WO2014034884A1 - Cryopreservable cell scaffold material - Google Patents

Abstract

The present invention provides a cryopreservative agent which enables the three-dimensional culture of cells. The present invention provides a hydrogel produced by the intermolecular crosslinking of an amphoteric electrolyte polymer in a physiological solution so that the amphoteric electrolyte polymer can be immobilized in the physiological solution that serves as a dispersion medium, wherein the amphoteric electrolyte polymer has both an amino group and a carboxyl group in the molecule and has a ratio of the content of the carboxyl group to the content of the amino group (i.e., a (carboxyl group)/(amino group) ratio) ranging from 0.45/0.55 to 0.95/0.05.

Description

Cryopreservable cell scaffold material

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.

In the field of regenerative medicine, tissue regeneration has achieved certain results, particularly with two-dimensional skin, cornea, and myocardium, and commercialization has partially started. In addition, 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. For example, a collagen gel is used as a scaffold material that enables such three-dimensional cell culture.

On the other hand, even in the field of regenerative medicine, even if cell and tissue regeneration and differentiation control technology has been established, if the regenerated cells and tissues cannot be stored or transported for a long period of time, industrial technology for regenerative medicine Dissemination is difficult. For this purpose, a promising technique is a cryopreservation technique for cells and tissues.

However, if cells and tissues are stored frozen as they are, they are usually easily destroyed, and it is difficult to recover live cells and tissues even after thawing. For example, even if the cells embedded in the collagen gel described above are cryopreserved as they are, their survival rate is very low. For this reason, freezing and thawing of such a three-dimensional structure is considered to be particularly difficult.

Conventionally, in order to enable cryopreservation of cells and tissues, it has been proposed to add a low molecular weight compound such as dimethyl sulfoxide (DMSO) as a cryopreservation agent for cells before freezing. In particular, this DMSO is a molecule widely recognized as a cryopreservation agent.

However, according to the study by the present inventor, such a low-permeability molecule has a drawback in that there is a concern that the toxicity to cells and the influence on differentiation are concerned, and further, removal will eventually be required. There are drawbacks. Therefore, the present inventor has proposed a technique using a polylysine derivative as a cryopreservation agent for cells (Patent Document 1: WO 2009/157209, Patent Document 2: JP 2011-30557 A).

PCT International Publication WO2009 / 157209 Japanese Patent Publication No. JP2011-30557

Even with such a polylysine derivative, cells were cryopreserved as a suspension and did not enable three-dimensional cell culture. Accordingly, an object of the present invention is to provide a cryopreservation agent that enables three-dimensional cell culture.

As a result of intensive research on the cryopreservation agent for cells, 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. In some cases, the inventors have found that high viability can be maintained even after thawing, and reached the present invention. According to 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. Among these three-dimensional structures, 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).

(1)
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. 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.
(2)
Intermolecular crosslinking
An azido group introduced into a molecule of an ampholyte polymer or a molecule of a polymer that is not an ampholyte, and
Intermolecular crosslinks formed by a triazole ring formation reaction with a carbon-carbon triple bond moiety introduced into another molecule of the ampholyte polymer or another molecule of the non-ampholyte polymer (provided that an azide group is introduced) And at least one of the molecules introduced with the carbon-carbon triple bond moiety is an ampholyte polymer),
Or an intermolecular crosslink formed by an amide bond forming reaction between the amino group of the ampholyte polymer and the N-hydroxysuccinimide group of the multi-arm PEG,
The hydrogel of (1).
(3)
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 ε-poly-L-lysine,
The hydrogel according to any one of (1) to (2).
(4)
The hydrogel according to any one of (1) to (3), wherein the amphoteric electrolyte polymer is contained in a physiological solution in an amount of 1 to 60% by mass.
(5)
A cell-embedded hydrogel comprising cells or tissues embedded in any one of (1) to (4).
(6)
A cell-embedded hydrogel for cryopreservation, wherein cells or tissues are embedded in any one of (1) to (4).
(7)
(6) A method for cryopreserving cells or tissues, wherein the cell-embedded hydrogel for cryopreservation is cryopreserved.

