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

Abstract

The present disclosure relates to a microcarrier for cell culture comprising polystyrene-based particles, a method for preparing the microcarrier for cell culture, and a cell culture composition using the same.

Description

Technical Field

[0001] This application claims the benefit of priority based on Korean Patent Application No. 10-2024-0067364, filed on May 23, 2024, and Korean Patent Application No. 10-2025-0067201, filed on May 23, 2025, the disclosure of which is incorporated herein by reference in its entirety.

[0002] This disclosure relates to microcarriers for cell culture, methods for preparing microcarriers for cell culture, and cell culture compositions using the same. Background Technology

[0003] With the development of the biopharmaceutical and regenerative medicine fields, the demand for large-scale cell culture technologies that can effectively produce cells, tissues, microorganisms, etc. is growing.

[0004] Adherent cells were cultured using microcarriers in a 3D bioreactor. Cells, culture medium, and microcarriers were placed in the bioreactor, and the medium was agitated to bring the cells into contact with the microcarriers, allowing the cells to adhere to the surface of the microcarriers and be cultured. Compared to 2D culture, this method is suitable for large-scale cell culture because the microcarriers used here provide a high surface area to volume ratio for cell attachment and growth.

[0005] Currently available commercially available microcarriers have a concentration of approximately 1.1 g / cm³. 3 Up to 1.3 g / cm 3 The density, and the cells have a density of approximately 1.2 g / cm³. 3 The density of cells is important. In this case, it is advantageous for cells to attach during the initial stage of culture in the bioreactor, but centrifugation is difficult when separating and recovering the cells after culture, and filtration methods based on microcarriers and cell size should be used. However, in this case, there are problems such as filter clogging or the process taking a long time, and the possibility of physical damage and contamination of cells, and potential cell loss.

[0006] To address these issues, a solution with a density below 1.0 g / cm³ has been developed. 3 Or higher than 1.3 g / cm 3 Materials have been used to fabricate microcarriers. However, the achievable density range is limited, and it is difficult to obtain sufficient yields of perfectly spherical microcarriers without damage or breakage. Summary of the Invention

[0007] Technical issues

[0008] This disclosure relates to microcarriers for cell culture, wherein the density of the microcarriers is controlled such that they can be separated from cells by density differences, while exhibiting improved dispersibility and enhanced cell adhesion in cell culture reactors or culture media.

[0009] This disclosure also relates to methods for preparing microcarriers for cell culture.

[0010] This disclosure also relates to cell culture compositions using the aforementioned cell culture microcarriers.

[0011] Technical solution

[0012] According to one embodiment of this disclosure, a microcarrier for cell culture is provided, comprising polystyrene-based particles, said polystyrene-based particles comprising a monomeric compound represented by the following chemical formula 1:

[0013] [Chemical Formula 1]

[0014]

[0015] In chemical formula 1, L0 is an arylene group or -(C=O)- with 6 or more carbon atoms, L1 and L2 are each independently an alkylene group with 1 or more carbon atoms, R1 is a reactive functional group capable of undergoing ring-opening reactions, and R... 10 It is hydrogen or an alkyl group having one or more carbon atoms, and n is an integer of 0 or greater.

[0016] According to another embodiment of this disclosure, a method for preparing microcarriers for cell culture is provided, comprising the steps of polymerizing polystyrene-based particles from a monomer mixture comprising a compound represented by the following chemical formula 1 and recovering the polystyrene-based particles:

[0017] [Chemical Formula 1]

[0018]

[0019] In chemical formula 1, L0 is an arylene group or -(C=O)- with 6 or more carbon atoms, L1 and L2 are each independently an alkylene group with 1 or more carbon atoms, R1 is a reactive functional group capable of undergoing ring-opening reactions, and R... 10 It is hydrogen or an alkyl group having one or more carbon atoms, and n is an integer of 0 or greater.

[0020] According to another embodiment of this disclosure, a cell culture composition is provided, comprising cells and the above-mentioned cell culture microcarriers.

[0021] Now, a cell culture microcarrier, a method for preparing the cell culture microcarrier, and a cell culture composition using the microcarrier will be described in more detail according to a specific embodiment of the present disclosure.

[0022] Unless otherwise specified herein, technical terms are used only to refer to a particular implementation and are not intended to limit the scope of this disclosure.

[0023] Unless the context clearly indicates otherwise, the singular form used in this document includes the plural form.

[0024] As used herein, the terms “comprising” or “including” specify the presence of the stated features, regions, integers, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and / or groups.

[0025] Furthermore, terms including ordinal numbers such as "first," "second," etc., are used only for the purpose of distinguishing one component from another and are not limited by ordinal numbers. For example, without departing from the scope of this disclosure, the first component may be referred to as the second component, or similarly, the second component may be referred to as the first component.

[0026] As used herein, the alkyl group can be straight-chain or branched; although there is no particular limitation on the number of carbon atoms, it is preferably 1 to 10. In another embodiment, the alkyl group contains 1 to 6 carbon atoms. Specific examples include, but are not limited to, methyl, ethyl, propyl, n-propyl, isopropyl, butyl, n-butyl, isobutyl, tert-butyl, sec-butyl, 1-methylbutyl, 1-ethylbutyl, 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, cyclohexylmethyl, octyl, n-octyl, tert-octyl, 1-methylheptyl, 2-ethylhexyl, 2-propylpentyl, n-nonyl, 2,2-dimethylheptyl, 1-ethylpropyl, 1,1-dimethylpropyl, isohexyl, 2-methylpentyl, 4-methylhexyl, and 5-methylhexyl.

[0027] As used herein, the cycloalkyl group is a monovalent substituent derived from cycloalkanes and can be monocyclic or polycyclic; although not particularly limited, it preferably has 3 to 20 carbon atoms. In another embodiment, the cycloalkyl group has 3 to 10 carbon atoms. Specific examples 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, and bicyclic [2.2.1]heptyl. The cycloalkyl group can be substituted or unsubstituted, and examples of substituents are as described above.

[0028] As used herein, heterocyclic alkyl means a cycloalkyl group containing at least one heteroatom other than carbon; the specific heteroatom may include one or more selected from O, N, Se and S.

[0029] This disclosure will be described in more detail.

[0030] According to one embodiment of this disclosure, a microcarrier for cell culture can be provided, comprising polystyrene-based particles, said polystyrene-based particles comprising a monomeric compound represented by chemical formula 1.

[0031] Through experiments, the inventors discovered that, in the case of the cell culture microcarrier of this embodiment, the inclusion of a compound represented by Chemical Formula 1 as a polystyrene-based monomer allows for control of the final density of the cell culture microcarrier, enabling separation from cells based on density differences, while improving dispersibility in the cell culture reactor or culture medium and enhancing cell adhesion, thus completing the present disclosure.

[0032] Conventional cell culture microcarriers control particle density by encapsulating non-reactive low-density oil inside the particles. However, a technical problem is that the encapsulated low-density oil leaks out when the particles are physically damaged.

[0033] Therefore, the inventors have determined that by including a compound represented by Chemical Formula 1 as a monomer in a polystyrene-based polymer used as a microcarrier for cell culture, the particle density can be adjusted to enable separation from cells based on density differences, while simultaneously improving dispersibility in a bioreactor or culture medium.

[0034] In addition, conventional cell culture microcarriers form a coating on the surface of polystyrene particles to enhance cell adhesion, but a technical problem exists that when the microcarriers are stirred in the culture medium, part of the coating may detach.

[0035] Therefore, the inventors have demonstrated that by including a compound represented by Formula 1 as a monomer in a polystyrene-based polymer used as a microcarrier for cell culture, reactive sites can be provided that allow cell adhesion ligands to be chemically immobilized on the surface of the microcarrier without the need for additional coating, thereby improving cell adhesion.

[0036] Specifically, in one embodiment of the microcarrier for cell culture, the microcarrier may comprise polystyrene-based particles, said polystyrene-based particles comprising a monomeric compound represented by the following chemical formula 1:

[0037] [Chemical Formula 1]

[0038]

[0039] In chemical formula 1, L0 is an arylene group or -(C=O)- with 6 or more carbon atoms, L1 and L2 are each independently an alkylene group with 1 or more carbon atoms, R1 is a reactive functional group capable of undergoing ring-opening reactions, and R... 10 It is hydrogen or an alkyl group having one or more carbon atoms, and n is an integer of 0 or greater.

[0040] Since the compound represented by Formula 1 contains reactive functional groups capable of ring-opening reactions, it can provide reactive sites that allow cell adhesion ligands to be chemically immobilized on the surface of microcarriers without the need for additional coating, thereby improving cell adhesion.

[0041] Reactive functional groups capable of ring-opening reactions are those that can participate in ring-opening processes, such as hydrolysis ring-opening.

[0042] Specifically, reactive functional groups capable of undergoing ring-opening reactions may include heterocyclic alkyl groups.

[0043] Heterocyclic alkyl groups can be cycloalkyl groups containing one or more non-carbon atoms, i.e., heteroatoms. Heteroatoms can include at least one atom selected from O, N, Se, and S, preferably O.

[0044] Heterocyclic alkyl groups are not particularly limited, but for example, they may include epoxy groups.

