Microcarriers for cell culture, methods for producing microcarriers for cell culture, and cell culture compositions using the same

Abstract

The present invention relates to a microcarrier for cell culture containing polystyrene particles, a method for producing a microcarrier for cell culture, and a cell culture composition using the same.

Description

[Technical Field]

[0001] This application claims priority rights under Korean Patent Application No. 10-2024-0067364 dated May 23, 2024, and Korean Patent Application No. 10-2025-0067201 dated May 23, 2025, and all content disclosed in the documents of said Korean Patent Applications is incorporated herein by reference.

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

[0003] With the expansion of the biopharmaceutical and regenerative medicine fields, there is a growing demand for mass cell culture technologies that can efficiently produce cells, tissues, microorganisms, and other microorganisms.

[0004] Adherent cells are cultured in a 3D bioreactor using microcarriers. Cells, culture medium, and microcarriers are placed in the bioreactor, and the culture medium is agitated to bring the cells and microcarriers into contact, causing the cells to adhere to the surface of the microcarriers and be cultured. The microcarriers used in this method offer a higher surface area ratio (surface area / volume) compared to 2D culture, allowing cells to adhere and proliferate, making it suitable for large-scale cell culture.

[0005] Currently commercially used microcarriers have a density of approximately 1.1–1.3 g / cm³. 3 The cell density is approximately 1.2 g / cm³. 3 This is the extent of the problem. In this case, it is advantageous for attaching initial culture cells to the bioreactor, but centrifugation is difficult when separating and recovering cells after culture, and filtering methods based on microcarrier and cell size must be used. However, in this case, there are problems such as the filter becoming clogged, the process time being long, physical damage and contamination of cells occurring easily, and cell loss.

[0006] To solve this problem, the density is 1.0 g / cm³. 3 It was even lower, or 1.3 g / cm³. 3 While microcarriers were manufactured by utilizing the superior material properties, this approach has the disadvantage of limiting the achievable density range and making it difficult to ensure a sufficient yield of perfectly spherical microcarriers without damage or fracture. [Overview of the project] [Problems that the invention aims to solve]

[0007] The present invention relates to a microcarrier for cell culture that allows for control of density and separation from cells based on density differences, while simultaneously improving dispersibility in cell culture reactors or culture media and enhancing cell adhesion.

[0008] Furthermore, the present invention relates to a method for producing the aforementioned microcarriers for cell culture.

[0009] Furthermore, the present invention relates to a cell culture composition using the aforementioned microcarriers for cell culture. [Means for solving the problem]

[0010] To solve the above problems, this specification provides a microcarrier for cell culture comprising polystyrene particles containing a compound represented by the following chemical formula 1 as a monomer compound.

[0011] [ka]

[0012] In the above chemical formula 1, L0 is an arylene group having 6 or more carbon atoms or -(C=O)-, L1 and L2 are each independently alkylene groups having 1 or more carbon atoms, R1 is a reactive functional group capable of ring-opening reactions, and R 10 n is a hydrogen atom or an alkyl group having one or more carbon atoms, and n is a non-negative integer.

[0013] This specification also provides a method for producing microcarriers for cell culture, comprising the step of polymerizing and recovering polystyrene particles from a monomer mixture containing a compound represented by the following chemical formula 1.

[0014] [ka]

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

[0016] This specification further provides cell culture compositions comprising cells and microcarriers for cell culture.

[0017] The following describes in more detail a microcarrier for cell culture, a method for producing a microcarrier for cell culture, and a cell culture composition using the same, according to specific embodiments of the invention.

[0018] In this specification, unless otherwise expressly stated, technical terms are used solely to refer to specific embodiments and are not intended to limit the invention.

[0019] As used herein, the singular form includes the plural form unless the wording explicitly indicates the opposite.

[0020] As used herein, "includes" embodies a particular characteristic, domain, integer, stage, operation, element and / or component, and does not exclude the presence or addition of other particular characteristics, domains, integers, stages, operations, elements, components and / or groups.

[0021] Furthermore, in this specification, terms including ordinal numbers such as “first” and “second” are used for the purpose of distinguishing one component from another, and are not limited by such ordinal numbers. For example, within the scope of the rights of the present invention, the first component may be named the second component, and similarly, the second component may be named the first component.

[0022] In this specification, the alkyl group may be linear or branched, and the number of carbon atoms is not particularly limited, but is preferably 1 to 10. According to another embodiment, the number of carbon atoms of the alkyl group is 1 to 6. Specific examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, n-propyl, isopropyl, butyl, n-butyl, isobutyl, tert-butyl, sec-butyl, 1-methyl-butyl, 1-ethyl-butyl, pentyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, n-hexyl, 1-methylpentyl, 2-methylpentyl, 4-methyl-2-pentyl, 3,3-dimethylbutyl, 2-ethylbutyl, heptyl, n-heptyl, 1-methylhexyl, cyclopentylmethyl, cyclohexylmethyl, octyl, n-octyl, tert-octyl, 1-methylheptyl, 2-ethylhexyl, 2-propylpentyl, n-nonyl, 2,2-dimethylheptyl, 1-ethyl-propyl, 1,1-dimethyl-propyl, isohexyl, 2-methylpentyl, 4-methylhexyl, and 5-methylhexyl.

