WO2014034884A1 - Matériau pour tuteur cellulaire pouvant faire l'objet d'une cryoconservation - Google Patents

Matériau pour tuteur cellulaire pouvant faire l'objet d'une cryoconservation Download PDF

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WO2014034884A1
WO2014034884A1 PCT/JP2013/073395 JP2013073395W WO2014034884A1 WO 2014034884 A1 WO2014034884 A1 WO 2014034884A1 JP 2013073395 W JP2013073395 W JP 2013073395W WO 2014034884 A1 WO2014034884 A1 WO 2014034884A1
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dextran
polymer
cell
group
cells
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PCT/JP2013/073395
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Japanese (ja)
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和明 松村
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国立大学法人北陸先端科学技術大学院大学
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Priority to JP2014533128A priority Critical patent/JP6270158B2/ja
Publication of WO2014034884A1 publication Critical patent/WO2014034884A1/fr

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    • 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
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • 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
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
    • C08B37/0021—Dextran, i.e. (alpha-1,4)-D-glucan; Derivatives thereof, e.g. Sephadex, i.e. crosslinked dextran
    • 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

Definitions

  • the present invention relates to a cell scaffold material capable of cryopreserving cells comprising a hydrogel obtained by crosslinking an ampholyte polymer having an amino group and a carboxyl group in the same molecule, and further to the cell scaffold material.
  • the present invention relates to a novel ampholyte polymer which can be suitably used.
  • tissue regeneration has achieved certain results, particularly with two-dimensional skin, cornea, and myocardium, and commercialization has partially started.
  • a technique for controlling differentiation by three-dimensionally culturing stem cells has been developed, and development of a regenerative tissue having a three-dimensional structure has been studied.
  • a collagen gel is used as a scaffold material that enables such three-dimensional cell culture.
  • DMSO dimethyl sulfoxide
  • Patent Document 1 WO 2009/157209
  • Patent Document 2 JP 2011-30557 A
  • an object of the present invention is to provide a cryopreservation agent that enables three-dimensional cell culture.
  • the present inventor has used a specific polymer cryopreservation agent as a cryopreservation agent for cells, and this polymer cryopreservation agent itself is subjected to a crosslinking reaction by a specific technique.
  • the cells suspended with the polymer cryopreservation agent were not damaged by the crosslinking reaction for gelation, and the cells embedded three-dimensionally in the gel were cryopreserved.
  • the inventors have found that high viability can be maintained even after thawing, and reached the present invention.
  • a three-dimensional structure capable of three-dimensional culture is formed from a suspension of cells, and the three-dimensional structure in which cells are embedded is frozen and thawed. Can do.
  • the polymer cryopreservation agent forms a gel, which provides a three-dimensional scaffold material for cell growth and / or differentiation. Accordingly, the present invention includes the following (1) to (1).
  • amphoteric electrolyte polymer having an amino group and a carboxyl group in the same molecule, wherein the ratio of carboxyl group to amino group (carboxyl group / amino group) is 0.45 / 0.55 to 0.95 / 0.05.
  • the ampholyte polymer in the range of In a physiological solution, intermolecularly cross-linked A hydrogel in which a physiological solution is fixed as a dispersion medium.
  • the ampholyte polymer is an aminocarboxy dextran in which an amino group and a carboxyl group are introduced into dextran, and the polymer that is not an ampholyte is dextran. Or the ampholyte polymer is carboxylated ⁇ -poly-L-lysine,
  • a cell-embedded hydrogel comprising cells or tissues embedded in any one of (1) to (4).
  • a cell-embedded hydrogel for cryopreservation wherein cells or tissues are embedded in any one of (1) to (4).
  • the hydrogel according to the present invention is a gel that can be formed from a suspension in which cells are dispersed without damaging the cells.
  • the hydrogel is a three-dimensional scaffold material for cell proliferation and / or differentiation. Furthermore, this hydrogel protects embedded cells in the process of freezing and thawing. Therefore, the present invention also includes a cell cryoprotectant and a composition for cell cryoprotection using the hydrogel, a three-dimensional scaffold material for cell cryoprotection, and a hydrogel for cell cryoprotection.
  • the present invention includes the following (11) to (11).
  • (11) An amphoteric electrolyte polymer having an amino group and a carboxyl group in the same molecule, wherein the ratio of carboxyl group to amino group (carboxyl group / amino group) is 0.45 / 0.55 to 0.95 / 0.05.
  • a method for producing a hydrogel comprising a step of gelling an amphoteric electrolyte polymer in the range of the above by intermolecular crosslinking in a physiological solution.
  • the ampholyte polymer is an aminocarboxy dextran in which an amino group and a carboxyl group are introduced into dextran, and the polymer that is not an ampholyte is dextran. Or the ampholyte polymer is carboxylated ⁇ -poly-L-lysine, (11) The production method of any one of (12). (14) The production method of any one of (11) to (14), wherein the ampholyte polymer is contained in a physiological solution in an amount of 1 to 60% by mass.
  • a step of dispersing or immersing cells or tissues together with an ampholyte polymer in a physiological solution comprising: (16) A step of cryopreserving the cell-embedded hydrogel produced by the production method of (15), A method for cryopreserving cells or tissues.
  • the present inventor uses the above-mentioned polymer cryopreservation agent to cryopreserve and thaw cells, and then gels by subjecting the obtained cell suspension to a crosslinking reaction by a specific technique. It was found that the cells suspended after being subjected to freezing and thawing were not damaged by the crosslinking reaction for gelation. According to such an embodiment, after the cell suspension is cryopreserved and thawed, it can be injected into a desired site for gelation. From the cell suspension at the desired site, A three-dimensional structure capable of original culture or three-dimensional growth can be formed. That is, an injectable gel material (injectable gel material) can be realized.