[0045] Specifically, the polystyrene-based particles may contain the reaction product of a compound represented by Formula 1 and an olefinic unsaturated crosslinking agent, and based on 100 parts by weight of the olefinic unsaturated crosslinking agent, may contain 1 part by weight or more and 15 parts by weight or less of the compound represented by Formula 1.

[0046] More specifically, the polystyrene-based particles may comprise the reaction product of a compound represented by Formula 1 and an olefinically unsaturated crosslinking agent, and based on 100 parts by weight of the olefinically unsaturated crosslinking agent, may comprise 1 or more, 1.5 or more, 15 or less, 10 or less, 5 or less, or 1 or more and 15 or less, 1.5 or more and 15 or less, 1 or more and 10 or less, 1.5 or more and 10 or less, 1 or more and 5 or less, or 1.5 or more and 5 or less of the compound represented by Formula 1.

[0047] When the compound represented by Formula 1 is included in too small an amount relative to 100 parts by weight of an olefinic unsaturated crosslinking agent, it provides an insufficient number of reactive sites capable of immobilizing cell adhesion ligands, potentially leading to poor cell adhesion. Conversely, when the compound represented by Formula 1 is included in too large an amount, the relative proportion of the high-density compound represented by Formula 1 increases, causing the particle density to become higher than the density of the cell culture medium, resulting in the loss of low-density properties.

[0048] Specifically, the compound represented by chemical formula 1 may include at least one compound selected from the compounds represented by chemical formulas 1-1 to 1-3 below.

[0049] [Chemical Formula 1-1]

[0050]

[0051] In chemical formula 1-1,

[0052] R 11 It is hydrogen or an alkyl group having one or more carbon atoms.

[0053] [Chemical Formula 1-2]

[0054]

[0055] In chemical formulas 1-2,

[0056] R 12 It is hydrogen or an alkyl group having at least one carbon atom.

[0057] [Chemical Formulas 1-3]

[0058]

[0059] Among them, in chemical formulas 1-4,

[0060] R 13 It is hydrogen or an alkyl group having one or more carbon atoms.

[0061] By including at least one compound selected from those represented by chemical formulas 1-1 to 1-3 as a monomer of a polystyrene-based polymer, the reactive sites that can be chemically bonded to the surface of a cell culture microcarrier without additional coating can be provided by adjusting the weight ratio between the styrene-based monomer and the compound represented by chemical formulas 1-1 to 1-3, thereby improving cell adhesion.

[0062] In addition to the compounds represented by Formula 1, polystyrene-based particles may also contain compounds represented by Formula 2 as monomer compounds:

[0063] [Chemical Formula 2]

[0064]

[0065] In chemical formula 2, R2 to R6 are each independently hydrogen or an alkyl group having one or more carbon atoms, and at least one of R2 to R6 is an alkyl group having one or more carbon atoms.

[0066] By including a compound represented by the following chemical formula 2 as a monomer for polystyrene-based particles, the density of microcarriers for cell culture can be precisely controlled by adjusting the content of the compound represented by chemical formula 2, thereby enabling separation from cells based on density differences and improving dispersibility in cell culture reactors or culture media.

[0067] Specifically, the compound represented by chemical formula 2 may include at least one compound selected from the compounds represented by chemical formulas 2-1 to 2-3 below.

[0068] [Chemical Formula 2-1]

[0069]

[0070] [Chemical Formula 2-2]

[0071]

[0072] [Chemical Formula 2-3]

[0073]

[0074] In chemical formulas 2-1 to 2-3, R 21 To R 26 Each is an alkyl group having one or more carbon atoms.

[0075] By including at least one compound selected from those represented by chemical formulas 2-1 to 2-3 as a monomer for polystyrene-based particles, the density of microcarriers for cell culture can be precisely controlled by adjusting the content of the compound represented by chemical formula 2, thereby enabling separation from cells based on density differences and simultaneously improving dispersibility in cell culture reactors or culture media.

[0076] More specifically, the compound represented by chemical formula 2 may include at least one compound selected from the compounds represented by chemical formulas 2-4 to 2-6:

[0077] [Chemical Formula 2-4]

[0078]

[0079] [Chemical Formula 2-5]

[0080]

[0081] [Chemical Formula 2-6]

[0082]

[0083] Among them, in chemical formulas 2-4 to 2-6, R 21 To R 26 Each is an alkyl group having one or more carbon atoms.

[0084] Meanwhile, the density of the compound represented by chemical formula 2 can be 0.92 g / cm³. 3 Or smaller.

[0085] Specifically, the density of the compound represented by chemical formula 2 can be 0.92 g / cm³. 3 Or smaller, 0.91 g / cm 3 or smaller, 0.906 g / cm 3 Or smaller, 0.9 g / cm³ 3 or smaller, 0.89 g / cm 3 Or smaller, 0.5 g / cm 3 or larger, 0.6 g / cm 3 or larger, 0.7 g / cm 3 or larger, 0.8 g / cm 3 or larger, 0.5 g / cm 3 Or larger and 0.92 g / cm 3 or smaller, 0.6 g / cm 3 Or larger and 0.92 g / cm 3 Or smaller, 0.7 g / cm 3Or larger and 0.92 g / cm 3 or smaller, 0.8 g / cm 3 Or larger and 0.92 g / cm 3 Or smaller, 0.5 g / cm 3 Or larger and 0.91 g / cm 3 or smaller, 0.6 g / cm 3 Or larger and 0.91 g / cm 3 Or smaller, 0.7 g / cm 3 Or larger and 0.91 g / cm 3 Or smaller, 0.8 g / cm 3 Or larger and 0.91 g / cm 3 Or smaller, 0.5 g / cm 3 Or larger and 0.906 g / cm 3 or smaller, 0.6 g / cm 3 Or larger and 0.906 g / cm³ 3 Or smaller, 0.7 g / cm 3 Or larger and 0.906 g / cm³ 3 or smaller, 0.8 g / cm 3 Or larger and 0.906 g / cm³ 3 Or smaller, 0.5 g / cm 3 Or larger and 0.9 g / cm 3 or smaller, 0.6 g / cm 3 Or larger and 0.9 g / cm 3 Or smaller, 0.7 g / cm 3 Or larger and 0.9 g / cm 3 or smaller, 0.8 g / cm 3 Or larger and 0.9 g / cm 3 Or smaller, 0.5 g / cm 3 Or larger and 0.89 g / cm 3 or smaller, 0.6 g / cm 3 Or larger and 0.89 g / cm 3 Or smaller, 0.7 g / cm 3 Or larger and 0.89 g / cm 3 or smaller, 0.8 g / cm 3 Or larger and 0.89 g / cm 3 Or smaller.

[0086] Since the density of the compound represented by chemical formula 2 is 0.92 g / cm³ 3Or even smaller, so the density of microcarriers for cell culture can be precisely controlled by adjusting the content of the compound represented by chemical formula 2, enabling separation from cells based on density differences, while improving dispersibility in cell culture bioreactors or culture media.

[0087] For example, the compound represented by Formula 2 may include at least one compound selected from the following: 4-methylstyrene, trimethylstyrene, 4-vinyl-2-methyl-1-(2-methylpropyl)benzene, 1-vinyl-2-methyl-4-(1-methylethyl)benzene, 1-vinyl-2,3-dimethylbenzene, 4-(1,1-dimethylethyl)-2-vinyl-1-methylbenzene, 2-vinyl-4-methyl-1-(1-methylethyl)benzene, 1-vinyl-2-(1-methylethyl)benzene, 2-tert-butylstyrene, 4-tert-butylstyrene, and 1-vinyl-3-(1-methylethyl)benzene.

[0088] In addition to compounds represented by chemical formula 1, polystyrene-based particles may also contain compounds represented by chemical formula 3 as monomers.

[0089] [Chemical Formula 3]

[0090]

[0091] In chemical formula 3, L 30 For -O(C=O)- or -(C=O)O-, R 30 It is a direct bond or an alkylene group having one or more carbon atoms, and R 31 It is an alkyl group having one or more carbon atoms.

[0092] That is, as a monomer component, polystyrene-based particles may contain compounds represented by chemical formula 1; or compounds represented by chemical formula 1 and chemical formula 2; or compounds represented by chemical formula 1 and chemical formula 3; or compounds represented by chemical formula 1, chemical formula 2 and chemical formula 3.

[0093] By incorporating a compound represented by chemical formula 3 as a polystyrene-based monomer, the substituents of the resulting cell culture microcarriers can be hydrolyzed, thereby improving dispersibility in cell culture reactors or culture media.

[0094] For example, compounds represented by chemical formula 3 may include acetoxystyrene.

[0095] Meanwhile, the microcarriers for cell culture may contain polystyrene-based particles. Preferably, the microcarriers for cell culture may consist of polystyrene-based particles.

[0096] Specifically, the apparent density of the polystyrene-based particles can range from 0.99 g / cm³ to 1.04 g / cm³. By maintaining this low density range, cells and microcarriers can be easily separated after culture when recovering the microcarriers and cells due to the difference in sedimentation velocities under gravity.

[0097] When the density of polystyrene-based particles exceeds 1.04 g / cm³ 3 At this density, the difference in density between cells and microcarriers decreases, which may make centrifugation difficult when separating and recovering cells after culture. Conversely, when the density is less than 0.99 g / cm³, the difference becomes smaller. 3 In the initial stage, microcarriers may simply float on the surface of the culture medium, making it difficult for cells to adhere.

