical treatment, etc., and adjusting the molecular weight to obtain the product. It may be a water-soluble polymer in the invention.
  • each monomer may be a naturally-occurring monomer, a naturally-occurring monomer may be modified or substituted for use, or a chemically synthesized monomer may be used.
  • the monomer contained in the water-soluble polymer in the present invention is a biological constituent component. It is considered that when an aqueous polymer solution containing a water-soluble polymer is used as a cryopreservation solution for biological samples, it has low cytotoxicity. Further, the process required for the conventional cryopreservation solution, such as washing and removing the cryoprotective agent contained in the cryopreservation solution from the biological sample after thawing, may be unnecessary or simplified.
  • the water-soluble polymer or salt thereof according to the present invention has a viscosity average molecular weight of more than 3000 and 500000 or less.
  • a water-soluble polymer having a viscosity average molecular weight of this level or a salt thereof when the aqueous solution is cooled, the amorphous glass state in the frozen state of the solvent is stabilized. Therefore, the cells are not easily damaged by cooling and freezing, and the cells can be stably and efficiently cryopreserved. Therefore, the viability of cells in the biological sample after thawing the biological sample after cryopreservation is high.
  • the viscosity average molecular weight of the water-soluble polymer is 3,000 or less, vitrification may not easily occur satisfactorily.
  • the viscosity average molecular weight of the water-soluble polymer is larger than 500000, the viscosity may remarkably increase, the solubility may decrease, or the prepared solution may foam and the handling property may deteriorate.
  • the viscosity average molecular weight is preferably 10,000 or more, for example.
  • the “viscosity average molecular weight” of the water-soluble polymer or salt used in the present invention is calculated by the following method and formula using the intrinsic viscosity ( ⁇ ) calculated from the above method and formula. Required by.
  • Viscosity average molecular weight is calculated from the intrinsic viscosity.
  • the viscosity average molecular weight M is obtained from the above-mentioned Mark Hoing Sakurada's formula by the intrinsic viscosity derived from the measurement and the values of K and ⁇ disclosed in the literature.
  • K the water-soluble polymer
  • K and ⁇ are numerical values that vary depending on the type of polymer, and the values of K and ⁇ are disclosed and published in many published documents such as “Handbook of Polymer Materials” (edited by The Society of Polymer Science, Japan).
  • the viscosity average molecular weight can be calculated from the intrinsic viscosity ( ⁇ ) by using the existing value.
  • the molecular weight calculated by this method is taken as the viscosity average molecular weight.
  • the molecular weight is clearly specified from the structural formula, so the molecular weight specified from the structural formula is simulated as the viscosity average molecular weight. And treat.
  • the hydrophilic group of the water-soluble polymer or salt thereof used in the present invention is not modified, or even if modified, 50% or less of the total number of hydrophilic groups, that is, a substituent is introduced into the polymer chain. It is desirable that the content of the hydrophilic groups is 50% or less of the total number of hydrophilic groups. It is presumed that the hydrophilic groups of the water-soluble polymer, particularly the OH group, NH 2 group, and COOH group, contribute to protection of the biological sample, vitrification of the solvent, and substitution with water molecules around the biological sample. This is because it is more advantageous to improve the survival rate of the biological sample if these functional groups are not modified.
  • the reagents used for modifying the hydrophilic group of the polymer may adversely affect the cryoprotective effect of the polymer, or may cause a biomedical problem for regenerative medicine. There is a risk that it may become unusable as a cryopreservation solution for cryopreservation of samples.
  • Examples of the water-soluble polymer salt used in the present invention include metal salts, halogen salts and sulfates.
  • the metal salt is preferably an alkali metal salt or an alkaline earth metal salt.
  • the alkali metal or alkaline earth metal sodium, potassium, calcium or the like is selected.
  • the halogen chlorine, bromine or the like can be used.
  • aqueous solvent for the aqueous polymer solution of the present invention examples include water, physiological solutions, or salt concentrations and sugars such as sodium ions, potassium ions, and calcium ions so as to be almost the same as the osmotic pressure of body fluids and cell fluids.
  • An isotonic aqueous solution whose concentration is adjusted is preferable.
  • physiological saline phosphate buffered saline (PBS) which is a physiological saline having a buffering effect
  • Dulbecco's phosphate buffered saline Tris buffered saline
  • examples include, but are not limited to, balanced salt solutions such as Tris Buffered Saline (TBS), HEPES buffered saline, and Hanks balanced salt solution, Ringer's solution, Ringer's lactate, Ringer's acetate, and Ringer's bicarbonate.
  • the solvent may contain other optional components such as an isotonicity agent, a chelating agent, and a solubilizing agent, as long as the effects of the present invention are not impaired.
  • the term “optional component” means a component that may or may not be included.
  • the solvent may be a 5% glucose aqueous solution or the like.
  • a medium for cell culture may be used as an aqueous solvent for preparing the aqueous polymer solution of the present invention.
  • the culture medium is not particularly limited and includes, for example, commercially available medium, D-MEM, E-MEM, ⁇ MEM, RPMI-1640 medium, Ham's F-12, Ham's F-10, M-199, etc.
  • the basal medium for animal cell culture, and general culture medium for various cells or tissues can be exemplified.
  • the biological sample such as cells after culturing may be suspended and cryopreserved in the polymer aqueous solution of the present invention, or
  • the water-soluble polymer or a salt thereof according to the invention may be added to a culture solution containing a biological sample such as a cell culture solution or a cell suspension after culturing at a desired concentration to carry out a cryopreservation step.
  • the pH of the aqueous polymer solution of the present invention can be adjusted, if necessary.
  • a hydrophilic group of the monomer having a hydrophilic group is a carboxylic acid group or the like
  • a polymer aqueous solution containing a water-soluble polymer obtained by polymerizing such a monomer may exhibit acidity.
  • the pH of the polymer aqueous solution to a neutral solution, the solution can be more suitable for the survival of the cells to be cryopreserved and the cell survival rate is improved.
  • the salt used for pH adjustment is not limited, and those commonly used for pH adjustment of aqueous solutions can be used.
  • the results of thermal analysis of the polymer aqueous solution of the present invention by differential scanning calorimetry (DSC) show that the polymer aqueous solution of the present invention has the following (1) to (2):
  • the heat generation amount of the exothermic peak in the temperature decreasing process is 0 J/g, or 65% or less of the corresponding heat generation amount of the reference liquid made of water, preferably water. Is 40% or less of the corresponding calorific value of the standard liquid consisting of.
  • DSC measurement conditions (1) Hold at 20°C for 1 minute, then cool down to -80°C at a cooling rate of 5°C/min, and (2) Hold at -80°C for 1 minute, then hold at 10°C/min to 20°C. Temperature rising.
  • the calorific value of the exothermic peak in the temperature-decreasing process in the DSC measurement is smaller than the corresponding calorific value of the reference solution made of water in the aqueous polymer solution of the present invention (less than 100%), that is, the frozen aqueous polymer solution. It means that bound water and free water that do not form ice crystals remain. That is, in the aqueous polymer solution of the present invention, the substitution of water for the water-soluble polymer and/or low-molecular-weight saccharide progresses, water molecules are trapped in the matrix of the water-soluble polymer, and the molecular motion of water is prevented. I have a limit.
  • cryopreservation of a biological sample using the polymer aqueous solution of the present invention can suppress or prevent the destruction of cells due to the formation of ice crystals.
  • the polymer aqueous solution of the present invention can not only suppress or prevent ice crystal formation in the cooling process, but also suppress recrystallization of water in the subsequent temperature rising process, that is, at the time of melting. That is, in the polymer aqueous solution of the present invention, it is considered that the frozen glass state of the biological sample is stabilized.
  • the polymer aqueous solution of the present invention does not require addition of protein components such as human- and bovine-derived serum and serum-derived components (eg, albumin) and has high cell protection. Show the effect. Therefore, it is considered that there is no concern about infectious diseases, and there is no effect of lot-to-lot differences due to biologics. It is possible to add a protein that is not contaminated with bacteria or viruses.
  • protein components such as human- and bovine-derived serum and serum-derived components (eg, albumin) and has high cell protection. Show the effect. Therefore, it is considered that there is no concern about infectious diseases, and there is no effect of lot-to-lot differences due to biologics. It is possible to add a protein that is not contaminated with bacteria or viruses.
  • the matrix contained in the frozen solidified body of the present invention shows that the matrix of the frozen solidified body of the present invention is analyzed by differential scanning calorimetry (DSC).
  • DSC differential scanning calorimetry
  • the endothermic peak endothermic amount in the temperature rising process is 0 J/g, or a standard consisting of water. It is 65% or less of the corresponding endothermic amount of the liquid, and preferably 40% or less of the corresponding endothermic amount of the reference liquid made of water.
  • DSC measurement conditions (1) Hold at 20°C for 1 minute, then cool down to -80°C at a cooling rate of 5°C/min, and (2) Hold at -80°C for 1 minute, then hold at 10°C/min to 20°C. Temperature rising.