[0023] In this specification, a cycloalkyl group is a monovalent functional group derived from a cycloalkane, and may be monocyclic or polycyclic, and is not particularly limited, but has 3 to 20 carbon atoms. According to another embodiment, the cycloalkyl group has 3 to 10 carbon atoms. Specifically, 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 bicyclo[2,2,1]heptyl. The cycloalkyl group may be substituted or unsubstituted, and if substituted, examples of substituents are as described above.

[0024] In this specification, heterocycloalkyl means a cycloalkyl group containing one or more non-carbon atoms or heteroatoms, and specifically, the heteroatoms may include one or more atoms selected from the group consisting of O, N, Se, and S.

[0025] The present invention will be described in more detail below.

[0026] According to one embodiment of the invention, a microcarrier for cell culture can be provided that includes polystyrene particles containing the compound represented by chemical formula 1 as a monomer compound.

[0027] The inventors have completed the invention by confirming that, in the case of the cell culture microcarrier of the above embodiment, by including the compound represented by chemical formula 1 as a polystyrene monomer, the density of the final cell culture microcarrier can be controlled, enabling separation from cells due to the density difference, improving dispersibility in the cell culture reactor or culture medium, and simultaneously improving cell adhesion.

[0028] Conventional microcarriers for cell culture controlled particle density by incorporating non-reactive, low-density oil inside the particles. However, a technical problem existed in that the low-density oil would leak out if the particles were physically damaged.

[0029] Therefore, the present inventors have confirmed that by including the compound represented by chemical formula 1 as a monomer in a polystyrene polymer used as a microcarrier for cell culture, the density of particles can be controlled, separation from cells due to the density difference can be achieved, and at the same time, dispersibility in the cell culture reactor or culture medium can be improved.

[0030] Furthermore, conventional microcarriers for cell culture have a coating layer formed on the surface of polystyrene particles to improve cell adhesion, but there was a technical problem in that some of the coating layer would detach when the microcarriers were stirred in the culture medium.

[0031] Therefore, the present inventors have confirmed that by including the compound represented by chemical formula 1 as a monomer in a polystyrene polymer used for cell culture microcarriers, reaction sites are provided on the surface of the cell culture microcarrier that allow cell-adhering ligands to be immobilized by chemical binding without the need for an additional coating layer, thereby improving cell adhesion.

[0032] Specifically, in the case of the microcarrier for cell culture according to one embodiment, it may include polystyrene particles containing a compound represented by the following chemical formula 1 as the monomer compound.

[0033] [ka]

[0034] In the above chemical formula 1, L0 is an arylene group having 6 or more carbon atoms or -(C=O)-, L1 and L2 are each independently alkylene groups having 1 or more carbon atoms, R1 is a reactive functional group capable of ring-opening reactions, and R 10n is a hydrogen atom or an alkyl group having one or more carbon atoms, and n is a non-negative integer.

[0035] By including a reactive functional group in the polystyrene polymer that can undergo ring-opening reaction with the compound represented by chemical formula 1, a reaction site is provided on the surface of the microcarrier for cell culture that allows cell-adhering ligands to be immobilized by chemical bonding without the need for an additional coating layer, thereby improving cell adhesion.

[0036] The aforementioned reactive functional group capable of ring-opening reactions can refer to a reactive functional group capable of ring-opening reactions such as hydrolyzed ring opening.

[0037] Specifically, the ring-opening reactive functional group may include heterocycloalkyl groups.

[0038] The heterocycloalkyl group can mean a cycloalkyl group containing one or more non-carbon atoms or heteroatoms. The heteroatoms can include one or more atoms selected from the group consisting of O, N, Se, and S, and preferably include O.

[0039] The heterocycloalkyl group is not particularly limited, but for example, it may include an epoxy group.

[0040] Specifically, the polystyrene particles contain the reaction product of the compound represented by chemical formula 1 and an ethylene-based unsaturated crosslinking agent, and the compound represented by chemical formula 1 may be present in an amount of 1 to 15 parts by weight per 100 parts by weight of the ethylene-based unsaturated crosslinking agent.

[0041] Specifically, the polystyrene-based particles contain a reaction product of the compound represented by Chemical Formula 1 and an ethylenically unsaturated crosslinking agent, and with respect to 100 parts by weight of the ethylenically unsaturated crosslinking agent, the compound represented by Chemical Formula 1 may be contained in an amount of 1 part by weight or more, 1.5 parts by weight or more, 15 parts by weight or less, 10 parts by weight or less, 5 parts by weight or less, or 1 part by weight or more and 15 parts by weight or less, 1.5 parts by weight or more and 15 parts by weight or less, 1 part by weight or more and 10 parts by weight or less, 1.5 parts by weight or more and 10 parts by weight or less, 1 part by weight or more and 5 parts by weight or less, 1.5 parts by weight or more and 5 parts by weight or less.

[0042] When the compound represented by Chemical Formula 1 is contained in an excessively small amount with respect to 100 parts by weight of the ethylenically unsaturated crosslinking agent, sufficient reaction sites for immobilizing the cell adhesion ligand are not provided, resulting in poor cell adhesion. When the compound is contained in an excessively large amount, the relative ratio of the compound represented by Chemical Formula 1 with a high density becomes high, and there may occur a problem that the density of the particles becomes higher than that of the cell culture medium, losing the low-density property.

[0043] Specifically, the compound represented by Chemical Formula 1 may contain any one of the compounds represented by Chemical Formula 1-1 to Chemical Formula 1-3 below.