  • the polymer cryopreservation agent forms a gel, which provides a three-dimensional scaffold material for cell growth and / or differentiation. Therefore, the present invention also exists in an injectable gel material, an injectable gel material for cell cryoprotection, a gelling cell cryoprotectant, and a gelling cell cryoprotection by the ampholyte polymer. Also in the composition.
  • modified dextran can be particularly suitably used as the polymer cryopreservation agent.
  • This modified dextran is excellent in that it does not damage cells even when gelled by a crosslinking reaction.
  • this modified dextran is excellent in that high viability can be maintained even when cells three-dimensionally embedded in a gel are cryopreserved and thawed.
  • this modified dextran is excellent in that it can maintain high viability in cells that have undergone cryopreservation and thawing even when used as a polymer cryopreservation agent that is added to a cell suspension without gelation.
  • the present invention includes the following (21) to (21).
  • a cell cryopreservation agent comprising the aminocarboxyl dextran of (21).
  • (21) aminocarboxyl dextran is A solution for cell cryopreservation, which is dissolved and contained in a physiological solution at a concentration of 1 to 60% by mass.
  • the present invention includes the following (31) to (31).
  • (31) The aminocarboxyl dextran of (21), wherein the aminocarboxyl dextran is a carboxyl group-introduced aminated dextran obtained by introducing a carboxyl group into an aminated dextran.
  • (32) The cell cryopreservation agent according to (22), wherein the aminocarboxydextran is a carboxyl group-introduced aminated dextran obtained by introducing a carboxyl group into an aminated dextran.
  • the present invention includes the following (41) to (41).
  • (41) A method for producing a cell cryopreservation solution by dissolving the aminocarboxyl dextran of (21) or (31) in a physiological solution at a concentration of 1 to 60% by mass.
  • (42) Use of the aminocarboxyl dextran of (21) or (31) for producing a cell cryopreservation solution.
  • the present invention includes the following (51) to (51). (51) (23) or (33) a step of dispersing or immersing cells or tissue in the cell cryopreservation solution; Freezing the dispersed or immersed cells or tissues; A method of cryopreserving a cell or tissue comprising (52) (51) In the method of cryopreserving a cell or tissue, After the step of freezing the dispersed or immersed cells or tissues, Storing frozen cells or tissues under freezing; Thawing cells or tissues stored under freezing, A method of cryopreserving a cell or tissue comprising
  • the present invention includes the following (61) to (61).
  • (61) The aminocarboxyl dextran of (21) or (31) is intermolecularly crosslinked, A hydrogel in which a physiological solution is fixed as a dispersion medium.
  • (62) Intermolecular crosslinking of aminocarboxy dextran (61) is an intermolecular bridge formed by a triazole ring formation reaction between an azide group introduced into a molecule of aminocarboxydextran and a carbon-carbon triple bond moiety introduced into another molecule of aminocarboxydextran. gel.
  • the carbon-carbon triple bond moiety introduced into the aminocarboxydextran molecule The hydrogel according to (62), which is a carbon-carbon triple bond portion of a terminal alkyne group or a cyclooctyne ring-containing compound group.
  • (64) (61) A cell-embedded hydrogel obtained by embedding cells or tissues in any one of the hydrogels.
  • the present invention includes the following (71) to (71).
  • (71) An azide-introduced aminocarboxyl dextran in which an azide group capable of triazole ring formation reaction is introduced into the aminocarboxyl dextran of (21) or (31).
  • (72) A carbon-carbon triple bond-introduced aminocarboxyl dextran in which a carbon-carbon triple bond moiety capable of triazole ring formation reaction is introduced into the aminocarboxyl dextran of (21) or (31).
  • (73) As introduction of azido group, The —OH group of aminocarboxydextran has the following formula (I):
  • Y 1 is a spacer that can be introduced at the position of the —OH group of aminocarboxydextran
  • Y 1 is the following: —O—CO—O— (CH 2 ) n — (Where n is an integer from 1 to 24) —O—CO—O— (CH 2 —CH 2 —O) m ⁇ 1 —CH 2 —CH 2 — (Where m is an integer from 1 to 24) —O—NH— (CH 2 ) j — (Where j is an integer from 1 to 24) —O—NH— (CH 2 —CH 2 —O) k-1 —CH 2 —CH 2 — (Where k is an integer from 1 to 24)
  • the carbon-carbon triple bond moiety introduced into the aminocarboxy dextran is The carbon-carbon triple bond-introduced aminocarboxyl dextran of (72), which is a terminal alkyne group or a cyclooctyne ring-containing compound group.
  • the —OH group of aminocarboxydextran is represented by the following formula (II) or formula (III):
  • Y 3 is a spacer that can be introduced at the position of the —OH group of aminocarboxydextran
  • R1 and R2 are Each independently hydrogen or a C1-C12 alkyl group, Alternatively, they integrally form a substituted or unsubstituted C6-C12 aromatic ring structure
  • R3 and R4 are Each independently hydrogen or a C1-C12 alkyl group, Alternatively, they together form a substituted or unsubstituted C6-C12 aromatic ring structure
  • a group of formula III is represented by the following formula IV:
  • Y 2 is, of the following: —O—CO—N (CH 3 ) — (CH 2 ) n — (Where n is an integer from 1 to 24) —O—CO—NH— (CH 2 —CH 2 —O) m-1 —CH 2 — (Where m is an integer from 1 to 24) —O—NH— (CH 2 ) j — (Where j is an integer from 1 to 24) —O—NH— (CH 2 —CH 2 —O) k-1 —CH 2 —CH 2 — (Where k is an integer from 1 to 24)
  • Y 3 is, of the following: —O—CO—N (CH 3 ) — (CH 2 ) n —CO— (Where n is an integer from 1 to 24) —O—CO—NH— (CH 2 —CH 2 —O) m ⁇ 1 —CH 2 —CO— (Where m is an integer from 1 to 24) —O—NH— (CH 2 ) j —CO— (Where j is an integer from 1 to 24) —O—NH— (CH 2 —CH 2 —O) k-1 —CH 2 —CO— (Where k is an integer from 1 to 24) A carbon-carbon triple bond-introduced aminocarboxyl dextran according to (73), wherein the spacer is a divalent group selected from the group consisting of (wherein the —N group of the cyclooctyne ring is bonded to the right end of each spacer).