  • the endothermic amount of the endothermic peak in the temperature rising process in the DSC measurement is smaller than the corresponding endothermic amount of the reference solution of water in the matrix of the present invention (less than 100%) means that ice crystals in the frozen matrix. It means that there is still bound water or free water that does not form. That is, in the aqueous solution of the water-soluble polymer that forms the matrix of the present invention, the substitution of water for the water-soluble polymer and/or low-molecular-weight saccharides proceeds, and water molecules are trapped in the matrix of the water-soluble polymer. That is, the molecular motion of water is restricted.
  • the frozen glass state of the biological sample contained in the frozen solidified body is stabilized. Since the glass state is more stabilized in this way, the biological sample stored in the frozen solidified body of the present invention requires the addition of a human or bovine-derived serum or a protein component such as a serum-derived component (eg, albumin). It shows a high cytoprotective effect. Therefore, it is considered that the biological sample after thawing does not have a risk of infectious diseases and is not affected by the difference between lots due to the biologics. It is possible to add a protein that is not contaminated with bacteria or viruses.
  • a human or bovine-derived serum or a protein component such as a serum-derived component (eg, albumin). It shows a high cytoprotective effect. Therefore, it is considered that the biological sample after thawing does not have a risk of infectious diseases and is not affected by the difference between lots due to the biologics. It is possible to add a protein that is not contaminated with bacteria or viruses.
  • the amount of heat generation and the amount of heat absorption are adjusted by selecting the molecular species of the water-soluble polymer and/or adjusting the amount added. For example, hyaluronic acid has a small calorific value and an endothermic amount, whereas pullulan has a large calorific value and an endothermic amount. Further, when the content of the water-soluble polymer is increased, both the calorific value and the endothermic value decrease.
  • the polymer aqueous solution for cryopreservation of the biological sample of the present invention is obtained by DSC in the DSC curve of the temperature rising process obtained by the following measurement conditions (1) to (2) using a differential scanning calorimeter (DSC).
  • the measured endothermic peak is not confirmed, or the temperature at which the endothermic peak is confirmed is preferably higher than -1.4°C and 1.1°C or lower.
  • DSC measurement conditions (1) Hold at 20°C for 1 minute, then cool down to -80°C at a cooling rate of 5°C/min, and (2) Hold at -80°C for 1 minute, then hold at 10°C/min to 20°C. Temperature rising.
  • the endothermic peak which is presumed to be caused by the melting of water is not observed in the process of raising the temperature of the polymer aqueous solution, or when observed, is -1.4°C.
  • the present inventors have found that it is desirable for the cryopreservation liquid to have the opposite characteristic of this technical common sense for good vitrification of the biological sample during freezing.
  • the phenomenon that the melting point (freezing point) is lower than that of water occurs when the solute is dispersed in the water matrix, but in the polymer aqueous solution of the present invention, conversely, it is water-soluble.
  • Water molecules are present in a polymer network matrix in an aqueous solvent formed of polymers, and water exists as free water in a state where water does not freeze in the matrix, or even if water freezes It freezes in the state of being caught and bound (water bound).
  • the endothermic peak of melting of water is not observed in the temperature rising process, or that it is observed at a temperature close to the temperature at which the endothermic peak of pure water is observed.
  • the present inventors adjusted the endothermic peak by adjusting the sugar solution having a lower molecular weight than the water-soluble polymer in the aqueous polymer solution containing the water-soluble polymer or a salt thereof and the frozen solidified product in the present invention. It has also been found that this can be done by adding the salt.
  • the low molecular weight saccharide or its salt is retained in the polymer matrix by hydrogen bonding due to the hydrophilic group in the water-soluble polymer of the present invention. It is considered that the water molecules near the cell membrane are replaced with the low molecular weight saccharides by the presence of the high molecular weight saccharides or polymers holding the salts around biological samples such as cells. Therefore, it is considered that the water discharged from the biological sample such as cells due to the osmotic pressure difference during freezing to replace the high molecular weight saccharides in addition to the polymer around the biological sample such as cells.
  • the addition of low molecular weight sugars or salts thereof aids the drainage of water from within the biological sample and, by substitution with water molecules, suppresses or prevents the formation and growth of ice crystals near the cell membrane.
  • the intrinsic viscosity of the polymer aqueous solution of the present invention to be in the range of 0.20 dL/g or more and 0.95 dL/g or less, the substitution of water molecules with low molecular weight saccharides can be promoted. As a result, cell membrane damage during freezing can be significantly suppressed.
  • the low molecular weight saccharide or its salt can function as a component for cell protection in the aqueous polymer solution and freeze-solidified product of the present invention.
  • the intrinsic viscosity of the aqueous polymer solution of the present invention is too low, low-molecular-weight saccharides diffuse in the aqueous polymer solution, and water molecules cannot be efficiently entangled and caught in the water-soluble polymer network.
  • the intrinsic viscosity of the aqueous polymer solution is too high, it is considered that the replacement of the low molecular weight sugar with the water molecule does not proceed efficiently.
  • the volume of the sample contracts as compared with the biological sample before freezing.
  • the orthogonal projection area of the biological sample in the state of being solidified at ⁇ 80° C. in the aqueous polymer solution of the present invention is compared with the orthogonal projection area of the biological sample in the culture solution (in the medium) before temperature decrease (before freezing). , 1/3.5 or more and less than 8/10.
  • the inside of the cell is well dehydrated during freezing. This significantly reduces the damage to the cells when frozen and the thawed cells show a high viability.
  • the "orthogonal projection area" of the biological sample when observing the biological sample on the microscope cooling stage, an image is taken from above the normal direction of the cooling stage, and the biological sample in the image is It means the area calculated by the image analysis software ImageJ (https://imagej.nih.gov/ij/).
  • the hydrophilic group of the water-soluble polymer or salt thereof used in the present invention is also unmodified or hydrophilic even if modified so as to retain the low-molecular-weight sugar or salt thereof in the matrix. It is desirable that 50% or less of the total number of groups, that is, 50% or less of the total number of hydrophilic groups, in which a substituent is not introduced into the polymer chain or even if it is introduced. If the hydrophilic group of the water-soluble polymer is modified, the retention effect of the low-molecular weight saccharide or its salt by the hydrophilic group will decrease, and even if the low-molecular weight saccharide coexists, the biological sample will survive. It may not be possible to sufficiently contribute to the improvement of the rate. Therefore, for example, it may not be preferable to modify the OH group or NH 2 group of the water-soluble polymer with a carboxylic acid or the like.
  • the low molecular weight saccharide or salt thereof in the present invention is, for example, a saccharide or salt thereof having a viscosity average molecular weight of 3000 or less.
  • a saccharide or salt thereof having a viscosity average molecular weight of 3000 or less.
  • it may be a monosaccharide, disaccharide, oligosaccharide or the like having a viscosity average molecular weight of 1,000 or less.
  • the low-molecular weight saccharide in the present invention may be, for example, the monosaccharide described above as a monomer constituting the water-soluble polymer in the present invention.
  • the low molecular weight saccharide is glucose, fructose, galactose or uronic acid in which the alcohol group is oxidized, or an amino sugar in which the alcohol group is substituted with an amino group, sucrose, trehalose, or a polymer or a combination thereof. .
  • the low molecular weight saccharide may also be, for example, a macromolecule such as a fragment of hyaluronic acid, dextran, pullulan, or chondroitin sulfate.
  • low-molecular-weight saccharides include, for example, monosaccharides constituting glycosaminoglycan cleavage products (fragments), that is, glycosaminoglycans, disaccharides thereof, or It can be the oligosaccharide.
  • fragments monosaccharides constituting glycosaminoglycan cleavage products (fragments), that is, glycosaminoglycans, disaccharides thereof, or It can be the oligosaccharide.
  • hyaluronic acid it is desirable to use one having a viscosity average molecular weight of 1000 or less.
  • the low molecular weight saccharide is a cleavage product of hyaluronic acid. Therefore, preferably, the low-molecular-weight saccharide or salt thereof in the present invention is glucuronic acid or N-acetylglucosamine, or a disaccharide or oligo consisting thereof, or a salt thereof.
  • the saccharide may be glucuronic acid or a modified compound thereof, or a disaccharide or oligosaccharide thereof, or a salt thereof.
  • the "cleavage product" in the present invention is a compound having a smaller molecular weight than the original polymer, which is considered to be obtained when the polymer is subjected to treatment such as hydrolysis, enzyme treatment, and subcritical treatment.
  • the polymer used in the present invention may be a water-soluble polymer obtained by treatment of a larger polymer compound as described above, and the low molecular weight saccharide used in the present invention is It may be a low molecular weight saccharide obtained by treating a water-soluble polymer.
  • the cleavage products may be monomers and/or polymers of varying degrees of polymerization of the monomers that are constituents of the original polymer and/or mixtures thereof.
  • “Subcritical treatment” means contacting a subcritical fluid as an extraction solvent in a subcritical state under a predetermined temperature and a predetermined pressure with a raw material to be extracted.
  • a subcritical fluid for example, water exhibits a state of being neither liquid nor gas when the pressure is raised to 22.12 MPa or higher and the temperature is raised to 374.15° C. or higher. This point is called the critical point of water, and hot water having a temperature and pressure near the critical point is called subcritical water.
  • the condition for subcritical treatment is, for example, a temperature of 150° C. or higher and 350° C.
  • the subcritical treatment pressure can be a saturated vapor pressure of each temperature or higher, for example, 0. It can be set to 0.5 MPa or more and 25 MPa or less.
