Cell culturing carrier and method for producing cell culturing carrier

Abstract

This cell culturing carrier contains a polysaccharide including carboxyl groups. The carboxyl groups include a carboxyl group crosslinked by a cation having a valence of two or more, and a carboxyl group to which a functional group is bonded.

Description

Cell culture carrier and method for producing a cell culture carrier

[0001] This disclosure relates to a cell culture carrier and a method for producing a cell culture carrier.

[0002] Microcarriers for cell culture are known in which cell adhesion proteins are immobilized on the surface of hydrogel microparticles made of hydrophilic polymers using a condensing agent (see, for example, Patent Documents 1 to 4).

[0003] In particular, hydrogel particles in which polysaccharide salts are ionically crosslinked by polyvalent cations such as calcium ions are frequently used due to their high safety and biocompatibility, high transparency which makes it easy to observe cells, and ease of cell recovery as they can be broken down and removed by degrading enzymes and chelating agents.

[0004] Hydrogel particles are produced by adding divalent or higher cations, such as calcium ions, to water droplets of an aqueous solution of a polysaccharide salt to induce gelation. Cell culture carriers containing hydrogel particles as a base material are stored and sold in a dry state for reasons such as storage stability and ease of sterilization, and are then used after adding water to swell them.

[0005] When drying microcarriers containing alginate, it has been reported that a desiccant should be impregnated into the microcarriers before drying them (see, for example, Patent Documents 5 and 6).

[0006] International Publication No. 2022 / 168872, Japanese Patent Publication No. 2021-153441, Japanese Patent Publication No. 2013-534404, Japanese Patent Publication No. 2013-500717, Japanese Patent Publication No. 2018-516578, International Publication No. 2022 / 239810

[0007] We have found that when conventional cell culture carriers are dried and the particles become smaller, the density of ionic crosslinks between the carboxyl groups of polysaccharides and calcium ions increases, forming new crosslink structures and making them less likely to swell. When the particles of cell culture carriers do not swell easily, the cell culture surface area of ​​the carrier may decrease, and the transparency of the carrier may decrease. Furthermore, while impregnating the carrier with a desiccant before drying makes the carrier particles swell more easily when water is added, impregnating the carrier with a desiccant requires immersing the carrier in a solution containing the desiccant for a long time, which is a complicated procedure.

[0008] Therefore, the problem that this disclosure aims to solve is to provide a cell culture carrier that can swell after drying without using a desiccant.

[0009] As a result of diligent research to solve the above problems, we discovered that by chemically modifying the carboxyl groups of the polysaccharides contained in the cell culture support, it is possible to swell the dried cell culture support to approximately the same size as before drying, without using a desiccant.

[0010] In other words, the present disclosure is as follows: [1] A cell culture carrier comprising a polysaccharide containing a carboxyl group, wherein the carboxyl group comprises a carboxyl group crosslinked with a divalent or higher cation and a carboxyl group to which a functional group is attached.

[0011] [2] The cell culture carrier according to [1], wherein the water content is 50% by mass or less.

[0012] [3] The cell culture carrier according to [1] or [2], wherein the average diameter increases by at least 100% when swollen with water, and / or the volume increases by at least 700% when swollen with water.

[0013] [4] A dry cell culture carrier according to any one of [1] to [3].

[0014] [5] A cell culture carrier according to any one of [1] to [4], wherein the functional group reacts with a primary amine.

[0015] [6] A cell culture carrier according to any one of [1] to [5], wherein the functional group is reactable with proteins and / or peptides.

[0016] [7] A cell culture carrier according to any one of [1] to [6], wherein the functional group is an N-hydroxysuccinimide group or an N-hydroxysulfosuccinimide group.

[0017] [8] The cell culture carrier according to any one of [1] to [7], wherein the polysaccharide is alginic acid and / or polygalacturonic acid.

[0018] [9] A cell culture carrier according to any one of [1] to [8], wherein the divalent or greater cation is a calcium ion.

[0019]

[10] A cell culture carrier according to any one of [1] to [9], wherein the carrier is a microcarrier.

[0020]

[11] A cell culture carrier comprising a polysaccharide containing a carboxyl group, wherein the carboxyl group comprises a carboxyl group crosslinked with a divalent or higher cation, and a carboxyl group to which a cell adhesion protein and / or cell adhesion peptide is bound.

[0021]

[12] The cell culture carrier according to

[11] , wherein the amino acid sequence of a cell adhesion protein and / or cell adhesion peptide includes a cell adhesion active sequence.

[0022]

[13] The cell culture carrier according to

[12] , wherein the cell adhesion active sequence is an RGD sequence.

[0023]

[14] A cell culture carrier according to any one of

[11] to

[13] , wherein the cell adhesion protein is laminin and / or vitronectin.

[0024]

[15] A cell culture carrier according to any one of

[11] to

[14] , wherein the carboxyl group contained in the polysaccharide further comprises a carboxyl group to which an amino acid is attached.

[0025]

[16] The cell culture carrier according to

[15] , wherein the amino acid is a monomer and / or the amino acid is contained in a peptide, and the molecular weight of the amino acid is smaller than the molecular weight of the cell adhesion protein and / or cell adhesion peptide.

[0026]

[17] The cell culture carrier according to

[15] , wherein the amino acid is glycine.

[0027]

[18] A cell culture carrier according to any one of

[11] to

[17] , wherein the water content is 50% by mass or less.

[0028]

[19] A cell culture carrier according to any one of

[11] to

[18] , wherein the average diameter increases by at least 100% when swollen with water, and / or the volume increases by at least 700% when swollen with water.

[0029]

[20] A dry cell culture carrier according to any one of

[11] to

[19] .

[0030]

[21] A cell culture carrier according to any one of

[11] to

[20] , further comprising a carboxyl group contained in a polysaccharide to which a functional group that reacts with a primary amine is attached.

[0031]

[22] The cell culture carrier according to any one of

[11] to

[21] , further comprising a carboxyl group in the polysaccharide to which a functional group capable of reacting with proteins and / or peptides is attached.

[0032]

[23] The cell culture carrier according to any one of

[11] to

[22] , wherein the carboxyl group contained in the polysaccharide further comprises a carboxyl group to which an N-hydroxysuccinimide group and / or an N-hydroxysulfosuccinimide group is bonded.

[0033]

[24] The cell culture carrier according to any one of

[11] to

[23] , wherein the polysaccharide is alginic acid and / or polygalacturonic acid.

[0034]

[25] A cell culture carrier according to any one of

[11] to

[24] , wherein the divalent or greater cation is a calcium ion.

[0035]

[26] The cell culture carrier according to any one of

[11] to

[25] , wherein the carrier is a microcarrier.

[0036]

[27] A method for producing a cell culture carrier, comprising: binding a functional group to a part of carboxyl groups of a carrier comprising a polysaccharide containing carboxyl groups; and reducing the water content of the carrier having the functional group bound to a part of the carboxyl groups.

[0037]

[28] The method for producing a cell culture carrier according to

[27] , wherein the carboxyl groups contained in the polysaccharide include carboxyl groups crosslinked with a divalent or higher cation.

[0038]

[29] The method for producing a cell culture carrier according to

[27] or

[28] , further comprising, after reducing the water content of the carrier, reacting a cell-adhesive protein and / or a cell-adhesive peptide with the functional group in an aqueous solution containing the cell-adhesive protein and / or the cell-adhesive peptide, and swelling the carrier with reduced water content while binding the cell-adhesive protein and / or the cell-adhesive peptide to a part of the carboxyl groups through the reaction.