  • the present invention includes the following (81) to (81).
  • (81) A dextran kit comprising any combination of the following (a) and (c), (a) and (d), (b) and (c): (A) the azide-introduced aminocarboxyl dextran of any of (71), (73) to (75), (B) Azide-introduced dextran (wherein azide introduction is as defined in any of (71), (73) to (74)), (C) the carbon-carbon triple bond-introduced aminocarboxyl dextran of any of (72), (75) to (79), (D) Carbon-carbon triple bond introduced dextran (however, carbon-carbon triple bond introduction is as defined in any of (72), (75) to (79)).
  • An aminocarboxyl dextran-containing composition comprising any combination of (81) (a) and (c), (a) and (d), and (b) and (c).
  • (83) (71) A cell cryopreservation agent comprising the aminocarboxyl dextran according to any one of (71) to (79).
  • (84) (71) to (79) any one of aminocarboxyl dextran is A solution for cell cryopreservation, which is dissolved and contained in a physiological solution at a concentration of 1 to 60% by mass.
  • the present invention includes the following (91) to (91).
  • (91) An additive for producing a cell-embedded hydrogel comprising the aminocarboxyl dextran according to any one of (71) to (79).
  • (92) A solution for producing a cell-embedded hydrogel, wherein the aminocarboxyl dextran according to any one of (71) to (79) is dissolved and contained in a physiological solution at a concentration of 1 to 60% by mass.
  • the solution for producing a cell-embedded hydrogel according to (92) wherein the physiological solution is physiological saline, cell culture liquid medium, or tissue culture liquid medium.
  • (94) (92) A cell-containing composition for producing a cell-embedded hydrogel, wherein cells or tissues are dispersed or immersed in the solution for producing a cell-embedded hydrogel according to any one of (92) to (93).
  • (95) (94) A cell-embedded hydrogel obtained by gelling the cell-containing composition for producing a cell-embedded hydrogel.
  • (96) (82) An additive for producing a cell-embedded hydrogel comprising the aminocarboxydextran-containing composition.
  • (97) (82) A solution for producing a cell-embedded hydrogel, comprising the aminocarboxydextran-containing composition dissolved in a physiological solution at a concentration of 1 to 60% by mass as the aminocarboxydextran concentration.
  • the present invention includes the following (101) to (101).
  • (101) A method for producing a solution for producing a cell-embedded hydrogel by dissolving the aminocarboxyl dextran of any one of (71) to (79) in a physiological solution at a concentration of 1 to 60% by mass.
  • (102) Use of the aminocarboxyl dextran of any of (71) to (79) for producing a solution for producing a cell-embedded hydrogel.
  • (103) A cell-containing composition for producing a cell-embedded hydrogel by dispersing or immersing cells or tissues in the solution for producing a cell-embedded hydrogel according to any one of (92) to (93) and (97) to (98) A method of manufacturing things.
  • the present invention includes the following (111) to (11). (111) (92) to (93), (97) to (98) Step of dispersing or immersing cells or tissue in the cell-embedded hydrogel production solution Freezing the dispersed or immersed cells or tissue
  • a method of cryopreserving a cell or tissue comprising (112) (111)
  • After the step of freezing the dispersed or immersed cells or tissues Storing frozen cells or tissues under freezing; Thawing cells or tissues stored under freezing
  • a method of cryopreserving a cell or tissue comprising (113) A cell-containing composition for producing a cell-embedded hydrogel by dispersing or immersing cells or tissues in the solution for producing a cell-embedded hydrogel according to any one of (92) to (93) and (97) to (98) Preparing a product, Freezing the prepared cell-containing composition; Storing the frozen cell-containing composition under freezing; Thawing a cell-containing composition stored under freezing, A step of preparing
  • the present invention includes the following (121) to (121).
  • (121) A cell-containing composition for producing a cell-embedded hydrogel by dispersing or immersing cells or tissues in the solution for producing a cell-embedded hydrogel according to any one of (92) to (93) and (97) to (98) Preparing a product, Preparing the cell-embedded hydrogel by gelling the prepared cell-containing composition by an intermolecular cross-linking reaction of aminocarboxydextran; A method for producing a cell-embedded hydrogel from a cell-containing composition.
  • the formation reaction of the intermolecular bridge of aminocarboxy dextran is The production method of (131), which is a triazole ring formation reaction between an azide moiety and an alkyne moiety of aminocarboxydextran.
  • (133) A cell-containing composition for producing a cell-embedded hydrogel by dispersing or immersing cells or tissues in the solution for producing a cell-embedded hydrogel according to any one of (92) to (93) and (97) to (98) Preparing a product, Preparing the cell-embedded hydrogel by gelling the prepared cell-containing composition by an intermolecular cross-linking reaction of aminocarboxydextran; Freezing the prepared cell-embedded hydrogel, A method of cryopreserving a cell or tissue comprising (134) (132) In the method for cryopreserving cells and tissues, After the step of freezing the prepared cell-embedded hydrogel, Storing the frozen cell-embedded hydrogel under freezing; Thawing a cell-
  • the present inventors have found that it is particularly suitable to use modified polylysine as the polymer cryopreservation agent and gel with a specific crosslinking agent. Gelation by this combination is excellent in that cells are not damaged even when cells coexist during the gelation reaction.