[0098] The cells are adherent animal cells, with no particular limitation, and may include at least one compound selected from, for example, fibroblasts, epithelial cells, osteoblasts, chondrocytes, hepatocytes, cells derived from human umbilical cord blood, mesenchymal stem cells derived from human bone marrow, CHO (Chinese hamster ovary) cells, kidney cells (HEK293, BHK21, MDCK, Vero cells, etc.), or a mixture of two or more thereof.

[0099] Furthermore, the density difference between the microcarriers used for cell culture and the cells can be 0.20 g / cm³. 3 Or smaller. Meets 0.20 g / cm³ 3 A smaller density difference allows cells and microcarriers to be easily separated when they are recovered after culture by taking advantage of the difference in their settling velocities under gravity, while also preventing the problem of cell attachment difficulties caused by microcarriers merely floating on the surface of the culture medium in the early stages of culture.

[0100] Polystyrene-based particles can contain reaction products of monomer mixtures and olefinically unsaturated crosslinking agents.

[0101] As described above, the monomer mixture may contain compounds represented by Chemical Formula 1. The monomer mixture may also contain compounds represented by Chemical Formula 1 and compounds represented by Chemical Formula 2. The monomer mixture may further contain compounds represented by Chemical Formula 1 and compounds represented by Chemical Formula 3. Furthermore, the monomer mixture may contain compounds represented by Chemical Formulas 1 to 3.

[0102] That is, the polystyrene-based particles may contain a compound represented by Formula 1, a reaction product of a mixture of styrene-based monomers and an olefinically unsaturated crosslinking agent, and the mixture of styrene-based monomers may contain a compound represented by Formula 2 or a compound represented by Formula 3.

[0103] Specifically, based on 100 parts by weight of the olefinic unsaturated crosslinking agent, the compound represented by Formula 2 may be included in an amount of 80 parts by weight or more and 150 parts by weight or less.

[0104] More specifically, based on 100 parts by weight of the olefinic unsaturated crosslinking agent, the compound represented by Formula 2 may be included in the following amounts: 80 parts by weight or more, 85 parts by weight or more, 90 parts by weight or more, 150 parts by weight or less, 125 parts by weight or less, 100 parts by weight or less, 99 parts by weight or less, 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.

[0105] By using 100 parts by weight of an olefinic unsaturated crosslinking agent, the compound represented by chemical formula 2 is contained in an amount of 80 parts by weight or more and 150 parts by weight or less. Due to the low density of the compound represented by chemical formula 2, the low density characteristics of the particles can be controlled, enabling separation from cells by density differences, and at the same time, improving dispersibility in cell culture reactors or culture media.

[0106] When the compound represented by Formula 2 is included in too small an amount relative to 100 parts by weight of the olefinic unsaturated crosslinking agent, the particle density increases, leading to the loss of low-density properties and resulting in sedimentation in the culture medium. Conversely, when it is included in excessive amounts, the particle density decreases to below 0.99 g / cm³. 3 High-speed agitation is required to disperse the particles in the culture medium, which may have an adverse effect on cell characteristics.

[0107] Furthermore, based on 100 parts by weight of a styrene-based monomer mixture, the compound represented by Formula 2 may be included in an amount of 90 parts by weight or more and 100 parts by weight or less.

[0108] Specifically, based on 100 parts by weight of the styrene-based monomer mixture, the compound represented by Formula 2 may be included in the following amounts: 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.

[0109] By including the compound represented by chemical formula 2 in the above amount relative to 100 parts by weight of a styrene-based monomer mixture, the density of microcarriers for cell culture can be precisely controlled, enabling separation from cells based on density differences, while improving dispersibility in cell culture bioreactors or culture media.

[0110] Furthermore, based on 100 parts by weight of the compound represented by Formula 2, the compound represented by Formula 1 may be included in an amount of 1 part by weight or more and 15 parts by weight or less.

[0111] Specifically, based on 100 parts by weight of the compound represented by Formula 2, the compound represented by Formula 1 may be included in the following amounts: 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.

[0112] When the compound represented by Formula 2 is included in 100 parts by weight in too small an amount, insufficient reactive sites for fixing cell adhesion ligands will be provided, which may result in poor cell adhesion; conversely, when it is included in too large an amount, the particle density may increase, leading to the technical problem of loss of low-density properties.

[0113] Furthermore, when a monomeric compound represented by Formula 3 is included, the compound represented by 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 olefinic unsaturated crosslinking agent.

[0114] More specifically, based on 100 parts by weight of the olefinic unsaturated crosslinking agent, the compound represented by Formula 3 may be included in the following amounts: 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 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 parts by weight or less, 0.5 parts by weight or more and 2 parts by weight or less, 1 part by weight or more and 2 parts by weight or less.

[0115] When a monomeric compound represented by chemical formula 3 is contained 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 an olefinic unsaturated crosslinking agent, dispersibility in a cell culture reactor or culture medium can be improved by hydrolyzing the substituents of the cell culture microcarrier.

[0116] When the olefinic unsaturated crosslinking agent, based on 100 parts by weight, is included in an amount exceeding 5 parts by weight of the compound represented by chemical formula 3, the particle density may increase, leading to the loss of low-density properties.

[0117] Furthermore, when the monomeric compound contains a compound represented by Formula 3, based on 100 parts by weight of a styrene-based monomer mixture, the compound represented by Formula 3 may be included in an amount of 0.1 parts by weight or more and 5 parts by weight or less.

[0118] Specifically, based on 100 parts by weight of a styrene-based monomer mixture, the compound represented by Formula 3 may be included in the following amounts: 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.

[0119] When the monomeric compound contains a compound represented by Formula 3, by including the compound represented by Formula 3 in an amount of 0.1 parts by weight or more and 5 parts by weight or less in a styrene-based monomer mixture based on 100 parts by weight, it is possible to maintain low-density properties while providing sufficient reactive sites for immobilization of cell adhesion ligands, thereby promoting cell adhesion.

[0120] Furthermore, when the monomeric compound contains a compound represented by Formula 3, the compound represented by 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 Formula 3.

[0121] Specifically, based on 100 parts by weight of the compound represented by Formula 3, the compound represented by Formula 1 may be included in the following amounts: 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, 200 parts by weight or less, 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, 150 parts by weight or more and 200 parts by weight or less.

[0122] When the monomer compound contains a compound represented by Formula 3, if, based on 100 parts by weight of the compound represented by Formula 3, less than 110 parts by weight of the compound represented by Formula 1 are included, the compound represented by Formula 1 may be lost before polymerization due to its relatively high water solubility and therefore may not be introduced onto the particle surface. Conversely, if it is included in an amount exceeding 500 parts by weight, the particle density may become high, thus losing the low-density properties.

[0123] In addition, based on 100 parts by weight of the styrene-based monomer mixture, the polystyrene-based particles may contain an olefinic unsaturated crosslinking agent in an amount of 60 parts by weight or more and 200 parts by weight or less.

[0124] Specifically, based on 100 parts by weight of the styrene-based monomer mixture, the polystyrene-based particles may contain an olefinically unsaturated crosslinking agent in amounts 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.

[0125] When an olefinic unsaturated crosslinking agent is included in a very small amount (less than 60 parts by weight) of 100 parts by weight of a styrene-based monomer mixture, the crosslinking density of the polystyrene-based polymer decreases, making it difficult to maintain a stable spherical shape of the particles.

[0126] Conversely, when an olefinic unsaturated crosslinking agent is included in an excessive amount of more than 200 parts by weight of a styrene-based monomer mixture based on 100 parts by weight, it is difficult to reduce the particle density to the desired level.

[0127] Examples of olefinically unsaturated crosslinking agents may include divinylbenzene.

[0128] The average diameter of polystyrene-based particles can range from 50 μm to 400 μm, or from 60 μm to 390 μm. When the average diameter of polystyrene-based particles falls within these ranges, cell adhesion and culture performance are excellent. When the average diameter is less than 50 μm, the surface area available for cell culture decreases, which may reduce culture efficiency; when it exceeds 400 μm, the interaction between adherent cells decreases and the cell density in the bioreactor decreases, which may also reduce culture efficiency.

[0129] The diameter of a polystyrene-based particle is the distance between the two points where a straight line passing through the particle's centroid meets its outermost surface, and the average diameter of a polystyrene-based particle can be determined by measuring the diameter of all polystyrene-based particles contained in a cell culture microcarrier under an optical microscope.

[0130] The polystyrene-based particles can be a group of individual particles with an average diameter of 50 μm to 400 μm or 60 μm to 390 μm, and the individual fine particles included in this group can each have an average diameter of 50 μm to 400 μm or 60 μm to 390 μm. More specifically, 95% or more, or 99% or more, of the individual fine particles included in this group can have a diameter of 50 μm to 400 μm or 60 μm to 390 μm.

[0131] In addition, polystyrene-based particles may have a percentage of perfectly spherical particles without damage or breakage, as defined by the following equation, greater than 90% and 100% or less, 92% or greater and 100% or less, 95% or greater and 100% or less, or 96% or greater and 99% or less.

[0132] [Equation]

[0133] .

[0134] For polystyrene-based granules, the percentage of perfectly spherical granules without damage or breakage, as determined by the above equation, can be obtained by counting the number of perfectly spherical granules without damage or breakage in all granules via SEM, and then calculating the percentage of these granules relative to the total number.