[0044]

Chemical formula

[0045] In Chemical Formula 1-1, R 11 is hydrogen or an alkyl group having 1 or more carbon atoms,

[0046]

Chemical formula

[0047] In Chemical Formula 1-2, R 12 is hydrogen or an alkyl group having 1 or more carbon atoms,

[0048]

Chemical formula

[0049] In the above chemical formulas 1 and 4, R 13 is hydrogen or an alkyl group having one or more carbon atoms.

[0050] By including one of the compounds represented by chemical formulas 1-1 to 1-3 as a monomer in a polystyrene polymer, reaction sites are provided on the surface of a microcarrier for cell culture that can be chemically fixed to the surface of the microcarrier by chemical binding, without the need for an additional coating layer, by adjusting the weight ratio of the styrene monomer to the compounds represented by chemical formulas 1-1 to 1-3, thereby improving cell adhesion.

[0051] On the other hand, the polystyrene particles may further contain, as monomeric compounds, the compound represented by the following chemical formula 2, in addition to the compound represented by chemical formula 1.

[0052] [ka]

[0053] In the above chemical formula 2, R2 to R6 are each independently hydrogen or an alkyl group having 1 or more carbon atoms, and at least one of R2 to R6 is an alkyl group having 1 or more carbon atoms.

[0054] By including the compound represented by chemical formula 2 as a polystyrene monomer, the density of the final cell culture microcarriers can be precisely controlled by adjusting the content of the compound represented by chemical formula 2, enabling separation from cells due to density differences, and simultaneously improving dispersibility in the cell culture reactor or culture medium.

[0055] Specifically, the compound represented by chemical formula 2 may include one or more compounds selected from the group consisting of compounds represented by chemical formula 2-1 to compounds represented by chemical formula 2-3.

[0056] [ka]

[0057] In the aforementioned chemical formulas 2-1 to 2-3, R 21 ~R 26 Each of these is independently an alkyl group having one or more carbon atoms.

[0058] By including one or more compounds selected from the group consisting of compounds represented by chemical formulas 2-1 to 2-3 as polystyrene monomers, the density of the final cell culture microcarriers can be precisely controlled by adjusting the content of the compound represented by chemical formula 2, enabling separation from cells due to density differences, and simultaneously improving dispersibility in cell culture reactors or culture media.

[0059] More specifically, the compound represented by chemical formula 2 may include one or more compounds selected from the group consisting of compounds represented by chemical formulas 2-4 to 6.

[0060] [ka]

[0061] In the aforementioned chemical formulas 2-4 to 2-6, R 21 ~R 26 Each of these is independently an alkyl group having one or more carbon atoms.

[0062] On the other hand, the compound represented by chemical formula 2 has a density of 0.92 g / cm³. 3 The following is also acceptable.

[0063] Specific には, the される compound shown in the chemical formula 2 mentioned above is は, and the density is 0.92g / cm 3 Below, 0.91g / cm 3 Below, 0.906 g / cm 3 Below, 0.9g / cm 3 Below, 0.89g / cm 3 Below, 0.5g / cm 3 Above, 0.6g / cm 3 Above, 0.7g / cm 3 Above, 0.8g / cm 3 Above, または0.5g / cm 3 Above 0.92g / cm 3 Below, 0.6g / cm 3 Above 0.92g / cm 3 Below, 0.7g / cm 3 Above 0.92g / cm 3 Below, 0.8g / cm 3 Above 0.92g / cm 3 Below, 0.5g / cm 3 Above 0.91g / cm 3 Below, 0.6g / cm 3 Above 0.91g / cm 3 Below, 0.7g / cm 3 Above 0.91g / cm 3 Below, 0.8g / cm 3 Above 0.91g / cm 3 Below, 0.5g / cm 3 Above 0.906 g / cm 3 Below, 0.6g / cm 3 Above 0.906 g / cm 3 Below, 0.7g / cm 3 Above 0.906 g / cm 3 Below, 0.8g / cm 3 Above 0.906 g / cm 3 Below, 0.5g / cm 3 Above 0.9g / cm 3 Below, 0.6g / cm 3 Above 0.9g / cm 3 Below, 0.7g / cm 3 Above 0.9g / cm 3 Below, 0.8g / cm 3 Above 0.9g / cm 3 Below, 0.5g / cm 3More than 0.89g / cm 3 Below, 0.6g / cm 3 More than 0.89g / cm 3 Below, 0.7g / cm 3 More than 0.89g / cm 3 Below, 0.8g / cm 3 More than 0.89g / cm 3 The following is also acceptable.

[0064] The density of the compound represented by the above chemical formula 2 is 0.92 g / cm³. 3 By satisfying the following conditions, the density of the final cell culture microcarriers can be precisely controlled by adjusting the content of the compound represented by chemical formula 2, enabling separation from cells due to density differences, and simultaneously improving dispersibility in the cell culture reactor or culture medium.

[0065] For example, the compound represented by chemical formula 2 may include 4-methylstyrene, trimethylstyrene, 4-ethenyl-2-methyl-1-(2-methylpropyl)benzene, 1-ethenyl-2-methyl-4-(1-methylethyl)benzene, 1-ethenyl-2,3-dimethylbenzene, 4-(1,1-dimethylethyl)-2-ethenyl-1-methylbenzene, 2-ethenyl-4-methyl-1-(1-methylethyl)benzene, 1-ethenyl-2-(1-methylethyl)benzene, 2-tert-butylstyrene, 4-tert-butylstyrene, and 1-ethenyl-3-(1-methylethyl)benzene.