  • This modified dextran is excellent in that high viability can be maintained even when cells three-dimensionally embedded in a gel are cryopreserved and thawed. Therefore, the present invention includes the following (141) to (141).
  • n is an integer such that the molecular weight of the compound of formula V is in the range of 1000 to 100,000
  • X is the following formula (VI):
  • (146) (144) A method for cryopreserving cells or tissues, wherein the cell-embedded hydrogel for cryopreservation according to (144) is cryopreserved.
  • the present invention includes the following (151) to (151).
  • (151) Carboxylated ⁇ -poly-L-lysine having a carboxyl group to amino group ratio (carboxyl group / amino group) in the range of 0.45 / 0.55 to 0.95 / 0.05,
  • 152) Physiology containing carboxylated ⁇ -poly-L-lysine in which the ratio of carboxyl group to amino group (carboxyl group / amino group) is in the range of 0.45 / 0.55 to 0.95 / 0.05.
  • Preparing a cell-containing composition by dispersing or immersing cells or tissues in an aqueous solution, The cell-containing composition is cross-linked by intermolecular cross-linking generated by an amide bond forming reaction between the amino group of ⁇ -poly-L-lysine and the N-hydroxysuccinimide group of multi-arm PEG to gel.
  • Preparing a cell-embedded hydrogel A method for producing a cell-embedded hydrogel comprising: (153) Physiology containing carboxylated ⁇ -poly-L-lysine in which the ratio of carboxyl group to amino group (carboxyl group / amino group) is in the range of 0.45 / 0.55 to 0.95 / 0.05.
  • Preparing a cell-containing composition by dispersing or immersing cells or tissues in an aqueous solution, Freezing the prepared cell-containing composition; Storing the frozen cell-containing composition under freezing; Thawing a cell-containing composition stored under freezing, The thawed cell-containing composition is cross-linked by intermolecular cross-linking generated by an amide bond forming reaction between the amino group of ⁇ -poly-L-lysine and the N-hydroxysuccinimide group of multi-arm PEG.
  • a step of gelling to prepare a cell-embedded hydrogel A method for producing a cell-embedded hydrogel comprising:
  • a three-dimensional structure containing cells can be safely frozen and thawed. Therefore, it becomes possible to store or transport cells and tissues that have been three-dimensionally cultured for a long period of time. Therefore, the present invention provides a technique for storing and transporting regenerated cells and tissues for a long period of time, which is necessary to realize the industrial spread of regenerative medicine.
  • FIG. 1 is a fluorescent photograph showing the results of cell viability determination after gelation of carboxylated polylysine.
  • FIG. 2 is a graph showing the survival rate of cells cryoprotected by Dex-PA solution.
  • FIG. 3 is a fluorescence photograph showing the results of viability determination of cells cryoprotected by a Dex-PA gel.
  • FIG. 4 is a fluorescence photograph observing the localization of Dex-PA in the vicinity of cells after freeze-thawing by FITC fluorescence.
  • FIG. 5 is a graph showing the concentration dependence of the cell cryoprotective activity of Dex-PA.
  • FIG. 6 is a photograph of the gel by Azide-Dex-PA and DBCO-Dex.
  • FIG. 1 is a fluorescent photograph showing the results of cell viability determination after gelation of carboxylated polylysine.
  • FIG. 2 is a graph showing the survival rate of cells cryoprotected by Dex-PA solution.
  • FIG. 3 is a flu
  • FIG. 7a is a fluorescent photograph showing the result of cell viability determination after freeze-thawing on a gel having an Azide: Alkyne ratio of 1: 4.
  • FIG. 7b is a fluorescence photograph showing the results of cell viability determination after freeze-thawing on a gel having an Azide: Alkyne ratio of 1: 6.
  • FIG. 7c is a fluorescence photograph showing the results of cell viability determination after freezing and thawing on a 1% collagen gel.
  • a hydrogel in which the ampholyte polymer in the range of .95 / 0.05 is intermolecularly crosslinked in a physiological solution, and the physiological solution is fixed as a dispersion medium can be obtained.
  • the hydrogel of the present invention can embed cells and tissues therein and protect them from damage caused by cryopreservation and thawing. Further, the hydrogel of the present invention itself provides a three-dimensional scaffold for cells. Three-dimensional scaffolds are considered very advantageous for cell growth and / or differentiation, and three-dimensional scaffolds that can cryoprotect cells are highly desirable. is there. In addition, the hydrogel of the present invention achieves a high cell survival rate because cell damage is minimized during the formation reaction of intermolecular crosslinks for gelation.
  • amphoteric electrolyte polymer The ampholyte polymer of the present invention has an amino group and a carboxyl group in the same molecule, and the ratio of carboxyl group to amino group (carboxyl group / amino group) is 0.45 / 0.55 to It is in the range of 0.95 / 0.05. This ratio is preferably in the range of 0.45 / 0.55 to 0.95 / 0.05, more preferably in the range of 0.50 / 0.50 to 0.90 / 0.10, or for example The range may be 70 / 0.30 to 0.75 / 0.25.
  • Introduction of an amino group and a carboxyl group into a polymer molecule can be performed by a known modification means.
  • the amino group is subjected to carboxylation or acetylation using, for example, carboxylic anhydride.
  • a carboxyl group can be introduce
  • examples of the polymer into which an amino group and a carboxyl group are introduced include dextran.
  • Dextran is a polysaccharide polymer having glucose as a structural unit, and includes ⁇ -1,6 bonds and ⁇ -1,4 bonds.