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.

Furthermore, 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.
(12)
The process of intermolecular crosslinking and gelation
An azido group introduced into a molecule of an ampholyte polymer or a molecule of a polymer that is not an ampholyte, and
Cross-linked by a triazole ring formation reaction with a carbon-carbon triple bond moiety introduced into another molecule of the ampholyte polymer or another molecule of the non-ampholyte polymer (however, an azide group was introduced) A molecule and a carbon-carbon triple bond moiety-introduced molecule, at least one of which is an ampholyte polymer), gelling step,
Or a step of gelation by intermolecular crosslinking by an amide bond forming reaction between the amino group of the ampholyte polymer and the N-hydroxysuccinimide group of the added multi-arm PEG,
The manufacturing method of (11).
(13)
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 ε-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.
(15)
In the production method of any one of (11) to (14), before the step of intermolecular crosslinking and gelation,
A step of dispersing or immersing cells or tissues together with an ampholyte polymer in a physiological solution;
A method for producing a cell-embedded hydrogel comprising:
(16)
A step of cryopreserving the cell-embedded hydrogel produced by the production method of (15),
A method for cryopreserving cells or tissues.
(17)
In the production method of any one of (11) to (14), before the step of intermolecular crosslinking and gelation,
A step of dispersing or immersing cells or tissues together with an ampholyte polymer in a physiological solution;
A step of cryopreserving the dispersed or immersed cells or tissues,
A method for producing a cell-embedded hydrogel after cryopreservation of 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. Among these three-dimensional structures, 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.

The present inventor has found that 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. In addition, 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. In addition, 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. ing. Accordingly, the present invention includes the following (21) to (21).

(21)
An aminocarboxyl dextran in which an amino group and a carboxyl group are introduced into dextran,
An aminocarboxyl dextran having a ratio of carboxyl group to amino group (carboxyl group / amino group) in the range of 0.45 / 0.55 to 0.95 / 0.05.
(22)
A cell cryopreservation agent comprising the aminocarboxyl dextran of (21).
(23)
(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.
(24)
The solution for cell cryopreservation according to (23), wherein the physiological solution is physiological saline, cell culture liquid medium, or tissue culture liquid medium.

Furthermore, 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.
(33)
The cell cryopreservation solution according to any one of (23) to (24), wherein the aminocarboxyl dextran is a carboxyl group-introduced aminated dextran obtained by introducing a carboxyl group into an aminated dextran.

Furthermore, 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.

Furthermore, 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

Furthermore, 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.
(63)
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.

Furthermore, 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 -N ₃ (I)

(Y ₁ is a spacer that can be introduced at the position of the —OH group of aminocarboxydextran)
The azide-introduced aminocarboxyl dextran of (71) substituted by the group represented by these.
(74)
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 azido-introduced aminocarboxyl dextran of (73), which is a divalent spacer selected from the group consisting of (wherein —N ₃ binds to the right end of each spacer).
(75)
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.
(76)
As the introduction of carbon-carbon triple bond moiety,
The —OH group of aminocarboxydextran is represented by the following formula (II) or formula (III):

-Y ₂ -C≡CH (II)

(Y ₂ is a spacer that can be introduced at the position of the —OH group of aminocarboxydextran)

(However, 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 carbon-carbon triple bond-introduced aminocarboxyl dextran of (72) or (75), which is substituted with a group represented by:
(77)
A group of formula III is represented by the following formula IV:

(Y ₃ is a spacer that can be introduced at the position of the —OH group of aminocarboxydextran)
A carbon-carbon triple bond-introduced aminocarboxyl dextran of (76), which is a group represented by:
(78)
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)
A carbon-carbon triple bond-introduced aminocarboxyl dextran according to (73), which is a divalent spacer selected from the group consisting of (wherein —C≡CH is bonded to the right end of each spacer).
(79)
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).

Furthermore, 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)).
(82)
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.