[0066] On the other hand, the polystyrene particles may further contain, as monomeric compounds, the compound represented by the following chemical formula 3, in addition to the compound represented by chemical formula 1.

[0067] [ka]

[0068] In the aforementioned chemical formula 3, L 30 is -O(C=O)- or -(C=O)O-, and R 30 R is a directly bonded alkylene group having 1 or more carbon atoms.31 It is an alkyl group having one or more carbon atoms.

[0069] In other words, the polystyrene particles may contain the compound represented by chemical formula 1 as a monomer compound, or contain the compound represented by chemical formula 1 and the compound represented by chemical formula 2, or contain the compound represented by chemical formula 1 and the compound represented by chemical formula 3, or contain the compound represented by chemical formula 1, the compound represented by chemical formula 2 and the compound represented by chemical formula 3.

[0070] By including the compound represented by chemical formula 3 as a polystyrene monomer, the substituents of the final manufactured cell culture microcarrier can be hydrolyzed, improving its dispersibility in the cell culture reactor or culture medium.

[0071] For example, the compound represented by chemical formula 3 may contain acetoxystyrene.

[0072] On the other hand, the cell culture microcarrier may include polystyrene particles. Preferably, the cell culture microcarrier may consist of polystyrene particles.

[0073] Specifically, the apparent density of the polystyrene-based particles is 0.99 g / cm³. 3 More than 1.04g / cm 3 The following may also be the case. By having the low-density range described above, when separating and recovering microcarriers from cells after cell culture, the difference in sedimentation velocity due to gravity allows for easy separation of cells and microcarriers.

[0074] The density of the polystyrene particles is 1.04 g / cm³. 3 If the value exceeds 0.99 g / cm³, the density difference between the cells and microcarriers is small, which can make centrifugation difficult when separating and recovering cells after culture. 3 If the microcarrier count is less than the minimum, a problem may occur in the early stages of culture where the microcarriers only float on the surface of the culture medium, making it difficult for cells to adhere.

[0075] The aforementioned cells are not limited to adherent animal cells, but may include, for example, fibroblasts, epithelial cells, osteoblasts, chondrocytes, hepatocytes, human umbilical cord blood cells, human bone marrow-derived mesenchymal stem cells, CHO (Chinese hamster ovary) cells, kidney cells (HEK293, BHK21, MDCK, vero cells, etc.), or mixtures of two or more of these.

[0076] Furthermore, the density difference between the cell culture microcarriers and the cells is 0.20 g / cm³. 3 The following may also apply: The density difference between the cell culture microcarrier and the cells is 0.20 g / cm³. 3 By satisfying the following conditions, when separating and recovering microcarriers from cells after cell culture, the difference in sedimentation rates due to gravity allows for easy separation of cells and microcarriers, while simultaneously preventing the problem in the early stages of culture where microcarriers float only on the surface of the culture medium, making it difficult for cells to adhere to them.

[0077] The polystyrene particles may include the reaction product of monomer mixtures and ethylene-based unsaturated crosslinking agents.

[0078] As described above, the monomer mixture may contain the compound represented by chemical formula 1. Furthermore, the monomer mixture may contain the compound represented by chemical formula 1 and the compound represented by chemical formula 2. Also, the monomer mixture may contain the compound represented by chemical formula 1 and the compound represented by chemical formula 3. Furthermore, the monomer mixture may contain the compound represented by chemical formula 3, ranging from the compound represented by chemical formula 1.

[0079] In other words, the polystyrene particles include the reaction product of the compound represented by chemical formula 1, a styrene monomer mixture, and an ethylene-based unsaturated crosslinking agent, and the styrene monomer mixture may include the compound represented by chemical formula 2 or the compound represented by chemical formula 3.

[0080] Specifically, the compound represented by chemical formula 2 may be included in an amount of 80 to 150 parts by weight per 100 parts by weight of the ethylene-based unsaturated crosslinking agent.

[0081] More specifically, the compound represented by chemical formula 2 may be included in amounts of 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, or 80 parts by weight or more and 150 parts by weight or less, 85 parts by weight or more and 150 parts by weight or less, 90 parts by weight or more and 150 parts by weight or less, 80 parts by weight or more and 125 parts by weight or less, 85 parts by weight or more and 125 parts by weight or less, 90 parts by weight or more and 125 parts by weight or less, 80 parts by weight or more and 100 parts by weight or less, 85 parts by weight or more and 100 parts by weight or less, 90 parts by weight or more and 100 parts by weight or less, 80 parts by weight or more and 99 parts by weight or less, 85 parts by weight or more and 99 parts by weight or less, and 90 parts by weight or more and 99 parts by weight or less per 100 parts by weight of the ethylene-based unsaturated crosslinking agent.

[0082] By including 80 to 150 parts by weight of the compound represented by chemical formula 2 with 100 parts by weight of the ethylene-based unsaturated crosslinking agent, the low density of the compound represented by chemical formula 2 can control the low density characteristics of the particles, enabling separation from cells due to the density difference, while simultaneously improving dispersibility in cell culture reactors or culture media.

[0083] If the compound represented by chemical formula 2 is included in an excessively small amount relative to 100 parts by weight of the ethylene-based unsaturated crosslinking agent, the particle density may increase, causing it to lose its low-density properties and resulting in sedimentation in the culture medium. If it is included in excessive amounts, the particle density may reach 0.99 g / cm³. 3 When the levels are below or very low, technical problems can arise where rapid agitation is required to disperse the cells in the culture medium, affecting their properties.