  • the hydrolysis or enzyme treatment is not particularly limited, and reagents and treatment methods that are commonly used can be used without problems.
  • the water-soluble polymer and the low-molecular-weight saccharide in the present invention may be simultaneously obtained by a single subcritical treatment. That is, the water-soluble polymer and the low molecular weight saccharide used in the present invention have a first molecular weight distribution in a molecular weight range of 3000 or more and 500000 or less as a viscosity average molecular weight, and a viscosity average molecular weight of 3000 or less. It may be a subcritically processed polymer compound having a second molecular weight distribution within the molecular weight range.
  • a polymer which is a polysaccharide having a viscosity average molecular weight of more than 500,000 is dissolved in water, and then extraction treatment is performed under subcritical conditions of water. Accordingly, the step of obtaining the water-soluble polymer and the low-molecular weight saccharide in the present invention may be included. However, of course, the water-soluble polymer and the low-molecular weight saccharide in the present invention may be used in combination in which they are treated in separate treatment steps and/or separated based on the molecular weight.
  • the endothermic peak of the low-molecular-weight saccharide or salt thereof in the present invention is observed at a temperature lower than the temperature (0.7° C.) at which the endothermic peak is observed in DSC measurement in pure water.
  • hyaluronic acid having a viscosity average molecular weight of 1000 or less has an endothermic peak temperature of -8.1°C, which is lower than the endothermic peak temperature of 0.7°C of pure water.
  • the water-soluble polymer or salt thereof in the present invention is, for example, hyaluronic acid having a viscosity average molecular weight of 15,000, an endothermic peak temperature of -1.4°C, and pullulan having a viscosity average molecular weight of 373,000 at 1.49°C. is there.
  • hyaluronic acid having a viscosity average molecular weight of 15,000
  • an endothermic peak temperature of -1.4°C an endothermic peak temperature of -1.4°C
  • pullulan having a viscosity average molecular weight of 373,000 at 1.49°C.
  • the endothermic peak temperature of the water-soluble polymer can be adjusted to be near the endothermic peak temperature of pure water of 0.7°C.
  • the low-molecular-weight saccharide or salt thereof having such an effect is not limited to hyaluronic acid having a viscosity-average molecular weight of 1,000 or less. If the viscosity average molecular weight is 1,000 or less, the same effect can be obtained with other saccharides such as sucrose and trehalose, instead of hyaluronic acid.
  • the low molecular weight saccharide used in the present invention exists between water molecules to inhibit water ice crystallization, or cooperates with a water-soluble polymer network to trap water in the network. It acts either in water or in free water) to inhibit ice crystallization of water in the first place, so that the polymer aqueous solution and the frozen solidified product of the present invention can behave like pure water. It is speculated that they are doing it.
  • the polymer aqueous solution and the frozen solidified product of the present invention do not contain a permeation type compound in cells, it is considered that the cytotoxicity when contacted with a biological sample for cryopreservation of the biological sample is low. Furthermore, since it is not necessary to increase the concentration of solute to limit the molecular movement of water, it is considered that the damage to cells is low. In addition, the low-molecular-weight saccharides or salts thereof added have a cytoprotective effect of rapidly replacing water discharged from the cells around the cells during freezing, so that the properties of the cells should not be changed during cryopreservation. Conceivable. Therefore, it is considered that the aqueous polymer solution and the freeze-solidified product of the present invention allow cryopreservation of a biological sample while maintaining the characteristics of cells.
  • the polymer aqueous solution and freeze-solidified product of the present invention do not require a large cooling rate for reducing osmotic shock during cooling. Therefore, according to the aqueous polymer solution and freeze-solidified product of the present invention, a biological sample can be efficiently cryopreserved by a simple method with reduced toxicity, and the cells thawed after cryopreservation have high survival. Indicates the rate.
  • the aqueous polymer solution and freeze-solidified product of the present invention contain a water-soluble polymer or a salt thereof at a concentration of about 0.1 w/v% or more and about 20 w/v% or less. If the concentration of the water-soluble polymer is too low, the solvent portion may not be vitrified well.
  • the concentration of the water-soluble polymer or its salt is preferably 1 w/v% or more, particularly preferably 5 w/v% or more. Further, at a concentration higher than 20 w/v%, the viscosity becomes too high, which may deteriorate the handling property.
  • the concentration of the polymer or its salt is 5 w/v% or more and 20 w/v% or less.
  • the content ratio of the low molecular weight saccharide or its salt in the aqueous polymer solution of the water-soluble polymer and the low molecular weight saccharide is about 10:1. It is preferable to include If the concentration of the saccharide or its salt is too low, the effect of cell protection by the low molecular weight saccharide may not be sufficiently obtained. Further, even if the saccharide is added at a concentration 1/10 or more that of the water-soluble polymer, it is difficult to obtain a further effect as a cell protective component.
  • the aqueous polymer solution and the frozen solidified body of the present invention showed that the glass transition temperature was around -23°C ⁇ 4°C. It is desirable to have In the polymer aqueous solution and freeze-solidified product of the present invention, vitrification is realized by entangling water molecules in a network of water-soluble polymers and capturing them during freezing, as described above. Therefore, it does not require a high cooling rate for cell protection during cooling, which is required in the conventional vitrification method. Therefore, after the biological sample is included in the aqueous polymer solution of the present invention, it is cooled to ⁇ 27° C.
  • aqueous polymer solution of the present invention containing a biological sample in a freezing treatment container or the like and leaving it in a deep freezer at ⁇ 80° C.
  • cells and tissues to be preserved are stably frozen and preserved. be able to.
  • a special container for liquid nitrogen cryopreservation and preparation of liquid nitrogen can be dispensed with. Therefore, the operation related to the cryopreservation of the biological sample can be significantly simplified.
  • the polymer aqueous solution and freeze-solidified product of the present invention can also suppress recrystallization of water during thawing
  • the use of the polymer aqueous solution of the present invention enables freezing, storage, and thawing of biological samples. A series of steps can be easily and efficiently performed without requiring a special procedure.
  • the polymer aqueous solution and the freeze-solidified product of the present invention function as a non-penetrating cryopreservation liquid and a freeze-solidified product obtained by freezing the cryopreservation liquid, it is frozen using the polymer aqueous solution and the freeze-solidified product of the present invention.
  • the biological sample to be stored is not particularly limited. It can be used for cryopreservation of various types of cells. Further, the origin of cells is not particularly limited. INDUSTRIAL APPLICABILITY
  • the aqueous polymer solution and freeze-solidified product of the present invention can effectively suppress or prevent ice crystal formation and recrystallization at the time of freezing and thawing, and thus can be favorably used for mammalian cells having a complicated structure. Can be done.
  • the polymer aqueous solution and the frozen solidified product of the present invention are known to have a large obstacle during freezing as compared with general cells for culture, and the so-called conventional slow freezing method causes a remarkable decrease in the survival rate. It can be particularly preferably used for cryopreservation of unavoidable stem cells, germ cells such as early embryos, eggs, sperms, and fertilized eggs.
  • the polymer aqueous solution and the freeze-solidified product of the present invention do not contain a chemical substance such as DMSO or ethylene glycol that can function as a differentiation factor, they can be used for preservation of cells that need to be maintained undifferentiated. Also, stem cells for regenerative medicine can be cryopreserved without the risk of differentiation.
  • the polymer aqueous solution and freeze-solidified product of the present invention are excellent in the effect of stabilizing the glass state in the frozen state, so that it is known that it is difficult to store cells such as eggs having a large cell size. It can also be used for cryopreservation of cells and fertilized eggs, and organized cell structures, tissues and tissue-like substances such as tissues obtained by regenerative medicine.
  • the aqueous polymer solution and freeze-solidified product of the present invention can be used for a biological sample selected from cells, tissues, or tissue-like substances such as membranes or aggregates, and can achieve high survival rate. ..
  • the polymer aqueous solution and freeze-solidified product of the present invention regardless of primary cells or established cells, are mesenchymal stem cells; hematopoietic stem cells; neural stem cells; somatic stem cells such as bone marrow stem cells and reproductive stem cells; blood cells
  • cryopreservation of primate stem cells which are considered to be less freeze-tolerant than mice, cryopreservation of tissues for transplantation, and cryopreservation of germ cells in reproductive medicine Can be used.
  • the water-soluble polymer in the polymer aqueous solution and the frozen solidified product of the present invention is, for example, a water-soluble polymer which is a biological constituent
  • it is used for cell administration as it is by thawing a frozen and preserved biological sample. It is also possible to use it as a solution.
  • a tissue or a tissue-like substance it is possible to add such a polymer aqueous solution of the present invention at the stage of assembling and freeze-store as it is. This will reduce post-transplant problems.
  • the preferred water-soluble polymer for the aqueous polymer solution and freeze-solidified product of the present invention is hyaluronic acid.
  • hyaluronic acid having a viscosity average molecular weight of more than 3,000, more preferably more than 5,000 and less than 60,000, more preferably less than 20,000.
  • the cryopreservation method using the polymer aqueous solution of the present invention comprises a step of including a biological sample in the polymer aqueous solution used for cryopreservation of the biological sample of the present invention, and cooling the polymer aqueous solution containing the biological sample. And the process of solidifying.
