Microcarrier for cell culture, method for preparing microcarrier for cell culture, and cell culture composition using same

Abstract

The present invention relates to: a microcarrier for a cell culture, the microcarrier containing polystyrene-based particles; a method for preparing the microcarrier for a cell culture; and a cell culture composition using same.

Description

Micro carrier for cell culture, method for manufacturing micro carrier for cell culture, and cell culture composition using the same

[0001] This application claims the benefit of priority to Korean Patent Application No. 10-2024-0067364, filed May 23, 2024, and Korean Patent Application No. 10-2025-0067201, filed May 23, 2025, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to a microcarrier for cell culture, a method for producing a microcarrier for cell culture, and a cell culture composition using the same.

[0003] As the fields of biopharmaceuticals and regenerative medicine expand, the demand for cell mass culture technologies that can efficiently produce cells, tissues, and microorganisms is increasing.

[0004] Adherent cells are cultured using microcarriers within a 3D bioreactor. Cells, culture medium, and microcarriers are placed within the bioreactor, and the culture medium is stirred to bring the cells and microcarriers into contact, allowing them to adhere to the surface of the microcarriers and be cultured. The microcarriers used here offer a high surface area / volume ratio (SAR) for cell attachment and proliferation compared to 2D culture, making them suitable for mass cell culture.

[0005] Currently commercially available microcarriers have densities of about 1.1 to 1.3 g / cm. 3 , and the density of cells is about 1.2 g / cm 3This is a degree of difficulty. While this is advantageous for initial cell attachment within the bioreactor, centrifugation is difficult for cell separation and recovery after culture, requiring the use of microcarriers and a size-based filtration method. However, this method poses problems such as filter clogging, prolonged processing times, potential physical damage and contamination of cells, and potential cell loss.

[0006] To solve this problem, the density is 1.0 g / cm 3 or lower than 1.3 g / cm 3 Microcarriers have been manufactured using the properties of higher materials, but in this case, the range of densities that can be achieved is limited, and it is difficult to sufficiently secure a yield of microcarriers with a perfect spherical shape without damage or destruction.

[0007] The present invention relates to providing a cell culture microcarrier that enables separation of cells by density difference by controlling density, while improving dispersibility in a cell culture reactor or medium and enhancing cell adhesion.

[0008] In addition, the present invention relates to a method for manufacturing the micro carrier for cell culture.

[0009] In addition, the present invention relates to a cell culture composition using the above cell culture microcarrier.

[0010] In order to solve the above problem, the present specification provides a cell culture microcarrier comprising a polystyrene particle including a compound represented by the following chemical formula 1 as a monomer compound.

[0011] [Chemical Formula 1]

[0012]

[0013] In the above chemical formula 1, L0 is an arylene group having 6 or more carbon atoms or -(C=O)-, L1 and L2 are each independently an alkylene group having 1 or more carbon atoms, R1 is a reactive functional group capable of a ring-opening reaction, and R 10 is hydrogen or an alkyl group having 1 or more carbon atoms, and n is an integer greater than or equal to 0.

[0014]

[0015] The present specification also provides a method for producing a cell culture microcarrier, comprising the steps of polymerizing and recovering polystyrene particles from a monomer mixture containing a compound represented by the following chemical formula 1.

[0016] [Chemical Formula 1]

[0017]

[0018] In the above chemical formula 1, L0 is an arylene group having 6 or more carbon atoms or -(C=O)-, L1 and L2 are each independently an alkylene group having 1 or more carbon atoms, R1 is a reactive functional group capable of a ring-opening reaction, and R 10 is hydrogen or an alkyl group having 1 or more carbon atoms, and n is an integer greater than or equal to 0.

[0019] Also provided herein is a cell culture composition comprising cells and microcarriers for cell culture.

[0020] Hereinafter, a cell culture microcarrier according to a specific embodiment of the invention, a method for manufacturing a cell culture microcarrier, and a cell culture composition using the same will be described in more detail.

[0021]

[0022] Unless explicitly stated otherwise in this specification, terminology is used only to describe specific embodiments and is not intended to limit the invention.

[0023] As used herein, the singular forms also include the plural forms unless the context clearly dictates otherwise.

[0024] As used herein, the term “including” means specifying a particular characteristic, region, integer, step, operation, element and / or component, but does not exclude the presence or addition of other particular characteristics, regions, integers, steps, operations, elements, components and / or groups.

[0025] In addition, terms including ordinal numbers, such as "first" and "second," in this specification are used to distinguish one component from another and are not limited by the ordinal numbers. For example, within the scope of the present invention, the first component may also be referred to as the second component, and similarly, the second component may be referred to as the first component.

[0026] In the present specification, the alkyl group may be straight or branched, and the number of carbon atoms is not particularly limited, but is preferably 1 to 10. According to another embodiment, the number of carbon atoms of the alkyl group is 1 to 6. Specific examples of alkyl groups include methyl, ethyl, propyl, n-propyl, isopropyl, butyl, n-butyl, isobutyl, tert-butyl, sec-butyl, 1-methyl-butyl, 1-ethyl-butyl, pentyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, n-hexyl, 1-methylpentyl, 2-methylpentyl, 4-methyl-2-pentyl, 3,3-dimethylbutyl, 2-ethylbutyl, heptyl, n-heptyl, 1-methylhexyl, cyclopentylmethyl, cyclohexetylmethyl, octyl, n-octyl, tert-octyl, 1-methylheptyl, 2-ethylhexyl, 2-propylpentyl, n-nonyl, 2,2-dimethylheptyl, 1-ethyl-propyl, 1,1-dimethyl-propyl, isohexyl, Examples include, but are not limited to, 2-methylpentyl, 4-methylhexyl, and 5-methylhexyl.

[0027] In the present specification, a cycloalkyl group is a monovalent functional group derived from cycloalkane, which may be monocyclic or polycyclic, and has, but is not particularly limited to, 3 to 20 carbon atoms. According to another embodiment, the cycloalkyl group has 3 to 10 carbon atoms. Specifically, examples thereof include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, 3-methylcyclopentyl, 2,3-dimethylcyclopentyl, cyclohexyl, 3-methylcyclohexyl, 4-methylcyclohexyl, 2,3-dimethylcyclohexyl, 3,4,5-trimethylcyclohexyl, 4-tert-butylcyclohexyl, cycloheptyl, cyclooctyl, bicyclo[2,2,1]heptyl, and the like. The cycloalkyl group may be substituted or unsubstituted, and when substituted, examples of the substituent are as described above.

[0028] In this specification, a heterocycloalkyl group means a cycloalkyl group containing one or more non-carbon atoms or heteroatoms, and specifically, the heteroatoms may contain one or more atoms selected from the group consisting of O, N, Se, and S.

[0029] Hereinafter, the present invention will be described in more detail.

[0030]

[0031] According to one embodiment of the invention, a cell culture microcarrier comprising a polystyrene particle comprising a compound represented by the above chemical formula 1 as a monomer compound can be provided.

[0032] The inventors of the present invention have confirmed that, in the case of the cell culture microcarrier of the above embodiment, by including a compound represented by the above chemical formula 1 as a polystyrene monomer, the density of the final cell culture microcarrier is controlled, thereby enabling separation by density difference with cells, while improving dispersibility in a cell culture reactor or medium, and enhancing cell adhesion, and have completed the invention.

[0033]

[0034] Conventional cell culture microcarriers controlled the density of particles by encapsulating non-reactive low-density oil inside the particles, but there was a technical problem in that the low-density oil encapsulated inside leaked out when the particles were physically damaged.

[0035] Accordingly, the inventors of the present invention confirmed that by including the compound represented by the above chemical formula 1 as a monomer in a polystyrene polymer used in a cell culture microcarrier, the density of the particles can be controlled, thereby enabling separation by density difference with cells, and at the same time, dispersibility in a cell culture reactor or medium can be improved.

[0036] In addition, conventional cell culture microcarriers formed a coating layer on the surface of polystyrene particles to improve cell adhesion, but there was a technical problem in that some of the coating layer was detached when the cell culture microcarrier was stirred in a culture medium.

[0037] Accordingly, the inventors of the present invention confirmed that by including the compound represented by the above chemical formula 1 as a monomer in a polystyrene polymer used in a cell culture microcarrier, a reaction site is provided on the surface of the cell culture microcarrier where a cell adhesive ligand can be fixed by chemical bonding without an additional coating layer, thereby improving cell adhesion.

[0038]

[0039] Specifically, in the case of the cell culture microcarrier of the above embodiment, it may include polystyrene particles including a compound represented by the following chemical formula 1 as a monomer compound.

[0040] [Chemical Formula 1]

[0041]

[0042] In the above chemical formula 1, L0 is an arylene group having 6 or more carbon atoms or -(C=O)-, L1 and L2 are each independently an alkylene group having 1 or more carbon atoms, R1 is a reactive functional group capable of a ring-opening reaction, and R 10 is hydrogen or an alkyl group having 1 or more carbon atoms, and n is an integer greater than or equal to 0.

[0043]

[0044] Since the compound represented by the above chemical formula 1 contains a reactive functional group capable of ring-opening reaction in the polystyrene polymer, a reactive site is provided on the surface of the cell culture microcarrier where a cell adhesion ligand can be fixed by chemical bonding without an additional coating layer, thereby improving cell adhesion.

[0045]

[0046] The reactive functional group capable of the above ring-opening reaction may refer to a reactive functional group capable of a ring-opening reaction such as hydrolyzed ring opening.

[0047] Specifically, the reactive functional group capable of the ring-opening reaction may include a heterocycloalkyl group.

[0048] The above heterocycloalkyl group may refer to a cycloalkyl group containing one or more non-carbon atoms or heteroatoms. The heteroatoms may contain one or more atoms selected from the group consisting of O, N, Se, and S, and preferably may contain O.

[0049] The above heterocycloalkyl group is not particularly limited, but may include, for example, an epoxy group.

[0050]

[0051] Specifically, the polystyrene particles include a reaction product of a compound represented by the chemical formula 1 and an ethylenically unsaturated crosslinking agent, and may include 1 part by weight or more and 15 parts by weight or less of the compound represented by the chemical formula 1 with respect to 100 parts by weight of the ethylenically unsaturated crosslinking agent.

[0052] Specifically, the polystyrene particles include a reaction product of a compound represented by the chemical formula 1 and an ethylenically unsaturated crosslinking agent, and may include the compound represented by the chemical formula 1 in an amount of 1 part by weight or more, 1.5 parts by weight or more, 15 parts by weight or less, 10 parts by weight or less, 5 parts by weight or less, or 1 part by weight or more and 15 parts by weight or less, 1.5 parts by weight or more and 15 parts by weight or less, 1 part by weight or more and 10 parts by weight or less, 1.5 parts by weight or more and 10 parts by weight or less, 1 part by weight or more and 5 parts by weight or less, or 1.5 parts by weight or more and 5 parts by weight or less, based on 100 parts by weight of the ethylenically unsaturated crosslinking agent.