[0084] Furthermore, the styrene monomer mixture may contain 90 to 100 parts by weight of the compound represented by chemical formula 2 per 100 parts by weight.

[0085] Specifically, the styrene monomer mixture may contain 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 per 100 parts by weight of the compound represented by chemical formula 2.

[0086] By including the compound represented by chemical formula 2 in the above-mentioned amount per 100 parts by weight of the styrene monomer mixture, the density of the final cell culture microcarriers can be precisely controlled, enabling separation from cells due to density differences, while simultaneously improving dispersibility in the cell culture reactor or culture medium.

[0087] Furthermore, the compound represented by chemical formula 1 may be included in an amount of 1 to 15 parts by weight per 100 parts by weight of the compound represented by chemical formula 2.

[0088] Specifically, per 100 parts by weight of the compound represented by chemical formula 2, the compound represented by chemical formula 1 may be included in amounts of 1 part by weight or more, 1.5 parts by weight or more, 15 parts by weight or less, 12 parts by weight or less, 10 parts by weight or less, 6 parts by weight or less, 1 part by weight or more and 15 parts by weight or less, 1.5 parts by weight or more and 15 parts by weight or less, 1 part by weight or more and 12 parts by weight or less, 1.5 parts by weight or more and 12 parts by weight or less, 1 part by weight or more and 10 parts by weight or less, 1 part by weight or more and 6 parts by weight or less, and 1.5 parts by weight or more and 6 parts by weight or less.

[0089] If the compound represented by chemical formula 1 is present in an excessively small amount relative to 100 parts by weight of the compound represented by chemical formula 2, there may not be enough reaction sites for the cell-adhering ligand to be immobilized, resulting in poor cell adhesion. Conversely, if the compound is present in an excessive amount, technical problems may arise where the particle density increases, causing a loss of low-density properties.

[0090] Furthermore, if the monomer compound contains a compound represented by the following chemical formula 3, the compound represented by chemical formula 3 may be present in an amount of 0.1 parts by weight or more and 5 parts by weight or less per 100 parts by weight of the ethylene-based unsaturated crosslinking agent.

[0091] More specifically, the compound represented by chemical formula 3 may be included in amounts of 0.1 parts by weight or more, 0.5 parts by weight or more, 1 part by weight or more, 5 parts by weight or less, 3 parts by weight or less, 2 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, or 1 part by weight or more and 2 parts by weight or less per 100 parts by weight of the ethylene-based unsaturated crosslinking agent.

[0092] When the monomer compound contains the compound represented by the following chemical formula 3, by including 0.1 to 5 parts by weight of the compound represented by chemical formula 3 per 100 parts by weight of the ethylene-based unsaturated crosslinking agent, the substituents of the final cell culture microcarrier can be hydrolyzed, thereby improving the dispersibility in the cell culture reactor or culture medium.

[0093] When the compound represented by chemical formula 3 is included in an amount exceeding 5 parts by weight per 100 parts by weight of the ethylene-based unsaturated crosslinking agent, technical problems may occur where the particle density increases and the low-density properties are lost.

[0094] Furthermore, if the monomer compound includes a compound represented by the following chemical formula 3, the styrene monomer mixture may contain 0.1 parts by weight or more and 5 parts by weight or less of the compound represented by the chemical formula 3 per 100 parts by weight.

[0095] Specifically, the styrene monomer mixture may contain, per 100 parts by weight, the compound represented by chemical formula 3 in amounts of 0.1 parts by weight or more, 0.5 parts by weight or more, 1 part by weight or more, 5 parts by weight or less, 3 parts by weight or less, 2.5 parts by weight or less, 0.1 parts by weight or more and 5 parts by weight or less, 0.5 parts by weight or more and 5 parts by weight or less, 1 part by weight or more and 5 parts by weight or less, 0.1 parts by weight or more and 3 parts by weight or less, 0.5 parts by weight or more and 3 parts by weight or less, 1 part by weight or more and 3 parts by weight or less, 0.1 parts by weight or more and 2.5 parts by weight or less, 0.5 parts by weight or more and 2.5 parts by weight or less, and 1 part by weight or more and 2.5 parts by weight or less.

[0096] When the monomer compound includes the compound represented by the following chemical formula 3, by including 0.1 parts by weight or more and 5 parts by weight or less of the compound represented by chemical formula 3 per 100 parts by weight of the styrene monomer mixture, sufficient reaction sites for immobilizing cell-adhering ligands are provided while maintaining low-density properties, thereby achieving an effect that facilitates cell adhesion.

[0097] Furthermore, if the monomer compound contains a compound represented by the following chemical formula 3, the compound represented by chemical formula 1 may be included in an amount of 110 to 500 parts by weight per 100 parts by weight of the compound represented by chemical formula 3.

[0098] Specifically, per 100 parts by weight of the compound represented by chemical formula 3, the compound represented by chemical formula 1 may be included in amounts of 110 parts by weight or more, 150 parts by weight or more, 500 parts by weight or less, 400 parts by weight or less, 300 parts by weight or less, 200 parts by weight or less, or 110 parts by weight or more and 500 parts by weight or less, 110 parts by weight or more and 400 parts by weight or less, 110 parts by weight or more and 300 parts by weight or less, 110 parts by weight or more and 200 parts by weight or less, 150 parts by weight or more and 500 parts by weight or less, 150 parts by weight or more and 400 parts by weight or less, 150 parts by weight or more and 300 parts by weight or less, or 150 parts by weight or more and 200 parts by weight or less.