  • the polymer aqueous solution of the present invention used in the cryopreservation method using the polymer aqueous solution of the present invention contains a water-soluble polymer or a salt thereof, and has a content of 0.20 dL/g or more and 0.95 dL/g or less.
  • the endothermic peak measured by DSC is confirmed in the DSC curve of the temperature rising process which has an intrinsic viscosity ( ⁇ ) and is obtained under the following measurement conditions (1) and (2) using a differential scanning calorimeter (DSC). It is desirable that the temperature not to be observed or the endothermic peak is confirmed exceeds ⁇ 1.4° C. and is 1.1° C. or less.
  • the orthogonal projection area of the biological sample in the state of being solidified at ⁇ 80° C. in the aqueous polymer solution is 8/compared with the orthogonal projection area of the biological sample in the culture solution (in the medium) before the temperature is lowered (before freezing). It is reduced to less than 10, preferably 1/3.5 or more.
  • the polymer aqueous solution of the present invention used in the cryopreservation method using the polymer aqueous solution of the present invention has a glass transition temperature in the vicinity of -23°C ⁇ 4°C. Since it is sometimes realized by entanglement of water molecules in a water-soluble polymer network, the cryopreservation method using the aqueous polymer solution of the present invention does not require a high cooling rate. Therefore, after the biological sample is included in the aqueous polymer solution of the present invention, it is cooled to ⁇ 27° C. or lower, which is the glass transition temperature of the aqueous polymer solution of the present invention, for example, the aqueous polymer solution of the present invention containing the biological sample. Simply put the cells in a freezing treatment container etc. and leave them in a deep freezer at -80°C to stably freeze the cells and tissues to be preserved while maintaining the viability, and store them as a frozen solidified body. can do.
  • cryopreservation temperature is not limited as long as it is ⁇ 27° C. or lower.
  • the upper limit is desirably ⁇ 70° C. or lower, preferably ⁇ 80° C. or lower.
  • the lower limit is desirably -196°C or higher, preferably -150°C or higher.
  • the cryopreservation method using the polymer aqueous solution of the present invention is the slow freezing method. That is, in the freezing method using the aqueous polymer solution of the present invention, the cooling rate is preferably 10° C./min or less. More preferably, the cooling rate is about 1° C./min or less. If the cooling rate is within this range, it is considered that intracellular dehydration is moderately carried out, vitrification of intracellular fluid is favorably carried out, and a high cell cryoprotective effect is obtained.
  • the pH of the polymer aqueous solution of the present invention may be adjusted, if necessary, before contact with a biological sample.
  • a hydrophilic group of the monomer having a hydrophilic group is a carboxylic acid group or the like
  • a polymer aqueous solution containing a water-soluble polymer obtained by polymerizing such a monomer may exhibit acidity.
  • the saccharide has a carboxylic acid group or the like
  • the aqueous solution of the polymer may be acidic due to such saccharide.
  • the solution can be more suitable for the survival of the cells to be cryopreserved and the cell survival rate is improved.
  • the salt used for pH adjustment is not limited, and those commonly used for pH adjustment of aqueous solutions can be used.
  • long-term stable storage refers to, for example, the survival rate of a biological sample such as cells after thawing when the aqueous polymer solution of the present invention is used, based on the survival rate of cells immediately before storage, After 5 months, less than 10%, preferably less than 5%, or after 6 months, less than 20%, preferably less than 10%, or after 12 months, less than 15% It means that the degree of reduction is preferably only about 30% or less.
  • long term stable storage means, for example, when cells are frozen and stored at ⁇ 80° C. for a long period of time and then thawed, and subsequently when the cells are stored at 4° C., after thawed. It means that even after 24 hours, the viability was reduced by less than 5% based on the cell viability immediately after thawing.
  • cryopreservation method using the polymer aqueous solution of the present invention it is considered that the cells can be cryopreserved under conditions with less stress as compared with the conventional cryopreservation method using the DMSO solution as the cryopreservation solution.
  • cryopreservation method of the present invention a very high cell viability after thawing can be obtained as compared with the cryopreservation method using a conventional cryopreservation solution such as a 10% DMSO solution. Furthermore, not only immediately after thawing, but also cells that have been refrigerated after thawing can show high cell viability. According to the cryopreservation method using the aqueous polymer solution of the present invention, cells can be stably cryopreserved for a long period of time without changing the properties of the cells.
  • the present invention will be specifically described based on examples, but the present invention is not limited to these.
  • the hyaluronic acid manufactured by lifecore biomedical is sodium hyaluronate, but is referred to as “hyaluronic acid” for simplification.
  • a water-soluble polymer which was a mixture of high molecular weight hyaluronic acid having a viscosity average molecular weight of about 10,000 and hyaluronic acid having a viscosity average molecular weight of 1,000, was obtained.
  • Example 1 When this water-soluble polymer was confirmed by HPLC (FIG. 12), the test sample of Example 1 was a mixture of a high molecular weight hyaluronic acid decomposition product and a low molecular weight hyaluronic acid decomposition product obtained by subcritical treatment of hyaluronic acid. Since it was considered, the high molecular weight hyaluronic acid was precipitated in ethanol, the low molecular weight hyaluronic acid was fractionated on the supernatant side, and the fractionated supernatant was re-analyzed by HPLC.
  • the viscosity average molecular weight of the low-molecular-weight hyaluronic acid component obtained in Example 1 was estimated to be 1000, since it was almost in agreement with the HPLC peaks of hyaluronic acid in Production Examples 1 to 3 described later (FIGS. 11A to 11C). ..
  • the intrinsic viscosity of high molecular weight hyaluronic acid was 0.49 dL/g, and the viscosity average molecular weight was 10,000.
  • the test sample solution of Example 1 was obtained by dissolving 1 g of the test sample of Example 1 in 10 mL of ⁇ MEM medium (manufactured by Gibco, product number C1257-1500BT, the solvent is water) as a solvent (that is, , The concentration of the hyaluronic acid sample in the test sample solution is 10 w/v %).
  • ⁇ MEM medium is used as a solvent in Examples
  • ultrapure water may be used as a solvent instead of the ⁇ MEM medium.
  • the solvent is ultrapure water instead of ⁇ MEM.
  • each test sample obtained by the following examples was prepared and used in a test for each evaluation described below so as to have a predetermined concentration using an appropriate solvent.
  • Example 1 The same operation as in Example 1 was performed except that the subcritical treatment was performed for a treatment time of 7 minutes to obtain a test sample which was a hyaluronic acid fragment having a viscosity average molecular weight of 1000.
  • the intrinsic viscosity was 0.08 dL/g.
  • Example 2 The same operation as in Example 1 was carried out except that the subcritical treatment was carried out for 5 minutes to obtain a test sample which was a hyaluronic acid fragment having a viscosity average molecular weight of 2000.
  • the intrinsic viscosity was 0.14 dL/g.
  • Example 3 The same operation as in Example 1 was performed except that the subcritical treatment was performed for a treatment time of 4 minutes to obtain a test sample which was a hyaluronic acid fragment having a viscosity average molecular weight of 3000.
  • the intrinsic viscosity was 0.19 dL/g.
  • DMSO manufactured by Nacalai Tesque, Inc., cell culture grade
  • a test sample solution of Comparative Example 1 was obtained by dissolving 1 mL of this test sample in 10 mL of a solvent ⁇ MEM medium (manufactured by Gibco, product number C1257-1500BT, solvent is water) (ie, test sample DMSO concentration of the test sample in the solution is 10 w/v %).
  • ⁇ MEM medium used as a solvent (manufactured by Gibco, product number C1257-1500BT, the solvent is water) was used as a test sample.
  • Comparative Example 3 A test sample of Comparative Example 3 was prepared by dissolving 1 g of gelatin (viscosity average molecular weight 315000, manufactured by Nitta Gelatin Co., Ltd.) in 10 mL of ⁇ MEM medium (manufactured by Gibco, product number C1257-1500BT, solvent is water) as a solvent. A solution was obtained (ie the gelatin concentration of the test sample in the test sample solution was 10 w/v %). The intrinsic viscosity was 0.50 dL/g.
  • gelatin viscosity average molecular weight 315000, manufactured by Nitta Gelatin Co., Ltd.
  • ⁇ MEM medium manufactured by Gibco, product number C1257-1500BT, solvent is water
  • ⁇ Comparative Example 4 Comparative Example 4 was prepared by dissolving 1 g of high-molecular-weight hyaluronic acid having a viscosity average molecular weight of 1,000,000 (manufactured by Changhai Easy Industrial Development Co., Ltd.) in 100 mL of ⁇ MEM medium (manufactured by Gibco, product number C1257-1500BT, solvent was water). The test sample solution of was obtained (that is, the hyaluronic acid concentration of the test sample in the test sample solution was 1 w/v %). The intrinsic viscosity was 17.2 dL/g.
  • Hyaluronic acid having a viscosity average molecular weight of 15,000 (made by lifecore biomedical; powder) was used as a test sample.
  • the test sample solution of Comparative Example 5 was obtained by dissolving 1 g of this test sample in 10 mL of a solvent ⁇ MEM medium (manufactured by Gibco, product number C1257-1500BT, solvent is water) (that is, the test sample).