[0053]

[0054] If the compound represented by the above chemical formula 1 is included in an excessively small amount relative to 100 parts by weight of the above ethylenically unsaturated crosslinking agent, sufficient reaction sites for fixing the cell adhesion ligand may not be provided, resulting in poor cell adhesion. In addition, if the compound represented by the above chemical formula 1 is included in an excessive amount, the relative proportion of the compound with a high density may increase, resulting in a problem in which the density of the particles becomes higher than that of the cell culture medium, thereby losing the low-density characteristic.

[0055] Specifically, the compound represented by the above chemical formula 1 may include any one of the compounds represented by the following chemical formula 1-1 to the compounds represented by the following chemical formula 1-3.

[0056] [Chemical Formula 1-1]

[0057]

[0058] In the above chemical formula 1-1,

[0059] R 11 is hydrogen or an alkyl group having 1 or more carbon atoms,

[0060] [Chemical Formula 1-2]

[0061]

[0062] In the above chemical formula 1-2,

[0063]

[0064] R 12 is hydrogen or an alkyl group having 1 or more carbon atoms,

[0065] [Chemical Formula 1-3]

[0066]

[0067] In the above chemical formula 1-4,

[0068] R 13 is hydrogen or an alkyl group having 1 or more carbon atoms.

[0069]

[0070] By including one of the compounds represented by the above chemical formula 1-1 to the above chemical formula 1-3 as a monomer in a polystyrene polymer, a reaction site is provided on which a cell adhesive ligand can be fixed by chemical bonding on the surface of a cell culture microcarrier without an additional coating layer by controlling the weight ratio of the styrene monomer and the compound represented by the above chemical formula 1-1 to the above chemical formula 1-3, thereby improving cell adhesion.

[0071]

[0072] Meanwhile, the polystyrene particles may further include a compound represented by the following chemical formula 2 in addition to the compound represented by the above chemical formula 1 as a monomer compound.

[0073] [Chemical Formula 2]

[0074]

[0075] In the above chemical formula 2, R2 to R6 are each independently hydrogen or an alkyl group having 1 or more carbon atoms, and at least one of R2 to R6 is It is an alkyl group with 1 or more carbon atoms.

[0076] By including the compound represented by the above chemical formula 2 as a polystyrene monomer, the density of the final cell culture microcarrier can be precisely controlled by adjusting the content of the compound represented by the above chemical formula 2, thereby enabling separation by density difference with cells, and at the same time, dispersibility in a cell culture reactor or medium can be improved.

[0077] Specifically, the compound represented by the above chemical formula 2 may include at least one compound selected from the group consisting of a compound represented by the following chemical formula 2-1 to a compound represented by the following chemical formula 2-3.

[0078] [Chemical Formula 2-1]

[0079]

[0080] [Chemical Formula 2-2]

[0081]

[0082] [Chemical Formula 2-3]

[0083]

[0084] In the above chemical formulas 2-1 to 2-3, R 21 Inland R 26 are each independently an alkyl group having 1 or more carbon atoms.

[0085]

[0086] By including at least one compound selected from the group consisting of a compound represented by the above chemical formula 2-1 to a compound represented by the above chemical formula 2-3 as a polystyrene monomer, the density of the final cell culture microcarrier can be precisely controlled by adjusting the content of the compound represented by the above chemical formula 2, thereby enabling separation by density difference with cells, and at the same time improving dispersibility in a cell culture reactor or medium.

[0087]

[0088] More specifically, the compound represented by the above chemical formula 2 may include at least one compound selected from the group consisting of a compound represented by the following chemical formula 2-4 to a compound represented by the following chemical formula 2-6.

[0089] [Chemical Formula 2-4]

[0090]

[0091] [Chemical Formula 2-5]

[0092]

[0093] [Chemical Formula 2-6]

[0094]

[0095] In the above chemical formulas 2-4 to 2-6, R 21 Inland R 26 are each independently an alkyl group having 1 or more carbon atoms.

[0096]

[0097] Meanwhile, the compound represented by the above chemical formula 2 has a density of 0.92 g / cm 3 It could be as follows:

[0098] Specifically, the compound represented by the above chemical formula 2 has a density of 0.92 g / cm 3 Below, 0.91 g / cm 3 Below, 0.906 g / cm 3 Below 0.9 g / cm 3 Below, 0.89 g / cm3 Below 0.5 g / cm 3 Above, 0.6 g / cm 3 Above, 0.7 g / cm 3 Above, 0.8 g / cm 3 or more than 0.5 g / cm 3 Above 0.92 g / cm 3 Below 0.6 g / cm 3 Above 0.92 g / cm 3 Below 0.7 g / cm 3 Above 0.92 g / cm 3 Below 0.8 g / cm 3 Above 0.92 g / cm 3 Below 0.5 g / cm 3 Above 0.91 g / cm 3 Below 0.6 g / cm 3 Above 0.91 g / cm 3 Below 0.7 g / cm 3 Above 0.91 g / cm 3 Below 0.8 g / cm 3 Above 0.91 g / cm 3 Below 0.5 g / cm 3 Above 0.906 g / cm 3 Below 0.6 g / cm 3 Above 0.906 g / cm 3 Below 0.7 g / cm 3 Above 0.906 g / cm 3 Below 0.8 g / cm 3 Above 0.906 g / cm 3 Below 0.5 g / cm 3 More than 0.9 g / cm 3 Below 0.6 g / cm 3 More than 0.9 g / cm 3 Below 0.7 g / cm 3 More than 0.9 g / cm 3 Below 0.8 g / cm 3 More than 0.9 g / cm 3 Below 0.5 g / cm 3 Above 0.89 g / cm 3 Below 0.6 g / cm 3 Above 0.89 g / cm3 Below 0.7 g / cm 3 Above 0.89 g / cm 3 Below 0.8 g / cm 3 Above 0.89 g / cm 3 It could be as follows:

[0099]

[0100] The density of the compound represented by the above chemical formula 2 is 0.92 g / cm 3 By satisfying the following, the density of the final cell culture microcarrier can be precisely controlled by adjusting the content of the compound represented by the above chemical formula 2, thereby enabling separation by density difference with cells, and at the same time, dispersibility in a cell culture reactor or medium can be improved.

[0101] For example, the compound represented by the above chemical formula 2 may include 4-methylstyrene, trimethylstyrene, 4-ethenyl-2-methyl-1-(2-methylpropyl)benzene, 1-ethenyl-2-methyl-4-(1-methylethyl)benzene, 1-ethenyl-2,3-dimethylbenzene, 4-(1,1-dimethylethyl)-2-ethenyl-1-methylbenzene, 2-ethenyl-4-methyl-1-(1-methylethyl)benzene, 1-ethenyl-2-(1-methylethyl)benzene, 2-tert-butylstyrene, 4-tert-butylstyrene, 1-ethenyl-3-(1-methylethyl)benzene.

[0102]

[0103] Meanwhile, the polystyrene particles may further include a compound represented by the following chemical formula 3 in addition to the compound represented by the above chemical formula 1 as a monomer compound.

[0104] [Chemical Formula 3]

[0105]

[0106] In the above chemical formula 3, L 30 is -O(C=O)- or -(C=O)O-, and R 30 is a direct bond or an alkylene group having 1 or more carbon atoms, and R 31 is an alkyl group having 1 or more carbon atoms.

[0107]

[0108] That is, the polystyrene particles may include a compound represented by the above chemical formula 1 as a monomer compound, or include a compound represented by the above chemical formula 1 and a compound represented by the above chemical formula 2, or include a compound represented by the above chemical formula 1 and a compound represented by the above chemical formula 3, or include a compound represented by the above chemical formula 1, a compound represented by the above chemical formula 2, and a compound represented by the above chemical formula 3.

[0109]

[0110] By including a compound represented by the above chemical formula 3 as a polystyrene monomer, the substituents of the finally manufactured cell culture microcarrier can be hydrolyzed, thereby improving dispersibility in a cell culture reactor or medium.

[0111]

[0112] For example, the compound represented by the above chemical formula 3 may include acetoxystyrene.

[0113]

[0114] Meanwhile, the cell culture microcarrier may include polystyrene particles. Preferably, the cell culture microcarrier may be made of polystyrene particles.

[0115] Specifically, the apparent density of the polystyrene particles is 0.99 g / cm 3 Above 1.04 g / cm 3 It may be as follows. As it has the low density range described above, when separating and recovering microcarriers and cells after cell culture, cells and microcarriers can be easily separated through the difference in sedimentation rate due to gravity.

[0116] The density of the above polystyrene particles is 1.04 g / cm 3If it exceeds 0.99 g / cm, the difference in density between cells and microcarriers is small, making centrifugation difficult when separating and recovering cells after culture. 3 If it is less than this, a problem may occur in the early stage of culture, where the microcarriers float only on the surface of the culture medium, making it difficult for cells to attach.

[0117] The above cells are not particularly limited to adherent animal cells, but may be, for example, fibroblasts, epithelial cells, osteoblasts, chondrocytes, hepatocytes, human-derived umbilical cord blood cells, human bone marrow-derived mesenchymal stem cells, CHO (Chinese hamster ovary) cells, kidney cells (HEK293, BHK21, MDCK, vero cells, etc.), or a mixture of two or more thereof.

[0118] In addition, the density difference between the cell culture microcarrier and the cell is 0.20 g / cm 3 It may be less than 0.20 g / cm. The density difference between the cell culture microcarrier and the cell is 0.20 g / cm. 3 By satisfying the following, when separating and recovering microcarriers and cells after cell culture, cells and microcarriers can be easily separated through the difference in sedimentation rate due to gravity, and at the same time, the problem of microcarriers floating only on the surface of the culture medium in the early stage of culture, making it difficult for cells to attach, can be prevented.

[0119]

[0120] The above polystyrene particles may include a reaction product of a monomer mixture and an ethylenically unsaturated crosslinking agent.

[0121] As described above, the monomer mixture may include a compound represented by the chemical formula 1. In addition, the monomer mixture may include a compound represented by the chemical formula 1 and a compound represented by the chemical formula 2. In addition, the monomer mixture may include a compound represented by the chemical formula 1 and a compound represented by the chemical formula 3. In addition, the monomer mixture may include a compound represented by the chemical formula 1 to a compound represented by the chemical formula 3.

[0122] That is, the polystyrene particles include a reaction product of a compound represented by the chemical formula 1, a styrene monomer mixture, and an ethylenically unsaturated crosslinking agent, and the styrene monomer mixture may include a compound represented by the chemical formula 2 or a compound represented by the chemical formula 3.

[0123]

[0124] Specifically, the compound represented by the chemical formula 2 may be included in an amount of 80 parts by weight or more and 150 parts by weight or less, based on 100 parts by weight of the ethylenically unsaturated crosslinking agent.