Furthermore, 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.
(93)
(92) 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.
(98)
The solution for producing a cell-embedded hydrogel according to (97), wherein the physiological solution is physiological saline, cell culture liquid medium, or tissue culture liquid medium.
(99)
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 (97) to (98).
(100)
(99) A cell-embedded hydrogel obtained by gelling the cell-containing composition for producing a cell-embedded hydrogel.

Furthermore, 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.
(104)
Use of the solution for producing a cell-embedded hydrogel according to any one of (92) to (93) and (97) to (98) for producing a cell-containing composition for producing a cell-embedded hydrogel.
(105)
A method for producing a cell-embedded hydrogel by gelling the cell-containing composition for producing a cell-embedded hydrogel according to (94) or (99).
(106)
Use of the cell-containing composition for producing a cell-embedded hydrogel according to (94) or (99) for producing a cell-embedded hydrogel.

Furthermore, 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 The process of
A method of cryopreserving a cell or tissue comprising
(112)
(111) In the method for cryopreserving cells and tissues,
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 a cell-embedded hydrogel by gelling the thawed cell-containing composition by an intermolecular cross-linking reaction of aminocarboxydextran;
A method for producing a cell-embedded hydrogel from a cryopreserved cell-containing composition.
(114)
The formation reaction of the intermolecular bridge of aminocarboxy dextran is
The production method of (113), which is a triazole ring formation reaction between an azide moiety and an alkyne moiety of aminocarboxydextran.

Furthermore, 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.
(132)
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-embedded hydrogel stored under freezing,
A method of cryopreserving a cell or tissue comprising
(135)
The formation reaction of the intermolecular bridge of aminocarboxy dextran is
The production method of any one of (133) to (134), which is a triazole ring formation reaction of an azide moiety and an alkyne moiety of aminocarboxydextran.

Furthermore, 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).

(141)
Carboxylated ε-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
In physiological solution, crosslinked by intermolecular crosslinking generated by an amide bond forming reaction between the amino group of ε-poly-L-lysine and the group of N-hydroxysuccinimide of multi-arm PEG,
A hydrogel in which a physiological solution is fixed as a dispersion medium.
(142)
Multi-arm PEG has the following formula (V):

C [CO— (CH ₂ —CH ₂ —O) _n —X] ₄ (V)

(Where 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):

Is a group represented by
(141) The hydrogel which is 4 arm PEG represented by these.
(143)
The hydrogel according to (141), wherein 1 to 60% by mass of carboxylated ε-poly-L-lysine is contained in a physiological solution.
(144)
A cell-embedded hydrogel in which cells or tissues are embedded in any of the hydrogels according to (141) to (142).
(145)
A cell-embedded hydrogel for cryopreservation, wherein cells or tissues are embedded in the hydrogel of any one of (141) to (142).
(146)
(144) A method for cryopreserving cells or tissues, wherein the cell-embedded hydrogel for cryopreservation according to (144) is cryopreserved.

Furthermore, the present invention includes the following (151) to (151).
(151)
Carboxylated ε-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,
In physiological solution, cross-linked by intermolecular cross-linking generated by amide bond formation reaction between amino group of ε-poly-L-lysine and N-hydroxysuccinimide group of multi-arm PEG to gel The process of
A method for producing a hydrogel, comprising:
(152)
Physiology containing carboxylated ε-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 ε-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 ε-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 ε-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:

According to the present invention, 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. 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.

The present invention will be described in detail below with specific embodiments. The present invention is not limited to the following specific embodiments and other embodiments.

According to the present invention, an ampholyte 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 / 0.55-0. 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. In a preferred embodiment, after introducing an amino group into the polymer or after preparing a polymer having an amino group, the amino group is subjected to carboxylation or acetylation using, for example, carboxylic anhydride. And a carboxyl group can be introduce | transduced so that it may become the range of the said ratio.

[Dextran]
In a preferred embodiment, 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 α-1,6 bonds and α-1,4 bonds. As 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.