  • Hyaluronic acid concentration of the test sample in the solution is 10 w/v %).
  • the intrinsic viscosity was 0.65 dL/g.
  • ⁇ Comparative example 6 Chondroitin sulfate sodium salt A (manufactured by Sigma) having a viscosity average molecular weight of 23,000 was used as a test sample.
  • the test sample solution of Comparative Example 6 was obtained by dissolving 1 g of this test sample in 10 mL of a solvent ⁇ MEM medium (manufactured by Gibco, product number C1257-1500BT, solvent is water) (that is, the test sample).
  • the chondroitin sulfate concentration of the test sample in the solution is 10 w/v %).
  • the intrinsic viscosity was 0.97 dL/g.
  • ⁇ Comparative Example 7 A test sample of Production Example 1 (a hyaluronic acid fragment sample having a viscosity average molecular weight of 1000) was added to carboxypolylysine having a 60% amino group carboxylation (viscosity average molecular weight 13400: manufactured by Bio Verde Co., Ltd., CryoScarless DMSO free). It was added in an amount of 1 w/v% to prepare a test sample solution of Comparative Example 7. The intrinsic viscosity was 0.11 dL/g.
  • fructan derived from lacquer tree was used as a test sample. Note that fructan was obtained by extracting and purifying by the following method according to Example 2 of JP2012-235728A.
  • the obtained purified solution was once sterilized by heating, and then activated carbon was added to the solution for decolorization again.
  • This purified liquid was sterilized by heating at 85° C. for 1 hour, freeze-dried, and pulverized to obtain 400 g of purified fructan powder.
  • the viscosity-average molecular weight of the obtained purified fructan powder was measured and found to be 12,000.
  • Comparative Example 9 Pullulan (produced by Tokyo Chemical Industry Co., Ltd.) having a viscosity average molecular weight of 373,000 was used as a test sample.
  • a test sample solution of Comparative Example 9 was obtained by dissolving 1 g of this test sample in 10 mL of ⁇ MEM medium (manufactured by Gibco, product number C1257-1500BT, solvent is water) as a solvent (that is, test sample solution).
  • Hyaluronic acid concentration of the test sample inside is 10 w/v %).
  • the intrinsic viscosity was 0.55 dL/g.
  • the test sample of Production Example 1 (a hyaluronic acid fragment sample having a viscosity-average molecular weight of 1000) was added to the test sample solution in 1) at a final concentration of 1 w/v %.
  • the pH of the obtained aqueous solution was adjusted to neutral with 10 mM Tris-HCl to prepare a test sample solution of Example 2.
  • the intrinsic viscosity was 0.65 dL/g.
  • Example 3 A test with a pullulan concentration of 10 w/v% in which 1 g of the test sample of Comparative Example 9 (pullulan having a viscosity average molecular weight of 373,000) was dissolved in 10 mL of an ⁇ MEM medium (manufactured by Gibco, product number C1257-1500BT, the solvent is water) as a solvent.
  • the test sample solution of Production Example 1 hyaluronic acid fragment sample having a viscosity average molecular weight of 1000
  • the intrinsic viscosity was 0.55 dL/g.
  • Example 4 Test with a pullulan concentration of 10 w/v% in which 1 g of the test sample of Comparative Example 9 (pullulan having a viscosity average molecular weight of 373,000) was dissolved in 10 mL of ⁇ MEM medium (manufactured by Gibco, product number C1257-1500BT, solvent is water) as a solvent
  • the test sample of Production Example 1 (a hyaluronic acid fragment sample having a viscosity-average molecular weight of 1000) was added to the sample solution at a final concentration of 5 w/v %.
  • the pH of the obtained aqueous solution was adjusted to neutral with 10 mM Tris-HCl to prepare a test sample solution of Example 4.
  • the intrinsic viscosity was 0.55 dL/g.
  • a high-pressure hyaluronic acid (Shanghai Easy Industrial Development Co., Ltd.) having an average molecular weight of 1,000,000 and water were mixed at 20:100 in a pressure-resistant container having a volume of 2 L, and a treatment temperature was 175° C., a treatment pressure was 0.89 MPa, and a treatment time was 3 times. Subcritical processing was performed in 0.5 minutes. Then, the subcritically processed product was dried by freeze drying or spray drying. Thus, a hyaluronic acid sample having a viscosity average molecular weight of about 6000 was obtained.
  • Hyaluronic acid concentration of 10 w/v% obtained by dissolving 1 g of the test sample of Comparative Example 5 (hyaluronic acid having a viscosity average molecular weight of 15,000) in 10 mL of ⁇ MEM medium (manufactured by Gibco, product number C1257-1500BT, solvent is water) which is a solvent.
  • the test sample of Production Example 2 (hyaluronic acid fragment sample having a viscosity average molecular weight of 2000) was added to the aqueous solution of Example 1 at a final concentration of 1 w/v% to obtain a test sample solution of Example 6.
  • the intrinsic viscosity was 0.47 dL/g.
  • ⁇ Test Example 1 Cryopreservation of primary human mesenchymal stem cells using a test sample solution and evaluation of its preservation effect> Cultured primary human mesenchymal stem cells (Lonza PT2501) at a concentration of 1 ⁇ 10 6 cells/mL and test sample solutions of Examples 1 and 5 and Comparative Examples 1, 2, 4, 5, 7 and 8 ( Serum-free).
  • the cell suspension containing each test sample solution was frozen in a slow cell freezer (Nalgene (registered trademark) Mr. Frosty) in a -80°C freezer to give a frozen solidified body.
  • the cell suspension containing each test sample frozen for 7 days was stored at -80°C and then rapidly thawed in a 37°C warm bath.
  • the cell viability of the cell suspension containing each test sample solution after thawing was evaluated by trypan blue staining immediately after thawing. The results are shown in FIG. 1 and Table 1 (results of Example 5 are only Table 1).
  • FIG. 1 shows cryopreservation in a medium to which a cryopreservation solution has not been added, that is, in a test sample solution of Comparative Example 2 or in a test sample solution of Example 1 and Comparative Examples 1, 4, 5, 7 and 8. 3 shows the cell viability of thawed cells after thawing.
  • the viscosity average molecular weight is about 10,000, and further, the viscosity average molecular weight is 1000, that is, less than 10,000.
  • the hyaluronic acid sample containing a cleavage product of hyaluronic acid having a molecular weight is a water-soluble polymer in an aqueous polymer solution, has an intrinsic viscosity of 0.49 dL/g, and has an exothermic heat in a DSC measurement during a temperature lowering process.
  • the test sample solution of Example 1 having an amount of 0 J/g (see Test Example 2 below)
  • a remarkably high preservative effect of more than 90% was obtained.
  • Comparative Example 7 containing carboxypolylysine as a test sample and having an intrinsic viscosity as small as 0.11 dL/g, the cell protective effect as in Example 1 or Comparative Example 1 using DMSO as a cryoprotectant was not observed.
  • the intrinsic viscosity of polylysine is low, water molecules that have moved to the outside of the cell and macromolecules or low molecular weight saccharides around the cells diffuse, resulting in water molecules and macromolecules or low molecules. It is presumed that the replacement of the molecular weight sugars and the like does not proceed and the water molecules remain around the cells to form ice crystals.
  • glucuronic acid and sucrose also have a function of adjusting the endothermic peak to around 0.7°C (the endothermic peak of hyaluronic acid having a viscosity average molecular weight of about 6000 is -1. 4°C).
  • the polymer aqueous solution of the present invention showed a high cell preservation effect even when prepared by using a culture medium such as ⁇ MEM medium instead of water as a solvent.
  • the cells can be suspended in the culture medium described above, frozen, and stored. It is believed that higher cell viability and maintenance of cell properties is possible without the need to centrifuge the stored cells.
  • ⁇ Test Example 2 Analysis of each test sample solution by a differential scanning calorimeter> The solvent of each test sample solution of Examples 1 to 6, Comparative Examples 1 to 9 and Production Examples 1 to 3 was changed from ⁇ MEM medium to ultrapure water to prepare a sample for differential scanning calorimetry. Each sample was scanned with a differential scanning calorimeter (DSC) as shown below. (1) After holding at 20°C for 1 minute, the temperature was lowered to -80°C at a speed free. (2) After holding at -80°C for 1 minute, the temperature was raised to 20°C at a temperature rising rate of 10°C/min.
  • DSC differential scanning calorimeter
  • FIG. 2A shows a DSC curve in the temperature rising process obtained under the above DSC measurement conditions in the case of Example 1, Comparative Example 5 and ultrapure water (Comparative Example 2).
  • FIG. 2B is an enlarged view of FIG. 2A in the vicinity of the glass transition temperature of the polymer aqueous solution of the present invention.
  • 3A to 3C show the DSC curves of Example 1, Comparative Example 1 and ultrapure water (Comparative Example 2), respectively.
  • Comparative Example 2 in which the test sample was ultrapure water (that is, a sample in which ultrapure water was used), only the ice crystal melting peak of free water was observed (FIG. 2A). ).
  • Comparative Example 5 in which the test sample was hyaluronic acid having a viscosity average molecular weight of 15,000, a shift of an ice crystal melting peak and a lowering of freezing point were observed as compared with Comparative Example 2 (FIG. 2A), and glass near -20° C. Metastasis was confirmed (Fig. 2B).