[0125] More specifically, with respect to 100 parts by weight of the ethylenically unsaturated crosslinking agent, the compound represented by the chemical formula 2 is present in an amount of 80 parts by weight or more, 85 parts by weight or more, 90 parts by weight or less, 150 parts by weight or less, 125 parts by weight or less, 100 parts by weight or less, 99 parts by weight or less, or 80 parts by weight or more and 150 parts by weight or less, 85 parts by weight or more and 150 parts by weight or less, 90 parts by weight or more and 150 parts by weight or less, 80 parts by weight or more and 125 parts by weight or less, 85 parts by weight or more and 125 parts by weight or less, 90 parts by weight or more and 125 parts by weight or less, 80 parts by weight or more and 100 parts by weight or less, 85 parts by weight or more and 100 parts by weight or less, 90 parts by weight or more and 100 parts by weight or less, 80 parts by weight or more and 99 parts by weight or less, 85 parts by weight or more and 99 parts by weight or less, 90 parts by weight or more and 99 parts by weight or less May be included below the weight limit.

[0126] By including 80 parts by weight or more and 150 parts by weight or less of the compound represented by the above chemical formula 2 relative to 100 parts by weight of the above ethylenically unsaturated crosslinking agent, the low density characteristics of the particles can be controlled due to the low density of the compound represented by the above chemical formula 2, thereby enabling separation by density difference from cells, and at the same time, dispersibility in a cell culture reactor or medium can be improved.

[0127] If the compound represented by the above chemical formula 2 is included in an excessively small amount relative to 100 parts by weight of the above ethylene-based unsaturated crosslinking agent, the density of the particles may increase, causing the particle to lose its low-density characteristics and settle in the medium. If the compound is included in an excessive amount, the density of the particles may be 0.99 g / cm. 3 If the concentration is very low, high-speed stirring to disperse the cells within the medium may cause technical problems that affect cell characteristics.

[0128]

[0129] In addition, the compound represented by the chemical formula 2 may be included in an amount of 90 parts by weight or more and 100 parts by weight or less, based on 100 parts by weight of the styrene-based monomer mixture.

[0130] Specifically, the compound represented by the above chemical formula 2 may be included in an amount of 90 parts by weight or more, 91 parts by weight or more, 100 parts by weight or less, 95 parts by weight or less, or 90 parts by weight or more and 100 parts by weight or less, 91 parts by weight or more and 100 parts by weight or less, 90 parts by weight or more and 95 parts by weight or less, or 91 parts by weight or more and 95 parts by weight or less, based on 100 parts by weight of the above styrene-based monomer mixture.

[0131] By including the compound represented by the above chemical formula 2 in the above content with respect to 100 parts by weight of the above styrene-based monomer mixture, the density of the final cell culture microcarrier can be precisely controlled, thereby enabling separation by density difference with cells, and at the same time, dispersibility in a cell culture reactor or medium can be improved.

[0132]

[0133] In addition, the compound represented by the chemical formula 1 may be included in an amount of 1 part by weight or more and 15 parts by weight or less, based on 100 parts by weight of the compound represented by the chemical formula 2.

[0134] Specifically, with respect to 100 parts by weight of the compound represented by the above chemical formula 2, the compound represented by the above chemical formula 1 may be included in an amount of 1 part by weight or more, 1.5 parts by weight or more, 15 parts by weight or less, 12 parts by weight or less, 10 parts by weight or less, 6 parts by weight or less, 1 part by weight or more and 15 parts by weight or less, 1.5 parts by weight or more and 15 parts by weight or less, 1 part by weight or more and 12 parts by weight or less, 1.5 parts by weight or more and 12 parts by weight or less, 1 part by weight or more and 10 parts by weight or less, 1.5 parts by weight or more and 10 parts by weight or less, 1 part by weight or more and 6 parts by weight or less, 1.5 parts by weight or more and 6 parts by weight or less.

[0135] If the compound represented by the above chemical formula 1 is included in an excessively small amount relative to 100 parts by weight of the compound represented by the above chemical formula 2, the cell adhesion may be poor because sufficient reaction sites for fixing the cell adhesion ligand are not provided, and if it is included in an excessive amount, the density of the particles may increase, resulting in a technical problem of losing the low-density characteristic.

[0136]

[0137] In addition, when the monomer compound includes a compound represented by the following chemical formula 3, the compound represented by the above chemical formula 3 may be included in an amount of 0.1 parts by weight or more and 5 parts by weight or less, based on 100 parts by weight of the ethylenically unsaturated crosslinking agent.

[0138] More specifically, the compound represented by the chemical formula 3 may be included in an amount of 0.1 part by weight or more, 0.5 part by weight or more, 1 part by weight or more, 5 parts by weight or less, 3 parts by weight or less, 2 parts by weight or less, 0.1 part by weight or more and 5 parts by weight or less, 0.5 part by weight or more and 5 parts by weight or less, 1 part by weight or more and 5 parts by weight or less, 0.1 part by weight or more and 3 parts by weight or less, 0.5 part by weight or more and 3 parts by weight or less, 1 part by weight or more and 3 parts by weight or less, 0.1 part by weight or more and 2 parts by weight or less, 0.5 part by weight or more and 2 parts by weight or less, 1 part by weight or more and 2 parts by weight or less.

[0139] When a compound represented by the following chemical formula 3 is included as a monomer compound, by including 0.1 to 5 parts by weight of the compound represented by the above chemical formula 3 relative to 100 parts by weight of the ethylenically unsaturated crosslinking agent, the substituents of the finally manufactured cell culture microcarrier can be hydrolyzed, thereby improving dispersibility in a cell culture reactor or medium.

[0140] If the compound represented by the above chemical formula 3 is included in an amount exceeding 5 parts by weight based on 100 parts by weight of the above ethylene-based unsaturated crosslinking agent, a technical problem may arise in which the density of the particles increases and the low-density characteristics are lost.

[0141]

[0142] In addition, when the monomer compound includes a compound represented by the following chemical formula 3, the compound represented by the above chemical formula 3 may be included in an amount of 0.1 parts by weight or more and 5 parts by weight or less with respect to 100 parts by weight of the styrene-based monomer mixture.

[0143] Specifically, with respect to 100 parts by weight of the styrene-based monomer mixture, the compound represented by the chemical formula 3 may be included in an amount of 0.1 parts by weight or more, 0.5 parts by weight or more, 1 part by weight or more, 5 parts by weight or less, 3 parts by weight or less, 2.5 parts by weight or less, 0.1 parts by weight or more and 5 parts by weight or less, 0.5 parts by weight or more and 5 parts by weight or less, 1 part by weight or more and 5 parts by weight or less, 0.1 parts by weight or more and 3 parts by weight or less, 0.5 parts by weight or more and 3 parts by weight or less, 1 part by weight or more and 3 parts by weight or less, 0.1 parts by weight or more and 2.5 parts by weight or less, 0.5 parts by weight or more and 2.5 parts by weight or less, 1 part by weight or more and 2.5 parts by weight or less.

[0144] When a compound represented by the following chemical formula 3 is included as a monomer compound, by including the compound represented by the above chemical formula 3 in an amount of 0.1 to 5 parts by weight based on 100 parts by weight of the styrene-based monomer mixture, sufficient reaction sites are provided to which a cell adhesion ligand can be fixed while maintaining low-density characteristics, thereby facilitating cell attachment.

[0145]

[0146] In addition, when the monomer compound includes a compound represented by the following chemical formula 3, the compound represented by the above chemical formula 1 may be included in an amount of 110 parts by weight or more and 500 parts by weight or less, based on 100 parts by weight of the compound represented by the above chemical formula 3.

[0147] Specifically, with respect to 100 parts by weight of the compound represented by the above chemical formula 3, the compound represented by the above chemical formula 1 may be included in an amount of 110 parts by weight or more, 150 parts by weight or more, 500 parts by weight or less, 400 parts by weight or less, 300 parts by weight or less, or 200 parts by weight or less, or 110 parts by weight or more and 500 parts by weight or less, 110 parts by weight or more and 400 parts by weight or less, 110 parts by weight or more and 300 parts by weight or less, 110 parts by weight or more and 200 parts by weight or less, 150 parts by weight or more and 500 parts by weight or less, 150 parts by weight or more and 400 parts by weight or less, 150 parts by weight or more and 300 parts by weight or less, or 150 parts by weight or more and 200 parts by weight or less.

[0148] When a compound represented by the following chemical formula 3 is included as a monomer compound, if the compound represented by the above chemical formula 1 is included in an amount of less than 110 parts by weight relative to 100 parts by weight of the compound represented by the above chemical formula 3, a problem may arise in which the compound represented by the above chemical formula 1 is not introduced to the particle surface as it is lost before polymerization due to the relatively high water solubility of the compound, and if it is included in an amount exceeding 500 parts by weight, a technical problem may arise in which the density of the particles increases and the low-density characteristic is lost.

[0149]

[0150] In addition, the polystyrene particles may contain 60 parts by weight or more and 200 parts by weight or less of the ethylenically unsaturated crosslinking agent with respect to 100 parts by weight of the styrene monomer mixture.

[0151] Specifically, the ethylene-based unsaturated crosslinking agent may be included in an amount of 60 parts by weight or more and 200 parts by weight or less, 60 parts by weight or more and 150 parts by weight or less, 60 parts by weight or more and 130 parts by weight or less, 100 parts by weight or more and 200 parts by weight or less, 100 parts by weight or more and 150 parts by weight or less, or 100 parts by weight or more and 130 parts by weight or less, based on 100 parts by weight of the styrene-based monomer mixture.

[0152] When the ethylene-based unsaturated crosslinking agent is included in an excessively small amount, less than 60 parts by weight, based on 100 parts by weight of the styrene-based monomer mixture, the crosslinking density of the polystyrene-based polymer decreases, making it difficult for the particle shape to stably maintain a spherical shape.

[0153] On the other hand, if the ethylene-based unsaturated crosslinking agent is included in an excessive amount exceeding 200 parts by weight with respect to 100 parts by weight of the styrene-based monomer mixture, there is a limitation in that it is difficult to lower the particle density to the target level.

[0154]

[0155] An example of the above ethylenically unsaturated crosslinking agent is divinylbenzene.

[0156]

[0157] The average diameter of the polystyrene particles may be 50 ㎛ to 400 ㎛, or 60 ㎛ to 390 ㎛. When the average diameter of the polystyrene particles satisfies the above-described range, cell attachment and culture performance are excellent. On the other hand, when the average diameter of the polystyrene particles is less than 50 ㎛, there is a concern that the surface area available for cell culture may be small, which may cause a problem of low culture efficiency, and when it exceeds 400 ㎛, the interaction between attached cells may be reduced, the cell density in the incubator may be lowered, and the cell culture efficiency may be lowered.

[0158] The diameter of the polystyrene particles refers to the distance between two points where a straight line passing through the center of gravity of the polystyrene particles meets the outermost surface of the polystyrene particles, and the average diameter of the polystyrene particles can be obtained by checking the diameter of all polystyrene particles included in the cell culture microcarrier using an optical microscope.

[0159] The polystyrene particles may be a group of individual particles having an average diameter of 50 μm to 400 μm, or 60 μm to 390 μm, and the individual particles included in the group may have an average diameter of 50 μm to 400 μm, or 60 μm to 390 μm. More specifically, 95%, or 99% of the individual particles included in the group may have a diameter of 50 μm to 400 μm, or 60 μm to 390 μm.