In order to obtain the ampholyte polymer of the present invention, amino groups and carboxyl groups are introduced in the above ratio with respect to dextran. In a preferred embodiment, after introducing 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. Examples of the carboxylic anhydride 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. Moreover, a carboxyl group may be introduce | transduced into an amino group as mentioned above, and you may block by acetylating an amino group by a well-known means so that an amino group and a carboxyl group may become the range of the said ratio. The introduced carboxyl group can be further partially aminated by reacting it with a compound such as diamine, triamine or polyamine. Examples of the 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.

[Polyamine]
In a preferred embodiment, a polyamine can be used as the polymer having an amino group. As the polyamine, for example, polyamino acids and aminated polysaccharides can be used. For example, polylysine, polyallylamine, polyarginine, polyarginine, polyglutamic acid, and polyaspartic acid can be used. In a preferred embodiment, ε-poly-L-lysine can be used as the polymer having an amino group. As such a polymer, 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.

In order to obtain the ampholyte polymer of the present invention, 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. Moreover, a carboxyl group may be introduce | transduced into an amino group as mentioned above, and you may block by acetylating an amino group by a well-known means so that an amino group and a carboxyl group may become the range of the said ratio. The introduced carboxyl group can be further partially aminated by reacting it with a compound such as diamine, triamine or polyamine.

[Physiological solution]
The hydrogel of the present invention is obtained by intermolecular crosslinking of the ampholyte polymer in a physiological solution. Examples of the physiological solution include physiological saline, cell culture liquid medium, tissue culture liquid medium, and serum-free medium. For example, Dulbecco's modified Eagle MEM medium (DMEM) can be mentioned as a preferable one. 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. In a preferred embodiment, 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. In the present invention, 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. However, if desired, a known cryoprotectant can be additionally added to the physiological solution and used. Examples of such known cryoprotective substances include dimethyl sulfoxide, glycerol, ethylene glycol, trehalose, sucrose, and the like, or an antioxidant. Examples of such an antioxidant include polyphenols such as catalase, peroxidase, superoxide dismutase, vitamin E, vitamin C, and epigallocatechin gallate, or glutathione.

[Intermolecular crosslinking: Triazole ring formation reaction]
In a preferred embodiment, 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). That is, first, 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. Next, 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. In a preferred embodiment, aminocarboxy dextran can be used as the ampholyte polymer and dextran can be used as the non-ampholyte polymer.

[Introduction of azido group]
In a preferred embodiment, 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. In a preferred embodiment, an atomic group having a terminal azide group can be introduced. For example, the following formula (I):

-Y ₁ -N ₃ (I)

Can be introduced. Y ₁ is a spacer inserted as desired. When 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. In a preferred embodiment, specifically, 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.

[Introduction of carbon-carbon triple bond moiety]
In a preferred embodiment, 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. In a preferred embodiment, the terminal alkyne group or the cyclooctyne ring-containing compound group can be used as the group having a carbon-carbon triple bond moiety. The following formula (II) or formula (III):

-Y ₂ -C≡CH (II)

Can be introduced. However, Y ₂ and Y ₃ are spacers inserted as desired. When 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.

In the above formula III, 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. In a preferred embodiment, 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. In a preferred embodiment, 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:

It can be set as the group represented by these.

In a preferred embodiment, 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.

In a preferred embodiment, 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.

An example of introduction of an azide group and an alkyne group into dextran is shown in the following scheme 2.

[Intermolecular crosslinking: Multi-arm PEG]
In a preferred embodiment, 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). , And can be. That is, multi-arm PEG (polyethylene glycol) serves as a cross-linking agent to form cross-links between molecules of the ampholyte polymer. Therefore, 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. In a preferred embodiment, 4-arm PEG can be used. For example, the multi-arm PEG has the following formula (V):

C [CO— (CH ₂ —CH ₂ —O) _n —X] ₄ (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):

It can be set as the group represented by these. In a preferred embodiment, the ampholyte polymer crosslinked by multi-arm PEG (polyethylene glycol) is a carboxylated polylysine, preferably a carboxylated ε-poly-L-lysine. The amino group of the carboxylated ε-poly-L-lysine is suitably cross-linked by multi-arm PEG (polyethylene glycol) to form a hydrogel.