[0135] Therefore, cell culture microcarriers can contain multiple polystyrene-based particles, and each of these particles can be visually determined by SEM to be a perfectly spherical particle without damage or breakage.

[0136] When the proportion of perfectly spherical particles without damage or breakage, as defined by the above equation, decreases to 90% or less, the number of irregular particles with non-uniform, concave surfaces increases, and these irregular particles remain suspended in the cell culture medium, exerting physical impact on the cultured cells. This may reduce cell culture efficiency to the point where cell culture is impossible to carry out successfully.

[0137] Specifically, the D50 particle size of microcarriers for cell culture can be in the ranges of 100 μm to 300 μm, 100 μm to 250 μm, 120 μm to 250 μm, or 130 μm to 250 μm. When the average diameter of the microcarriers for cell culture falls within the aforementioned range, cell attachment and culture performance are excellent. Conversely, when the D50 particle size of the microcarriers for cell culture is less than 100 μm, the available surface area for cell culture becomes limited, which may reduce culture efficiency; when it exceeds 300 μm, the interaction between adhered cells deteriorates and the cell density in the bioreactor decreases, which may also lead to a decrease in culture efficiency.

[0138] Meanwhile, cell culture microcarriers may include a cell attachment induction layer formed on polystyrene-based particles.

[0139] The cell adhesion induction layer contains cell adhesion material that provides sites where transmembrane proteins can bind, thereby enabling adhered cells to attach, spread, and be cultured stably.

[0140] There are no particular limitations on the polymers that form the cell attachment induction layer, and they may include one or more polymers selected from the following: gelatin, collagen, fibronectin, chitosan, polylysine, fibronectin, peptides containing RGD, lignin, cationic dextran, dihydroxyphenylalanine (DOPA), dopamine, norepinephrine, epinephrine, epigallocatechin gallate and its derivatives.

[0141] The epoxy content of the cell culture microcarriers in the implementation scheme can be in the range of 40 µmol / g to 100 µmol / g.

[0142] Specifically, the epoxy content of the cell culture microcarriers in the implementation scheme can be 40 µmol / g or more, 45 µmol / g or more, 49 µmol / g or more, 49.5 µmol / g or more, or 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, or 49.5 µmol / g or more and 100 µmol / g or less.

[0143] After adding cell culture microcarriers to an acidic solution, introducing an indicator solution, and titrating with NaOH, the epoxy content can be calculated using the following equation.

[0144] [Equation]

[0145]

[0146] In this equation, V0 is the volume of NaOH added to the control containing deionized water rather than particles, V is the volume of NaOH added to the actual sample, and C NaOH This indicates the concentration of NaOH used in the titration.

[0147] When the microcarriers used for cell culture meet the above epoxy content requirements, excellent cell adhesion can be achieved. However, excessively low epoxy content may lead to technical problems such as reduced particle dispersibility and decreased efficiency in introducing surface modifiers.

[0148] Meanwhile, according to another embodiment of this disclosure, a method for preparing microcarriers for cell culture can be provided, the method comprising the steps of: polymerizing polystyrene-based particles from a monomer mixture comprising a compound represented by the following chemical formula 1 and recovering the polystyrene-based particles.

[0149] In the method for preparing cell culture microcarriers according to this embodiment, the description of polystyrene-based particles and compounds represented by chemical formula 1 covers all the above-mentioned matters.

[0150] Specifically, the compound represented by chemical formula 1 may include at least one compound selected from the compounds represented by chemical formulas 1-1 to 1-3 below.

[0151] [Chemical Formula 1-1]

[0152]

[0153] In chemical formula 1-1,

[0154] R 11It is hydrogen or an alkyl group having one or more carbon atoms.

[0155] [Chemical Formula 1-2]

[0156]

[0157] In chemical formulas 1-2,

[0158] R 12 It is hydrogen or an alkyl group having at least one carbon atom.

[0159] [Chemical Formulas 1-3]

[0160]

[0161] Among them, in chemical formulas 1-4,

[0162] R 13 It is hydrogen or an alkyl group having one or more carbon atoms.

[0163] By including at least one compound selected from those represented by chemical formulas 1-1 to 1-3 as a monomer of a polystyrene-based polymer, and by adjusting the weight ratio between the styrene-based monomer and the compound represented by chemical formulas 1-1 to 1-3, reactive sites can be provided that allow cell adhesion ligands to be immobilized onto the surface of a cell culture microcarrier by chemical bonding without the need for additional coating, thereby improving cell adhesion.

[0164] In addition to the compounds represented by Formula 1, polystyrene-based particles may also contain compounds represented by Formula 2 as monomer compounds:

[0165] [Chemical Formula 2]

[0166]

[0167] In chemical formula 2, R2 to R6 are each independently hydrogen or an alkyl group having one or more carbon atoms, and at least one of R2 to R6 is an alkyl group having one or more carbon atoms.

[0168] By including a compound represented by the following chemical formula 2 as a monomer for polystyrene-based particles, the density of microcarriers for cell culture can be precisely controlled by adjusting the content of the compound represented by chemical formula 2, thereby enabling separation from cells based on density differences and improving dispersibility in cell culture reactors or culture media.

[0169] Specifically, the compound represented by chemical formula 2 may include at least one compound selected from the compounds represented by chemical formulas 2-1 to 2-3 below.

[0170] [Chemical Formula 2-1]

[0171]

[0172] [Chemical Formula 2-2]

[0173]

[0174] [Chemical Formula 2-3]

[0175]

[0176] In chemical formulas 2-1 to 2-3, R 21 To R 26 Each is an alkyl group having one or more carbon atoms.

[0177] By including at least one compound selected from those represented by chemical formulas 2-1 to 2-3 as a monomer for polystyrene-based particles, the density of microcarriers for cell culture can be precisely controlled by adjusting the content of the compound represented by chemical formula 2, thereby enabling separation from cells based on density differences and simultaneously improving dispersibility in cell culture reactors or culture media.

[0178] More specifically, the compound represented by chemical formula 2 may include at least one compound selected from the compounds represented by chemical formulas 2-4 to 2-6:

[0179] [Chemical Formula 2-4]

[0180]

[0181] [Chemical Formula 2-5]

[0182]

[0183] [Chemical Formula 2-6]

[0184]

[0185] Among them, in chemical formulas 2-4 to 2-6, R 21 To R 26 Each is an alkyl group having one or more carbon atoms.

[0186] Meanwhile, the density of the compound represented by chemical formula 2 can be 0.92 g / cm³. 3 Or smaller.

[0187] Specifically, the density of the compound represented by chemical formula 2 can be 0.92 g / cm³. 3 Or smaller, 0.91 g / cm3 or smaller, 0.906 g / cm 3 Or smaller, 0.9 g / cm³ 3 or smaller, 0.89 g / cm 3 Or smaller, 0.5 g / cm 3 or larger, 0.6 g / cm 3 or larger, 0.7 g / cm 3 or larger, 0.8 g / cm 3 or larger, 0.5 g / cm 3 Or larger and 0.92 g / cm 3 or smaller, 0.6 g / cm 3 Or larger and 0.92 g / cm 3 Or smaller, 0.7 g / cm 3 Or larger and 0.92 g / cm 3 or smaller, 0.8 g / cm 3 Or larger and 0.92 g / cm 3 Or smaller, 0.5 g / cm 3 Or larger and 0.91 g / cm 3 or smaller, 0.6 g / cm 3 Or larger and 0.91 g / cm 3 Or smaller, 0.7 g / cm 3 Or larger and 0.91 g / cm 3 or smaller, 0.8 g / cm 3 Or larger and 0.91 g / cm 3 Or smaller, 0.5 g / cm 3 Or larger and 0.906 g / cm³ 3 or smaller, 0.6 g / cm 3 Or larger and 0.906 g / cm³ 3 Or smaller, 0.7 g / cm 3 Or larger and 0.906 g / cm³ 3 or smaller, 0.8 g / cm 3 Or larger and 0.906 g / cm³ 3 Or smaller, 0.5 g / cm 3 Or larger and 0.9 g / cm 3 or smaller, 0.6 g / cm 3 Or larger and 0.9 g / cm 3 Or smaller, 0.7 g / cm 3 Or larger and 0.9 g / cm 3 or smaller, 0.8 g / cm 3 Or larger and 0.9 g / cm3 Or smaller, 0.5 g / cm 3 Or larger and 0.89 g / cm 3 or smaller, 0.6 g / cm 3 Or larger and 0.89 g / cm 3 Or smaller, 0.7 g / cm 3 Or larger and 0.89 g / cm 3 or smaller, 0.8 g / cm 3 Or larger and 0.89 g / cm 3 Or smaller.

[0188] Since the density of the compound represented by chemical formula 2 is 0.92 g / cm³ 3 Or even smaller, so the density of microcarriers for cell culture can be precisely controlled by adjusting the content of the compound represented by chemical formula 2, enabling separation from cells based on density differences, while improving dispersibility in cell culture bioreactors or culture media.

[0189] For example, compounds represented by Formula 2 may include 4-methylstyrene, trimethylstyrene, 4-vinyl-2-methyl-1-(2-methylpropyl)benzene, 1-vinyl-2-methyl-4-(1-methylethyl)benzene, 1-vinyl-2,3-dimethylbenzene, 4-(1,1-dimethylethyl)-2-vinyl-1-methylbenzene, 2-vinyl-4-methyl-1-(1-methylethyl)benzene, 1-vinyl-2-(1-methylethyl)benzene, 2-tert-butylstyrene, 4-tert-butylstyrene, and 1-vinyl-3-(1-methylethyl)benzene.