  • Example 1 containing the cleavage product of hyaluronic acid (low molecular weight hyaluronic acid having a viscosity average molecular weight of 1000) in addition to hyaluronic acid having a viscosity average molecular weight of about 10,000, the peak of ice crystal melting was It was extremely small, almost not observed (FIG. 2A), and only a glass transition was confirmed at around ⁇ 23° C. ⁇ 4° C. (FIG. 2B).
  • hyaluronic acid low molecular weight hyaluronic acid having a viscosity average molecular weight of 1000
  • Comparative Example 2 in which the test sample was ultrapure water, a very large exothermic peak due to ice crystal formation was observed in the temperature decreasing process. A large amount of heat of melting associated with melting of ice crystals was also observed in the temperature rising process.
  • Comparative Example 1 in which the test sample was DMSO as shown in FIG. 3B, only peaks associated with small ice crystal formation were observed, and almost no peaks associated with ice crystal melting were observed. This indicates that the cryopreservation liquid containing the test sample (DMSO) of Comparative Example 1 is close to the vitrified state in the frozen state.
  • Example 5 the temperature at which the endothermic peak was observed (endothermic peak temperature) was ⁇ 1.4° C. in Comparative Example 5 (the test sample was hyaluronic acid having a viscosity average molecular weight of 15,000), but in Comparative Example 5, Production Example 1 was used.
  • Example 2 in which a hyaluronic acid fragment having a viscosity average molecular weight of 1,000 or less was mixed, the temperature was ⁇ 0.4° C., which was close to the endothermic peak appearance temperature of pure water of 0.7° C.
  • the endothermic peak temperature of Production Example 1 is ⁇ 8.1° C., and it is expected that the endothermic peak appearing temperature will be further lowered by adding it to the water-soluble polymer whose endothermic peak appearing temperature is lower than that of pure water.
  • the endothermic peak appearance temperature can be increased to approach the endothermic peak appearance temperature of pure water or adjusted so that the endothermic peak or the exothermic peak itself does not appear.
  • the endothermic peak appearance temperature of Comparative Example 9 (the test sample is pullulan having a viscosity average molecular weight of 373,000) was 1.49° C., which was higher than the endothermic peak appearance temperature of pure water of 0.7° C.
  • the endothermic peak appearance temperature was lowered rather than raised to give pure water.
  • the endothermic peak appearance temperature was 0.83°C, which was close to 0.7°C.
  • the endothermic peak temperature of the hyaluronic acid fragment having a viscosity average molecular weight of 2000 or less in Production Example 2 was -0.3°C as shown in Table 1.
  • the low molecular weight sample of Production Example 2 was mixed with Comparative Example 5 (the endothermic peak appearance temperature was ⁇ 1.4° C.), and the endothermic peak appearance temperature of Example 6 was lowered to ⁇ 1.6° C.
  • the exothermic amount of the exothermic peak in the temperature decreasing process of the test sample solutions of Examples 1 to 6 was smaller than the corresponding exothermic amount of the reference liquid made of water (Comparative Example 2) and was 65% or less. Further, the endothermic amount of the endothermic peak in the temperature rising process of the test sample solutions of Examples 1 to 6 was also less than the corresponding endothermic amount of the reference liquid made of water (Comparative Example 2), which was 65% or less. It can be seen that in the polymer aqueous solution of the present invention, water is solidified in the glass state without being crystallized.
  • the endothermic peak temperature of the test sample solutions of Examples 1 to 6 is around 0.7° C. of the endothermic peak temperature of water, that is, the behavior is close to that of pure water, and the exothermic amount of the exothermic peak is water.
  • the fact that the calorific value of the exothermic peak observed in the temperature-decreasing process is smaller than that in the frozen aqueous polymer solution of the present invention indicates that bound water or free water that does not form ice crystals remains. It is considered that the water-soluble polymer in the present invention realizes vitrification by trapping water molecules in its matrix during freezing.
  • Example 3 Evaluation of long-term preservation effect in cryopreservation using a test sample solution of primary human mesenchymal stem cells> Cultured primary human mesenchymal stem cells (Lonza PT2501) were suspended in the test sample solutions (serum-free) of Example 1 and Comparative Example 1 at a concentration of 1 ⁇ 10 6 cells/mL. Then, the cell suspension containing each test sample solution was frozen in a slow cell freezer (Nalgene (registered trademark) Mr. Frosty) in a -80°C freezer to give a frozen solidified body. The frozen cell suspension containing each test sample solution was stored at ⁇ 80° C.
  • a slow cell freezer Nealgene (registered trademark) Mr. Frosty
  • the cryopreservation using the polymer aqueous solution of the present invention showed only a decrease in the survival rate of less than 10%, while the cryopreservation using the sample solution of Comparative Example 1 resulted in The survival rate was reduced to about 25%. Furthermore, in the cryopreservation with the polymer aqueous solution of the present invention, even after 12 months of cryopreservation, the high cell viability was still maintained, and the decrease in cell viability was less than 15%.
  • the aqueous polymer solution of the present invention enables cells to be cryopreserved stably for a long period of time with a high cell viability.
  • the cells that survived after thawing were the cells that substantially proceeded to normal growth, and that the polymer aqueous solution of the present invention was stored during storage. It was confirmed that the polymer aqueous solution of the present invention has a very excellent cell preservation effect.
  • Example 4 Evaluation of properties of primary human mesenchymal stem cells after cryopreservation using a test sample solution> Cultured primary human mesenchymal stem cells (Lonza PT2501) were suspended in the test sample solutions (serum-free) of Example 1 and Comparative Example 1 at a concentration of 1 ⁇ 10 6 cells/mL. Then, the cell suspension containing each test sample solution was frozen in a slow cell freezer (Nalgene (registered trademark) Mr. Frosty) in a -80°C freezer to give a frozen solidified body. The frozen cell suspension containing each test sample solution was frozen and stored at ⁇ 80° C. for 6 months.
  • a slow cell freezer Nealgene (registered trademark) Mr. Frosty
  • HGF and IL-10 concentrations in the culture medium were quantified.
  • a dedicated kit Quantikine (registered trademark) ELISA Human HGF, catalog number DHG00, manufactured by R&D
  • Quantikine registered trademark
  • ELISA Human IL-10 catalog number D100B, R&D
  • Figures 5A and 5B The results are shown in Figures 5A and 5B.
  • HGF production in cells after thawing was high in cryopreservation in the presence of the sample solution of Comparative Example 1 containing DMSO as a cryopreservative.
  • the test sample solution of Example 1 of the present invention which is a hyaluronic acid having a viscosity average molecular weight of 10,000 containing a cleavage product of hyaluronic acid
  • HGF production in the cells after thawing was low, and Comparative Example It was 1/3 or less of 1.
  • the results in the presence of DMSO indicate that the cells were in a stressed state during storage in the presence of DMSO.
  • Such high HGF production was not observed in cell preservation using the polymer aqueous solution of the present invention, which indicates that the cells were stably protected by the polymer aqueous solution of the present invention.
  • the amount of IL-10 produced was high in the cells cryopreserved using the test sample solution of Example 1, and was high in the cells cryopreserved using the test sample solution of Comparative Example 1. So it was very low. From these results, it can be seen that the aqueous polymer solution of the present invention can favorably store cells frozen while maintaining the function of the cells.
  • the polymer aqueous solution of the present invention does not contain a compound having cytotoxicity such as DMSO and ethylene glycol, the polymer aqueous solution of the present invention is different from the conventional cryopreservation liquid without exposing the biological sample to a stressed state. It has a remarkable effect that it can be cryopreserved while maintaining its properties.
  • Example 5 Evaluation of properties (undifferentiated) of primary human mesenchymal stem cells after cryopreservation using a test sample solution> Cultured primary human mesenchymal stem cells (Lonza PT2501) were suspended in the test sample solutions (serum-free) of Example 1 and Comparative Example 1 at a concentration of 1 ⁇ 10 6 cells/mL. Then, the cell suspension containing each test sample solution was frozen in a slow cell freezer (Nalgene (registered trademark) Mr. Frosty) in a -80°C freezer to give a frozen solidified body. The frozen cell suspension containing each test sample solution was frozen and stored at ⁇ 80° C. for 6 months.
  • a slow cell freezer Nealgene (registered trademark) Mr. Frosty
  • CD90, CD44, and CD105 are typical surface proteins expressed on undifferentiated mesenchymal stem cells, and are used as undifferentiated markers for mesenchymal stem cells.
  • expression of all undifferentiated biomarkers of CD90, CD44, and CD105 was cryopreserved with the test sample solution of Comparative Example 1 in the cells cryopreserved with the aqueous polymer solution of Example 1. was higher than the cells. Therefore, it can be seen that the cells cryopreserved with the polymer aqueous solution of Example 1 maintain the expression of the undifferentiated marker. That is, the cells cryopreserved with the polymer aqueous solution of Example 1 can maintain the undifferentiated state. It can be seen that the cryopreservation using the polymer aqueous solution of the present invention can reduce the influence on the differentiation state as seen when the test aqueous solution of Comparative Example 1 is stored.