[0160] In addition, the polystyrene particles may have a perfect spherical particle ratio of more than 90% and less than 100%, or more than 92% and less than 100%, or more than 95% and less than 100%, or more than 96% and less than 99% without damage or destruction according to the following mathematical formula.

[0161] [Mathematical formula]

[0162] Percentage of perfectly spherical particles without damage or destruction (%) = (Number of polystyrene particles with perfectly spherical shapes without damage or destruction / Number of total polystyrene particles) x 100.

[0163] The percentage of perfectly spherical particles without damage or destruction according to the above mathematical formula can be obtained by measuring the number of particles having a perfectly spherical shape without damage or destruction among all particles of the polystyrene particles using SEM and calculating the percentage ratio of the number of perfectly spherical particles without damage or destruction compared to all particles.

[0164] That is, the above cell culture microcarrier may contain a plurality of polystyrene particles, and it can be visually determined through SEM whether or not the plurality of polystyrene particles have a completely spherical shape without damage or destruction.

[0165] If the ratio of perfectly spherical particles without damage or destruction according to the above mathematical formula decreases to less than 90%, there is a concern that the number of irregular particles with uneven and sunken particle surfaces will increase, and the irregular particles will float in the cell culture medium, causing physical impact on the cells being cultured, thereby lowering the cell culture efficiency to the point where cell culture becomes impossible.

[0166]

[0167] Specifically, the D50 particle diameter of the cell culture microcarrier may be 100 ㎛ to 300 ㎛, or 100 ㎛ to 250 ㎛, or 120 ㎛ to 250 ㎛, or 130 ㎛ to 250 ㎛. When the average diameter of the cell culture microcarrier satisfies the above-described range, cell attachment and culture performance are excellent. On the other hand, when the D50 particle diameter of the cell culture microcarrier is less than 100 ㎛, there is a concern that the surface area available for cell culture may be small, thereby lowering the culture efficiency, and when it exceeds 300 ㎛, the interaction between attached cells may be reduced, the cell density in the incubator may be lowered, thereby lowering the cell culture efficiency.

[0168]

[0169] Meanwhile, the cell culture microcarrier may include a cell adhesion inducing layer formed on the polystyrene particles.

[0170] The above cell adhesion inducing layer is composed of cell adhesive substances, which serve to provide a site where transmembrane proteins of cells can bind, thereby enabling adherent cells to stably attach, spread, and be cultured.

[0171] The polymer forming the cell adhesion-inducing layer may include, but is not particularly limited to, one or more selected from the group consisting of gelatin, collagen, fibronectin, chitosan, polylysine, vitronectin, peptides including RGD, lignin, cationic dextran, dihydroxyphenylalanine (DOPA), dopamine, norepinephrine, epinephrine, epigallocatechin, and derivatives thereof.

[0172]

[0173] A cell culture microcarrier manufactured according to the above method for manufacturing a cell culture microcarrier may have an epoxy content of 40 μmol / g or more and 100 μmol / g or less.

[0174] Specifically, the above cell culture microcarrier may have an epoxy content of 40 μmol / g or more, 45 μmol / g or more, 49 μmol / g or more, 49.5 μmol / g or more, 40 μmol / g or more and 100 μmol / g or less, 45 μmol / g or more and 100 μmol / g or less, 49 μmol / g or more and 100 μmol / g or less, 49.5 μmol / g or more and 100 μmol / g or less.

[0175] The above epoxy content can be calculated by adding the above cell culture microcarrier to an acidic solution, then adding an indicator solution and titrating with NaOH, using the following formula.

[0176] [Mathematical formula]

[0177] Epoxy content (μmol / g) = [V0(ml)-V(ml)]*C NaOH (mol / L)

[0178] In the above equation, V0 is the volume of NaOH added to the control group containing deionized water instead of particles, V is the amount of NaOH added to the actual sample, and C NaOH refers to the concentration of NaOH used in titration.

[0179] As the above cell culture microcarrier satisfies the above epoxy content, excellent cell adhesion can be achieved. If the epoxy content is excessively reduced, technical problems such as reduced particle dispersion and reduced introduction efficiency of surface modification materials may arise.

[0180]

[0181] Meanwhile, according to another embodiment of the invention, a method for manufacturing a microcarrier for cell culture may be provided, comprising the steps of polymerizing and recovering polystyrene particles from a monomer mixture containing a compound represented by the chemical formula 1.

[0182]

[0183] In the method for manufacturing a cell culture microcarrier of the above embodiment, the contents of the polystyrene particles and the compound represented by the chemical formula 1 include all of the contents described above.

[0184]

[0185] Specifically, the compound represented by the above chemical formula 1 may include any one of the compounds represented by the following chemical formula 1-1 to the compounds represented by the following chemical formula 1-3.

[0186] [Chemical Formula 1-1]

[0187]

[0188] In the above chemical formula 1-1,

[0189] R 11is hydrogen or an alkyl group having 1 or more carbon atoms,

[0190] [Chemical Formula 1-2]

[0191]

[0192] In the above chemical formula 1-2,

[0193] R 12 is hydrogen or an alkyl group having 1 or more carbon atoms,

[0194] [Chemical Formula 1-3]

[0195]

[0196] In the above chemical formula 1-4,

[0197] R 13 is hydrogen or an alkyl group having 1 or more carbon atoms.

[0198]

[0199] By including one of the compounds represented by the above chemical formula 1-1 to the above chemical formula 1-3 as a monomer in a polystyrene polymer, and by including an epoxy group in the compound represented by the above chemical formula 1-1 to the above chemical formula 1-3, a reaction site is provided on the surface of a cell culture microcarrier where a cell adhesive ligand can be fixed by chemical bonding without an additional coating layer, thereby improving cell adhesion.

[0200]

[0201] Meanwhile, the polystyrene particles may include a compound represented by the following chemical formula 2 as a monomer compound.

[0202] [Chemical Formula 2]

[0203]

[0204] In the above chemical formula 2, R2 to R6 are each independently hydrogen or an alkyl group having 1 or more carbon atoms, and at least one of R2 to R6 is It is an alkyl group with 1 or more carbon atoms.

[0205] By including the compound represented by the above chemical formula 2 as a polystyrene monomer, the density of the final cell culture microcarrier can be precisely controlled by adjusting the content of the compound represented by the above chemical formula 2, thereby enabling separation by density difference from cells after completion of cell culture, and at the same time, dispersibility in a cell culture reactor or medium can be improved.

[0206] Specifically, the compound represented by the above chemical formula 2 may include at least one compound selected from the group consisting of a compound represented by the following chemical formula 2-1 to a compound represented by the following chemical formula 2-3.

[0207] [Chemical Formula 2-1]

[0208]

[0209] [Chemical Formula 2-2]

[0210]

[0211] [Chemical Formula 2-3]

[0212]

[0213] In the above chemical formulas 2-1 to 2-3, R 21 Inland R 26 are each independently an alkyl group having 1 or more carbon atoms.

[0214]

[0215] By including at least one compound selected from the group consisting of a compound represented by the above chemical formula 2-1 to a compound represented by the above chemical formula 2-3 as a polystyrene monomer, the density of the final cell culture microcarrier can be precisely controlled by adjusting the content of the compound represented by the above chemical formula 2, thereby enabling separation by density difference from cells after completion of cell culture, and at the same time, improving dispersibility in a cell culture reactor or medium.

[0216]

[0217] More specifically, the compound represented by the above chemical formula 2 may include at least one compound selected from the group consisting of a compound represented by the following chemical formula 2-4 to a compound represented by the following chemical formula 2-6.

[0218] [Chemical Formula 2-4]

[0219]

[0220] [Chemical Formula 2-5]

[0221]

[0222] [Chemical Formula 2-6]

[0223]

[0224] In the above chemical formulas 2-4 to 2-6, R 21 Inland R 26 are each independently an alkyl group having 1 or more carbon atoms.

[0225]

[0226] Meanwhile, the compound represented by the above chemical formula 2 has a density of 0.92 g / cm 3 It could be as follows:

[0227] Specifically, the compound represented by the above chemical formula 2 has a density of 0.92 g / cm 3 Below, 0.91 g / cm 3 Below, 0.906 g / cm 3 Below 0.9 g / cm 3 Below, 0.89 g / cm 3 Below 0.5 g / cm 3 Above, 0.6 g / cm 3 Above, 0.7 g / cm 3 Above, 0.8 g / cm 3 or more than 0.5 g / cm 3 Above 0.92 g / cm 3 Below 0.6 g / cm 3 Above 0.92 g / cm 3 Below 0.7 g / cm 3 Above 0.92 g / cm 3Below 0.8 g / cm 3 Above 0.92 g / cm 3 Below 0.5 g / cm 3 Above 0.91 g / cm 3 Below 0.6 g / cm 3 Above 0.91 g / cm 3 Below 0.7 g / cm 3 Above 0.91 g / cm 3 Below 0.8 g / cm 3 Above 0.91 g / cm 3 Below 0.5 g / cm 3 Above 0.906 g / cm 3 Below 0.6 g / cm 3 Above 0.906 g / cm 3 Below 0.7 g / cm 3 Above 0.906 g / cm 3 Below 0.8 g / cm 3 Above 0.906 g / cm 3 Below 0.5 g / cm 3 More than 0.9 g / cm 3 Below 0.6 g / cm 3 More than 0.9 g / cm 3 Below 0.7 g / cm 3 More than 0.9 g / cm 3 Below 0.8 g / cm 3 More than 0.9 g / cm 3 Below 0.5 g / cm 3 Above 0.89 g / cm 3 Below 0.6 g / cm 3 Above 0.89 g / cm 3 Below 0.7 g / cm 3 Above 0.89 g / cm 3 Below 0.8 g / cm 3 Above 0.89 g / cm 3 It could be as follows:

[0228]

[0229] The density of the compound represented by the above chemical formula 2 is 0.92 g / cm 3By satisfying the following, the density of the final cell culture microcarrier can be precisely controlled by adjusting the content of the compound represented by the above chemical formula 2, thereby enabling separation by density difference from cells after completion of cell culture, and at the same time, dispersibility in a cell culture reactor or medium can be improved.

[0230] For example, the compound represented by the above chemical formula 2 may include 4-methylstyrene, trimethylstyrene, 4-ethenyl-2-methyl-1-(2-methylpropyl)benzene, 1-ethenyl-2-methyl-4-(1-methylethyl)benzene, 1-ethenyl-2,3-dimethylbenzene, 4-(1,1-dimethylethyl)-2-ethenyl-1-methylbenzene, 2-ethenyl-4-methyl-1-(1-methylethyl)benzene, 1-ethenyl-2-(1-methylethyl)benzene, 2-tert-butylstyrene, 4-tert-butylstyrene, 1-ethenyl-3-(1-methylethyl)benzene.

[0231]

[0232] Meanwhile, the polystyrene particles may further include a compound represented by the following chemical formula 3 in addition to the compound represented by the above chemical formula 1 as a monomer compound.