  • dextran for example, commercially available dextran can be used.
  • the number average molecular weight includes, for example, a range of 1000 to 10,000,000, such as a range of 5000 to 1,000,000, such as a range of 10,000 to 500,000, such as a range of 10,000 to 100,000. Can be used.
  • amino groups and carboxyl groups are introduced in the above ratio with respect to dextran.
  • an atomic group having an amino group with respect to the hydroxyl group of dextran an atomic group having a carboxyl group is introduced with respect to a certain ratio of the introduced amino group, and the amino group And a carboxyl group can be introduced into dextran to form an ampholyte polymer.
  • Such an amino group can be introduced by a known means, for example, using carbonyldiimidazole (CDI) and a diamine compound.
  • the introduction of the carboxyl group to the amino group thus introduced can be carried out by a known means, for example, using carboxylic anhydride.
  • carboxylic anhydride examples include acetic anhydride, citric anhydride, succinic anhydride, glutaric anhydride, malic anhydride, fumaric anhydride, and maleic anhydride. Of these, succinic anhydride and acetic anhydride are preferred, and succinic anhydride is particularly preferred.
  • a carboxyl group may be introduce
  • the introduced carboxyl group can be further partially aminated by reacting it with a compound such as diamine, triamine or polyamine.
  • a compound such as diamine, triamine or polyamine.
  • diamine used in this way include ethylenediamine.
  • An example of introduction of an amino group and a carboxyl group into dextran is shown in the following scheme 1.
  • a polyamine can be used as the polymer having an amino group.
  • polyamino acids and aminated polysaccharides can be used.
  • polylysine, polyallylamine, polyarginine, polyarginine, polyglutamic acid, and polyaspartic acid can be used.
  • ⁇ -poly-L-lysine can be used as the polymer having an amino group.
  • the number average molecular weight is, for example, in the range of 1000 to 10,000,000, such as in the range of 5000 to 1,000,000, such as in the range of 10,000 to 500,000, such as 10,000 to 100. Those containing the range of 1,000 can be used.
  • a carboxyl group is introduced so that the amino group and the carboxyl group are in the above ratio with respect to the polymer having an amino group.
  • the introduction of the carboxyl group with respect to the amino group can be carried out by a known means. For example, it can be introduced using the carboxylic anhydride described above. Of these, succinic anhydride and acetic anhydride are preferred, and succinic anhydride is particularly preferred.
  • a carboxyl group may be introduce
  • the introduced carboxyl group can be further partially aminated by reacting it with a compound such as diamine, triamine or polyamine.
  • the hydrogel of the present invention is obtained by intermolecular crosslinking of the ampholyte polymer in a physiological solution.
  • the physiological solution include physiological saline, cell culture liquid medium, tissue culture liquid medium, and serum-free medium.
  • DMEM Dulbecco's modified Eagle MEM medium
  • the intermolecular cross-linking of the present invention can form a hydrogel by a cross-linking reaction even in a solution containing such various physiological substances and cells themselves.
  • the ampholyte polymer can be added to the physiological solution so as to have a concentration of, for example, 1 to 60% by mass, for example, 5 to 30% by mass.
  • the amphoteric electrolyte polymer and the hydrogel formed by crosslinking the amphoteric electrolyte polymer exhibit excellent cryoprotective effects themselves, and only this is suitably used as a cryoprotectant.
  • a known cryoprotectant can be additionally added to the physiological solution and used.
  • known cryoprotective substances include dimethyl sulfoxide, glycerol, ethylene glycol, trehalose, sucrose, and the like, or an antioxidant.
  • an antioxidant include polyphenols such as catalase, peroxidase, superoxide dismutase, vitamin E, vitamin C, and epigallocatechin gallate, or glutathione.
  • the intermolecular cross-linking is carried out by introducing an azido group introduced into a molecule of an ampholyte polymer or a molecule of a polymer that is not an ampholyte and another molecule of the ampholyte polymer or a non-ampholyte polymer.
  • An intermolecular bridge formed by a triazole ring-forming reaction with a carbon-carbon triple bond moiety introduced into another molecule of the molecule (however, a molecule introduced with an azide group and a molecule introduced with a carbon-carbon triple bond moiety) Among them, at least one of the molecules is an ampholyte polymer).
  • an azide group or a carbon-carbon triple bond moiety is introduced into the ampholyte polymer that is responsible for cell protection.
  • Cells are dispersed or tissue is immersed in the solution of the ampholyte polymer modified in this way.
  • a molecule having a carbon-carbon triple bond moiety or an azide group introduced so as to enable a triazole ring formation reaction with this modified ampholyte polymer molecule is added as if it were a cross-linking agent, and a triazole ring was added.
  • the formation reaction can proceed to form intermolecular crosslinks and gelation can be performed.
  • the molecule added as if it were a crosslinking agent may be an ampholyte polymer molecule or a polymer molecule that is not an ampholyte. This is because the total amount may be adjusted so that the amount of the ampholyte polymer in the finally formed gel becomes a desired amount or concentration.
  • aminocarboxy dextran can be used as the ampholyte polymer and dextran can be used as the non-ampholyte polymer.
  • an azide group is introduced into a molecule of an amphoteric electrolyte polymer or a molecule of a polymer that is not an ampholyte so that a triazole ring formation reaction is possible.
  • an atomic group having a terminal azide group can be introduced. For example, the following formula (I):
  • Y 1 is a spacer inserted as desired.
  • aminocarboxydextran is used as the ampholyte polymer or dextran is used as the polymer that is not an ampholyte, it is preferably a group that can be introduced into the hydroxyl group of the glucose ring of dextran.