[0190] In addition to compounds represented by chemical formula 1, polystyrene-based particles may also contain compounds represented by chemical formula 3 as monomers.

[0191] [Chemical Formula 3]

[0192]

[0193] In chemical formula 3, L 30 For -O(C=O)- or -(C=O)O-, R 30 It is a direct bond or an alkylene group having one or more carbon atoms, and R 31 It is an alkyl group having one or more carbon atoms.

[0194] That is, as a monomer component, polystyrene-based particles may contain compounds represented by chemical formula 1; or compounds represented by chemical formula 1 and chemical formula 2; or compounds represented by chemical formula 1 and chemical formula 3; or compounds represented by chemical formula 1, chemical formula 2 and chemical formula 3.

[0195] By incorporating a compound represented by chemical formula 3 as a polystyrene-based monomer, the substituents of the resulting cell culture microcarriers can be hydrolyzed, thereby improving dispersibility in cell culture reactors or culture media.

[0196] For example, compounds represented by chemical formula 3 may include acetoxystyrene.

[0197] Meanwhile, based on 100 parts by weight of the monomer mixture, the monomer mixture may contain the compound represented by chemical formula 2 in an amount of 20 parts by weight or more and 99 parts by weight or less.

[0198] The monomer mixture may contain both a compound represented by chemical formula 1 and a styrene-based monomer mixture.

[0199] Specifically, based on 100 parts by weight of the monomer mixture, the compound represented by Formula 2 may be included in the following amounts: 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.

[0200] When the compound represented by Formula 2 is included in a monomer mixture of 100 parts by weight in an amount of less than 20 parts by weight, the particle density may increase, resulting in a loss of low-density properties. On the other hand, when it is included in an amount of more than 99 parts by weight, the particle density may decrease to below 0.99 g / cm³. 3 High-speed agitation is required to disperse the cells in the culture medium, which may have an adverse effect on cell characteristics.

[0201] Meanwhile, based on 100 parts by weight of the monomer mixture, the monomer mixture may contain a compound represented by chemical formula 3 in an amount of 0.1 parts by weight or more and 5 parts by weight or less.

[0202] The monomer mixture may include all of the compounds represented by Formula 1, styrene-based monomer mixtures, and olefinic unsaturated crosslinking agents.

[0203] Specifically, based on 100 parts by weight of the monomer mixture, the monomer mixture may contain the compound represented by chemical formula 3 in the following amounts: 0.1 parts 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 parts by weight or more and 5 parts by weight or less, 0.1 parts by weight or more and 4 parts by weight or less, 0.1 parts by weight or more and 3 parts by weight or less, 0.1 parts 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.

[0204] When a compound represented by Formula 3 is included in a monomer mixture of 100 parts by weight in an amount of less than 0.1 parts by weight, the surface hydrophilicity may be insufficient, resulting in reduced dispersibility in culture vessels. Conversely, when it is included in an amount of more than 5 parts by weight, the particle density may increase, leading to technical problems such as loss of low-density properties.

[0205] Furthermore, according to one embodiment of this disclosure, the step of polymerizing polystyrene-based particles from a monomer mixture comprising a compound represented by the following chemical formula 1 and recycling the polystyrene-based particles may include the step of polymerizing polystyrene-based particles and recycling the polystyrene-based particles by reacting the monomer mixture comprising a compound represented by the following chemical formula 1 with an olefinic unsaturated crosslinking agent.

[0206] Alkenyl-bonded unsaturated crosslinking agents include all of the above.

[0207] In the method for preparing microcarriers for cell culture, based on 100 parts by weight of an olefinic unsaturated crosslinking agent, the compound represented by chemical formula 2 may be included in an amount of 80 parts by weight or more and 150 parts by weight or less.

[0208] More specifically, based on 100 parts by weight of the olefinic unsaturated crosslinking agent, the compound represented by Formula 2 may be included in the following amounts: 80 parts by weight or more, 85 parts by weight or more, 90 parts by weight or more, 150 parts by weight or less, 125 parts by weight or less, 100 parts by weight or less, 99 parts by weight or less, 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.

[0209] By using 100 parts by weight of an olefinic unsaturated crosslinking agent, the compound represented by chemical formula 2 is contained in an amount of 80 parts by weight or more and 150 parts by weight or less. Due to the low density of the compound represented by chemical formula 2, the low density characteristics of the particles can be controlled, enabling separation from cells by density differences, and at the same time, improving dispersibility in cell culture reactors or culture media.

[0210] When the compound represented by Formula 2 is included in too small an amount relative to 100 parts by weight of the olefinic unsaturated crosslinking agent, the particle density increases, leading to the loss of low-density properties and sedimentation in the culture medium. Conversely, when it is included in excessive amounts, the particle density decreases to below 0.99 g / cm³. 3 High-speed agitation is required to disperse the particles in the culture medium, which may have an adverse effect on cell characteristics.

[0211] The monomer mixture may contain compounds represented by chemical formula 1 and styrene-based monomer mixtures.

[0212] Based on 100 parts by weight of a styrene-based monomer mixture, the compound represented by Formula 2 may be included in an amount of 90 parts by weight or more and 100 parts by weight or less.

[0213] Specifically, based on 100 parts by weight of the styrene-based monomer mixture, the compound represented by Formula 2 may be included in the following amounts: 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.

[0214] By using a styrene-based monomer mixture of 100 parts by weight, containing the compound represented by chemical formula 2 in the aforementioned amount, the density of microcarriers for cell culture can be precisely controlled, enabling separation from cells based on density differences, while improving dispersibility in cell culture bioreactors or culture media.

[0215] Furthermore, based on 100 parts by weight of the compound represented by Formula 2, the compound represented by Formula 1 may be included in an amount of 1 part by weight or more and 15 parts by weight or less.

[0216] Specifically, based on 100 parts by weight of the compound represented by Formula 2, the compound represented by Formula 1 may be included in the following amounts: 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.

[0217] When the compound represented by Formula 2 is included in 100 parts by weight in too small an amount, insufficient reactive sites for fixing cell adhesion ligands will be provided, which may result in poor cell adhesion; conversely, when it is included in too large an amount, the particle density may increase, leading to the technical problem of loss of low-density properties.

[0218] Furthermore, when a monomeric compound represented by Formula 3 is included, the compound represented by 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 olefinic unsaturated crosslinking agent.

[0219] More specifically, based on 100 parts by weight of the olefinic unsaturated crosslinking agent, the compound represented by Formula 3 may be included in the following amounts: 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 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 parts by weight or less, 0.5 parts by weight or more and 2 parts by weight or less, 1 part by weight or more and 2 parts by weight or less.

[0220] When a monomeric compound represented by chemical formula 3 is 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 an olefinic unsaturated crosslinking agent, dispersibility in a cell culture reactor or culture medium can be improved by hydrolyzing the substituents of the cell culture microcarrier.

[0221] When the olefinic unsaturated crosslinking agent, based on 100 parts by weight, is included in an amount exceeding 5 parts by weight of the compound represented by chemical formula 3, the particle density may increase, leading to the loss of low-density properties.

[0222] Furthermore, when the monomeric compound contains a compound represented by Formula 3, based on 100 parts by weight of a styrene-based monomer mixture, the compound represented by Formula 3 may be included in an amount of 0.1 parts by weight or more and 5 parts by weight or less.

[0223] Specifically, based on 100 parts by weight of a styrene-based monomer mixture, the compound represented by Formula 3 may be included in the following amounts: 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.

[0224] When the monomeric compound contains a compound represented by Formula 3, by including the compound represented by Formula 3 in an amount of 0.1 parts by weight or more and 5 parts by weight or less in a styrene-based monomer mixture based on 100 parts by weight, the substituents of the resulting cell culture microcarrier can be hydrolyzed, thereby improving dispersibility in cell culture reactors or culture media.

[0225] Furthermore, when the monomeric compound contains a compound represented by Formula 3, the compound represented by 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 Formula 3.

[0226] Specifically, based on 100 parts by weight of the compound represented by Formula 3, the compound represented by Formula 1 may be included in the following amounts: 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, 200 parts by weight or less, 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, 150 parts by weight or more and 200 parts by weight or less.

[0227] When the monomer compound contains a compound represented by Formula 3, if, based on 100 parts by weight of the compound represented by Formula 3, the compound represented by Formula 1 is included in an amount of less than 110 parts by weight, the compound represented by Formula 1 may be lost before polymerization due to its relatively high water solubility and therefore may not be introduced onto the particle surface. Conversely, if it is included in an amount of more than 500 parts by weight, the particle density may become high, thus losing the low-density properties.

[0228] In the method for preparing cell culture microcarriers according to the embodiment, the step of polymerizing polystyrene-based particles from a monomer mixture containing a compound represented by chemical formula 1 and recovering the polystyrene-based particles may include the step of performing a suspension polymerization reaction of the monomer composition and recovering the suspension polymerization reaction product.