[0099] When the monomer compound contains the compound represented by the following chemical formula 3, if the compound represented by chemical formula 1 is included in an amount of less than 110 parts by weight per 100 parts by weight of the compound represented by chemical formula 3, a problem may occur where the compound represented by chemical formula 1 is washed away before polymerization due to its relatively high water solubility and not introduced to the particle surface. If it is included in an amount exceeding 500 parts by weight, a technical problem may occur where the particle density becomes high and the low-density properties are lost.

[0100] Furthermore, the polystyrene particles may contain 60 to 200 parts by weight of the ethylene-based unsaturated crosslinking agent per 100 parts by weight of the styrene monomer mixture.

[0101] Specifically, the ethylene-based unsaturated crosslinking agent may be included in amounts of 60 to 200 parts by weight, 60 to 150 parts by weight, 60 to 130 parts by weight, 100 to 200 parts by weight, 100 to 150 parts by weight, or 100 to 130 parts by weight per 100 parts by weight of the styrene-based monomer mixture.

[0102] When the ethylene-based unsaturated crosslinking agent is present in an excessively small amount (less than 60 parts by weight) per 100 parts by weight of the styrene-based monomer mixture, the crosslinking density of the polystyrene polymer decreases, making it difficult for the particles to stably maintain a spherical shape.

[0103] In contrast, when the ethylene-based unsaturated crosslinking agent is excessively present in an amount exceeding 200 parts by weight per 100 parts by weight of the styrene-based monomer mixture, there is a limit to how low the particle density can be to the target level.

[0104] Divinylbenzene is an example of the ethylene-based unsaturated crosslinking agent mentioned above.

[0105] The average diameter of the polystyrene particles may be 50 μm to 400 μm, or 60 μm to 390 μm. When the average diameter of the polystyrene particles satisfies the above range, cell adhesion and culture performance are excellent. On the other hand, if the average diameter of the polystyrene particles is less than 50 μm, there is a risk of problems such as a small surface area for cell culture and low culture efficiency, and if it exceeds 400 μm, the interaction between adhered cells decreases, the cell density in the culture vessel decreases, and problems such as low cell culture efficiency may occur.

[0106] The diameter of the polystyrene particles refers to the distance between two points where a straight line passing through the centroid of the polystyrene particles intersects the outermost surface of the polystyrene particles. The average diameter of the polystyrene particles is determined by checking the diameter of all polystyrene particles contained in the cell culture microcarrier using an optical microscope.

[0107] The polystyrene particles may be a group of individual particles having an average diameter of 50 μm to 400 μm or 60 μm to 390 μm, and the individual fine particles included in such a group may have an average diameter of 50 μm to 400 μm or 60 μm to 390 μm. More specifically, 95% or 99% of the individual fine particles included in the group may have a diameter of 50 μm to 400 μm or 60 μm to 390 μm.

[0108] Furthermore, the polystyrene particles may have a ratio of perfectly spherical particles without damage or destruction according to the following formula, which is greater than 90% but less than or equal to 100%, or 92% or more but less than or equal to 100%, or 95% or more but less than or equal to 100%, or 96% or more but less than or equal to 99%.

[0109] [Mathematical formula] The percentage of perfectly spherical particles without damage or destruction (%) = (Number of polystyrene particles that are perfectly spherical without damage or destruction / Total number of polystyrene particles) × 100.

[0110] The ratio of perfectly spherical particles without damage or destruction according to the above formula is determined by measuring the number of perfectly spherical particles without damage or destruction among all polystyrene particles using SEM, and calculating the percentage ratio of the number of perfectly spherical particles without damage or destruction to the total number of particles.

[0111] In other words, the cell culture microcarrier can contain multiple polystyrene particles, and whether or not these multiple polystyrene particles have a perfect spherical shape without damage or destruction can be determined visually using a scanning electron microscope (SEM).

[0112] If the proportion of perfectly spherical particles decreases to 90% or less without damage or destruction as described in the above formula, the number of amorphous particles with uneven or concave surfaces will increase. These amorphous particles may float in the cell culture medium, physically impacting the cultured cells and potentially reducing cell culture efficiency to such an extent that cell culture becomes impossible.

[0113] Specifically, the D50 particle diameter of the cell culture microcarrier may be 100 μm to 300 μm, or 100 μm to 250 μm, or 120 μm to 250 μm, or 130 μm to 250 μm. When the average diameter of the cell culture microcarrier satisfies the above range, it exhibits excellent cell adhesion and culture performance. On the other hand, if the D50 particle diameter of the cell culture microcarrier is less than 100 μm, there is a risk of problems arising such as a small surface area for cell culture and low culture efficiency. If it exceeds 300 μm, the interaction between adhered cells decreases, the cell density in the incubator decreases, and problems arising such as low cell culture efficiency may occur.

[0114] On the other hand, the cell culture microcarrier may include a cell adhesion induction layer formed on the polystyrene particles.

[0115] The cell adhesion-inducing layer is composed of cell-adhering substances, which provide sites where transmembrane proteins of cells can bind, enabling adherent cells to stably adhere, spread, and culture.