[Intermolecular crosslinking reaction]
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. In addition to this, after dispersing cells or immersing the tissue in a solution for producing a hydrogel, freezing it in a solution state without forming a hydrogel, thawing it, and then 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. In a preferred embodiment, intermolecular crosslinking can be performed under temperature conditions that allow cell culture, for example, at 35 to 38 ° C., for example, 37 ° C.

When it is desired to accelerate 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. For example, 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. For example, 7: 1 to 1: 7, 7: 1 to 1: 5, 7 : 1 to 1: 3, 6: 1 to 1: 6, 6: 1 to 1: 4, 6: 1 to 1: 2, 4: 1 to 1: 4, 4: 1 to 1: 2, 2: 1 It can be in the range of ~ 1: 2. Alternatively, when it is desired to accelerate the intermolecular crosslinking reaction by the amide bond formation reaction using the NHS-multi-arm PEG, 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. Accordingly, 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. Therefore, an azide group-introduced aminocarboxyl dextran and a carbon-carbon triple bond moiety-introduced aminocarboxyl dextran are themselves excellent cell cryopreservation agents. The concentration of these dextran derivatives (polymer concentration, dextran concentration) 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%.

[Cells and tissues]
In the present invention, 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. In preferred embodiments, 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. In a preferred embodiment, 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.

Hereinafter, the present invention will be described in detail with reference to examples. The present invention is not limited to the examples illustrated below.
[Example 1]
[Hydrogel with carboxylated polylysine]
[Synthesis of carboxylated polylysine]
5 mL of 25% aqueous solution of ε-poly-L-lysine (Chisso, molecular weight 4000) was added, 0.5-0.9 g of succinic anhydride (Wako Pure Chemical Industries) was added, and reacted at 50 ° C for 1 hour to prepare carboxylated polylysine . The amount of carboxyl group introduced was determined by calculating from the amount of amino group decrease using the TNBS method, which is amino group quantification. A product in which 50% of a carboxyl group is introduced with respect to the number of moles of amino group is expressed as PLL (0.50).

[Cryopreservation of cells with carboxylated polylysine]
1 × 10 ⁶ mouse fibroblast L929 cells in 1 mL of 10% PLL (0.50) medium solution (serum-free medium, Dulbecco's Modified Eagle Medium (DMEM), Sigma) in cryovials (Simport Plastics) And stored in a freezer at −80 ° C. for cryopreservation. Thawing was immediately thawed in a 37 ° C. hot water bath, washed with a medium, and then judged to be alive by trypan blue staining. As a result, a survival rate of 95% or more was confirmed.

[Gelation of carboxylated polylysine]
The thawed suspension is then transferred to a 24-well culture plate and 10% tetrafunctional polyethylene glycol (4-armed functional PEG) (N-hydroxysuccinimide end) (NOF, Sunbright PTE-100S, molecular weight) 10,000) It was made to gel quickly by adding a medium solution. When the culture medium was added to the top of the gel and the cell viability was determined using the Live-dead assay kit (formerly invitrogen, now Life Technologies) after 6 hours in a 37 ° C incubator, the survival rate was 80% or higher. It was confirmed. FIG. 1 is a fluorescent photograph showing this result. FIG. 1 is a photograph of double fluorescence staining of green and red in a color photograph, where green fluorescence indicates cells that are alive and red fluorescence indicates cells that are not alive. . Viability was calculated by counting these. Further, by leaving the gel in an incubator, it was confirmed that all the gel was dissolved after 48 hours and the cells were adhered to the culture plate.

The experimental procedure from synthesis of the above carboxylated polylysine to hydrogel formation and culture is summarized in the following scheme 3.