[0229] More specifically, the suspension polymerization reaction of the monomer composition may include: mixing the monomer composition with an aqueous dispersion, applying shear force to homogenize the monomer composition into droplets in the aqueous dispersion; and carrying out suspension polymerization of the homogenized monomer composition at a stirring speed of 300 rpm to 1000 rpm.

[0230] During the step of homogenizing the monomer composition into droplets in an aqueous dispersion, stirring can be performed at a speed of 300 rpm to 1000 rpm or 400 rpm to 800 rpm.

[0231] In the suspension polymerization step of the monomer composition homogenized at stirring speeds of 300 rpm to 1000 rpm or 400 rpm to 800 rpm, the particle structure of polystyrene, due to the structure of the compound represented by Formula 1, can further reduce the density of the microcarriers while still being able to produce microcarriers with a high proportion of intact, perfectly spherical particles without damage or breakage.

[0232] In the suspension polymerization step of homogenizing the monomer composition at a stirring speed of 300 rpm to 1000 rpm or 400 rpm to 800 rpm, there are no particular limitations on the suspension polymerization conditions; for example, the process can be carried out for 3 to 18 hours in the range of 50°C to 100°C.

[0233] Meanwhile, the method for preparing microcarriers for cell culture may also include a washing and drying step after the step of polymerizing polystyrene-based particles from a monomer mixture containing a compound represented by chemical formula 1 and recovering the polystyrene-based particles.

[0234] Specifically, the washing step may include filtering the reaction product through a sieve of 30 μm to 100 μm, followed by stirring it in 100% ethanol 5 to 7 times at room temperature.

[0235] The drying step may include placing the washed product in a vacuum oven and drying it under vacuum at room temperature; however, this is not limiting and any conventionally known drying method may be used without particular limitation.

[0236] Furthermore, in this disclosure, cell adhesion materials can be introduced onto the particle surface via chemical bonding. For example, the method may include modifying the particles with a solution containing one or more compounds selected from: gelatin, collagen, fibronectin, chitosan, poly-L-lysine, hyalin, peptides containing RGD, lignin, cationic dextran, dihydroxyphenylalanine (DOPA), dopamine, norepinephrine, epinephrine, epigallocatechin gallate, and derivatives thereof.

[0237] Solutions containing one or more compounds selected from gelatin, collagen, fibronectin, chitosan, poly-L-lysine, hyalin, peptides containing RGD, lignin, cationic dextran, dihydroxyphenylalanine (DOPA), dopamine, norepinephrine, epinephrine, epigallocatechin, and their derivatives can act as adhesion factors to bind cells to microcarriers, thereby enhancing cell-microcarrier adhesion and making them more suitable for large-scale cell culture.

[0238] Specifically, the step of introducing a cell attachment induction layer may include immersing the surface of polystyrene-based particles in a solution containing one or more compounds selected from gelatin, collagen, fibronectin, chitosan, poly-L-lysine, hyalin, peptides containing RGD, lignin, cationic dextran, dihydroxyphenylalanine (DOPA), dopamine, norepinephrine, epinephrine, epigallocatechin gallate, and their derivatives, and allowing the reaction to proceed for 10 to 20 hours, or 15 to 20 hours, or 17 to 19 hours.

[0239] The epoxy content of the cell culture microcarriers prepared by the above methods can be in the range of 40 µmol / g to 100 µmol / g.

[0240] Specifically, the epoxy content of the cell culture microcarriers in the implementation scheme can be 40 µmol / g or more, 45 µmol / g or more, 49 µmol / g or more, 49.5 µmol / g or more, or 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, or 49.5 µmol / g or more and 100 µmol / g or less.

[0241] After adding cell culture microcarriers to an acidic solution, introducing an indicator solution, and titrating with NaOH, the epoxy content can be calculated using the following equation.

[0242] [Equation]

[0243]

[0244] In this equation, V0 is the volume of NaOH added to the control containing deionized water rather than particles, V is the volume of NaOH added to the actual sample, and C NaOH This indicates the concentration of NaOH used in the titration.

[0245] When the microcarriers used for cell culture meet the above epoxy content requirements, excellent cell adhesion can be achieved. However, excessively low epoxy content may lead to technical problems such as reduced particle dispersibility and decreased efficiency in introducing surface modifiers.

[0246] According to another embodiment of this disclosure, a cell culture composition comprising cells and the cell culture microcarriers of the foregoing embodiments can be provided. All details regarding the cell culture microcarriers described in the above embodiments are incorporated herein.

[0247] The cells are adherent animal cells, with no particular limitation, and may include at least one compound selected from, for example, fibroblasts, epithelial cells, osteoblasts, chondrocytes, hepatocytes, cells derived from human umbilical cord blood, mesenchymal stem cells derived from human bone marrow, CHO (Chinese hamster ovary) cells, kidney cells (HEK293, BHK21, MDCK, Vero cells, etc.), or a mixture of two or more thereof.

[0248] Furthermore, the density difference between the microcarriers used for cell culture and the cells can be 0.20 g / cm³. 3 Or smaller. Meets 0.20 g / cm³ 3 A smaller density difference allows cells and microcarriers to be easily separated when they are recovered after culture by taking advantage of the difference in their settling velocities under gravity, while also preventing the problem of cell attachment difficulties caused by microcarriers merely floating on the surface of the culture medium in the early stages of culture.

[0249] The cell culture composition may also contain a culture medium solution. This culture medium solution may contain various additives that adequately meet nutritional and environmental conditions, such as pH, temperature, and osmotic pressure, closely mimicking physiological conditions based on body fluids (e.g., plasma or lymph). Any of the various substances known in the field of cell culture technology may be used without limitation.

[0250] For example, in one embodiment, the cell culture microcarrier has a lower density than the culture medium solution, so it remains suspended in the solution when introduced with stirring. As the number of cells attached to the surface of the low-density microcarrier increases, the density of the cell-loaded microcarrier (hereinafter referred to as the "microcarrier-cell conjugate") gradually increases, causing it to settle in the culture medium solution.

[0251] Therefore, after treating the cell-loaded microcarriers (microcarrier-cell conjugates) with cell-detachment enzymes, they can be separated by centrifugation. The cultured cells can then be easily collected by detaching them from the microcarrier-cell conjugates.

[0252] Beneficial effects

[0253] According to this disclosure, microcarriers for cell culture, methods for preparing microcarriers, and cell culture methods using the same can be provided, wherein the microcarriers for cell culture have high surface hydrophilicity and enhanced cell adhesion properties, and improved dispersibility in cell culture reactors or culture media. Attached Figure Description

[0254] Figure 1 The image shows a SEM image of the surface morphology of the cell culture microcarrier according to Example 1. Detailed Implementation

[0255] In the following description, embodiments of the present disclosure will be described in more detail with reference to the following examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present disclosure.

[0256] Example 1

[0257] A water-based dispersion was prepared by dissolving polyvinyl alcohol (molecular weight 85 K to 124 K, 87% to 89% hydrolyzed) in distilled water at a concentration of 2% by weight and stirring it at room temperature for 20 minutes.

[0258] Styrene, glycidyl methacrylate, and tert-butylstyrene as monomers, and divinylbenzene as a crosslinking agent were mixed and completely dissolved in a weight ratio of 0.05:0.05:0.9:1; then 2% by weight of initiator V-65 (based on the total weight of monomers and crosslinking agent) was added to 25 g of the mixture and stirred for another 5 minutes to obtain a monomer composition.

[0259] 600 g of an aqueous dispersion was charged into a 1 L reactor, and a monomer composition was added thereto. Shear was applied to the aqueous dispersion and monomer composition at 400 rpm at room temperature, causing the monomer composition to disperse as fine droplets into the aqueous phase, thereby homogenizing the mixture.

[0260] The homogenized mixture was stirred at 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 recovered by drying in an oven at 80°C. The recovered polystyrene particles were used as microcarriers for cell culture.

[0261] The physical properties of polystyrene particles are as follows.

[0262] Average diameter: 178 μm (D50 measured using a PSA device)

[0263] Apparent density: 0.99 g / cm³ to 1.04 g / cm³

[0264] Example 2

[0265] Cell culture microcarriers were prepared in the same manner as in Example 1, except that the weight ratio of styrene, glycidyl methacrylate, tert-butylstyrene as monomers, and divinylbenzene as crosslinking agent was adjusted to 0.07:0.03:0.9:1.

[0266] Example 3

[0267] Cell culture microcarriers were prepared in the same manner as in Example 1, except that the weight ratio of styrene, glycidyl methacrylate, tert-butylstyrene as monomers, and divinylbenzene as crosslinking agent was adjusted to 0.085:0.015:0.9:1.

[0268] Example 4

[0269] Cell culture microcarriers were prepared in the same manner as in Example 1, except that the weight ratio of styrene, glycidyl methacrylate, tert-butylstyrene, acetoxystyrene (density: 1.06 g / cm³) as monomers and divinylbenzene as crosslinking agent was adjusted to 0.06:0.02:0.91:0.01:1.

[0270] Example 5

[0271] Cell culture microcarriers were prepared in the same manner as in Example 1, except that the weight ratio of styrene, glycidyl methacrylate, tert-butylstyrene, acetoxystyrene (density: 1.06 g / cm³) as monomers and divinylbenzene as crosslinking agent was adjusted to 0.05:0.03:0.90:0.02:1.