[0039]

[30] The method for producing a cell culture carrier according to

[29] , wherein the cell-adhesive protein is laminin and / or vitronectin.

[0040]

[31] The method for producing a cell culture carrier according to

[29] or

[30] , further comprising reducing the water content of the swollen carrier.

[0041]

[32] The method for producing a cell culture carrier according to any one of

[29] to

[31] , further comprising binding an amino acid to a part of the carboxyl groups contained in the polysaccharide comprised in the swollen carrier.

[0042]

[33] The method for producing a cell culture carrier according to

[32] , wherein the amino acid is a monomer, and / or the amino acid is contained in a peptide, and the molecular weight of the amino acid is smaller than the molecular weight of the cell-adhesive protein and / or the cell-adhesive peptide.

[0043]

[34] A method for producing a cell culture carrier according to

[32] or

[33] , wherein the amino acid is glycine.

[0044]

[35] A method for producing a cell culture carrier according to any one of

[27] to

[34] , wherein the functional group reacts with a primary amine.

[0045]

[36] A method for producing a cell culture carrier according to any one of

[27] to

[35] , wherein the functional group is reactable with proteins and / or peptides.

[0046]

[37] A method for producing a cell culture carrier according to any one of

[27] to

[36] , wherein the functional group is an N-hydroxysuccinimide group or an N-hydroxysulfosuccinimide group.

[0047]

[38] A method for producing a cell culture carrier according to any one of

[27] to

[37] , wherein the polysaccharide is alginic acid and / or polygalacturonic acid.

[0048]

[39] A method for producing a cell culture carrier according to any one of

[28] to

[38] , wherein the divalent or greater cation is a calcium ion.

[0049]

[40] A method for producing a cell culture carrier according to any one of

[27] to

[39] , wherein the carrier is a microcarrier.

[0050] A cell culture carrier manufactured by any of the methods described in

[41]

[27] to

[38] .

[0051]

[42] A method for culturing cells, comprising: swelling a cell culture carrier containing a polysaccharide containing a carboxyl group in an aqueous solution; adhering cells to the swollen carrier; and culturing cells on the swollen carrier, wherein the carboxyl group of the polysaccharide comprises a carboxyl group crosslinked with a divalent or higher cation, and a carboxyl group to which a cell adhesion protein and / or cell adhesion peptide is bound.

[0052]

[43] The culture method according to

[42] , wherein the amino acid sequence of a cell adhesion protein and / or cell adhesion peptide includes a cell adhesion active sequence.

[0053]

[44] The culture method according to

[42] , wherein the cell adhesion active sequence is an RGD sequence.

[0054]

[45] The cell culture method according to any one of

[42] to

[44] , wherein the cell adhesion protein is laminin and / or vitronectin.

[0055]

[46] The culture method according to any one of

[42] to

[45] , wherein the carboxyl group contained in the polysaccharide further comprises a carboxyl group to which an amino acid is attached.

[0056]

[47] The culture method according to

[46] , wherein the amino acid is contained in a peptide as one amino acid and / or an amino acid, and the molecular weight of the amino acid is smaller than the molecular weight of the cell adhesion protein and / or peptide.

[0057]

[48] ​​The culture method according to

[46] , wherein the amino acid is glycine.

[0058]

[49] The culture method according to any one of

[42] to

[45] , wherein the carboxyl group contained in the polysaccharide further comprises a carboxyl group to which an N-hydroxysuccinimide group and / or an N-hydroxysulfosuccinimide group is bonded.

[0059]

[50] The culture method according to any one of

[42] to

[49] , wherein the polysaccharide is alginic acid and / or polygalacturonic acid.

[0060]

[51] The culture method according to any one of

[42] to

[50] , wherein the divalent or greater cation is a calcium ion.

[0061]

[52] A culture method according to any one of

[42] to

[51] , wherein the culture method comprises dissolving a swollen carrier and recovering the cells.

[0062]

[53] The culture method according to

[52] , wherein dissolving the carrier is a mixture of the swollen carrier and a chelating agent.

[0063]

[54] The culture method according to

[53] , wherein the chelating agent is ethylenediaminetetraacetic acid.

[0064]

[55] A culture method according to any one of

[52] to

[54] , wherein the culture method further comprises subculturing cells, and subculturing further comprises attaching the recovered cells to a newly swollen carrier.

[0065]

[56] The culture method according to any one of

[42] to

[55] , wherein the carrier is a microcarrier.

[0066] According to this disclosure, it is possible to provide a cell culture carrier that can swell after drying without using a desiccant.

[0067] This is a schematic diagram of a method for producing a cell culture carrier according to an embodiment. This is a schematic diagram of a method for producing a cell culture carrier according to an embodiment. This is a schematic diagram of a method for producing a cell culture carrier according to an embodiment.

[0068] The following describes the forms for implementing this disclosure (hereinafter sometimes referred to as "embodiments"). This disclosure is not limited to the embodiments described below, and can be implemented in various ways within the scope of its gist. Furthermore, the embodiments shown below are illustrative of methods, etc., for realizing the technical idea of ​​this disclosure, and are not limited to these examples.

[0069] The cell culture carrier according to this embodiment is a cell culture carrier comprising a polysaccharide containing a carboxyl group, wherein the carboxyl group comprises a carboxyl group crosslinked with a divalent or higher cation and a carboxyl group to which a functional group is attached. In this embodiment, a carboxyl group is defined as COOH or COO － Or it refers to both.

[0070] In this embodiment, the cell culture carrier may be a microcarrier. In this specification, "carrier" refers to a substrate designed to promote cell adhesion, proliferation, differentiation, or transplantation, and its shape is not particularly limited. The carrier may be spherical, plate-shaped, polygonal, conical, pyramidal, needle-shaped, film-shaped, or ellipsoidal, and may have a porous structure. Furthermore, from the viewpoint of providing a wide surface area for cell adsorption and suppressing cell damage, the cell culture carrier may be spherical particles.

[0071] In this embodiment, the polysaccharide containing a carboxyl group is not particularly limited, but may be at least one selected from the group consisting of alginic acid, polygalacturonic acid, carrageenan, gellan gum, and pectin. The polysaccharide containing a carboxyl group may be alginic acid and / or polygalacturonic acid, or it may be alginic acid.

[0072] In this embodiment, at least a portion of the carboxyl groups contained in the polysaccharide are crosslinked by a divalent or higher cation. The divalent or higher cation is not particularly limited, but may be one or more selected from the group consisting of calcium ions, magnesium ions, ferrous ions, ferric ions, ferrous ions, ferrous ions, copper ions, zinc ions, selenium ions, and other essential trace element ions for humans. Examples of essential trace element ions for humans include manganese ions, chromium ions, and cobalt ions. The divalent or higher cation may also be a calcium ion.