[0116] The polymers forming the cell adhesion-inducing layer are not limited in scope, but may include one or more selected from the group consisting of gelatin, collagen, fibronectin, chitosan, polylysine, vitronectin, peptides containing RGD, lignin, cationic dextran, dihydroxyphenylalanine (DOPA), dopamine, norepinephrine, epinephrine, epigallocatechin, and derivatives thereof.

[0117] The cell culture microcarriers produced by the method for producing cell culture microcarriers described above may have an epoxy content of 40 μmol / g or more and 100 μmol / g or less.

[0118] Specifically, the microcarrier for cell culture may have an epoxy content of 40 μmol / g or more, 45 μmol / g or more, 49 μmol / g or more, 49.5 μmol / g or more, 40 μmol / g or more and 100 μmol / g or less, 45 μmol / g or more and 100 μmol / g or less, 49 μmol / g or more and 100 μmol / g or less, or 49.5 μmol / g or more and 100 μmol / g or less.

[0119] The epoxy content can be calculated by adding the cell culture microcarrier to an acidic solution, then adding an indicator solution, and titrating with NaOH, using the following formula.

[0120] [Mathematical formula] Epoxy content (μmol / g) = [V0(ml) - V(ml)] × C NaOH (mol / L)

[0121] In the above formula, V0 is the volume of NaOH added to the control group which contains deionized water instead of particles, V is the amount of NaOH added to the actual sample, and C NaOH This indicates the concentration of NaOH used in the titration.

[0122] By satisfying the epoxy content requirement, the aforementioned microcarriers for cell culture can achieve excellent cell adhesion. If the epoxy content is excessively reduced, technical problems may arise such as a decrease in particle dispersion and a reduction in the efficiency of introducing surface modifiers.

[0123] On the other hand, according to another embodiment of the invention, a method for producing microcarriers for cell culture can be provided, which includes the step of polymerizing and recovering polystyrene particles from a monomer mixture containing the compound represented by chemical formula 1.

[0124] In the method for producing microcarriers for cell culture according to the above embodiment, the details regarding polystyrene particles and the compound represented by chemical formula 1 include all of the above-mentioned details.

[0125] Specifically, the compound represented by chemical formula 1 may contain any one of the compounds represented by chemical formulas 1-1 through 1-3 below.

[0126] [ka]

[0127] In the above chemical formula 1-1, R 11 is hydrogen or an alkyl group having 1 or more carbon atoms,

[0128] [ka]

[0129] In the above chemical formulas 1 and 2, R 12 is hydrogen or an alkyl group having 1 or more carbon atoms,

[0130] [ka]

[0131] In the above chemical formulas 1 and 3, R 13 is hydrogen or an alkyl group having one or more carbon atoms.

[0132] By including one of the compounds represented by chemical formulas 1-1 to 1-3 as a monomer in a polystyrene polymer, and by including an epoxy group in the compounds represented by chemical formulas 1-1 to 1-3, reaction sites are provided on the surface of a microcarrier for cell culture where cell-adhering ligands can be chemically fixed without an additional coating layer, thereby improving cell adhesion.

[0133] On the other hand, the polystyrene particles may contain a monomer compound represented by the following chemical formula 2.

[0134] [ka]

[0135] In the above chemical formula 2, R2 to R6 are each independently hydrogen or an alkyl group having 1 or more carbon atoms, and at least one of R2 to R6 is an alkyl group having 1 or more carbon atoms.

[0136] By including the compound represented by chemical formula 2 as a polystyrene monomer, the density of the final cell culture microcarriers can be precisely controlled by adjusting the content of the compound represented by chemical formula 2, enabling separation from cells after cell culture is complete based on the density difference, while simultaneously improving dispersibility in the cell culture reactor or culture medium.

[0137] Specifically, the compound represented by chemical formula 2 may include one or more compounds selected from the group consisting of compounds represented by chemical formula 2-1 to compounds represented by chemical formula 2-3.

[0138] [ka]

[0139] In the aforementioned chemical formulas 2-1 to 2-3, R 21 ~R 26 Each of these is independently an alkyl group having one or more carbon atoms.

[0140] By including one or more compounds selected from the group consisting of compounds represented by chemical formulas 2-1 to 2-3 as polystyrene monomers, the density of the final cell culture microcarriers can be precisely controlled by adjusting the content of the compound represented by chemical formula 2, enabling separation from cells after cell culture is complete based on the density difference, while simultaneously improving dispersibility in the cell culture reactor or culture medium.

[0141] More specifically, the compound represented by chemical formula 2 may include one or more compounds selected from the group consisting of compounds represented by chemical formulas 2-4 to 6.

[0142] [ka]

[0143] In the aforementioned chemical formulas 2-4 to 2-6, R 21 ~R 26 Each of these is independently an alkyl group having one or more carbon atoms.

[0144] On the other hand, the compound represented by chemical formula 2 has a density of 0.92 g / cm³. 3 The following is also acceptable.