  • Y 1 is, for example: —O—CO—O— (CH 2 ) n — (Where n is an integer from 1 to 24) —O—CO—O— (CH 2 —CH 2 —O) m ⁇ 1 —CH 2 —CH 2 — (Where m is an integer from 1 to 24) —O—NH— (CH 2 ) j — (Where j is an integer from 1 to 24) —O—NH— (CH 2 —CH 2 —O) k-1 —CH 2 —CH 2 — (Where k is an integer from 1 to 24) Or a divalent spacer selected from the group consisting of (wherein —N 3 is bound to the right end of each spacer).
  • N, m, j, and k can be independently set to desired values. For example, they can be integers of 1 to 24, integers of 1 to 12, and integers of 1 to 6.
  • a carbon-carbon triple bond moiety is introduced into a molecule of an amphoteric electrolyte polymer or a non-ampholyte polymer molecule so that a triazole ring formation reaction is possible.
  • the terminal alkyne group or the cyclooctyne ring-containing compound group can be used as the group having a carbon-carbon triple bond moiety.
  • Y 2 and Y 3 are spacers inserted as desired.
  • aminocarboxydextran is used as the ampholyte polymer or dextran is used as the polymer that is not an ampholyte, it is preferably a group that can be introduced into the hydroxyl group of the glucose ring of dextran.
  • R1 and R2 are each independently hydrogen or a C1-C12 alkyl group, or together, a substituted or unsubstituted C6-C12 aromatic cyclic structure.
  • R3 and R4 are each independently hydrogen, a C1-C12 alkyl group, or together, a substituted or unsubstituted C6-C12 fragrance. It can be a group forming a group cyclic structure.
  • the substituted or unsubstituted C6-C12 aromatic ring structure may be a heterocyclic ring.
  • the substituted or unsubstituted C6-C12 aromatic ring structure can be a substituted or unsubstituted benzene ring.
  • the substituent on the benzene ring can be, for example, a C1-C12 alkyl group.
  • R 1 and R 2, R 3 and R 4 can be joined together to form an unsubstituted benzene ring, ie, a group represented by formula III can have the following formula IV:
  • Y 2 is the following: —O—CO—N (CH 3 ) — (CH 2 ) n — (Where n is an integer from 1 to 24) —O—CO—NH— (CH 2 —CH 2 —O) m-1 —CH 2 — (Where m is an integer from 1 to 24) —O—NH— (CH 2 ) j — (Where j is an integer from 1 to 24) —O—NH— (CH 2 —CH 2 —O) k-1 —CH 2 —CH 2 — (Where k is an integer from 1 to 24) A divalent spacer selected from the group consisting of (wherein —C ⁇ CH 2 is bonded to the right end of each spacer).
  • N, m, j, and k can be independently set to desired values. For example, they can be integers of 1 to 24, integers of 1 to 12, and integers of 1 to 6.
  • said Y 3 is: —O—CO—N (CH 3 ) — (CH 2 ) n —CO— (Where n is an integer from 1 to 24) —O—CO—NH— (CH 2 —CH 2 —O) m ⁇ 1 —CH 2 —CO— (Where m is an integer from 1 to 24) —O—NH— (CH 2 ) j —CO— (Where j is an integer from 1 to 24) —O—NH— (CH 2 —CH 2 —O) k-1 —CH 2 —CO— (Where k is an integer from 1 to 24) A divalent spacer selected from the group consisting of (wherein the —N group of the cyclooctyne ring is bound to the right end of each spacer).
  • N, m, j, and k can be independently set to desired values. For example, they can be integers of 1 to 24, integers of 1 to 12, and integers of 1 to 6.
  • the intermolecular crosslink is generated by an amide bond forming reaction between the amino group of the ampholyte polymer and the N-hydroxysuccinimide group of multi-arm PEG (polyethylene glycol).
  • multi-arm PEG polyethylene glycol
  • the number of branches (arms) meaning multi-arm can be selected according to the number of molecules intended for crosslinking, and can be 2 to 16, 2 to 8, 2 to 4, for example.
  • 4-arm PEG can be used.
  • the multi-arm PEG has the following formula (V):
  • the 4-arm PEG represented by N in the formula (V) may be an integer such that the molecular weight of the compound of the formula V is in the range of 1,000 to 100,000, for example, in the range of 1,000 to 50,000.
  • X in the above formula (V) represents the following formula (VI):
  • the ampholyte polymer crosslinked by multi-arm PEG is a carboxylated polylysine, preferably a carboxylated ⁇ -poly-L-lysine.
  • the amino group of the carboxylated ⁇ -poly-L-lysine is suitably cross-linked by multi-arm PEG (polyethylene glycol) to form a hydrogel.
  • the intermolecular cross-linking reaction for forming the hydrogel of the present invention proceeds rapidly under physiological conditions under atmospheric pressure, and further, coexistence of various molecules on the cell surface and components in the cell culture medium. Proceeds quickly even under. Therefore, after dispersing the cells or immersing the tissue in the solution for producing the hydrogel of the present invention, an intermolecular cross-linking reaction is performed to form a hydrogel, and a desired three-dimensional structure is obtained. It can also be cryopreserved with the tissue embedded in a hydrogel.
  • a desired three-dimensional structure can be formed in a living body by injecting into a site, for example, a site where a wound is desired to be healed, and performing an intermolecular crosslinking reaction in the physiological state to form a hydrogel.
  • intermolecular crosslinking can be performed under temperature conditions that allow cell culture, for example, at 35 to 38 ° C., for example, 37 ° C.
  • the intermolecular crosslinking reaction due to the formation of the triazole ring it can be accelerated by adopting known suitable conditions for the triazole ring formation reaction by the azide group and the carbon-carbon triple bond.
  • copper ions and ascorbic acid can be added in the reaction between the terminal alkyne group and the azide group.