[0233] [Chemical Formula 3]

[0234]

[0235] In the above chemical formula 3, L 30 is -O(C=O)- or -(C=O)O-, and R 30 is a direct bond or an alkylene group having 1 or more carbon atoms, and R 31 is an alkyl group having 1 or more carbon atoms.

[0236]

[0237] That is, the polystyrene particles may include a compound represented by the above chemical formula 1 as a monomer compound, or include a compound represented by the above chemical formula 1 and a compound represented by the above chemical formula 2, or include a compound represented by the above chemical formula 1 and a compound represented by the above chemical formula 3, or include a compound represented by the above chemical formula 1, a compound represented by the above chemical formula 2, and a compound represented by the above chemical formula 3.

[0238]

[0239] By including a compound represented by the above chemical formula 3 as a polystyrene monomer, the substituents of the finally manufactured cell culture microcarrier can be hydrolyzed, thereby improving dispersibility in a cell culture reactor or medium.

[0240]

[0241] For example, the compound represented by the above chemical formula 3 may include acetoxystyrene.

[0242]

[0243] Meanwhile, the monomer mixture may contain 20 parts by weight or more and 99 parts by weight or less of the compound represented by the chemical formula 2 based on 100 parts by weight of the monomer mixture.

[0244] The above monomer mixture may include both the compound represented by the above chemical formula 1 and a styrene-based monomer mixture.

[0245] Specifically, the monomer mixture may contain the compound represented by the chemical formula 2 in an amount of 20 parts by weight or more, 30 parts by weight or more, 50 parts by weight or more, 75 parts by weight or more, 80 parts by weight or more, 90 parts by weight or more, 99 parts by weight or less, 95 parts by weight or less, 20 parts by weight or more and 99 parts by weight or less, 30 parts by weight or more and 99 parts by weight or less, 50 parts by weight or more and 99 parts by weight or less, 75 parts by weight or more and 99 parts by weight or less, 80 parts by weight or more and 99 parts by weight or less, 90 parts by weight or more and 99 parts by weight or less, 20 parts by weight or more and 95 parts by weight or less, 30 parts by weight or more and 95 parts by weight or less, 50 parts by weight or more and 95 parts by weight or less, 75 parts by weight or more and 95 parts by weight or less, 80 parts by weight or more and 95 parts by weight or less, 90 parts by weight or more and 95 parts by weight or less. there is.

[0246] If the compound represented by the above chemical formula 2 is included in less than 20 parts by weight with respect to 100 parts by weight of the above monomer mixture, the density of the particles may increase, resulting in a technical problem of losing the low-density characteristic, and if it is included in more than 99 parts by weight, the density of the particles may be 0.99 g / cm 3 Technical issues may arise that affect cell characteristics due to high-speed stirring for dispersion within the medium, which may lower the pH below.

[0247]

[0248] Meanwhile, the monomer mixture may contain 0.1 parts by weight or more and 5 parts by weight or less of the compound represented by the chemical formula 3 based on 100 parts by weight of the monomer mixture.

[0249] The above monomer mixture may include all of the compound represented by the above chemical formula 1, the styrene monomer mixture, and the ethylenically unsaturated crosslinking agent.

[0250] Specifically, the monomer mixture may contain the compound represented by the chemical formula 3 in an amount of 0.1 part by weight or more, 1 part by weight or more, 5 parts by weight or less, 4 parts by weight or less, 3 parts by weight or less, 2 parts by weight or less, 0.1 part by weight or more and 5 parts by weight or less, 0.1 part by weight or more and 4 parts by weight or less, 0.1 part by weight or more and 3 parts by weight or less, 0.1 part by weight or more and 2 parts by weight or less, 1 part by weight or more and 5 parts by weight or less, 1 part by weight or more and 4 parts by weight or less, 1 part by weight or more and 3 parts by weight or less, 1 part by weight or more and 2 parts by weight or less, based on 100 parts by weight of the monomer mixture.

[0251] If the compound represented by the above chemical formula 3 is included in less than 0.1 parts by weight relative to 100 parts by weight of the above monomer mixture, the hydrophilicity of the surface may be insufficient, resulting in low dispersibility within the incubator. If the compound is included in more than 5 parts by weight, the density of the particles may increase, resulting in a technical problem of losing the low-density characteristic.

[0252]

[0253] Meanwhile, according to the above-described embodiment of the invention, the step of polymerizing and recovering polystyrene particles from a monomer mixture including a compound represented by the above-described chemical formula 1 may include a step of polymerizing and recovering polystyrene particles by reacting a monomer mixture including a compound represented by the above-described chemical formula 1 and an ethylenically unsaturated crosslinking agent.

[0254] The above ethylenically unsaturated crosslinking agent includes all of the above-described contents.

[0255]

[0256] In the method for manufacturing the above cell culture microcarrier, the compound represented by the above chemical formula 2 may be included in an amount of 80 parts by weight or more and 150 parts by weight or less, based on 100 parts by weight of the above ethylenically unsaturated crosslinking agent.

[0257] More specifically, with respect to 100 parts by weight of the ethylenically unsaturated crosslinking agent, the compound represented by the chemical formula 2 is present in an amount of 80 parts by weight or more, 85 parts by weight or more, 90 parts by weight or less, 150 parts by weight or less, 125 parts by weight or less, 100 parts by weight or less, 99 parts by weight or less, or 80 parts by weight or more and 150 parts by weight or less, 85 parts by weight or more and 150 parts by weight or less, 90 parts by weight or more and 150 parts by weight or less, 80 parts by weight or more and 125 parts by weight or less, 85 parts by weight or more and 125 parts by weight or less, 90 parts by weight or more and 125 parts by weight or less, 80 parts by weight or more and 100 parts by weight or less, 85 parts by weight or more and 100 parts by weight or less, 90 parts by weight or more and 100 parts by weight or less, 80 parts by weight or more and 99 parts by weight or less, 85 parts by weight or more and 99 parts by weight or less, 90 parts by weight or more and 99 parts by weight or less May be included below the weight limit.

[0258] By including 80 parts by weight or more and 150 parts by weight or less of the compound represented by the above chemical formula 2 relative to 100 parts by weight of the above ethylenically unsaturated crosslinking agent, the low density characteristics of the particles can be controlled due to the low density of the compound represented by the above chemical formula 2, thereby enabling separation by density difference from cells, and at the same time, dispersibility in a cell culture reactor or medium can be improved.

[0259] If the compound represented by the above chemical formula 2 is included in an excessively small amount relative to 100 parts by weight of the above ethylene-based unsaturated crosslinking agent, the density of the particles may increase, causing the particle to lose its low-density characteristics and settle in the medium. If the compound is included in an excessive amount, the density of the particles may be 0.99 g / cm. 3 If the concentration is very low, high-speed stirring to disperse the cells within the medium may cause technical problems that affect cell characteristics.

[0260]

[0261] The above monomer mixture may include a compound represented by the above chemical formula 1 and a styrene-based monomer mixture.

[0262] The compound represented by the above chemical formula 2 may be included in an amount of 90 parts by weight or more and 100 parts by weight or less, based on 100 parts by weight of the above styrene-based monomer mixture.

[0263] Specifically, the compound represented by the above chemical formula 2 may be included in an amount of 90 parts by weight or more, 91 parts by weight or more, 100 parts by weight or less, 95 parts by weight or less, or 90 parts by weight or more and 100 parts by weight or less, 91 parts by weight or more and 100 parts by weight or less, 90 parts by weight or more and 95 parts by weight or less, or 91 parts by weight or more and 95 parts by weight or less, based on 100 parts by weight of the above styrene-based monomer mixture.

[0264] By including the compound represented by the above chemical formula 2 in the above content with respect to 100 parts by weight of the above styrene-based monomer mixture, the density of the final cell culture microcarrier can be precisely controlled, thereby enabling separation by density difference with cells, and at the same time, dispersibility in a cell culture reactor or medium can be improved.

[0265]

[0266] In addition, the compound represented by the chemical formula 1 may be included in an amount of 1 part by weight or more and 15 parts by weight or less, based on 100 parts by weight of the compound represented by the chemical formula 2.

[0267] Specifically, with respect to 100 parts by weight of the compound represented by the above chemical formula 2, the compound represented by the above chemical formula 1 may be included in an amount of 1 part by weight or more, 1.5 parts by weight or more, 15 parts by weight or less, 12 parts by weight or less, 10 parts by weight or less, 6 parts by weight or less, 1 part by weight or more and 15 parts by weight or less, 1.5 parts by weight or more and 15 parts by weight or less, 1 part by weight or more and 12 parts by weight or less, 1.5 parts by weight or more and 12 parts by weight or less, 1 part by weight or more and 10 parts by weight or less, 1.5 parts by weight or more and 10 parts by weight or less, 1 part by weight or more and 6 parts by weight or less, 1.5 parts by weight or more and 6 parts by weight or less.

[0268] If the compound represented by the above chemical formula 1 is included in an excessively small amount relative to 100 parts by weight of the compound represented by the above chemical formula 2, the cell adhesion may be poor because sufficient reaction sites for fixing the cell adhesion ligand are not provided, and if it is included in an excessive amount, the density of the particles may increase, resulting in a technical problem of losing the low-density characteristic.

[0269]

[0270] In addition, when the monomer compound includes a compound represented by the following chemical formula 3, the compound represented by the above chemical formula 3 may be included in an amount of 0.1 parts by weight or more and 5 parts by weight or less, based on 100 parts by weight of the ethylenically unsaturated crosslinking agent.

[0271] More specifically, the compound represented by the chemical formula 3 may be included in an amount of 0.1 part by weight or more, 0.5 part by weight or more, 1 part by weight or more, 5 parts by weight or less, 3 parts by weight or less, 2 parts by weight or less, 0.1 part by weight or more and 5 parts by weight or less, 0.5 part by weight or more and 5 parts by weight or less, 1 part by weight or more and 5 parts by weight or less, 0.1 part by weight or more and 3 parts by weight or less, 0.5 part by weight or more and 3 parts by weight or less, 1 part by weight or more and 3 parts by weight or less, 0.1 part by weight or more and 2 parts by weight or less, 0.5 part by weight or more and 2 parts by weight or less, 1 part by weight or more and 2 parts by weight or less.

[0272] When a compound represented by the following chemical formula 3 is included as a monomer compound, by including 0.1 to 5 parts by weight of the compound represented by the above chemical formula 3 relative to 100 parts by weight of the ethylenically unsaturated crosslinking agent, the substituents of the finally manufactured cell culture microcarrier can be hydrolyzed, thereby improving dispersibility in a cell culture reactor or medium.

[0273] If the compound represented by the above chemical formula 3 is included in an amount exceeding 5 parts by weight based on 100 parts by weight of the above ethylene-based unsaturated crosslinking agent, a technical problem may arise in which the density of the particles increases and the low-density characteristics are lost.

[0274]

[0275] In addition, when the monomer compound includes a compound represented by the following chemical formula 3, the compound represented by the above chemical formula 3 may be included in an amount of 0.1 parts by weight or more and 5 parts by weight or less with respect to 100 parts by weight of the styrene-based monomer mixture.