  • cryopreservation method using the polymer aqueous solution of the present invention can be suitably applied to the cryopreservation of stem cells, which is important to cryopreserve in an undifferentiated state.
  • ⁇ Test Example 6 Evaluation of a polymer aqueous solution containing various samples as a cryopreservation liquid> Cultured primary dog mesenchymal stem cells (cyagen C160) at a concentration of 1 ⁇ 10 6 cells/mL and test sample solutions of Examples 1 to 4 and 6 and Comparative Examples 3, 6 and 9 (serum-free) (However, the concentration of the hyaluronic acid sample in the test sample solution of Example 1 was 20 w/v %). Then, the cell suspension containing each test sample solution was frozen in a slow cell freezer (Nalgene (registered trademark) Mr. Frosty) in a -80°C freezer to give a frozen solidified body.
  • a slow cell freezer Nealgene (registered trademark) Mr. Frosty
  • the water-soluble polymer in the present invention is not limited to hyaluronic acid, and even if other water-soluble polymer is used, its high It can be seen that if the aqueous solution of the molecule has a predetermined intrinsic viscosity and endothermic peak temperature, a high cytoprotective effect is exhibited.
  • Comparative Example 9 and Examples 3 and 4 Comparative Example 9 with the addition of Production Example 1
  • Example 6 is a test sample in which the low molecular weight saccharide of Production Example 2 (a hyaluronic acid fragment sample having a viscosity average molecular weight of 2000) was added to a high molecular weight aqueous solution of hyaluronic acid having a viscosity average molecular weight of 15,000 (Comparative Example 5).
  • the test sample solution prepared by adding a saccharide or a salt thereof is acidic, by making the test sample solution neutral by adjusting the pH, a good cytoprotective effect can be obtained. Was obtained. It is considered that the pH adjustment made the conditions more appropriate for cell survival.
  • the added low molecular weight saccharide or salt thereof to act as a cell protective component to further improve the cell viability of the cells after cryopreservation, the low molecular weight saccharide or salt thereof in a polymer aqueous solution is It was necessary to be added in an amount of 1 to 5 w/v% with respect to the water-soluble polymer. On the other hand, even if the amount of the low-molecular-weight saccharide or its salt added was more than 10 w/v% with respect to the water-soluble polymer, no further effect was observed on the effect of improving cell viability.
  • the intrinsic viscosity ( ⁇ ) of the test sample solution is outside the range of 0.20 dL/g or more and 0.95 dL/g or less, or the endothermic peak temperature of the DSC curve is ⁇ 1.4° C.
  • the temperature is higher than 1.1°C, the cell viability after cryopreservation decreases.
  • the desired effect high cell viability
  • the aqueous polymer solution helps the discharge of water molecules from the inside of the biological sample, and the discharged water molecules are efficiently increased around the biological sample. It is believed that it can replace molecular and/or low molecular sugars. Furthermore, it is considered that the endothermic peak temperature exceeding ⁇ 1.4° C. and about 1.1° C. or less indicates that the aqueous polymer solution can retain water molecules in the polymer matrix during freezing.
  • Example 7 Evaluation of cells in frozen test sample solution (evaluation of intracellular vitrification state)>
  • the cultured primary dog mesenchymal stem cells (cyagen C160) were suspended in the test sample solutions (serum-free) of Example 1 and Comparative Example 1 at a concentration of 1 ⁇ 10 6 cells/mL.
  • a control suspension was prepared in the same manner, in which cells were suspended in the test sample solution of Comparative Example 2 consisting of an ⁇ MEM medium containing no cryopreservative. Then, 5 ⁇ L of the cell suspension containing each test sample solution and the control suspension was added to a hard glass sample plate (16 ⁇ 0.12 mm), and a hard glass cover glass (12 ⁇ 0.12 mm) was added.
  • FIG. 7A shows the results of the control suspension, and it can be seen that in the medium alone, the cells are darkened because ice crystals are generated in the cells and light is diffusely reflected.
  • FIG. 7B shows the results of Comparative Example 1 in which DMSO is the test sample. Also in FIG. 7B, it can be seen that the cells are dark and micro ice crystals are formed.
  • FIG. 7C is the result of an aqueous polymer solution containing the test sample of Example 1. It can be seen that the cells are turning bright and the inside of the cells vitrifies into an amorphous state.
  • the polymer aqueous solution of the present invention freezes the inside of the cells in a vitrified state. It was also found that the glass state is formed more stably by including the saccharide that is a cleavage product of hyaluronic acid. It is considered that vitrification occurred stably and efficiently because the formation of ice crystals around the cells was suppressed by the saccharides having a low molecular weight.
  • ⁇ Test Example 8 Evaluation of intracellular vitrification state using difference in brightness> Cultured primary dog mesenchymal stem cells (cyagen C160) were suspended in the test sample solutions (serum-free) of Examples 1 to 6 and Comparative Examples 1 to 9 at a concentration of 1 ⁇ 10 6 cells/mL. ..
  • a control suspension was prepared in the same manner, in which cells were suspended in the test sample solution of Comparative Example 2 consisting of an ⁇ MEM medium containing no cryopreservative. Then, 5 ⁇ L of the cell suspension containing each test sample solution and the control suspension was added to a hard glass sample plate (16 ⁇ 0.12 mm), and a hard glass cover glass (12 ⁇ 0.12 mm) was added.
  • Example 1 and Comparative Examples 1 and 2 are shown in FIGS. 8A and 8B.
  • the difference (absolute value) between the intracellular and extracellular brightness of the observed image shown in FIG. 8A was analyzed using the image analysis software ImageJ.
  • ImageJ image analysis software
  • the lightness of the solvent region and the lightness inside the cell and the Munsell lightness were read under the same conditions, and the read data of the lightness of the solvent region and the lightness inside the cell were compared with the read data of each Munsell lightness.
  • the Munsell lightness of the closest read data was adopted as the lightness of the solvent region and the lightness inside the cell to quantify the lightness of the solvent and the lightness inside the cell (Munsell value conversion).
  • the read value of the brightness of the intracellular region or the solvent region read by the image analysis software is a darker value than the read value of the darkest Munsell value of 0, the Munsell of the intracellular region or the solvent region will be used for convenience. If the value is set to 0 and the lightness reading value of the intracellular region or solvent region read by the image analysis software is a brighter value than the brightest value 10 of the Munsell lightness value, the intracellular region or the solvent is conveniently used. The Munsell lightness value of the area is set to 10.
  • the Munsell brightness is 2 or more in the solvent region and the intracellular region.
  • the Munsell brightness having such a value indicates that ice crystals are not formed in the solvent region and the intracellular region, that is, the glass is satisfactorily vitrified.
  • the Munsell lightness of the solvent region was 4, the frozen cells were bright, and the Munsell value of the intracellular region was 2. Therefore, it was found that ice crystals were not formed in either region.
  • the difference in brightness between the Munsell lightness in the solvent region and the Munsell value in the intracellular region was 2 in Examples 2 to 6 as in Example 1. That is, in the example, the brightness of the intracellular region and the brightness of the solvent region are close to each other. This result indicates that in the cryopreservation using the polymer aqueous solution containing the test sample of the example, the frozen state is almost the same in the matrix region and the intracellular region, that is, ice crystals are present in the region in the biological sample. It has not been formed.
  • the difference in brightness between the intracellular region and the solvent region is preferably 3 or less. Further, it is desirable that the lightness value of the solvent region is greater than or equal to the lightness value of the intracellular region.
  • ⁇ Test Example 9 Evaluation of cells in frozen sample solution> Cultured primary dog mesenchymal stem cells (cyagen C160) at a concentration of 1 ⁇ 10 6 cells/mL were used as test sample solutions of Examples 1 to 6, Comparative Examples 1 to 9 and Production Examples 1 to 3 (serum-free). Suspension). Thereafter, 5 ⁇ L of a cell suspension containing each test sample solution was added to a hard glass sample plate (16 ⁇ 0.12 mm), and the cell suspension was covered with a hard glass cover glass (12 ⁇ 0.12 mm), and linKam was used. It was cooled to ⁇ 80° C.
  • the cells frozen with the test sample solutions of Examples 1 to 6 exhibited cell contraction rates of 38%, 33%, 67%, 76%, 40% and 50%, respectively.
  • drainage of water from the frozen cells was promoted, and the cells were well dehydrated during freezing.
  • the water-soluble polymer and/or the low-molecular-weight saccharide of the present invention existed sufficiently close to the periphery of the cell, and the intracellular fluid was Water molecules that have moved from the inside to the outside of the cell due to the osmotic pressure difference that occurs between the polymer and the aqueous solution of the polymer are trapped in the matrix of the water-soluble polymer to further promote the discharge of water from the inside of the cell.
  • the intracellular water is supercooled due to the increased concentration of proteins and sugars in the cells, and the vapor pressure difference between the water and the supercooled water further promotes the movement of water molecules to the outside of the cell through the cell membrane. Prevents or suppresses freezing and growth of ice crystals inside.
  • the cells are contracting, it can be seen that the water-soluble polymer or the like does not enter the cells.
  • the endothermic peak temperature of the DSC curve deviates from the endothermic peak temperature of water of about 0.7° C.
  • the test sample solutions of 1.4°C or lower and higher than 1.1°C Comparative Examples 5 and 9
  • sufficient cell contraction was not observed.