  • the molar ratio of the azide group (azi group) to the terminal alkyne group can be used as long as it can be gel-formed.
  • It can be in the range of ⁇ 1: 2.
  • known suitable conditions for the amide bond formation reaction between an amino group and a group of N-hydroxysuccinimide This can be accelerated.
  • amphoteric electrolyte dextran The inventor of the present invention, when an ampholyte dextran (Dex-PA) obtained by introducing an amino group and a carboxyl group into dextran is gelled suitably without damaging cells and tissues when cross-linked by the above means. When it was found that the obtained hydrogel had an excellent cryoprotective effect, the ampholyte dextran (Dex-PA) was used as a solution without hydrogelation. Also found that it has an excellent cryoprotective effect.
  • Dex-PA ampholyte dextran
  • the present invention provides an aminocarboxyl dextran in which an amino group and a carboxyl group are introduced into dextran, wherein the ratio of the carboxyl group to the amino group (carboxyl group / amino group) is 0.45 / 0.55 to There is also a cell cryopreservation agent consisting of aminocarboxy dextran in the range of 0.95 / 0.05.
  • This ampholyte dextran (Dex-PA) has an excellent cryoprotective effect both when the azide group for the triazole ring formation reaction is introduced or when a carbon-carbon triple bond moiety is introduced. It was what you are doing.
  • an azide group-introduced aminocarboxyl dextran and a carbon-carbon triple bond moiety-introduced aminocarboxyl dextran are themselves excellent cell cryopreservation agents.
  • concentration of these dextran derivatives can be appropriately selected within a range in which a cytoprotective effect is exhibited. For example, 5-20%, 7-20%, 7-15%, It can be in the range of 8-14%, 10-13%, 10-12%.
  • a hydrogel can be formed by performing a crosslinking reaction under physiological conditions using a physiological solution and also at a temperature and atmospheric pressure. Therefore, there are no particular limitations on the cells and tissues that can be embedded in the hydrogel. Furthermore, in the present invention, the amphoteric electrolyte polymer and the hydrogel based on the amphoteric electrolyte polymer exhibit cryoprotective effects without penetrating into the cells. Therefore, there are no particular limitations on the cells and tissues that can exhibit the effect of cryoprotection.
  • such cells can include, for example, established cells for culture, fertilized eggs and egg cells of animals including humans, and also include, for example, sperm cells, ES cells, iPS cells, Examples include stem cells such as leaf stem cells, hematopoietic stem cells, neural stem cells, umbilical cord blood cells, animal cells or plant cells including humans such as hepatocytes, nerve cells, cardiomyocytes, vascular endothelial cells, vascular smooth muscle cells, blood cells, etc. Can do.
  • examples of such tissues / organs include skin, nerve, blood vessel, cartilage, cornea, liver, kidney, heart, islet, and the like, and further include cells derived therefrom. Can do.
  • the amphoteric electrolyte polymer PLL (0.50) aqueous solution is a polymer solution that can cryopreserve cells with high survival rate, and the cells are dispersed by the amino groups of the amphoteric electrolyte polymer PLL (0.50). It was found that it was possible to form a hydrogel while maintaining the cells with a high survival rate. In other words, the cells can be frozen and then thawed, and the gel can be immediately gelled without the need to remove the cell cryopreservation agent. It was found that it can be formed. Further, this hydrogel had biodegradability that biodegraded in 24 to 48 hours under culture conditions.
  • injectable gel three-dimensionally embedded cells engraft as it is
  • aminated dextran was made into a 10% solution, succinic anhydride was added, and reacted at 50 ° C. for 1 hour to prepare various carboxyl group-introduced aminated dextran (dextran ampholyte, (Dex-PA)).
  • Dex-PA carboxyl group-introduced aminated dextran
  • FIG. 2 is a bar graph showing the results.
  • the horizontal axis of FIG. 2 shows the ratio (%) of carboxyl groups to all amino groups in the modified dextran.
  • EDA-introduced azidated dextran and azide-introduced ampholyte dextran were synthesized by the same method as the above-described aminated dextran synthesis method.
  • FIG. 3 is a fluorescent photograph showing the result.
  • FIG. 3 is a photograph of double fluorescence staining of green and red in a color photograph, in which green fluorescence indicates cells that are alive and red fluorescence indicates cells that are not alive. . Viability was calculated by counting these.
  • Dex-PA by introducing an azide group and an alkyl group to cause a cross-linking reaction, and the cross-linking reaction and gelation also damage the cells embedded therein. And found a high survival rate. Furthermore, the Dex-PA hydrogel produced by the cross-linking reaction does not damage the cells even when cryopreserved with the cells embedded, that is, the Dex-PA hydrogel is embedded. The cells were found to have a high cryoprotective effect.
  • the amphoteric electrolyte polymer Dex-PA aqueous solution is a polymer solution capable of cryopreserving cells with high survival rate
  • the amphoteric electrolyte polymer Dex-PA has an azide group and a carbon-carbon triple bond.
  • a hydrogel is formed by cross-linking of the ampholyte polymer Dex-PA, a three-dimensional structure capable of three-dimensional culture is formed from the cell suspension, and the cells are embedded in the tertiary.
  • the original structure can be cryopreserved and thawed, so to speak, it provides a three-dimensional scaffold material for cell growth and / or differentiation through the process of cryopreservation and thawing. all right.
  • FIG. 4 is a photograph of the localization of Dex-PA in the vicinity of the cells observed by FITC fluorescence after freezing and thawing.
  • fluorescence was observed only around the cell membrane, and it was confirmed that the amphoteric electrolyte did not enter the cytoplasm. That is, from this result, it was found that the ampholyte polymer Dex-PA of the present invention cryoprotects cells without penetrating into the cells. That is, in the present invention, it was found that the cells were cryoprotected without penetrating into the cells by the scaffold material as the extracellular matrix.