[Summary of results]
From the above experiments, 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. That is, for example, after injecting cell fluid into the affected area, it is gelled, and within a few days, the gel itself is biodegraded, and the so-called injectable gel (three-dimensionally embedded cells engraft as it is) It was found that it was possible to realize an injectable gel).

[Example 2]
[Hydrogel by Dextran]
[Synthesis of aminated dextran]
1.5 g of dextran (name sugar industry, molecular weight 70,000) was dissolved in 90 mL of dimethyl sulfoxide (DMSO), 3.0 g of carbonyldiimidazole (CDI) was added, and the mixture was reacted at 50 ° C. for 15 minutes. Then, 1.35 mL of ethylenediamine (EDA) was added and reacted at 50 ° C. for 18 hours to obtain an aminated dextran (Amino-Dex) with an introduction rate of 64.1% (the number of EDA introduced per unit sugar residue). . The obtained 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)). The synthetic procedure is summarized in Scheme 4 below.

In order to investigate the cell cryoprotective activity of these Dex-PA, 1.0 × 10 ⁶ L929 cells were suspended in 1 mL of 10% Dex-PA solution, frozen and thawed in the same way as the above freezing method, and the survival rate was increased. evaluated. As a result, it was confirmed that a high survival rate of almost 80% or more was obtained when the ratio of the carboxyl group to the amino group was 50-90%, and that Dex-PA also had cell cryoprotective activity. 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. The vertical axis in FIG. 2 represents the cell viability (%) after freezing L929 cells in a solution containing 10% of Dex-PA into which carboxyl groups were introduced at different ratios (ratio). From this result, it was found that modified dextran (Dex-PA), which was converted into an amphoteric polymer electrolyte by introducing an amino group and a carboxyl group, exhibited a high cell cryoprotective effect.

[Synthesis of azide group-containing dextran]
[Synthesis of 3-azidopropanol]
Dissolve 2.34 g sodium azide (Tokyo Kasei) and 0.05 g tetrabutylammonium hydrogen sulfate (Tokyo Kasei) in 6 ml water, add 1.5 mL 3-chloropropanol (Tokyo Kasei) The reaction was allowed to proceed for 24 hours, and then allowed to stand at room temperature for 12 hours. The solution extracted with diethyl ether was dried to obtain 3-azidopropanol.

[Synthesis of Azidodextran (Az-Dex)]
0.1 g of dextran was dissolved in 6 mL of DMSO, 0.06 g of CDI (carbamoyldiimidazole) was added and reacted at 50 ° C. for 2 hours. Thereafter, 0.018 g of 3-azidopropanol was added and allowed to react for 24 hours. The azide introduction rate was evaluated by NMR, and the result was 10% (per unit sugar residue) (compound B in Scheme 3 described later). The procedure for synthesizing this azidodextran (Az-Dex) is shown in Scheme 3 below. Thereafter, EDA-introduced azidated dextran and azide-introduced ampholyte dextran (Az-Dex-PA) were synthesized by the same method as the above-described aminated dextran synthesis method.

[Synthesis of alkyne-containing dextran]
0.5 g of dextran was dissolved in 30 mL of DMSO, 0.1 g of CDI (carbamoyldiimidazole) was added and reacted at 50 ° C. for 2 hours. Thereafter, 52.1 μL of N-methylpropargylamine (Tokyo Kasei) was added and reacted for 24 hours to synthesize alkyne-substituted dextran (Alk-Dex). The alkyne introduction rate was evaluated by NMR, and the introduction rate was NMR˜12.4% (per unit sugar residue) (compound C of Scheme 3 described later). The procedure for synthesizing this alkyne-containing dextran (Alk-Dex) is shown in Scheme 5 below.

[Hydrogel formation]
When 10% Az-Dex-PA medium solution and 10% Alk-Dex are mixed in an equivalent amount and CuSO4 is added as copper ions at a concentration of 0.2 mg / mL and ascorbic acid at a concentration of 0.8 mg / mL or higher, gelation may occur quickly. It could be confirmed. A schematic diagram illustrating the reaction of triazole ring formation resulting in this gelation is shown in Scheme 6.