[0276] Specifically, with respect to 100 parts by weight of the styrene-based monomer mixture, the compound represented by the chemical formula 3 may be included in an amount of 0.1 parts by weight or more, 0.5 parts by weight or more, 1 part by weight or more, 5 parts by weight or less, 3 parts by weight or less, 2.5 parts by weight or less, 0.1 parts by weight or more and 5 parts by weight or less, 0.5 parts by weight or more and 5 parts by weight or less, 1 part by weight or more and 5 parts by weight or less, 0.1 parts by weight or more and 3 parts by weight or less, 0.5 parts by weight or more and 3 parts by weight or less, 1 part by weight or more and 3 parts by weight or less, 0.1 parts by weight or more and 2.5 parts by weight or less, 0.5 parts by weight or more and 2.5 parts by weight or less, 1 part by weight or more and 2.5 parts by weight or less.

[0277] When the monomer compound includes a compound represented by the following chemical formula 3, by including the compound represented by the above chemical formula 3 in an amount of 0.1 to 5 parts by weight based on 100 parts by weight of the styrene-based monomer mixture, the substituents of the finally manufactured cell culture microcarrier can be hydrolyzed, thereby improving dispersibility in a cell culture reactor or medium.

[0278]

[0279] In addition, when the monomer compound includes a compound represented by the following chemical formula 3, the compound represented by the above chemical formula 1 may be included in an amount of 110 parts by weight or more and 500 parts by weight or less, based on 100 parts by weight of the compound represented by the above chemical formula 3.

[0280] Specifically, with respect to 100 parts by weight of the compound represented by the above chemical formula 3, the compound represented by the above chemical formula 1 may be included in an amount of 110 parts by weight or more, 150 parts by weight or more, 500 parts by weight or less, 400 parts by weight or less, 300 parts by weight or less, or 200 parts by weight or less, or 110 parts by weight or more and 500 parts by weight or less, 110 parts by weight or more and 400 parts by weight or less, 110 parts by weight or more and 300 parts by weight or less, 110 parts by weight or more and 200 parts by weight or less, 150 parts by weight or more and 500 parts by weight or less, 150 parts by weight or more and 400 parts by weight or less, 150 parts by weight or more and 300 parts by weight or less, or 150 parts by weight or more and 200 parts by weight or less.

[0281] When the compound represented by the above chemical formula 1 is included in less than 110 parts by weight relative to 100 parts by weight of the compound represented by the above chemical formula 3, a problem may arise in which the compound represented by the above chemical formula 1 is lost before polymerization due to its relatively high water solubility and is not introduced to the particle surface. When the compound is included in more than 500 parts by weight, a technical problem may arise in which the density of the particles increases and the low-density characteristics are lost.

[0282]

[0283] In the method for manufacturing the above cell culture microcarrier, the step of polymerizing and recovering the polystyrene particles may include a step of performing a suspension polymerization reaction of a monomer composition and recovering the suspension polymerization reaction result.

[0284]

[0285] More specifically, the suspension polymerization reaction of the monomer composition may include a step of mixing the monomer composition into an aqueous dispersion and applying a shear force to homogenize the monomer composition into droplets in the aqueous dispersion; and a step of suspension polymerizing the homogenized monomer composition at a stirring speed of 300 rpm to 1000 rpm.

[0286] In the step of homogenizing the above monomer composition into a droplet form in an aqueous dispersion, stirring may be performed at a stirring speed of 300 rpm to 1000 rpm, or 400 rpm to 800 rpm.

[0287] In the step of suspension polymerizing the homogenized monomer composition at a stirring speed of 300 rpm to 1000 rpm, or 400 rpm to 800 rpm, the density of the microcarrier can be further reduced by the structure of the compound represented by the chemical formula 1 during the formation of the particle structure of polystyrene, while producing a microcarrier with a high percentage of perfectly spherical particles without damage or destruction.

[0288] In the step of suspension polymerizing the homogenized monomer composition at a stirring speed of 300 rpm to 1000 rpm, or 400 rpm to 800 rpm, examples of the suspension polymerization conditions are not particularly limited, but for example, it may be performed at a temperature of 50°C to 100°C for 3 to 18 hours.

[0289]

[0290] Meanwhile, the method for manufacturing the micro carrier for cell culture may further include a washing step and a drying step after the step of polymerizing and recovering polystyrene particles from a monomer mixture containing the compound represented by the chemical formula 1.

[0291] Specifically, the washing step may include a step of filtering the reaction product through a sieve of 30 μm to 100 μm and then stirring it 5 to 7 times in 100% ethanol at room temperature.

[0292] The above drying step includes a step of placing the product in a vacuum oven and vacuum drying it at room temperature. However, the present invention is not limited thereto, and any commonly known drying method can be used without any particular restrictions.

[0293]

[0294] Meanwhile, the present invention can introduce a cell adhesion substance to the particle surface through chemical bonding. For example, the method can include a step of modifying the particle with a solution containing at least one selected from the group consisting of gelatin, collagen, fibronectin, chitosan, poly L-lysine, vitronectin, peptides including RGD, lignin, cationic dextran, dihydroxyphenylalanine (DOPA), dopamine, norepinephrine, epinephrine, epigallocatechin, and derivatives thereof.

[0295] The solution containing at least one selected from the group consisting of gelatin, collagen, fibronectin, chitosan, poly L-lysine, vitronectin, peptides including RGD, lignin, cationic dextran, dihydroxyphenylalanine (DOPA), dopamine, norepinephrine, epinephrine, epigallocatechin, and derivatives thereof can act as an adhesive factor for adhering cells and microcarriers, thereby increasing the adhesion of cells and microcarriers, and thus making it more suitable for mass culture of cells.

[0296] Specifically, the step of introducing a cell adhesion inducing layer may include a step of reacting by immersing the surface of the polystyrene particles in a solution containing at least one selected from the group consisting of gelatin, collagen, fibronectin, chitosan, poly L-lysine, vitronectin, peptides including RGD, lignin, cationic dextran, dihydroxyphenylalanine (DOPA), dopamine, norepinephrine, epinephrine, epigallocatechin, and derivatives thereof for 10 to 20 hours, or 15 to 20 hours, or 17 to 19 hours.

[0297]

[0298] A cell culture microcarrier manufactured according to the above method for manufacturing a cell culture microcarrier may have an epoxy content of 40 μmol / g or more and 100 μmol / g or less.

[0299] Specifically, the above cell culture microcarrier may have an epoxy content of 40 μmol / g or more, 45 μmol / g or more, 49 μmol / g or more, 49.5 μmol / g or more, 40 μmol / g or more and 100 μmol / g or less, 45 μmol / g or more and 100 μmol / g or less, 49 μmol / g or more and 100 μmol / g or less, 49.5 μmol / g or more and 100 μmol / g or less.

[0300] The above epoxy content can be calculated by adding the above cell culture microcarrier to an acidic solution, then adding an indicator solution and titrating with NaOH, using the following formula.

[0301] [Mathematical formula]

[0302] Epoxy content (μmol / g) = [V0(ml)-V(ml)]*C NaOH (mol / L)

[0303] In the above equation, V0 is the volume of NaOH added to the control group containing deionized water instead of particles, V is the amount of NaOH added to the actual sample, and C NaOH refers to the concentration of NaOH used in titration.

[0304] As the above cell culture microcarrier satisfies the above epoxy content, excellent cell adhesion can be achieved. If the epoxy content is excessively reduced, technical problems such as reduced particle dispersion and reduced introduction efficiency of surface modification materials may arise.

[0305]

[0306] According to another embodiment of the invention, a cell culture composition comprising cells and a cell culture microcarrier of the above embodiment may be provided. The contents of the cell culture microcarrier include all of the contents described above in the above embodiment.

[0307] The above cells are not particularly limited to adherent animal cells, but may be, for example, fibroblasts, epithelial cells, osteoblasts, chondrocytes, hepatocytes, human-derived umbilical cord blood cells, human bone marrow-derived mesenchymal stem cells, CHO (Chinese hamster ovary) cells, kidney cells (HEK293, BHK21, MDCK, vero cells, etc.), or a mixture of two or more thereof.

[0308]

[0309] In addition, the density difference between the cell culture microcarrier and the cell is 0.20 g / cm 3 It may be less than 0.20 g / cm. The density difference between the cell culture microcarrier and the cell is 0.20 g / cm. 3By satisfying the following, when separating and recovering microcarriers and cells after cell culture, cells and microcarriers can be easily separated through the difference in sedimentation rate due to gravity, and at the same time, the problem of microcarriers floating only on the surface of the culture medium in the early stage of culture, making it difficult for cells to attach, can be prevented.

[0310]

[0311] The above cell culture composition may further include a medium solution. The medium solution may include various additives to sufficiently satisfy environmental conditions such as pH, temperature, and osmotic pressure, as well as nutrients similar to those of a living organism based on body fluids such as plasma or lymph. Various materials widely known in the art of cell culture may be used without limitation.

[0312] For example, the cell culture microcarrier of the above embodiment has a density lower than that of the medium solution, and is injected into the medium solution and floats within the medium solution under stirring conditions. Thereafter, as the number of cells attached to the surface of the low-density microcarrier increases, the density of the cell-attached microcarrier (hereinafter referred to as a "microcarrier-cell complex") gradually increases, and the microcarrier gradually sinks within the medium solution.

[0313] Accordingly, the cultured cells can be easily obtained by separating the cells from the microcarrier-cell complex by centrifuging the microcarrier (microcarrier-cell complex) to which the cells are attached after adding a cell detachment enzyme.

[0314]

[0315] According to the present invention, a cell culture microcarrier having high surface hydrophilicity and enhanced cell adhesion, and improved dispersibility in a cell culture reactor or medium, a method for producing a cell culture microcarrier, and a cell culture method using the same can be provided.

[0316] Figure 1 is a surface shape SEM image of the cell culture microcarrier of Example 1.

[0317] The invention is described in more detail in the following examples. However, the following examples are merely illustrative of the present invention, and the scope of the present invention is not limited by the following examples.

[0318] Example 1

[0319] Polyvinyl alcohol (molecular weight 85-124K, 87-89% hydrolysis) was dissolved in distilled water at 2% to prepare an aqueous dispersion, which was then stirred at room temperature for 20 minutes.

[0320] A monomer composition was prepared by adding 25 g of a mixture in which the monomers styrene, glycidyl methacrylate, and t-butyl styrene and the crosslinking agent divinylbenzene were sufficiently dissolved in a weight ratio of 0.05:0.05:0.9:1 to 2 wt% of V-65 initiator (initiator input amount: based on the total of monomers and crosslinking agent) and stirring for an additional 5 minutes.

[0321] 600 g of an aqueous dispersion was added to a 1 L reactor, and the monomer composition was added thereto. A shear force was applied to the aqueous dispersion and the monomer composition at a speed of 400 rpm at room temperature to disperse the monomer composition in the aqueous dispersion in the form of fine droplets and homogenize the dispersion.