[0073] "Crosslinking" is a form of bonding in which multiple identical or different molecules are reversibly or irreversibly bonded together, with or without the involvement of other substances. Unless otherwise defined in this disclosure, "chemical crosslinking" and similar expressions refer to irreversible bonding of multiple molecules via covalent bonds, while "physical crosslinking" and similar expressions refer to reversible bonding of multiple molecules via bonds other than covalent bonds. Furthermore, "physical crosslinking" may or may not involve chemical bonding. Examples of physical crosslinking with chemical bonding include bonding of multiple molecules via intermolecular electrostatic attraction such as inter-ionic interactions, hydrogen bonds, dipole interactions, van der Waals forces, and other chemical or intermolecular forces. Examples of physical crosslinking without chemical bonding include mechanical adhesion or mechanical bonding between molecules. For example, "alginic acid crosslinked by divalent or higher cations" refers to the reversible or irreversible bonding of multiple alginic acid molecules via divalent or higher cations, and such crosslinking is physical crosslinking with chemical bonding.

[0074] In this embodiment, a functional group is attached to at least some of the carboxyl groups contained in the polysaccharide. "A functional group is attached to at least some of the carboxyl groups" means that at least some of the hydrogen atoms of the carboxyl groups are substituted, and a functional group is introduced via a covalent bond. In the polysaccharide, for example, the carboxyl groups to which the functional group is attached are not crosslinked by divalent or higher cations. The functional group is not particularly limited as long as it can bind to primary amines or proteins and / or peptides, but may be selected from the group consisting of isothiocyanates, isocyanates, acyl azides, N-hydroxysuccinimide esters (NHS esters), N-hydroxysulfosuccinimide esters (sulfon-NHS esters), sulfonyl chlorides, aldehydes, glyoxal, epoxides, oxiranes, carbonates, aryl halides, imide esters, carbodiimides, anhydrides, fluoroesters, fluorobenzenes, pentafluorophenyl esters, and derivatives thereof. The functional group may be an N-hydroxysuccinimide ester or an N-hydroxysulfosuccinimide ester.

[0075] The protein and / or peptide to which the functional group reacts may be a cell adhesion protein and / or cell adhesion peptide. The cell adhesion protein and / or cell adhesion peptide is not particularly limited, but may have cell adhesion activity. Examples of cell adhesion proteins and / or cell adhesion peptides having cell adhesion activity include proteins that form the extracellular matrix, such as laminin, vitronectin, fibronectin, collagen, and gelatin, and / or peptides having an RGD sequence known as a cell adhesion activity sequence. The protein may be laminin or vitronectin. The RGD sequence refers to a sequence represented by Arg-Gly-Asp (arginine residue - glycine residue - aspartic acid residue). Furthermore, the cell adhesion protein may also be an antibody. For example, antibodies that can bind to antigens present on the cell membrane, such as CD antibodies, and antibodies that can bind to cell adhesion substances are examples of cell adhesion proteins.

[0076] The cell culture carrier according to this embodiment may be in the form of a dry powder. "Dry" in the cell culture carrier may mean that its moisture content is 50% by mass or less, 30% by mass or less, or 15% by mass or less. The moisture content is measured by the following method: Measure the weight of the cell culture carrier before and after heat treatment. Divide the difference in weight between the two states by the weight of the cell culture carrier before heat treatment and multiply by 100 to obtain the moisture content (in units of mass%). The heat treatment conditions are to leave the carrier in a dryer set to an ambient temperature of 100°C and dry for 2 hours. Furthermore, "dry" in the cell culture carrier may mean that its moisture content is 0% by mass or more, 0.1% by mass or more, 1% by mass or more, 2% by mass or more, 5% by mass or more, or 10% by mass or more. Keeping cell culture media dry is expected to suppress performance degradation due to changes in chemical structure and microbial growth during storage. Ideal storage conditions include keeping the media dry to suppress performance degradation and microbial growth due to changes in chemical structure, and the temperature should be below 25°C, preferably below 4°C, preferably below -30°C, and preferably below -80°C. Furthermore, when storing in a frozen state, a low moisture content in the cell culture media reduces the risk of damage due to water expansion. It also reduces the risk of hydrolysis of unstable substances such as NHS esters that are easily hydrolyzed by water.

[0077] The dried cell culture carrier may have an average diameter increased by at least 100%, optionally at least 125%, and optionally at least 150% when immersed in a sufficient amount of water for 5 minutes to swell. The average diameter is the average value of the equivalent volume spherical diameters of any 20 cell culture carriers observed under a phase-contrast microscope. In addition, the dried cell culture carrier may have a volume increased by at least 700%, optionally at least 1500%, and optionally at least 2000% when swollen with water. The volume is calculated from the aforementioned average diameter using the formula for the volume of a sphere.

[0078] The cell culture carrier of the present embodiment, in a swollen state, may have an average diameter of 20 μm to 1 mm, optionally 50 μm to 500 μm, optionally 100 μm to 300 μm, and optionally 150 μm to 250 μm. The cell culture carrier of the present embodiment, in a swollen state, has a surface area of 1000 μm ２ to 3200000 μm ２ , optionally 8000 μm ２ to 800000 μm ２ , optionally 30000 μm ２ to 300000 μm ２ , and optionally 70000 μm ２ to 200000 μm ２ . The surface area is calculated from the aforementioned average diameter using the formula for the surface area of a sphere.

[0079] The cell culture carrier according to the present embodiment may be a cell culture carrier containing a polysaccharide having carboxyl groups, wherein the carboxyl groups include carboxyl groups crosslinked by divalent or higher cations, and carboxyl groups that form amide bonds with cell-adhesive proteins and / or cell-adhesive peptides.

[0080] As the polysaccharide containing carboxyl groups, the divalent or higher cations, and the cell-adhesive proteins and / or cell-adhesive peptides, the same ones as described above may be used.

[0081] In this embodiment, at least some of the carboxyl groups in the polysaccharide may have amino acids or peptides attached to them via amide bonds. In the polysaccharide, for example, the carboxyl groups to which amino acids or peptides are attached via amide bonds do not have cell adhesion proteins and / or cell adhesion peptides attached via amide bonds. Furthermore, the carboxyl groups to which amino acids or peptides are attached via amide bonds are not crosslinked by divalent or higher cations. The amino acids may be monomers. The molecular weight of the amino acid or peptide is not particularly limited as long as it is smaller than the molecular weight of the cell adhesion protein and / or cell adhesion peptide. By having a molecular weight of amino acid or peptide smaller than the molecular weight of the cell adhesion protein and / or cell adhesion peptide, it is expected that the amino acid or peptide can enter the gaps inside the cell culture carrier where cell adhesion proteins and / or cell adhesion peptides cannot sterically occupy, and can bind to carboxyl groups inside the cell culture carrier. Amino acids are used because they have an amino group, are inexpensive, highly safe, and small in size.

[0082] The amino acids may be the 20 amino acids known to make up proteins: glycine, valine, isoleucine, leucine, methionine, lysine, phenylalanine, tryptophan, threonine, histidine, arginine, alanine, serine, tyrosine, cysteine, asparagine, glutamine, proline, aspartic acid, and glutamic acid. These 20 amino acids are particularly safe and inexpensive. By binding amino acids to carboxyl groups that are not bound to cell adhesion proteins and / or cell adhesion peptides, the decrease in solubility of cell culture carriers resulting from cross-linking can be suppressed.