Therefore, 1.0 × 10 ⁶ L929 were suspended in 10% Az-Dex-PA medium solution, gelled with 10% Alk-Dex, CuSO4, ascorbic acid, frozen at -80 ° C, and thawed When the subsequent survival rate was confirmed by Live-Dead assay in the same manner as in Example 1, the survival rate was about 70%. 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. From this result, it is possible to gel 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.

[Summary of results]
From the above experiments, the amphoteric electrolyte polymer Dex-PA aqueous solution is a polymer solution capable of cryopreserving cells with high survival rate, and the amphoteric electrolyte polymer Dex-PA has an azide group and a carbon-carbon triple bond. By introducing a group and crosslinking by a triazole ring formation reaction, it is possible to form a hydrogel while maintaining the cells at a high survival rate in a dispersed state, and of the amphoteric electrolyte polymer Dex-PA in which the cells are embedded It was found that the hydrogel preserves and protects cells with a high survival rate even when frozen and thawed. In other words, if 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.

[Comparative Example 1]
[Cryopreservation of cells in collagen gel]
Cell matrix type IA (Nitta Gelatin) was used to disperse L929 in the collagen gel. When it was frozen as it was at −80 ° C. and the survival rate was examined after thawing, all cells were killed and the survival rate was 0%. From this result, it was found that it was very difficult to freeze cells in a general gel used conventionally.

[Example 3]
[Synthesis of cyclooctyne-containing dextran]
In accordance with Example 2, in place of alkyne-containing dextran of Example 2, dibenzylcyclooctanoic acid (DBCO-acid) was introduced into dextran instead of N-methylpropargylamine, and dextran having a cyclooctyne group Was synthesized (DBCO-Dex). The procedure of this synthesis is shown in the following scheme 7.

Schematic diagram illustrating the triazole ring formation reaction that occurs between DBCO-Dex and Az-Dex-PA is shown in Scheme 8.

[Example 4]
[Interaction of ampholyte polymer Dex-PA with cells]
In order to investigate the interaction between the ampholyte polymer Dex-PA and cells, the following experiment was conducted.

L929 was frozen with Dex-PA made from dextran introduced with FITC (Dojindo), and after thawing, it was observed with a eutectic point laser microscope. The obtained fluorescence photograph is shown in FIG. 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. As a result, 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]
In order to further investigate the cell cryoprotective activity of Dex-PA, the following experiment was performed. In Example 2, 1 × 10 ⁶ L929 cells were suspended in 1 ml of Dex-PA solution (concentration 10%) with various 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.

[Scheme 9]

[Gelification of a mixed solution of Azide-Dex-PA and DBCO-Dex]
The conditions for gelation of a mixed solution of Azide-Dex-PA and DBCO-Dex were examined.
Regarding gelation, the gelation time and hardness vary depending on the polymer concentration, the azide introduction rate of azide-Dex-PA and the DBCO introduction rate of DBCO-Dex. Gelation time was defined as the time when the mixed solution in a test tube solidified and lost fluidity when turned upside down. Table 1 below 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.

As described above, when the polymer concentration was low, the gelation time was long, and at 30 mg / mL or less, gelation did not occur even after 1 hour (indicated as x in Table 1). Moreover, the tendency for gelation time to become long was seen when the quantity of the azide with respect to DBCO (alkyne) was increased. The longer the gel time, the softer the gel.

[Creation of cell-embedded gel and examination of cryoprotective activity]
Next, 1 × 10 ⁶ L929 cells were suspended in a mixed solution of Azide-Dex-PA and DBCO-Dex, waited for gelation, and frozen by leaving the gel in a freezer at −80 ° C. . The survival rate after thawing was confirmed by the Live / Dead assay in the same manner as in FIG. FIG. 7A shows a gel having a ratio of Azide to DBCO (Alkyne) of 1: 4 (polymer concentration 10%), and 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%, and the survival rate in FIG. 7C was 0%.

According to the present invention, 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.