[0272] Example 6

[0273] Cell culture microcarriers were prepared in the same manner as in Example 1, except that 2-(2-epoxyethylene methoxy)ethyl methacrylate (density: 1.1 g / cm³) was used. 3 It can replace the monomer glycidyl methacrylate.

[0274] Example 7

[0275] Cell culture microcarriers were prepared in the same manner as in Example 1, except that styrene as a monomer was omitted, and 4-vinylphenyl glycidyl ether was used instead of glycidyl methacrylate. Furthermore, tert-butylstyrene and 4-vinylphenyl glycidyl ether (density: 1.1 g / cm³) were used as monomers. 3 The weight ratio of ) and divinylbenzene as a crosslinking agent is adjusted to 0.9:0.1:1.

[0276] Example 8

[0277] Cell culture microcarriers were prepared in the same manner as in Example 1, except that the weight ratio of styrene, glycidyl methacrylate, tert-butylstyrene as monomers, and divinylbenzene as crosslinking agent was adjusted to 0.1:0.1:0.8:1.

[0278] Example 9

[0279] Cell culture microcarriers were prepared in the same manner as in Example 1, except that the weight ratio of styrene, glycidyl methacrylate, tert-butylstyrene as monomers, and divinylbenzene as crosslinking agent was adjusted to 0.15:0.05:0.8:1.

[0280] Example 10

[0281] Cell culture microcarriers were prepared in the same manner as in Example 1, except that the weight ratio of styrene, glycidyl methacrylate, tert-butylstyrene as monomers, and divinylbenzene as crosslinking agent was adjusted to 0.075:0.075:0.85:1.

[0282] Example 11

[0283] Cell culture microcarriers were prepared in the same manner as in Example 1, except that the weight ratio of styrene, glycidyl methacrylate, tert-butylstyrene as monomers, and divinylbenzene as crosslinking agent was adjusted to 0.1:0.05:0.85:1.

[0284] Example 12

[0285] Cell culture microcarriers were prepared in the same manner as in Example 1, except that the weight ratio of styrene, glycidyl methacrylate, tert-butylstyrene as monomers, and divinylbenzene as crosslinking agent was adjusted to 0.02:0.08:0.9:1.

[0286] Example 13

[0287] Cell culture microcarriers were prepared in the same manner as in Example 1, except that the weight ratio of styrene, glycidyl methacrylate, tert-butylstyrene as monomers, and divinylbenzene as crosslinking agent was adjusted to 0.9:0.1:0:1.

[0288] Comparative Example 1

[0289] Cell culture microcarriers were prepared in the same manner as in Example 1, except that the weight ratio of styrene, glycidyl methacrylate, tert-butylstyrene as monomers, and divinylbenzene as crosslinking agent was adjusted to 0.1:0:0.9:1.

[0290] Experimental Example: Measurement of the Physical Properties of Microcarriers for Cell Culture

[0291] For the cell culture microcarriers obtained in the examples and comparative examples, their physical properties were measured by the following methods, and the results are shown in Tables 1 and 2.

[0292] Experiment 1. Average particle size (unit: μm)

[0293] For the cell culture microcarriers obtained in the examples and comparative examples, the particles were dispersed in ethanol at a level of 10% by weight, and then the D50 particle size (corresponding to the particle size at which the cumulative distribution percentage reaches 50%) was measured using a PSA (particle size analysis) instrument.

[0294] Experiment 2. Epoxy Content Analysis

[0295] 0.1 g of the cell culture microcarriers prepared in the examples and comparative examples were dispersed in an HCl / acetone solution (volume ratio = 1:80) and sonicated for 4 minutes.

[0296] After adding two drops of indicator solution (0.1 wt% cresol red + 0.1 wt% thymol blue, volume ratio 1:3, pH 7, 0.01 M NaOH), titrate with 0.1 M NaOH.

[0297] Calculate the epoxy content (μmol / g) using the following equation.

[0298] [Equation]

[0299]

[0300] In this equation, V0 is the volume of NaOH added to the control containing 0.1 g DIW (not particles), V is the volume of NaOH added to the actual sample, and C... NaOH This represents the concentration of NaOH used in the titration.

[0301] Experiment 3. Dispersibility of particles in a bioreactor

[0302] 1 g of the cell culture microcarriers prepared in the examples and comparative examples were dispersed in 4 mL of 1×PBS, followed by the addition of 10 mg of GRGDSK peptide and incubation for 18 hours. The peptide-coated microcarriers were then washed three times with 1×PBS.

[0303] Prior to culturing, the required amount of these microcarriers were pre-dispersed in culture medium in 20 mL glass vials and moistened for approximately 10 to 18 hours. The pre-dispersed microcarriers were filtered through a cell filter and introduced into a 100 mL 3D bioreactor along with 60 mL of culture medium, and culture medium containing mesenchymal stem cells (density: 1.05 g / cm³) was added. 3 The mixture was then incubated for 24 hours. (Cultivation conditions: 37℃, 5% CO2 incubator, bioreactor with stirring at 25 rpm)

[0304] After 24 hours of incubation, remove any particles that remain undispersed and float on the surface of the culture medium by filtration. Thoroughly wash, dry, and weigh the dispersed particles in the incubator with DPBS or water, and calculate the dispersibility as a percentage of the weight of the dispersed particles relative to the total weight. Evaluate according to the following criteria.

[0305] Excellent: 90% or higher, up to 100% dispersion

[0306] Good: 80% or higher, up to 90% dispersion

[0307] Medium: Dispersion of 60% or higher but less than 80%

[0308] Poor: Dispersion less than 60%

[0309] Experiment 4. Initial Cell Attachment

[0310] 1 g of the cell culture microcarriers prepared in the examples and comparative examples were dispersed in 4 mL of 1×PBS, followed by the addition of 10 mg of GRGDSK peptide and incubation for 18 hours. The peptide-coated microcarriers were then obtained by washing three times with 1×PBS.

[0311] Before culturing, 1 g of the required microcarriers was pre-dispersed in culture medium in a 20 mL glass vial and moistened for approximately 10 to 18 hours. The pre-dispersed microcarriers were then passed through a cell filter, combined with 60 mL of culture medium, and transferred to a 100 mL 3D bioreactor. Culture medium containing 1,800,000 mesenchymal stem cells (density: 1.05 g / cm³) was added. 3 The cells were cultured for 24 hours in a bioreactor at 37°C, 5% CO2, and with stirring at 25 rpm.

[0312] After 24 hours of culture, 1 mL of particulate suspension was collected from the bioreactor vessel. Using a Nucleocounter NC-200 (Chemometec), the percentage of cells attached to the microcarriers relative to the total cell number was determined and evaluated according to the following criteria:

[0313] Excellent: 93% to 100% of total cells attach to the microcarrier.

[0314] Good: Total cells 85% to 93% adhered to the microcarrier

[0315] Medium: Total cells 60% to 85% adhered to the microcarrier

[0316] Poor: Total cells 60% adhered to the microcarrier

[0317] [Table 1]

[0318]

[0319] As shown in Table 1, the cell culture microcarriers of the implementation scheme exhibit excellent cell adhesion due to the improved particle dispersion under culture conditions and their ability to react with ligand peptides for cell attachment.

[0320] In Comparative Example 1, it was found that both the dispersion and initial cell attachment were inferior to those of the implementation scheme.

[0321] Experiment 5. Apparent density (unit: g / cm³)

[0322] The cell culture microcarriers prepared in the above examples and comparative examples were added to distilled water (density: 0.99 g / cm³) at room temperature (25°C) and atmospheric pressure (1 atm). 3 ) or cell culture medium (density: 1.01 g / cm³) 3 Apparent density was assessed by observing whether the particles floated or settled. The cell culture medium was prepared from 94.9% by weight Advanced MEM, 5% by weight fetal bovine serum, and 1% by weight gentamicin.

[0323] For the cell culture microcarriers prepared in the examples and comparative examples, the apparent density was evaluated according to the following criteria by adding the particles to aqueous solutions of ethanol with densities of 0.985 g / cm³, 0.99 g / cm³ and 0.997 g / cm³ and aqueous solutions of glycerol with densities of 1.02 g / cm³ and 1.04 g / cm³ at room temperature (25°C) and atmospheric pressure (1 atm). The particles were observed to float or sink.

[0324] 1) 0.985 g / cm 3 < d < 0.99 g / cm 3 (greater than 0.985 g / cm)3 And less than 0.99 g / cm 3 )

[0325] It sinks in an aqueous ethanol solution with a density of 0.985 g / cm³ and floats in an aqueous ethanol solution with a density of 0.99 g / cm³.

[0326] 2) 0.99 g / cm 3 < 1.02 g / cm 3 (greater than 0.99 g / cm) 3 And less than 1.02 g / cm 3 )

[0327] It sinks in an aqueous solution of ethanol with a density of 0.99 g / cm³ and floats in an aqueous solution of glycerol with a density of 1.02 g / cm³.

[0328] 3) 0.99 g / cm 3 < 1.04 g / cm 3 (greater than 0.99 g / cm) 3 And less than 1.04 g / cm 3 )

[0329] It sinks in an aqueous solution of ethanol with a density of 0.99 g / cm³ and floats in an aqueous solution of glycerol with a density of 1.04 g / cm³.

[0330] 4) 0.99 g / cm 3 < d < 1.01 g / cm 3 (greater than 0.99 g / cm) 3 And less than 1.01 g / cm 3 )

[0331] It sinks in distilled water with a density of 0.99 g / cm³ and floats in cell culture medium with a density of 1.01 g / cm³.