[0083] The peptide may be a homopolymer of amino acids. For example, it may be polylysine or polyornithine. When the peptide is polylysine or polyornithine, it can promote cell adhesion and proliferation. By having an amino acid or peptide attached to at least some of the carboxyl groups contained in a polysaccharide via an amide bond, the properties of the amino acid residue can be imparted to the cell culture carrier. For example, by attaching lysine, arginine, histidine, or peptides containing these to at least some of the carboxyl groups contained in a polysaccharide via an amide bond, a positive charge can be imparted to the cell culture carrier, which is expected to improve the cell adhesion of negatively charged cells. For example, by attaching glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, proline, methionine, or peptides containing these to at least some of the carboxyl groups contained in a polysaccharide via an amide bond, the hydrophobicity of the cell culture carrier can be increased, and hydrophobic interactions are expected to promote the adsorption of cell adhesion substances to the surface of the cell culture carrier, thereby improving cell adhesion.

[0084] Furthermore, even if cell adhesion proteins and / or cell adhesion peptides are not bound to functional groups, the presence of these amino acids or peptides bound to at least some of the carboxyl groups can impart cell adhesion and swelling properties to the cell culture carrier. In this embodiment, the cell culture carrier may have amino acids or peptides bound to at least some of the carboxyl groups contained in the polysaccharides present inside the cell culture carrier via amide bonds. The binding of amino acids or peptides to at least some of the carboxyl groups contained in the polysaccharides present inside the cell culture carrier via amide bonds can suppress the formation of new cross-linking structures and the resulting decrease in solubility when the cell culture carrier is dried.

[0085] The mechanism by which the solubility of cell culture carriers decreases due to crosslinking may be as follows: Cell adhesion proteins and cell adhesion peptides contain multiple primary amines, and these multiple primary amines react and bind to functional groups bound to the polysaccharides of the carrier, thereby indirectly forming crosslinks between polysaccharides. After some of the primary amines of the cell adhesion proteins and cell adhesion peptides react and bind to some of the functional groups bound to the polysaccharides, if there are unreacted functional groups due to steric barriers caused by the size of the cell adhesion proteins and cell adhesion peptides, as time passes, the unreacted primary amines of the cell adhesion proteins and cell adhesion peptides may react and bind further to the unreacted functional groups bound to the polysaccharides, thereby further forming indirect crosslinks between polysaccharides and potentially decreasing the solubility of the cell culture carrier.

[0086] After attaching cell adhesion proteins and / or cell adhesion peptides, the unreacted functional groups may be reduced by reacting them with amino acids, thereby suppressing the decrease in solubility.

[0087] Furthermore, cell adhesion proteins and peptides are very expensive, making it costly to react them with functional groups at high concentrations. Therefore, using cell adhesion proteins and peptides at low concentrations increases the risk of unreacted functional groups remaining. On the other hand, amino acids are inexpensive, so even at high concentrations, the cost remains low. By reacting the functional groups of the support with high concentrations of amino acids, the amount of unreacted functional groups can be reduced.

[0088] The method for producing a cell culture carrier according to this embodiment includes attaching a functional group to some of the carboxyl groups of a carrier containing a polysaccharide containing carboxyl groups, and reducing the water content of the carrier to which the functional group is attached to some of the carboxyl groups.

[0089] The method for producing a cell culture carrier according to this embodiment includes, schematically shown in Figure 1, attaching a functional group 10 to a portion of the carboxyl groups of a carrier 1 containing a polysaccharide containing carboxyl groups, and drying the carrier 2 to which the functional group 10 is attached to obtain a dried carrier 3. Figure 1 illustrates the case where the functional group 10 is an NHS ester. The method for producing a cell culture carrier according to this embodiment may further include swelling the dried carrier 3 again to obtain carrier 2.

[0090] For example, a method for producing a cell culture carrier containing alginic acid includes extruding alginic acid in droplet form into air or oil from a thin nozzle, mixing the droplet with an aqueous solution containing divalent or higher cations to gel the droplet, bonding functional groups to the carboxyl groups contained in the alginic acid, and reducing the water content of the gel.

[0091] The carboxyl groups contained in polysaccharides may include carboxyl groups crosslinked with divalent or higher cations.

[0092] Polysaccharides containing carboxyl groups, cations with a valency of 2 or higher, and functional groups may be the same as those described above.

[0093] The attachment of functional groups to some of the carboxyl groups of a support containing a polysaccharide containing carboxyl groups can be carried out by known methods, for example, by immersing the support containing a polysaccharide containing carboxyl groups in a solution containing functional groups.

[0094] Reducing the moisture content of a support to which functional groups are bonded to some carboxyl groups may be done by known methods, such as vacuum drying, freeze-drying, or dry heat drying, or a combination thereof. The drying time for vacuum drying may be 10 minutes or more, 30 minutes or more, or 60 minutes or more. In freeze-drying, the temperature may be -160°C or higher and -20°C or lower, or -120°C or higher and -30°C or lower, and the pressure may be 1 Pa or higher and 60 Pa or lower, or 10 Pa or higher and 40 Pa or lower. In dry heat drying, the temperature may be 100°C or higher and 200°C or 120°C or higher and 160°C or lower, and the time may be 0.5 hours or higher and 6 hours or higher, or 1 hour or higher and 4 hours or lower.

[0095] The method for producing a cell culture carrier according to this embodiment may further include reducing the water content of the carrier, and then reacting the cell adhesion protein and / or cell adhesion peptide with the functional groups bonded to some of the carboxyl groups in an aqueous solution containing the cell adhesion protein and / or cell adhesion peptide, thereby forming amide bonds between the cell adhesion protein and / or cell adhesion peptide and some of the carboxyl groups, while swelling the carrier with reduced water content.

[0096] The method for producing a cell culture carrier according to this embodiment may include, as schematically shown in Figure 2, immersing a dried carrier 3 in an aqueous solution containing a cell adhesion protein and / or cell adhesion peptide 4 to react the functional groups 10 of the carrier with the cell adhesion protein and / or cell adhesion peptide 4, causing the carrier to swell while forming amide bonds between the cell adhesion protein and / or cell adhesion peptide 4 and the carboxyl groups through the reaction, thereby obtaining a carrier 6 to which the cell adhesion protein and / or cell adhesion peptide 4 is bound. Figure 2 illustrates the case where the functional groups are NHS esters.

[0097] In this embodiment, by immersing a carrier with reduced water content in an aqueous solution containing cell adhesion proteins and / or cell adhesion peptides, the cell adhesion proteins and / or cell adhesion peptides react with the functional groups bonded to some of the carboxyl groups, and the cell culture carrier can be swelled while amide bonds are formed between the cell adhesion proteins and / or cell adhesion peptides and some of the carboxyl groups through this reaction.

[0098] For example, if a carrier is swollen with an aqueous solution that does not contain cell adhesion proteins and / or cell adhesion peptides, and then the carrier is placed in an aqueous solution containing cell adhesion proteins and / or cell adhesion peptides to bind the cell adhesion proteins and / or cell adhesion peptides to the carrier, the aqueous solution used to swell the carrier remains inside the carrier, which can dilute the aqueous solution containing the cell adhesion proteins and / or cell adhesion peptides and potentially decrease the concentration of the cell adhesion proteins and / or cell adhesion peptides. In contrast, by reacting the cell adhesion proteins and / or cell adhesion peptides with functional groups bonded to some carboxyl groups, thereby forming amide bonds between the cell adhesion proteins and / or cell adhesion peptides and some carboxyl groups, and simultaneously swelling the cell culture carrier, the swelling ability of the carrier is increased, and the aqueous solution containing cell adhesion proteins and / or cell adhesion peptides is not diluted, allowing the cell adhesion proteins and / or cell adhesion peptides to react efficiently with the functional groups. Furthermore, uneven amide bond formation can be suppressed even without stirring the aqueous solution.