  • Example 5 [Concentration dependence of cell cryoprotective activity of Dex-PA]
  • Dex-PA solution concentration 10%
  • carboxyl group introduction amounts frozen, thawed, and the same as the evaluation of survival rate.
  • the cryoprotective activity of a solution in which the concentration was changed from 5% to 15% using Azide-Dex-PA with a carboxyl group introduction amount of 65% was also examined. As a result, as shown in FIG. 5, it was found that 12% showed the highest cryoprotective activity.
  • Example 6 [Cell-embedded gel with Azide-Dex-PA and DBCO-Dex] As described later, according to the procedure of Scheme 9, a cell (L929) suspension was gelled to prepare a cell-embedded gel, and an experiment was conducted to examine its cytoprotective activity. A photograph of the gel obtained in this experiment is shown in FIG. In FIG. 6, the bottom of the container placed upside down is in the range surrounded by the upper elliptical line, and the resulting gel is observed there.
  • Table 1 shows the Azide-Dex-PA concentration A (mg / mL), DBCO-Dex concentration B (mg / mL), and the ratio in the mixed solution of azide and DBCO (Azide: Alkyne mole). Ratio) and gelation time.
  • FIG. 7A shows a gel having a ratio of Azide to DBCO (Alkyne) of 1: 4 (polymer concentration 10%)
  • FIG. 7B shows a gel having a ratio of Azide: Alkyne 1: 6 (polymer concentration of 10). %)
  • (C) of FIG. 7 shows the state of cells frozen and thawed in a 1% collagen gel.
  • the survival rate in FIG. 7A was 93%
  • the survival rate in FIG. 7B was 94%
  • the survival rate in FIG. 7C was 0%.
  • a three-dimensional structure containing cells can be safely frozen and thawed. Therefore, it becomes possible to store or transport cells and tissues that have been three-dimensionally cultured for a long period of time.
  • the present invention provides a technique for storing and transporting regenerated cells and tissues for a long period of time, which is necessary to realize the industrial spread of regenerative medicine.
  • the present invention is an industrially useful invention.

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Abstract

La présente invention concerne un agent de cryoconservation permettant la culture en trois dimensions de cellules. La présente invention concerne un hydrogel obtenu par réticulation intermoléculaire d'un polymère de type électrolyte amphotère dans une solution physiologique de façon à ce que ledit polymère de type électrolyte amphotère puisse être immobilisé dans la solution physiologique servant de milieu de dispersion, ledit polymère de type électrolyte amphotère comportant à la fois un groupe amine et un groupe carboxyle dans sa molécule et le rapport entre la teneur en groupes carboxyle et en groupes amine (c'est-à-dire rapport (groupes carboxyle)/(groupes amine)) variant de 0,45/0,55 à 0,95/0,05.
PCT/JP2013/073395 2012-08-31 2013-08-30 Matériau pour tuteur cellulaire pouvant faire l'objet d'une cryoconservation WO2014034884A1 (fr)

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CN110029084A (zh) * 2019-04-12 2019-07-19 河海大学常州校区 一种局部交联强度可调控的非匀质葡聚糖3d凝胶、制备方法及其应用方法
CN110049789A (zh) * 2016-12-13 2019-07-23 米伦纽姆医药公司 生物表面的共形涂层
JP2020022501A (ja) * 2019-11-01 2020-02-13 学校法人明治大学 生体試料保存容器
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JP2016002432A (ja) * 2014-06-19 2016-01-12 テルモ株式会社 細胞を分散したゲルの製造方法およびその製造方法により製造された細胞分散ゲル
US11655343B2 (en) 2016-03-24 2023-05-23 Takeda Pharmaceutical Company Limited Alginate hydrogel compositions
CN106075599A (zh) * 2016-07-06 2016-11-09 青岛大学 一种可细胞原位包覆的多孔冻凝胶及其制备方法
JP7125398B2 (ja) 2016-12-13 2022-08-24 ミレニアム ファーマシューティカルズ， インコーポレイテッド 生体表面のコンフォーマルコーティング
CN110049789A (zh) * 2016-12-13 2019-07-23 米伦纽姆医药公司 生物表面的共形涂层
JP2020501646A (ja) * 2016-12-13 2020-01-23 ミレニアム ファーマシューティカルズ， インコーポレイテッドＭｉｌｌｅｎｎｉｕｍ Ｐｈａｒｍａｃｅｕｔｉｃａｌｓ， Ｉｎｃ． 生体表面のコンフォーマルコーティング
US11998002B2 (en) 2018-03-16 2024-06-04 University Of Warwick Cryopreserving processes
EP3789488A4 (fr) * 2018-03-30 2021-12-29 Ajinomoto Co., Inc. Composition contenant un analogue de polylysine et favorisant la croissance cellulaire
CN110029084B (zh) * 2019-04-12 2023-04-07 河海大学常州校区 一种局部交联强度可调控的非匀质葡聚糖3d凝胶、制备方法及其应用方法
CN110029084A (zh) * 2019-04-12 2019-07-19 河海大学常州校区 一种局部交联强度可调控的非匀质葡聚糖3d凝胶、制备方法及其应用方法
JP2020022501A (ja) * 2019-11-01 2020-02-13 学校法人明治大学 生体試料保存容器
JP2020156523A (ja) * 2020-07-06 2020-10-01 学校法人明治大学 生体試料保存容器
EP4306549A1 (fr) * 2022-07-13 2024-01-17 Adocia Hydrogels pour thérapie cellulaire
WO2024013355A1 (fr) * 2022-07-13 2024-01-18 Adocia Hydrogels pour thérapie cellulaire

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