Claims (19)

1. 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. 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.
2. Intermolecular crosslinking
   An azido group introduced into a molecule of an ampholyte polymer or a molecule of a polymer that is not an ampholyte, and
   Intermolecular crosslinks formed by a triazole ring formation reaction with a carbon-carbon triple bond moiety introduced into another molecule of the ampholyte polymer or another molecule of the non-ampholyte polymer (provided that an azide group is introduced) And at least one of the molecules introduced with the carbon-carbon triple bond moiety is an ampholyte polymer),
   Or an intermolecular crosslink formed by an amide bond forming reaction between the amino group of the ampholyte polymer and the N-hydroxysuccinimide group of the multi-arm PEG,
   The hydrogel according to claim 1, wherein
3. 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 ε-poly-L-lysine,
   The hydrogel according to any one of claims 1 and 2.
4. The hydrogel according to any one of claims 1 to 3, wherein the ampholyte polymer is contained in a physiological solution in an amount of 1 to 60% by mass.
5. A cell-embedded hydrogel comprising cells or tissues embedded in the hydrogel according to any one of claims 1 to 4.
6. A cell-embedded hydrogel for cryopreservation, wherein cells or tissues are embedded in the hydrogel according to any one of claims 1 to 4.
7. A method for cryopreserving cells or tissues, wherein the cell-embedded hydrogel for cryopreservation according to claim 6 is cryopreserved.
8. 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.
9. The process of intermolecular crosslinking and gelation
   An azido group introduced into a molecule of an ampholyte polymer or a molecule of a polymer that is not an ampholyte, and
   Cross-linked by a triazole ring formation reaction with a carbon-carbon triple bond moiety introduced into another molecule of the ampholyte polymer or another molecule of the non-ampholyte polymer (however, an azide group was introduced) A molecule and a carbon-carbon triple bond moiety-introduced molecule, at least one of which is an ampholyte polymer), gelling step,
   Or a step of gelation by intermolecular crosslinking by an amide bond forming reaction between the amino group of the ampholyte polymer and the N-hydroxysuccinimide group of the added multi-arm PEG,
   The manufacturing method according to claim 8, wherein
10. 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 ε-poly-L-lysine,
    The production method according to any one of claims 8 to 9.
11. 11. The production method according to claim 8, wherein the ampholyte polymer is contained in 1 to 60% by mass in the physiological solution.
12. In the production method according to any one of claims 8 to 11, before the step of intermolecular crosslinking and gelation,
    A step of dispersing or immersing cells or tissues together with an ampholyte polymer in a physiological solution;
    A method for producing a cell-embedded hydrogel comprising:
13. A step of cryopreserving the cell-embedded hydrogel produced by the production method according to claim 12,
    A method for cryopreserving cells or tissues.
14. In the production method according to any one of claims 8 to 12, before the step of intermolecular crosslinking and gelation,
    A step of dispersing or immersing cells or tissues together with an ampholyte polymer in a physiological solution;
    A step of cryopreserving the dispersed or immersed cells or tissues,
    A method for producing a cell-embedded hydrogel after cryopreservation of cells or tissues.
15. An aminocarboxyl dextran in which an amino group and a carboxyl group are introduced into dextran,
    An aminocarboxyl dextran having a ratio of carboxyl group to amino group (carboxyl group / amino group) in the range of 0.45 / 0.55 to 0.95 / 0.05.
16. An azido-introduced aminocarboxyl dextran in which an azide group capable of triazole ring formation reaction is introduced into the aminocarboxyl dextran according to claim 15.
17. A carbon-carbon triple bond-introduced aminocarboxyl dextran in which a carbon-carbon triple bond part capable of triazole ring formation reaction is introduced into the aminocarboxyl dextran according to claim 15.
18. A cell cryopreservation agent comprising the aminocarboxyl dextran according to any one of claims 15 to 17.
19. The aminocarboxyl dextran according to any one of claims 15 to 17,
    A solution for cell cryopreservation, which is dissolved and contained in a physiological solution at a concentration of 1 to 60% by mass.

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