[0099] Cell adhesion proteins and / or cell adhesion peptides may be selected in the same manner as described above.

[0100] The method for producing a cell culture carrier according to this embodiment may further include reducing the water content of a cell culture carrier that has been swollen while binding cell adhesion proteins and / or cell adhesion peptides to it, and then further including swelling the carrier with reduced water content again in an aqueous solution.

[0101] The method for producing a cell culture carrier according to this embodiment may include drying a carrier 6, in which a carboxyl group and a cell adhesion protein and / or cell adhesion peptide 4 form an amide bond, to obtain a dried carrier 7, as schematically shown in Figure 3. Figure 3 illustrates the case where the functional group 10 is an NHS ester. The method for producing a cell culture carrier according to this embodiment may further include swelling the dried carrier 7 again to obtain a carrier 6.

[0102] Reducing the moisture content can be done in the same way as described above.

[0103] The method for producing a cell culture carrier according to this embodiment may further include reacting an amino acid with at least some of the functional groups bonded to carboxyl groups in the polysaccharides contained in the swollen carrier, thereby forming an amide bond between the amino acid and at least some of the carboxyl groups.

[0104] In this embodiment, the swollen support may be immersed in an aqueous solution containing amino acids to react with at least some of the functional groups bonded to the carboxyl groups, thereby forming amide bonds between the amino acids and at least some of the carboxyl groups. The amino acids may be selected in the same manner as described above.

[0105] The method for producing a cell culture carrier according to this embodiment may include, as schematically shown in Figure 2, immersing a dried carrier 3 in an aqueous solution containing a cell adhesion protein and / or cell adhesion peptide 4 to react the functional groups 10 of the carrier with the cell adhesion protein and / or cell adhesion peptide 4, causing the carrier to swell while forming amide bonds between the cell adhesion protein and / or cell adhesion peptide and the carboxyl groups, thereby obtaining a carrier 6 to which the cell adhesion protein and / or cell adhesion peptide 4 is bound; and reacting the functional groups 10 to which the cell adhesion protein and / or cell adhesion peptide 4 is not bound with an amino acid 5 to which an amide bond is formed between the amino acid and the carboxyl groups.

[0106] In this embodiment, the swollen carrier may be immersed in water or an aqueous solution to hydrolyze the functional groups bonded to the carboxyl groups. Hydrolyzing the functional groups bonded to the carboxyl groups can suppress the formation of crosslinks by components in the culture medium, such as proteins, which can reduce solubility during cell culture. Hydrolysis of the functional groups by immersion in water or an aqueous solution may be performed after the step of immersing the carrier in an aqueous solution containing cell adhesion protein and / or cell adhesion peptide 4 as described above. Hydrolysis of the functional groups by immersion in water or an aqueous solution may be performed after the step of immersing the carrier in an aqueous solution containing amino acids as described above. By immersing the carrier in an aqueous solution after the step of immersing the carrier in an aqueous solution containing amino acids, it is expected that the number of unreacted functional groups of the carrier will be reduced, improving storage stability and suppressing the decrease in solubility. Hydrolysis of the functional groups by immersion in water or an aqueous solution may be performed before cell culture, as described later.

[0107] The water or aqueous solution into which the swollen support is immersed may be any solution capable of hydrolyzing the functional groups. In the case of an aqueous solution, it may contain phosphate ions, sodium ions, calcium ions, potassium ions, etc. The concentration of calcium ions in the aqueous solution may be 0.01% by mass or more and 1% by mass or less. By having a calcium ion concentration of 0.01% by mass or more and 1% by mass or less in the aqueous solution, the cell culture support can be stabilized. The pH of the water or aqueous solution may be 4 or more and 10 or less, 5 or more and 9 or less, or 6 or more and 8 or less. The time for which the swollen support is immersed in water or aqueous solution may be any time for hydrolysis to proceed, and may be 1 hour or more, or 10 hours or more.

[0108] The cell culture method according to this embodiment includes swelling a cell culture carrier containing a polysaccharide containing a carboxyl group in an aqueous solution, adhering cells to the swollen carrier, and culturing the cells on the swollen carrier. In the cell culture method according to this embodiment, the carboxyl groups of the polysaccharide contained in the cell culture carrier include carboxyl groups crosslinked with a divalent or higher cation, and carboxyl groups to which cell adhesion proteins and / or cell adhesion peptides are bound.

[0109] The cells are not particularly limited, but preferably they are adherent cells that can be cultured on the carrier surface. Examples of such cells include hepatocytes (parenchymal cells of the liver), Kupffer cells, endothelial cells such as vascular endothelial cells and corneal endothelial cells, epidermal cells such as fibroblasts, osteoblasts, osteoclasts, periodontal ligament-derived cells and epidermal keratinocytes, epithelial cells such as tracheal epithelial cells, gastrointestinal epithelial cells, cervical epithelial cells and corneal epithelial cells, mammary gland cells, pericytes, myoblasts, myotubes, satellite cells, muscle cells such as smooth muscle cells and cardiomyocytes, renal cells, pancreatic islet cells, nerve cells such as peripheral nerve cells and optic nerve cells, chondrocytes, and osteocytes. These cells may be primary cells directly collected from tissues or organs, or they may be passaged several times. Furthermore, these cells may be undifferentiated cells such as embryonic stem cells or induced pluripotent stem cells (iPS cells), somatic stem cells such as mesenchymal stem cells with differentiation potential, unipotent stem cells such as vascular endothelial progenitor cells with single differentiation potential, or cells whose differentiation has been completed. The cells may also be CHO cells, HEK293 cells, 3T3 cells, Vero cells, MRC5 cells, HeLa cells, hybridomas, etc., which are widely used as cell substrates for the production of biopharmaceuticals and viral vectors, or cell lines derived from these. In addition, a single type of cell may be cultured, or two or more types of cells may be co-cultured. By culturing such cells using the cell culture method according to this embodiment, medical products and foods that are expected to have therapeutic effects can be manufactured. Cell culture may be carried out by known methods.

[0110] The conditions for cell culture are not particularly limited, and appropriate conditions are selected depending on the cells being cultured. Culture may be carried out under static conditions or by suspending the cell culture carrier through agitation or other means. For example, if the cells are mesenchymal stem cells or iPS cells, they can be cultured at 37°C in a 5% CO2 atmosphere.

[0111] Before culturing cells, the cell culture carrier may be subjected to sterilization or disinfection treatment to prevent microbial contamination. Examples of sterilization or disinfection methods include autoclave sterilization, gamma ray sterilization, electron beam sterilization, EOG gas sterilization, dry heat sterilization, ethanol sterilization, UV sterilization, heat sterilization, or suppressing microbial contamination during manufacturing and performing sterility testing in the final step to confirm that there are no or very few microorganisms.

[0112] In this embodiment, the culture period may be appropriately determined depending on the cells being cultured, for example, the cells may be cultured for 12 hours to 21 days, 24 hours to 14 days, or 2 days to 10 days.

[0113] After culturing, the cell culture carrier may be dissolved to separate and recover the cells from the carrier. When the cell culture carrier of this embodiment is reacted with a chelating agent, divalent or higher cations that crosslink the carboxyl groups of the polysaccharide are removed, and the cell culture carrier can be dissolved.