[0322] The homogenized mixture was stirred at a stirring speed of 400 rpm and reacted at 85°C for 6 hours under nitrogen purging to produce polystyrene particles. The particles were washed twice with distilled water at 60°C and five times with ethanol, and then dried in an oven at 80°C to recover the particles. The recovered polystyrene particles were used as microcarriers for cell culture.

[0323] The properties of the above polystyrene particles are as follows.

[0324]

[0325] Average diameter: 178 ㎛ (based on D50 measured using PSA equipment)

[0326] Apparent density: 0.99 ~ 1.04 g / cm 3

[0327]

[0328] Example 2

[0329] A cell culture microcarrier was manufactured in the same manner as in Example 1, except that the weight ratio of the monomers styrene, glycidyl methacrylate, and t-butyl styrene and the crosslinking agent divinylbenzene was adjusted to 0.07:0.03:0.9:1.

[0330]

[0331] Example 3

[0332] A cell culture microcarrier was manufactured in the same manner as in Example 1, except that the weight ratio of the monomers styrene, glycidyl methacrylate, and t-butyl styrene and the crosslinking agent divinylbenzene was adjusted to 0.085:0.015:0.9:1.

[0333]

[0334] Example 4

[0335] In the above Example 1, the monomers styrene, glycidyl methacrylate, t-butylstyrene, and acetoxystyrene (density: 1.06 g / cm 3 ) and the crosslinking agent divinylbenzene were adjusted to be 0.06:0.02:0.91:0.01:1, and a cell culture microcarrier was manufactured in the same manner as in Example 1.

[0336]

[0337] Example 5

[0338] In the above Example 1, the monomers styrene, glycidyl methacrylate, t-butylstyrene, and acetoxystyrene (density: 1.06 g / cm 3) and the crosslinking agent divinylbenzene were adjusted to be 0.05:0.03:0.90:0.02:1, and a cell culture microcarrier was manufactured in the same manner as in Example 1.

[0339]

[0340] Example 6

[0341] In the above Example 1, instead of the monomer glycidyl methacrylate, 2-(2-oxiranylmethoxy)ethyl methacrylate (density: 1.1 g / cm 3 A cell culture microcarrier was manufactured in the same manner as in Example 1, except that 2-(2-Oxiranylmethoxy)ethyl methacrylate) was used.

[0342]

[0343] Example 7

[0344] In the above Example 1, without adding the monomer styrene, 4-vinylphenyl glycidyl ether was used instead of glycidyl methacrylate, and t-butylstyrene, 4-vinylphenyl glycidyl ether (density: 1.1 g / cm 3 A cell culture microcarrier was manufactured in the same manner as in Example 1, except that the weight ratio of the crosslinking agent (4-vinylphenylglycidyl ether) and divinylbenzene was adjusted to 0.9:0.1:1.

[0345]

[0346] Example 8

[0347] A cell culture microcarrier was manufactured in the same manner as in Example 1, except that the weight ratio of the monomers styrene, glycidyl methacrylate, and t-butyl styrene and the crosslinking agent divinylbenzene was adjusted to 0.1:0.1:0.8:1.

[0348]

[0349] Example 9

[0350] A cell culture microcarrier was manufactured in the same manner as in Example 1, except that the weight ratio of the monomers styrene, glycidyl methacrylate, and t-butyl styrene and the crosslinking agent divinylbenzene was adjusted to 0.15:0.05:0.8:1.

[0351]

[0352] Example 10

[0353] A cell culture microcarrier was manufactured in the same manner as in Example 1, except that the weight ratio of the monomers styrene, glycidyl methacrylate, and t-butyl styrene and the crosslinking agent divinylbenzene was adjusted to 0.075:0.075:0.85:1.

[0354]

[0355] Example 11

[0356] A cell culture microcarrier was manufactured in the same manner as in Example 1, except that the weight ratio of the monomers styrene, glycidyl methacrylate, and t-butyl styrene and the crosslinking agent divinylbenzene was adjusted to 0.1:0.05:0.85:1.

[0357]

[0358] Example 12

[0359] A cell culture microcarrier was manufactured in the same manner as in Example 1, except that the weight ratio of the monomers styrene, glycidyl methacrylate, and t-butyl styrene and the crosslinking agent divinylbenzene was adjusted to 0.02:0.08:0.9:1.

[0360]

[0361] Example 13

[0362] A cell culture microcarrier was manufactured in the same manner as in Example 1, except that the weight ratio of the monomers styrene, glycidyl methacrylate, and t-butyl styrene and the crosslinking agent divinylbenzene was adjusted to 0.9:0.1:0:1.

[0363]

[0364] Comparative Example 1

[0365] A cell culture microcarrier was manufactured in the same manner as in Example 1, except that the weight ratio of the monomers styrene, glycidyl methacrylate, and t-butyl styrene and the crosslinking agent divinylbenzene was adjusted to 0.1:0:0.9:1.

[0366]

[0367] <Experimental Example: Measurement of Physical Properties of Microcarriers for Cell Culture>

[0368] The physical properties of the cell culture microcarriers obtained in the above examples and comparative examples were measured by the following methods, and the results are shown in Tables 1 and 2.

[0369]

[0370] Experiment 1. Average particle size (unit: ㎛)

[0371] For the cell culture microcarriers obtained in the above examples and comparative examples, they were dispersed in ethanol at a level of 10 wt%, and then the particle diameter D50 (particle size value corresponding to the cumulative distribution percentage reaching 50%) was measured using PSA (Particle size analysis) equipment.

[0372]

[0373] Experiment 2. Analysis of epoxy content

[0374] 0.1 g of the cell culture microcarriers manufactured in the above examples and comparative examples were dispersed in a HCl / acetone (volume ratio = 1:80) solution and then sonicated for 4 minutes.

[0375] After adding 2 drops of indicator solution (0.1 wt% cresol red + 0.1 wt% thymol blue, volume ratio 1:3, pH 7, 0.01 M NaOH), titration was performed with 0.1 M NaOH.

[0376] The epoxy content (μmol / g) was calculated by the following formula.

[0377] [Mathematical formula]

[0378] Epoxy content (μmol / g) = [V0(ml)-V(ml)]*C NaOH (mol / L)

[0379] In the above equation, V0 is the volume of NaOH added to the control group containing 0.1 g of DIW instead of particles, V is the amount of NaOH added to the actual sample, and C NaOH refers to the concentration of NaOH used in titration.

[0380]

[0381] Experiment 3. Particle dispersion in the incubator

[0382] 1 g of the above cell culture micro carrier was dispersed in 4 ml of 1x PBS, 10 mg of GRGDSK peptide was added, and the mixture was reacted for 18 hours. Then, the mixture was washed three times in 1x PBS to produce a cell culture micro carrier with a peptide attached thereto.

[0383] Before culturing, the above cell culture micro carriers were pre-dispersed in a 20 mL glass vial with the medium and wetting was performed for 10 to 18 hours. After the pre-dispersed cell culture micro carriers were filtered through a cell strainer, they were placed in a 100 mL 3D bioreactor with 60 mL of medium, and mesenchymal stem cells (density: 1.05 g / cm 3 ) was filled with a culture medium containing the culture medium and cultured for 24 hours. (Culture conditions: 37 ℃, 5% CO2 incubator, bioreactor 25 rpm stirring operation)

[0384] After 24 hours of culture, the particles that were not dispersed inside the incubator but were floating on the upper interface of the medium were filtered out and removed, the particles dispersed inside the incubator were thoroughly washed with DPBS or water, dried, and weighed. The percentage ratio of the weight of the dispersed particles to the total weight was calculated to calculate the degree of dispersion, and the evaluation was performed under the following criteria.

[0385] Best: Dispersion ≥90% and ≤100%

[0386] Top: Dispersion 80% or more and 90% or less

[0387] Medium: Dispersion 60% or more and less than 80%

[0388] Ha: Dispersion less than 60%

[0389]

[0390] Experiment 4. Initial cell adhesion

[0391] 1 g of the above cell culture micro carrier was dispersed in 4 ml of 1x PBS, 10 mg of GRGDSK peptide was added, and the mixture was reacted for 18 hours. Then, the mixture was washed three times in 1x PBS to produce a cell culture micro carrier with a peptide attached thereto.

[0392] Before culturing, 1 g of the above cell culture micro carriers required for cell culture were pre-dispersed in a 20 mL glass vial with medium and wetting was performed for 10 to 18 hours. After filtering the pre-dispersed cell culture micro carriers through a cell strainer, they were placed in a 100 mL 3D bioreactor with 60 mL of medium, and mesenchymal stem cells (density: 1.05 g / cm) were cultured. 3 ) was filled with a culture medium containing 1,800,000 cells and cultured for 24 hours. (Culture conditions: 37 ℃, 5% CO2 incubator, bioreactor 25 rpm stirring operation)

[0393] After 24 hours of incubation, 1 mL of the particle dispersion in the bioreactor vessel was recovered. Using a Nucleocounter NC-200 (Chemometec), the percentage of cells attached to the cell culture microcarriers relative to the total number of cells was calculated and evaluated according to the following criteria.

[0394] Best: The percentage of cells attached to the microcarriers relative to the total number of cells is 93% or more and 100% or less.

[0395] Top: Percentage of cells attached to microcarriers compared to total cell count: 85% or more and less than 93%

[0396] Medium: The percentage of cells attached to the microcarriers compared to the total number of cells is 60% or more and less than 85%.

[0397] Ha: Percentage of cells attached to microcarriers compared to total cell number less than 60%

[0398]

[0399] ClassificationAverage particle size (㎛, D50)Epoxy content (μmol / g)Dispersity (%)Initial cell adhesion (%)Example 117897.8BestBestExample 215575.1BestBestExample 316249.7SupportiveExample 418451.1SupportiveExample 518972.9SupportiveExample 619077.5BestSupportiveExample 721049.4BestBestExample 817249.3SupportiveMediumExample 918019.3SupportiveMediumExample 1016899.3SupportiveMediumExample 1117779.7SupportiveMediumExample 1217259.8SupportiveExample 13182170.1SupportiveMediumComparativeExample 11630HaHa

[0400] As shown in Table 1 above, it was confirmed that the cell culture microcarrier of the example exhibited excellent cell adhesion as the particle dispersion was improved under cell culture conditions and the cell attachment ligand peptide reaction was possible.

[0401] In the case of Comparative Example 1, it was confirmed that the dispersion and initial cell adhesion were poor compared to the examples.

[0402]

[0403] Experiment 5. Apparent density (unit: g / cm) 3 )

[0404] For the cell culture microcarriers manufactured in the above examples and comparative examples, under room temperature (25 ℃) and atmospheric pressure (1 atm), the density was 0.99 g / cm 3 Distilled water, or having a density of 1.01 g / cm 3 The apparent density was evaluated by adding each of the particles to the cell culture medium and checking whether the particles floated or settled. The cell culture medium was prepared with 94.9 wt% Advanced MEM, 5 wt% fetal bovine serum, and 1 wt% gentamicin.