  • the cryopreservation of the test sample solutions of these comparative examples the dehydration of the cells did not occur or was not sufficient. Therefore, the viability of the cells after the cryopreservation was also excellent. It is thought that it was lower than that of ⁇ 6.
  • Comparative Examples 7 and 8 the orthogonal projected area of the cells was larger than that before the freezing. In Comparative Example 8, it was observed that cell rupture also occurred due to freezing. In Comparative Examples 7 and 8, the water-soluble polymer is considered to have penetrated into the cells. The cell viability of the cells after cryopreservation in Comparative Examples 7 and 8 was also low. Such a cell-permeable water-soluble polymer expands cells by freezing and damages the cells, and is therefore suitable as a water-soluble polymer for an aqueous polymer solution used for cryopreservation of biological samples. I don't think there is. Further, in Production Examples 1 to 3 having a small intrinsic viscosity ( ⁇ ), no change was observed in the orthographic projected area of the cells, and it was estimated that the dehydration of the cells did not occur.
  • ⁇ intrinsic viscosity
  • the intracellular dehydration is favorably dehydrated at the time of freezing, so that the damage to the cells at the time of freezing is significantly reduced, whereby the thawed cells have a high survival rate. It is considered to indicate.
  • ⁇ Test Example 10 Evaluation of cryoprotection for various cells>
  • Each of the cultured mouse-derived macrophage-like cell line (RAW264), human colon cancer-derived cells (Caco-2) and primary human lung microvascular endothelial cells (HMVEC) was administered at a concentration of 1 ⁇ 10 6 cells/mL. 1 and the test sample solution of Comparative Example 1 (serum-free).
  • the cell suspension containing each test sample solution was frozen in a slow cell freezer (Nalgene (registered trademark) Mr. Frosty) in a -80°C freezer to give a frozen solidified body.
  • the cell suspension containing each test sample solution was taken out and rapidly thawed in a 37°C warm bath.
  • the cell viability of the cell suspension containing each test sample solution after thawing was evaluated by trypan blue staining. The results are shown in Fig. 9.
  • Example 11 Evaluation of effect of low molecular weight saccharides on cell viability>
  • the cultured primary dog mesenchymal stem cells (cyagen C160) were suspended in each test sample solution (serum-free) of Production Examples 1 to 3 at a concentration of 1 ⁇ 10 6 cells/mL. Then, the cell suspension containing each test sample solution was frozen in a slow cell freezer (Nalgene (registered trademark) Mr. Frosty) in a -80°C freezer to give a frozen solidified body. After frozen storage for 7 days, the cell suspension containing each test sample solution was taken out and rapidly thawed in a 37°C warm bath. The cell viability of the cell suspension containing each test sample solution after thawing was evaluated by trypan blue staining. The results are shown in FIG. 10 and Table 1.
  • the intrinsic viscosities ( ⁇ ) of low-molecular-weight sugars or their salts (viscosity average molecular weights of 1,000, 2000 and 3000, respectively) having a smaller molecular weight than the water-soluble polymer in the present invention are respectively , 0.08, 0.14, and 0.19, which are outside the desired range of 0.20 dL/g or more and 0.95 dL/g or less.
  • This Test Example 11 revealed that the cell survival effect cannot be obtained only with such a low molecular weight saccharide or a salt thereof.
  • a cell viability of about 10% was observed, which is presumed to be due to hyaluronic acid having a higher molecular weight than 3000 that may be contained in the test sample of Production Example 3.
  • ⁇ Test Example 12 HPLC analysis of subcritically processed hyaluronic acid sample> 1 wt% aqueous solutions of the test samples of Production Examples 1 to 3 were prepared and filtered with a 0.45 ⁇ m membrane filter (manufactured by Millipore), and then the components of each test sample were analyzed by HPLC.
  • a solution 16 mM NaH 2 PO 4 aqueous solution
  • B solution 800 mM NaH 2 with PO 4 aqueous solution
  • ZORBAX NH 2 (Agilent Technologies Co., column size ⁇ 4.6 ⁇ 250mm, particle size 5 [mu] m)
  • the components were separated at a flow rate of 1.0 mL/min, a column temperature of 40° C., and a detection wavelength of 210 nm.
  • Gradient conditions were: mobile phase B concentration 0% (0 minutes) ⁇ mobile phase B concentration 100% (60 minutes). The results are shown in Figures 11A-C.
  • disaccharide HA02, tetrasaccharide HA04, hexasaccharide HA06, octasaccharide HA08, and decasaccharide HA10 (all manufactured by Idron) at a concentration of 0.2 wt% were prepared.
  • An aqueous solution containing each oligosaccharide of hyaluronic acid was prepared and subjected to HPLC analysis in the same manner. The result is shown in FIG. 11D.
  • FIG. 11A shows the analysis results of a test sample containing a cleavage product of hyaluronic acid of Production Example 1 having a viscosity average molecular weight of 1000, and a peak corresponding to a monosaccharide near a retention time of 3.5 minutes. , A peak corresponding to a disaccharide having a retention time of about 9 minutes is observed. That is, it is considered that the test sample of Production Example 1 contains such monosaccharide and disaccharide components and contributes to the effect of improving the cell viability.
  • Example 13 HPLC analysis of test sample of Example 1>
  • test sample of Example 1 a hyaluronic acid sample having a viscosity average molecular weight of about 10,000 obtained by subcritical treatment
  • Example 1 peaks corresponding to monosaccharides and disaccharides are observed in the test sample of Example 1 near retention times of 3.5 minutes and 9 minutes, respectively. That is, the test sample of Example 1 also contains such monosaccharide and disaccharide components, and it is considered that a high cell viability effect is obtained by this.
  • the inside of the biological sample is stably vitrified and the growth of ice crystals in the biological sample is prevented or suppressed, whereby DMSO, ethylene glycol, etc. It is remarkable that a biological sample can be cryopreserved with high cell viability without basically requiring the addition of a cell-permeable and cytotoxic compound and/or serum or a serum-derived protein. It can be seen that it has various effects. The cells are well protected and their properties are maintained.
  • freeze-solidified product of the present invention can be inferred by DSC measurement, lightness measurement, and measurement of the orthogonal projection area of cells to determine whether or not the cells are satisfactorily cryopreserved without thawing. I also have.

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Abstract

Le but de la présente invention est de fournir : une solution macromoléculaire aqueuse pour la cryoconservation d'échantillons biologiques qui a un taux de survie cellulaire élevé ; et un corps solidifié contenant un échantillon biologique. La présente invention concerne une solution macromoléculaire aqueuse utilisée pour la cryoconservation d'échantillons biologiques et contenant, dans un solvant aqueux, une macromolécule hydrosoluble ou un sel associé, la solution macromoléculaire aqueuse étant caractérisée en ce que : la viscosité limite (η) de la solution macromoléculaire aqueuse est de 0,20 dL/g à 0,95 dL/g ; la zone de projection orthogonale de l'échantillon biologique dans un état solidifié à -80 °C diminue jusqu'à une taille qui est inférieure à 8/10 celle de ladite zone avant la solidification ; et sur une courbe DSC d'un procédé d'abaissement de température obtenue à l'aide d'un calorimètre à balayage différentiel (DSC), la quantité de génération de chaleur d'un pic de génération de chaleur dans le processus d'abaissement de température est de 0 J/g ou n'est pas supérieure à 65 % de la quantité de génération de chaleur correspondante pour un liquide de référence comprenant de l'eau. L'invention concerne également un corps solidifié congelé de ladite solution macromoléculaire aqueuse.
PCT/JP2020/005870 2019-02-15 2020-02-14 Solution macromoléculaire aqueuse pour la cryoconservation et corps solidifié contenant un échantillon biologique WO2020166712A1 (fr)

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JP2020130164A (ja) * 2019-02-15 2020-08-31 イビデン株式会社 凍結保存用高分子水溶液

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Publication number Priority date Publication date Assignee Title
WO1992021234A1 (fr) * 1991-06-03 1992-12-10 Vetrepharm, Inc. Composition et procede de culture et de congelation de cellules et de tissus
JP2012235728A (ja) * 2011-05-11 2012-12-06 Univ Of Fukui 細胞の凍結保存液および凍結保存方法
WO2018084228A1 (fr) * 2016-11-04 2018-05-11 国立大学法人東京大学 Solution pour cryoconservation, produit de cryogénisation, et procédé de cryoconservation de cellules animales ou tissus animaux

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WO1992021234A1 (fr) * 1991-06-03 1992-12-10 Vetrepharm, Inc. Composition et procede de culture et de congelation de cellules et de tissus
JP2012235728A (ja) * 2011-05-11 2012-12-06 Univ Of Fukui 細胞の凍結保存液および凍結保存方法
WO2018084228A1 (fr) * 2016-11-04 2018-05-11 国立大学法人東京大学 Solution pour cryoconservation, produit de cryogénisation, et procédé de cryoconservation de cellules animales ou tissus animaux

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2020130164A (ja) * 2019-02-15 2020-08-31 イビデン株式会社 凍結保存用高分子水溶液
JP7391528B2 (ja) 2019-02-15 2023-12-05 イビデン株式会社 凍結保存用高分子水溶液

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