[0114] Examples of chelating agents used to dissolve cell culture carriers include ethylenediaminetetraacetic acid (EDTA), glycol etherdiaminetetraacetic acid (EGTA), 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), N'-(2-hydroxyethyl)ethylenediamine-N,N,N'-triacetic acid (HEDTA), and nitrilotriacetic acid (NTA), with EDTA being a suitable choice. Furthermore, adding a proteolytic enzyme in addition to the chelating agent can hydrolyze hydrolyzed hydrophilic polymers, leading to faster dissolution. However, from the perspective of minimizing damage to cells, ensuring safety, and reducing costs, it is preferable to omit the use of proteolytic enzymes to minimize the amount of added substances.

[0115] After culturing, the cell culture carrier may be dissolved and the recovered cells may be subcultured and cultured further, or the recovered cells may be attached to a new cell culture carrier and cultured.

[0116] The Disclosure is described in more detail below, but is not limited to this Disclosure and may be modified without exceeding the scope of the Disclosure.

[0117] <Example 1> 1-1: Preparation of dry microparticles made of alginate A 1 wt% sodium alginate aqueous solution was extruded from a nozzle with a diameter of 120 μm to form minute droplets, which were then dropped into a 1 wt% calcium chloride aqueous solution to gel as calcium alginate, thereby producing hydrogel-like microparticles with a diameter of approximately 200 μm. Next, 0.1 mL of the prepared sodium alginate was gelled to produce the microparticles, which were then autoclaved for sterilization. After removing the solution other than the fine particles using a 30G syringe, the fine particles were immersed in an aqueous solution prepared by mixing 30 mg of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC, Sigma-Aldrich, E7750), 18 mg of N-hydroxysuccinimide (NHS, Sigma-Aldrich, 130672), 40 μL of PBS (Sigma-Aldrich, D8537), and 360 μL of 0.11 wt% aqueous calcium chloride solution to form an NHS ester on the carboxyl group of alginic acid. The mixture was reacted at room temperature for 1 hour with stirring. After removing the solution other than the fine particles using a 30G syringe, the fine particles were washed with 1 mL of water to remove the solution. The fine particles were then washed with 1 mL of ethanol to remove the solution, and dried by vacuum drying for 30 minutes to remove water, thereby producing dried fine particles. The size of the dried fine particles at this time was approximately 70 μm in diameter. The moisture content of the prepared dried microparticles was measured to be 17.2%. When 1 mL of water was added to the prepared dried microparticles and the absorbance was measured from 200 to 350 nm, an absorption peak was detected around 280 nm. The measuring instrument used was a SpectraMax M2 manufactured by Molecular Devices. 100 μl of the aqueous solution containing the microparticles dispersed in 1 mL of water was placed in a flat-bottomed 96-well plate, and the plate was set in the measuring instrument to measure the absorbance. An absorption peak was detected at a similar position in the NHS aqueous solution, but no absorption peak was observed in the hydrogel microparticles that had not been chemically modified with calcium alginate. From the above, it was shown that the dried microparticles prepared in this way contain NHS and that NHS esters are formed.

[0118] 1-2: Verification of the swelling properties of dried microparticles 1 mL of water was added to dried microparticles with a diameter of approximately 70 μm prepared in Example 1-1. After a few minutes, the diameter of the microparticles increased to approximately 200 μm, which is about the same size as before drying, demonstrating that the microparticles swell well after drying even without the use of a desiccant. Furthermore, after removing the solution from the microparticles after swelling, the microparticles were washed with 1 mL of ethanol, the solution was removed again, and then the microparticles were vacuum-dried for 30 minutes to remove moisture once more to prepare dried microparticles. When 1 mL of water was added to the dried microparticles, they swelled in the same way as the first swelling, demonstrating that drying and swelling can be repeated.

[0119] Furthermore, hydrogel microparticles that were not chemically modified with NHS ester from calcium alginate were dried using the same method to produce dried microparticles. The dried microparticles showed almost no change in size and hardly swelled even after adding 1 mL of water and waiting for several minutes.

[0120] <Example 2> 2-1: Swelling and fixation of dried microparticles with an aqueous solution of cell adhesion protein (human vivornectin) When 40 μL of a 0.05 mg / mL aqueous solution of human vivornectin (Thermo Fisher Scientific, A14700 human vivornectin aqueous solution, 4 μL, PBS, and 32 μL of water) was added to the dried microparticles prepared in Example 1-1, the microparticles swelled rapidly to a diameter of approximately 200 μm. The NHS ester of the microparticles was reacted with human vivornectin at room temperature for 1 hour without stirring to prepare microparticles with immobilized human vivornectin.

[0121] 2-2: Swelling and Immobilization of Dried Microparticles with Aqueous Solution of Cell Adhesion Protein (Human Laminin) The dried microparticles prepared in Example 1-1 were swollen with 40 μL of an aqueous solution of 0.05 mg / mL human laminin fragments (4 μL of iMatrix-511 aqueous solution, 4 μL of PBS, and 32 μL of water, manufactured by Matrixome Co., Ltd.). After a few minutes, the microparticles swelled to a diameter of approximately 200 μm. The NHS ester of the microparticles and the human laminin fragments were reacted at room temperature for 1 hour without stirring to produce microparticles with the human laminin fragments immobilized.

[0122] 2-3: Immobilization of amino acids (glycine) The microparticles immobilized with human vivonectin prepared in Example 2-1 were washed with 1 mL of 0.05 wt% calcium chloride aqueous solution to remove the human vivonectin aqueous solution, and then immersed in 1 mL of 6 wt% glycine aqueous solution (60 mg of glycine, 100 μL of PBS, and 900 μL of 0.056 wt% calcium chloride aqueous solution) to prepare microparticles immobilized with human vivonectin and glycine.

[0123] 2-4: Preparation of dried microparticles For the microparticles immobilized with human vivornectin, microparticles immobilized with human laminin fragments, and microparticles immobilized with human vivornectin and glycine, prepared in Examples 2-1, 2-2, and 2-3, the solution other than the microparticles was removed using a 30G syringe, and then the microparticles were washed with 1 mL of water to further remove the solution. The solution was further removed by washing with 1 mL of ethanol, and the microparticles were vacuum-dried for 30 minutes to remove water from the microparticles and prepare dried microparticles. The diameter of the dried microparticles at this time was approximately 70 μm.

[0124] 2-5: Verification of the swelling properties of dried microparticles When 1 mL of water was added to the dried microparticles with a diameter of approximately 70 μm prepared in Example 2-4, the diameter increased to approximately 200 μm within a few minutes, and microparticles that had swelled to a size similar to that before drying were obtained. This indicates that good swelling after drying can be achieved even without the use of a desiccant.

[0125] 2-6: Measurement of the moisture content of dried microparticles The moisture content of the dried microparticles with human vivonectin and glycine immobilized, prepared in Example 2-4, was measured to be 17.9%.

[0126] 2-7: Preparation of heat-treated dried microparticles The dried microparticles prepared in Example 2-4 were heat-treated at 100°C for 2 hours and then dried further. The moisture content of the dried microparticles was measured to be 3.3%. When the swelling properties of these dried microparticles were verified using the same method as in Example 2-5, microparticles that swelled to a size similar to that before drying were obtained. This indicates that even when dried to a moisture content of 3.3%, good swelling is possible.