[0405] For the cell culture microcarriers manufactured in the above examples and comparative examples, the density was 0.985 g / cm under room temperature (25 ℃) and atmospheric pressure (1 atm). 3 , 0.99 g / cm 3 , 0.997 g / cm 3 In ethanol aqueous solution and density 1.02 g / cm 3 , 1.04 g / cm 3 The apparent density was evaluated under the following criteria by adding each to an aqueous glycerol solution and checking whether the particles floated or settled.

[0406] 1) 0.985 g / cm 3 < d < 0.99 g / cm 3 (0.985 g / cm 3 Exceeding 0.99 g / cm 3 under)

[0407] Density is 0.985 g / cm 3 It precipitates in an ethanol solution and has a density of 0.99 g / cm. 3 Floating in aqueous ethanol solution

[0408] 2) 0.99 g / cm 3 < 1.02 g / cm 3(0.99 g / cm 3 Exceeding 1.02 g / cm 3 less than )

[0409] Density is 0.99 g / cm 3 It precipitates in an ethanol solution and has a density of 1.02 g / cm. 3 Suspended in aqueous glycerol solution

[0410] 3) 0.99 g / cm 3 < 1.04 g / cm 3 (0.99 g / cm 3 Exceeding 1.04 g / cm 3 less than )

[0411] Density is 0.99 g / cm 3 It precipitates in an ethanol solution and has a density of 1.04 g / cm. 3 Suspended in aqueous glycerol solution

[0412] 4) 0.99 g / cm 3 < d < 1.01 g / cm 3 (0.99 g / cm 3 Exceeding 1.01 g / cm 3 under)

[0413] Density is 0.99 g / cm 3 It settles in distilled water and has a density of 1.01 g / cm 3 Suspended in human cell culture medium

[0414] 5) d > 1.04 g / cm 3 (1.04 g / cm 3 over)

[0415] Density is 1.04 g / cm 3 Sedimentation in aqueous glycerol solution

[0416]

[0417] Experiment 6. Separation by density difference

[0418] Regarding the cell culture microcarriers manufactured in the above examples and comparative examples, whether separation of cells and the cell culture microcarriers by density difference was possible under room temperature (25°C) and atmospheric pressure (1 atm) conditions was evaluated by centrifuging a mixture of cell culture medium and microcarriers as follows.

[0419] O: No particles settled at the bottom after centrifugation.

[0420] X: There are particles that have settled at the bottom after centrifugation.

[0421]

[0422] Experiment 7. Surface shape observation

[0423] For the cell culture microcarrier manufactured in Example 1 above, the surface shape was observed using SEM (JEOL JSM7610F) equipment, and each of these is shown in Fig. 1.

[0424]

[0425] Apparent density (g / cm) 3 ) Density difference separation possible or notExample 10.99~1.04OExample 20.99~1.04OExample 30.99~1.04OExample 40.99~1.04OExample 50.99~1.04OExample 60.99~1.04OExample 70.99 - 1.04OExample 8Exceeding 1.04XExample 9Exceeding 1.04XExample 10Exceeding 1.04XExample 11Exceeding 1.04XExample 12Exceeding 1.04XExample 13Exceeding 1.04XComparative example Less than 10.99O

[0426] As shown in Table 2 above, the cell culture microcarrier of Example 1-7 has a density of 1.04 g / cm 3 It is suitable for cell culture at low densities below, and it was confirmed that separation of microcarriers by density difference is possible under cell culture conditions.

[0427] On the other hand, in the case of Examples 8 to 13, it was confirmed that separation of microcarriers by density difference was impossible because the density of the particles exceeded the density of the cell culture medium and did not have low-density characteristics.

[0428] In the case of Comparative Example 1, it was confirmed that the apparent density was too low, which could cause a problem in which the microcarriers floated only on the surface of the culture medium in the early stage of culture, making it difficult for cells to attach.

Claims

1. A microcarrier for cell culture, comprising: a polystyrene particle comprising a compound represented by the following chemical formula 1 as a monomer compound; [Chemical Formula 1] In the above chemical formula 1, L0 is an arylene group having 6 or more carbon atoms or -(C=O)-, L1 and L2 are each independently an alkylene group having 1 or more carbon atoms, R1 is a reactive functional group capable of ring-opening reaction, R 10 is hydrogen or an alkyl group having 1 or more carbon atoms, n is an integer greater than or equal to 0.

2. In paragraph 1, The above polystyrene particles include a reaction product of a compound represented by the above chemical formula 1 and an ethylenically unsaturated crosslinking agent, A cell culture microcarrier comprising 1 part by weight or more and 15 parts by weight or less of a compound represented by the chemical formula 1 based on 100 parts by weight of the ethylenically unsaturated crosslinking agent.

3. In paragraph 1, A cell culture microcarrier comprising a compound represented by the above chemical formula 1, wherein the compound represented by the following chemical formula 1-1 to the compound represented by the following chemical formula 1-3: [Chemical Formula 1-1] In the above chemical formula 1-1, R 11 is hydrogen or an alkyl group having 1 or more carbon atoms, [Chemical Formula 1-2] In the above chemical formula 1-2, R 12 is hydrogen or an alkyl group having 1 or more carbon atoms, [Chemical Formula 1-3] In the above chemical formula 1-4, R 13 is hydrogen or an alkyl group having 1 or more carbon atoms.

4. In paragraph 1, The above polystyrene particles are, A cell culture microcarrier comprising a compound represented by the following chemical formula 2 as a monomer compound: [Chemical Formula 2] In the above chemical formula 2, R2 to R6 are each independently hydrogen or an alkyl group having 1 or more carbon atoms, At least one of the above R2 to R6 It is an alkyl group with 1 or more carbon atoms.

5. In paragraph 4, A cell culture microcarrier comprising at least one compound selected from the group consisting of a compound represented by the chemical formula 2 below to a compound represented by the chemical formula 2-3 below, wherein the compound represented by the chemical formula 2 above is: [Chemical Formula 2-1] [Chemical Formula 2-2] [Chemical Formula 2-3] In the above chemical formulas 2-1 to 2-3, R 21 Inland R 26 are each independently an alkyl group having 1 or more carbon atoms.

6. In paragraph 4, The compound represented by the above chemical formula 2 has a density of 0.92 g / cm 3 Below, micro carriers for cell culture.

7. In paragraph 4, The above polystyrene particles include a reaction product of a compound represented by the above chemical formula 1, a styrene monomer mixture, and an ethylenically unsaturated crosslinking agent, A cell culture microcarrier comprising 80 parts by weight or more and 150 parts by weight or less of a compound represented by the chemical formula 2 based on 100 parts by weight of the ethylenically unsaturated crosslinking agent.

8. In paragraph 4, The above polystyrene particles include a reaction product of a compound represented by the above chemical formula 1, a styrene monomer mixture, and an ethylenically unsaturated crosslinking agent, A cell culture microcarrier comprising 90 parts by weight or more and 100 parts by weight or less of a compound represented by the chemical formula 2 based on 100 parts by weight of the styrene-based monomer mixture.

9. In paragraph 4, The above polystyrene particles include a reaction product of a compound represented by the above chemical formula 1, a styrene monomer mixture, and an ethylenically unsaturated crosslinking agent, A cell culture microcarrier comprising 1 part by weight or more and 15 parts by weight or less of a compound represented by the chemical formula 1 based on 100 parts by weight of a compound represented by the chemical formula 2.

10. In paragraph 1, A microcarrier for cell culture comprising a compound represented by the following chemical formula 3 as a monomer compound: [Chemical Formula 3] In the above chemical formula 3, L 30 is -O(C=O)- or -(C=O)O-, R 30 is a direct bond or an alkylene group having 1 or more carbon atoms, R 31 is an alkyl group having 1 or more carbon atoms.

11. In paragraph 10, The above polystyrene particles include a reaction product of a compound represented by the above chemical formula 1, a styrene monomer mixture, and an ethylenically unsaturated crosslinking agent, A cell culture microcarrier comprising 0.1 to 5 parts by weight of a compound represented by the chemical formula 3 based on 100 parts by weight of the ethylenically unsaturated crosslinking agent.

12. In paragraph 10, The above polystyrene particles include a reaction product of a compound represented by the above chemical formula 1, a styrene monomer mixture, and an ethylenically unsaturated crosslinking agent, A cell culture microcarrier comprising 0.1 to 5 parts by weight of a compound represented by the chemical formula 3 based on 100 parts by weight of the styrene-based monomer mixture.

13. In paragraph 10, The above polystyrene particles include a reaction product of a compound represented by the above chemical formula 1, a styrene monomer mixture, and an ethylenically unsaturated crosslinking agent, A cell culture microcarrier comprising 110 to 500 parts by weight of a compound represented by the chemical formula 1 based on 100 parts by weight of a compound represented by the chemical formula 3.

14. In paragraph 1, The above cell culture microcarriers are A cell culture microcarrier comprising a cell adhesion inducing layer formed on the polystyrene particles.

15. In paragraph 1, The apparent density of the microcarrier for cell culture is 0.99 g / cm 3 Exceeding 1.04 g / cm 3 Micro carriers for cell culture, less than 100 μm.

16. In paragraph 1, A cell culture microcarrier having a D50 particle diameter of 100 ㎛ to 300 ㎛.

17. A method for manufacturing a microcarrier for cell culture, comprising: a step of polymerizing and recovering polystyrene particles from a monomer mixture containing a compound represented by the following chemical formula 1; [Chemical Formula 1] In the above chemical formula 1, L0 is an arylene group having 6 or more carbon atoms or -(C=O)-, L1 and L2 are each independently an alkylene group having 1 or more carbon atoms, R1 is a reactive functional group capable of ring-opening reaction, R 10 is hydrogen or an alkyl group having 1 or more carbon atoms, n is an integer greater than or equal to 0.

18. In paragraph 17, A method for producing a microcarrier for cell culture, wherein the monomer mixture comprises a compound represented by the following chemical formula 2: [Chemical Formula 2] In the above chemical formula 2, R2 to R6 are each independently hydrogen or an alkyl group having 1 or more carbon atoms, At least one of the above R2 to R6 It is an alkyl group with 1 or more carbon atoms.

19. In paragraph 17, A method for producing a microcarrier for cell culture, wherein the monomer mixture comprises a compound represented by the following chemical formula 3: [Chemical Formula 3] In the above chemical formula 3, L 30 is -O(C=O)- or -(C=O)O-, R 30 is a direct bond or an alkylene group having 1 or more carbon atoms, R 31 is an alkyl group having 1 or more carbon atoms.

20. In paragraph 17, A method for manufacturing a cell culture microcarrier, further comprising the step of applying a cell adhesion inducing layer on the polystyrene particles.

21. A cell culture composition comprising cells and a cell culture microcarrier of claim 1.

22. In paragraph 21, A cell culture composition comprising at least one compound selected from the group consisting of fibroblasts, epithelial cells, osteoblasts, chondrocytes, hepatocytes, umbilical cord blood cells, mesenchymal stem cells, CHO cells, and kidney cells.

23. In paragraph 21, The difference in apparent density between the above cell culture microcarrier and the above cells is 0.20 g / cm 3 Below, cell culture composition.

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