[0332] 5) d > 1.04 g / cm 3 (greater than 1.04 g / cm) 3 )

[0333] At a density of 1.04 g / cm³ 3 It sank in an aqueous solution of glycerol.

[0334] Experiment 6. The possibility of separation by density difference

[0335] For the cell culture microcarriers prepared in the examples and comparative examples, the possibility of separating cells from the cell culture microcarriers by means of density difference was evaluated by centrifuging the mixture of cell culture medium and microcarriers at ambient temperature (25°C) and atmospheric pressure (1 atmosphere) as follows.

[0336] No particles settled at the bottom after centrifugation.

[0337] After centrifugation, the particles settle at the bottom.

[0338] Experiment 7. Observation of Surface Morphology

[0339] The surface morphology of the cell culture microcarriers prepared in Example 1 was examined using a scanning electron microscope (SEM, JSM-7610F, JEOL), and the results are shown in... Figure 1 middle.

[0340] [Table 2]

[0341]

[0342] As shown in Table 2, the density of Examples 1 to 7 is 1.04 g / cm³. 3 Smaller microcarriers for cell culture are suitable for cell culture and can be separated under culture conditions based on density differences.

[0343] In contrast, the particles in Examples 8 to 13 had a density exceeding that of the culture medium and therefore lacked low-density properties, making separation by density impossible.

[0344] In Comparative Example 1, the apparent density was too low, so during the initial culture phase, the microcarriers merely floated on the surface of the culture medium, which may have hindered cell attachment.

Claims

1. A microcarrier for cell culture comprising polystyrene-based particles, said polystyrene-based particles comprising a monomeric compound represented by chemical formula 1: [Chemical Formula 1] ， In chemical formula 1, L0 is an arylene group with 6 or more carbon atoms or -(C=O)-. L1 and L2 are each independently an alkylene group having one or more carbon atoms. R1 is a reactive functional group capable of undergoing ring-opening reactions. R 10 It is hydrogen or an alkyl group having one or more carbon atoms, and n is an integer of 0 or greater.

2. The cell culture microcarrier according to claim 1, wherein: The polystyrene-based particles comprise the reaction product of the compound represented by Formula 1 and an olefinically unsaturated crosslinking agent, and Based on 100 parts by weight of the olefinic unsaturated crosslinking agent, the polystyrene-based particles contain 1 part by weight or more and 15 parts by weight or less of the compound represented by chemical formula 1.

3. The microcarrier for cell culture according to claim 1, wherein: The compound represented by chemical formula 1 includes at least one compound selected from the compounds represented by chemical formulas 1-1 to 1-3: [Chemical Formula 1-1] ， In chemical formula 1-1, R 11 It is hydrogen or an alkyl group having one or more carbon atoms. [Chemical Formula 1-2] ， In chemical formulas 1-2, R 12 It is hydrogen or an alkyl group having at least one carbon atom. [Chemical Formulas 1-3] ， Among them, in chemical formulas 1-4, R 13 It is hydrogen or an alkyl group having one or more carbon atoms.

4. The cell culture microcarrier according to claim 1, wherein: The polystyrene-based particles contain a compound represented by the following chemical formula 2 as a monomer: [Chemical Formula 2] ， In chemical formula 2, R2 to R6 are each independently hydrogen or an alkyl group having one or more carbon atoms, and at least one of R2 to R6 is an alkyl group having one or more carbon atoms.

5. The cell culture microcarrier according to claim 4, wherein: The compound represented by chemical formula 2 includes at least one compound selected from the compounds represented by chemical formulas 2-1 to 2-3: [Chemical Formula 2-1] ， [Chemical Formula 2-2] ， [Chemical Formula 2-3] ， Among them, in chemical formulas 2-1 to 2-3, R 21 To R 26 Each is an alkyl group having one or more carbon atoms.

6. The cell culture microcarrier according to claim 4, wherein: The density of the compound represented by chemical formula 2 is 0.92 g / cm³. 3 Or smaller.

7. The cell culture microcarrier according to claim 4, wherein: The polystyrene-based particles comprise the reaction product of the compound represented by Formula 1, a mixture of styrene-based monomers, and an olefinically unsaturated crosslinking agent, and Based on 100 parts by weight of the olefinic unsaturated crosslinking agent, the polystyrene-based particles contain the compound represented by chemical formula 2 in an amount of 80 parts by weight or more and 150 parts by weight or less.

8. The cell culture microcarrier according to claim 4, wherein: The polystyrene-based particles comprise the reaction product of the compound represented by Formula 1, a mixture of styrene-based monomers, and an olefinically unsaturated crosslinking agent, and Based on 100 parts by weight of the styrene-based monomer mixture, the polystyrene-based particles contain the compound represented by chemical formula 2 in an amount of 90 parts by weight or more and 100 parts by weight or less.

9. The microcarrier for cell culture according to claim 4, wherein: The polystyrene-based particles comprise the reaction product of the compound represented by Formula 1, a mixture of styrene-based monomers, and an olefinically unsaturated crosslinking agent, and Based on 100 parts by weight of the compound represented by chemical formula 2, the polystyrene-based particles contain the compound represented by chemical formula 1 in an amount of 1 part by weight or more and 15 parts by weight or less.

10. The microcarrier for cell culture according to claim 1, wherein: The polystyrene-based particles contain a compound represented by the following chemical formula 3 as a monomer: [Chemical Formula 3] ， In chemical formula 3, L 30 It is -O(C=O)- or -(C=O)O-. R 30 For direct bonds or alkylene groups having one or more carbon atoms, and R 31 It is an alkyl group having one or more carbon atoms.

11. The microcarrier for cell culture according to claim 10, wherein: The polystyrene-based particles comprise the reaction product of the compound represented by Formula 1, a mixture of styrene-based monomers, and an olefinically unsaturated crosslinking agent, and Based on 100 parts by weight of the olefinic unsaturated crosslinking agent, the polystyrene-based particles contain the compound represented by chemical formula 3 in an amount of 0.1 parts by weight or more and 5 parts by weight or less.

12. The microcarrier for cell culture according to claim 10, wherein: The polystyrene-based particles comprise the reaction product of the compound represented by Formula 1, a mixture of styrene-based monomers, and an olefinically unsaturated crosslinking agent, and Based on 100 parts by weight of the styrene-based monomer mixture, the polystyrene-based particles contain the compound represented by chemical formula 3 in an amount of 0.1 parts by weight or more and 5 parts by weight or less.

13. The microcarrier for cell culture according to claim 10, wherein: The polystyrene-based particles comprise the reaction product of the compound represented by Formula 1, a mixture of styrene-based monomers, and an olefinically unsaturated crosslinking agent, and Based on 100 parts by weight of the compound represented by chemical formula 3, the polystyrene-based particles contain 110 parts by weight or more and 500 parts by weight or less of the compound represented by chemical formula 1.

14. The microcarrier for cell culture according to claim 1, wherein: This includes a cell attachment induction layer formed on the polystyrene-based particles.

15. The microcarrier for cell culture according to claim 1, wherein: The apparent density of the cell culture microcarriers is in the range of 0.99 g / cm³ to 1.04 g / cm³.

16. The microcarrier for cell culture according to claim 1, wherein: The D50 particle size of the microcarriers used for cell culture is in the range of 100 μm to 300 μm.

17. A method for preparing microcarriers for cell culture, The process includes the steps of polymerizing polystyrene-based particles from a monomer mixture comprising a compound represented by the following chemical formula 1 and recycling the polystyrene-based particles: [Chemical Formula 1] ， In chemical formula 1, L0 is an arylene group with 6 or more carbon atoms or -(C=O)-. L1 and L2 are each independently an alkylene group having one or more carbon atoms. R1 is a reactive functional group capable of undergoing ring-opening reactions. R 10 It is hydrogen or an alkyl group having one or more carbon atoms, and n is an integer of 0 or greater.

18. The method for preparing microcarriers for cell culture according to claim 17, wherein: The monomer mixture comprises a compound represented by the following chemical formula 2: [Chemical Formula 2] ， In chemical formula 2, R2 to R6 are each independently hydrogen or an alkyl group having one or more carbon atoms, and at least one of R2 to R6 is an alkyl group having one or more carbon atoms.

19. The method for preparing microcarriers for cell culture according to claim 17, wherein: The monomer mixture comprises a compound represented by the following chemical formula 3: [Chemical Formula 3] ， In chemical formula 3, L 30 It is -O(C=O)- or -(C=O)O-. R 30 For direct bonds or alkylene groups having one or more carbon atoms, and R 31 It is an alkyl group having one or more carbon atoms.

20. The method for preparing microcarriers for cell culture according to claim 17, wherein: The step includes introducing a cell attachment inducing layer onto the polystyrene-based particles.

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

22. The cell culture composition according to claim 21, wherein: The cells include at least one compound selected from fibroblasts, epithelial cells, osteoblasts, chondrocytes, hepatocytes, cells derived from human umbilical cord blood, mesenchymal stem cells derived from human bone marrow, CHO cells, and kidney cells.

23. The cell culture composition according to claim 21, wherein: The difference in apparent density between the cell culture microcarrier and the cells is 0.20 g / cm³ or less.

Images