[0127] <Example 3> Cell Culture Evaluation 3-1: Cell Culture Evaluation using Human Mesenchymal Stem Cells Microparticles immobilized with human vivonectin, microparticles immobilized with human laminin fragments, and microparticles immobilized with human vivonectin and glycine, as prepared in Examples 2-1, 2-2, and 2-3, microparticles swollen after drying prepared in Example 2-5, and microparticles swollen from heat-treated dried microparticles prepared in Example 2-7 were each used as microcarriers for cell culture. After removing the solution other than the microcarriers with a 30G syringe, 2 mL of culture medium was added, and human mesenchymal stem cells (Lonza Corporation, PT-2501, bone marrow-derived) were seeded and cultured at 37°C in 5% CO2. ２ Cells were cultured under specific concentrations, and cell adhesion and proliferation were observed using a phase-contrast microscope. Lonza Corporation's PT3001 culture medium was used, and 0.05 w% CaCl was added to stabilize the microcarriers. ２ The substance was added to the culture medium. In all microcarrier conditions, cells were adhering to a portion of the microcarrier surface on day 1 of culture. Furthermore, by day 7 of culture, cells had proliferated on the microcarrier surface to the point where they completely covered the surface of the microcarrier.

[0128] 3-2: Evaluation of cell culture using human iPS cells. The microparticles immobilized with human vivonectin, the microparticles immobilized with human laminin fragments, and the microparticles immobilized with human vivonectin and glycine, prepared in Examples 2-1, 2-2, and 2-3, as well as the microparticles that swelled after drying, prepared in 2-5, were used as microcarriers for cell adhesion. After removing the solution other than the microcarriers using a 30G syringe, 2 mL of culture medium was added, and human iPS cells were seeded at 37°C and 5% CO2. ２ Cells were cultured under specific concentrations, and cell adhesion and proliferation were observed using a phase-contrast microscope. Thermo Fisher Scientific A3349401 culture medium was used, with 0.025 w% CaCl added to stabilize the microcarriers. ２The substance was added to the culture medium. Additionally, 0.01 mmol / L of Y-27632 manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. was added to the medium only on the first day, and not thereafter. Under all conditions, cells were adhering to a portion of the microcarrier surface on day 1 of culture. Furthermore, by day 7 of culture, cells had proliferated on the microcarrier surface to the point where they completely covered the microcarrier surface.

[0129] <Example 4> Evaluation of Microcarrier Solubility The microparticles immobilized with human vivonectin and glycine, prepared in Example 2-3, and the microparticles obtained by drying and swelling them as in Example 2-5, were used as microcarriers in the same manner as in Example 3, and human mesenchymal stem cells and human iPS cells were cultured for 7 days. After culturing, the solution was removed using a 30G syringe. To dissolve the microcarriers, 1 mL of 10 mmol / L EDTA aqueous solution was added to the microparticles and left at 37°C for 2 minutes. Almost all microcarriers dissolved to the point where they could no longer be seen under a phase-contrast microscope. Similarly, the microparticles immobilized with human vivonectin, prepared in Example 2-1, and the microparticles obtained by drying and swelling them as in Example 2-5, were cultured as microcarriers. After culturing, when the dissolution procedure was performed, the microcarriers did not dissolve completely, indicating that the microcarriers with amino acids attached had higher solubility.

[0130] Furthermore, the solubility of microcarriers that were not attached to cells was evaluated. The procedure was carried out under the same conditions as above, except that cells were not used, and similar results were obtained. That is, microcarriers bound with amino acids dissolved completely, while microcarriers not bound with amino acids did not dissolve completely.

[0131] <Example 5> Gelatin-Immobilized Microparticles When 40 μL of a 0.05 mg / mL gelatin aqueous solution (4 μL of a 0.5 mg / mL aqueous solution of Sigma-Aldrich G9391 gelatin, 4 μL of PBS, and 32 μL of water) was added to the dried microparticles prepared in Example 1-1, the microparticles swelled rapidly to a diameter of approximately 200 μm. The NHS ester of the microparticles and the gelatin were reacted at room temperature for 1 hour without stirring to produce microparticles with immobilized gelatin. Then, using these microparticles, microparticles with immobilized gelatin and glycine were prepared in the same manner as in Example 2-3. Then, dried microparticles with a diameter of approximately 70 μm were prepared in the same manner as in Examples 2-4 and 2-5. When 1 mL of water was added to these dried microparticles, the diameter increased to approximately 200 μm within a few minutes. Furthermore, when these swollen microparticles were used as microcarriers for cell culture and cultured using human mesenchymal stem cells in the same manner as in Example 3-1, cells were found to be adhering to a portion of the microcarrier surface on day 1 of culture. On day 7 of culture, cells proliferated on the surface of the microcarriers, and the cells completely covered the surface of the microcarriers. In addition, after culturing, the solution was removed using a 30G syringe, and 1 mL of 10 mmol / L EDTA aqueous solution was added and left at 37°C for 2 minutes, at which point almost all of the microcarriers dissolved to the point where they could no longer be seen under a phase-contrast microscope.

[0132] <Example 6> Microparticles immobilized with human vivonectin and arginine or human vivonectin and alanine Microparticles immobilized with human vivonectin and arginine, and microparticles immobilized with human vivonectin and alanine, respectively, were prepared in the same manner as in Example 2-3, except that arginine or alanine was used instead of glycine. Then, in the same manner as in Examples 2-4 and 2-5, dried microparticles with a diameter of approximately 70 μm were prepared, and when 1 mL of water was added to each of these dried microparticles, the diameter increased to approximately 200 μm within a few minutes. Furthermore, these swollen microparticles were used as microcarriers for cell culture, and culture evaluation with human mesenchymal stem cells was performed in the same manner as in Example 3-1. On the first day of culture, cells were adhering to a portion of the surface of both microcarriers. On the seventh day of culture, cells had proliferated to the point where they completely covered the surface of both microcarriers. Furthermore, after culturing, the solutions were removed using a 30G syringe, and 1 mL of 10 mmol / L EDTA aqueous solution was added to each, and the mixture was left at 37°C for 2 minutes. Almost all of the microcarriers dissolved to the point where they could no longer be seen under a phase-contrast microscope. The dried microparticles prepared in Example 1-1 and whose water content was measured were stored frozen (-30°C) for one month, and then their swelling properties were verified using the same method as in Example 1-2. Good swelling was confirmed, similar to Example 1-2. Next, using the dried microparticles stored frozen (-30°C) as described above, vitronectin and glycine were fixed and dried using the same method as in Example 2 to prepare dried microparticles, and their swelling properties and cell culture evaluation were performed using the same method as in Example 2-5 and Example 3-1. Good swelling and cell adhesion and proliferation were confirmed, similar to Example 2-5 and Example 3-1. Solubility was also evaluated after culturing using the same method as in Example 4. High solubility was similarly confirmed.

[0133] <Example 7> Evaluation of Storage Stability The microparticles prepared in Example 2-4 and whose water content was measured were stored for one month at room temperature, refrigerated (4°C), and frozen (-30°C). Swelling properties and cell culture evaluation were then performed using the same methods as in Examples 2-5 and 3-1. Good swelling and cell adhesion / proliferation were confirmed at all storage temperatures, similar to Examples 2-5 and 3-1. Furthermore, solubility was evaluated after culturing using the same method as in Example 4. High solubility was similarly confirmed.

[0134] <Example 8> Evaluation of microparticles produced by a hydrolysis method. Microparticles immobilized with human vivonectin, prepared in Example 2-1, were immersed in 1 mL of aqueous solution (a mixed aqueous solution of 100 μL of PBS and 900 μL of 0.1 wt% calcium chloride aqueous solution) and left to stand at room temperature for 12 hours to allow the hydrolysis of the NHS ester contained in the microparticles to proceed. Subsequently, cell culture evaluation was performed in the same manner as in Example 3-1, and good cell adhesion and proliferation were confirmed in the same manner as in Example 3-1. Furthermore, the solubility of the microparticles was evaluated in the same manner as in Example 4 after culturing, and high solubility was similarly confirmed. In addition, the solubility of the microparticles in which the above NHS ester hydrolysis had progressed was evaluated under the same conditions as above, except that cells were not used, and high solubility was similarly confirmed. Furthermore, regarding the microparticles in which the above NHS ester hydrolysis had progressed, dried microparticles with a diameter of approximately 70 μm were prepared in the same manner as in Examples 2-4 and 2-5, and when 1 mL of water was added to these dried microparticles, the diameter increased to approximately 200 μm within a few minutes. Furthermore, when these swollen microparticles were used as microcarriers for cell culture, and cell culture evaluation and solubility were assessed in the same manner as in Examples 3-1 and 4, good cell adhesion and proliferation and high solubility were similarly confirmed. In addition, when solubility was evaluated under the same conditions as above, except that cells were not used, high solubility was similarly confirmed.

[0135] 1, 2, 3...carriers, 4...cell adhesion proteins and / or cell adhesion peptides, 5...amino acids, 6...carriers, 7...dried carriers, 10...functional groups

Claims

1. A cell culture carrier comprising a polysaccharide containing a carboxyl group, wherein the carboxyl group comprises a carboxyl group crosslinked with a divalent or higher cation and a carboxyl group to which a functional group is attached.

2. The cell culture carrier according to claim 1, wherein the water content is 50% by mass or less.

3. The cell culture carrier according to claim 1, wherein the average diameter increases by at least 100% when swollen with water, and / or the volume increases by at least 700% when swollen with water.

4. The cell culture carrier according to claim 1, wherein the cell culture carrier is dry.

5. The cell culture carrier according to claim 1, wherein the functional group reacts with a primary amine.

6. The cell culture carrier according to claim 1, wherein the functional group is capable of reacting with proteins and / or peptides.

7. The cell culture carrier according to claim 1, wherein the functional group is an N-hydroxysuccinimide group or an N-hydroxysulfosuccinimide group.

8. The cell culture carrier according to claim 1, wherein the polysaccharide is alginic acid and / or polygalacturonic acid.

9. The cell culture carrier according to claim 1, wherein the divalent or greater cation is a calcium ion.

10. The cell culture carrier according to claim 1, wherein the carrier is a microcarrier.

11. A cell culture carrier comprising a polysaccharide containing a carboxyl group, wherein the carboxyl group comprises a carboxyl group crosslinked with a divalent or higher cation, and a carboxyl group to which a cell adhesion protein and / or cell adhesion peptide is bound.

12. The cell culture carrier according to claim 11, wherein the amino acid sequence of the cell adhesion protein and / or the cell adhesion peptide includes a cell adhesion active sequence.

13. The cell culture carrier according to claim 12, wherein the cell adhesion active sequence is an RGD sequence.

14. The cell culture carrier according to claim 11, wherein the cell adhesion protein is laminin and / or vitronectin.

15. The cell culture carrier according to claim 11, wherein the carboxyl group contained in the polysaccharide further comprises a carboxyl group to which an amino acid is bound.

16. The cell culture carrier according to claim 15, wherein the amino acid is a monomer and / or the amino acid is contained in a peptide, and the molecular weight of the amino acid is smaller than the molecular weight of the cell adhesion protein and / or the cell adhesion peptide.

17. The cell culture carrier according to claim 15, wherein the amino acid is glycine.

18. The cell culture carrier according to claim 11, wherein the water content is 50% by mass or less.

19. The cell culture carrier according to claim 11, wherein the average diameter increases by at least 100% when swollen with water, and / or the volume increases by at least 700% when swollen with water.

20. The cell culture carrier according to claim 11, wherein the cell culture carrier is dry.

21. The cell culture carrier according to claim 11, wherein the carboxyl group contained in the polysaccharide further comprises a carboxyl group to which a functional group that reacts with a primary amine is attached.

22. The cell culture carrier according to claim 11, wherein the carboxyl group contained in the polysaccharide further comprises a carboxyl group to which a functional group capable of reacting with proteins and / or peptides is attached.

23. The cell culture carrier according to claim 11, wherein the carboxyl group contained in the polysaccharide further comprises a carboxyl group to which an N-hydroxysuccinimide group and / or an N-hydroxysulfosuccinimide group is bonded.

24. The cell culture carrier according to claim 11, wherein the polysaccharide is alginic acid and / or polygalacturonic acid.

25. The cell culture carrier according to claim 11, wherein the divalent or greater cation is a calcium ion.

26. The cell culture carrier according to claim 11, wherein the carrier is a microcarrier.

27. A method for producing a cell culture carrier, comprising: attaching a functional group to a portion of the carboxyl groups of a carrier containing a polysaccharide containing carboxyl groups; and reducing the water content of the carrier to which the functional group is attached to a portion of the carboxyl groups.

28. The method for producing a cell culture carrier according to claim 27, wherein the carboxyl group contained in the polysaccharide includes a carboxyl group crosslinked with a divalent or higher cation.

29. A method for producing a cell culture carrier according to claim 27, further comprising: reducing the water content of the carrier; reacting the functional groups with the cell adhesion protein and / or cell adhesion peptide in an aqueous solution containing the cell adhesion protein and / or cell adhesion peptide; and swelling the carrier with reduced water content while the reaction causes the protein and / or peptide to bond to some of the carboxyl groups.

30. The method for producing a cell culture carrier according to claim 29, wherein the cell adhesion protein is laminin and / or vitronectin.

31. A method for producing a cell culture carrier according to claim 29, further comprising reducing the water content of the swollen carrier.

32. A method for producing a cell culture carrier according to claim 29, further comprising attaching an amino acid to a portion of the carboxyl groups contained in the polysaccharide contained in the swollen carrier.

33. The method for producing a cell culture carrier according to claim 32, wherein the amino acid is a monomer and / or the amino acid is contained in a peptide, and the molecular weight of the amino acid is smaller than the molecular weight of the cell adhesion protein and / or the cell adhesion peptide.

34. The method for producing a cell culture carrier according to claim 32, wherein the amino acid is glycine.

35. The method for producing a cell culture carrier according to claim 27, wherein the functional group reacts with a primary amine.

36. The method for producing a cell culture carrier according to claim 27, wherein the functional group is reactable with proteins and / or peptides.

37. The method for producing a cell culture carrier according to claim 27, wherein the functional group is an N-hydroxysuccinimide group or an N-hydroxysulfosuccinimide group.

38. The method for producing a cell culture carrier according to claim 27, wherein the polysaccharide is alginic acid and / or polygalacturonic acid.

39. The method for producing a cell culture carrier according to claim 28, wherein the divalent or greater cation is a calcium ion.

40. The method for producing a cell culture carrier according to claim 27, wherein the carrier is a microcarrier.

41. A cell culture carrier manufactured by the method described in any one of claims 27 to 38.

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