[0145] Specific には, the される compound shown in the chemical formula 2 mentioned above is は, and the density is 0.92g / cm 3 Below, 0.91g / cm 3 Below, 0.906 g / cm 3 Below, 0.9g / cm 3 Below, 0.89g / cm 3 Below, 0.5g / cm 3 Above, 0.6g / cm 3 Above, 0.7g / cm 3 Above, 0.8g / cm 3 Above, または0.5g / cm 3 Above 0.92g / cm 3 Below, 0.6g / cm 3 Above 0.92g / cm 3 Below, 0.7g / cm 3 Above 0.92g / cm 3 Below, 0.8g / cm 3 Above 0.92g / cm 3 Below, 0.5g / cm 3 Above 0.91g / cm 3 Below, 0.6g / cm 3 Above 0.91g / cm 3 Below, 0.7g / cm 3 Above 0.91g / cm 3 Below, 0.8g / cm 3 Above 0.91g / cm 3 Below, 0.5g / cm 3 Above 0.906 g / cm 3 Below, 0.6g / cm 3 Above 0.906 g / cm 3 Below, 0.7g / cm 3 Above 0.906 g / cm 3 Below, 0.8g / cm 3 Above 0.906 g / cm 3 Below, 0.5g / cm 3 Above 0.9g / cm 3 Below, 0.6g / cm 3 Above 0.9g / cm 3 Below, 0.7g / cm 3 Above 0.9g / cm 3 Below, 0.8g / cm 3 Above 0.9g / cm 3 Below, 0.5g / cm 3More than 0.89g / cm 3 Below, 0.6g / cm 3 More than 0.89g / cm 3 Below, 0.7g / cm 3 More than 0.89g / cm 3 Below, 0.8g / cm 3 More than 0.89g / cm 3 The following is also acceptable.

[0146] The density of the compound represented by the above chemical formula 2 is 0.92 g / cm³. 3 By satisfying the following conditions, the density of the microcarriers for final cell culture can be precisely controlled by adjusting the content of the compound represented by chemical formula 2, enabling separation from cells after cell culture is complete due to the density difference, while simultaneously improving dispersibility in the cell culture reactor or culture medium.

[0147] For example, the compound represented by chemical formula 2 may include 4-methylstyrene, trimethylstyrene, 4-ethenyl-2-methyl-1-(2-methylpropyl)benzene, 1-ethenyl-2-methyl-4-(1-methylethyl)benzene, 1-ethenyl-2,3-dimethylbenzene, 4-(1,1-dimethylethyl)-2-ethenyl-1-methylbenzene, 2-ethenyl-4-methyl-1-(1-methylethyl)benzene, 1-ethenyl-2-(1-methylethyl)benzene, 2-tert-butylstyrene, 4-tert-butylstyrene, and 1-ethenyl-3-(1-methylethyl)benzene.

[0148] On the other hand, the polystyrene particles may further contain, as monomeric compounds, the compound represented by the following chemical formula 3, in addition to the compound represented by chemical formula 1.

[0149] [ka]

[0150] In the aforementioned chemical formula 3, L 30 is -O(C=O)- or -(C=O)O-, and R 30R is a directly bonded alkylene group having 1 or more carbon atoms. 31 It is an alkyl group having one or more carbon atoms.

[0151] In other words, the polystyrene particles may contain the compound represented by chemical formula 1 as a monomer compound, or contain the compound represented by chemical formula 1 and the compound represented by chemical formula 2, or contain the compound represented by chemical formula 1 and the compound represented by chemical formula 3, or contain the compound represented by chemical formula 1, the compound represented by chemical formula 2 and the compound represented by chemical formula 3.

[0152] By including the compound represented by chemical formula 3 as a polystyrene monomer, the substituents of the final manufactured cell culture microcarrier can be hydrolyzed, improving its dispersibility in the cell culture reactor or culture medium.

[0153] For example, the compound represented by chemical formula 3 may contain acetoxystyrene.

[0154] On the other hand, the monomer mixture may contain 20 to 99 parts by weight of the compound represented by chemical formula 2 per 100 parts by weight of the monomer mixture.

[0155] The monomer mixture may include all of the compounds represented by chemical formula 1 and the styrene monomer mixture.

[0156] Specifically, the monomer mixture may contain, per 100 parts by weight, the compound represented by chemical formula 2 in amounts of 20 parts by weight or more, 30 parts by weight or more, 50 parts by weight or more, 75 parts by weight or more, 80 parts by weight or more, 90 parts by weight or more, 99 parts by weight or less, 95 parts by weight or less, 20 parts by weight or more and 99 parts by weight or less, 30 parts by weight or more and 99 parts by weight or less, 50 parts by weight or more and 99 parts by weight or less, 75 parts by weight or more and 99 parts by weight or less, 80 parts by weight or more and 99 parts by weight or less, 90 parts by weight or more and 99 parts by weight or less, 20 parts by weight or more and 95 parts by weight or less, 30 parts by weight or more and 95 parts by weight or less, 50 parts by weight or more and 95 parts by weight or less, 75 parts by weight or more and 95 parts by weight or less, 80 parts by weight or more and 95 parts by weight or less, and 90 parts by weight or more and 95 parts by weight or less.

[0157] If the monomer mixture contains less than 20 parts by weight of the compound represented by chemical formula 2 per 100 parts by weight, a technical problem may occur where the particle density becomes high and the low-density properties are lost. If it contains more than 99 parts by weight, the particle density becomes 0.99 g / cm³. 3 When the temperature drops below a certain level, technical problems can arise where rapid agitation for dispersion within the culture medium can affect cell properties.

[0158] On the other hand, the monomer mixture may contain 0.1 parts by weight or more and 5 parts by weight or less of the compound represented by chemical formula 3, per 100 parts by weight of the monomer mixture.

[0159] The monomer mixture may include all of the compounds represented by chemical formula 1, the styrene monomer mixture, and the ethylene-based unsaturated crosslinking agent.

[0160] Specifically, the monomer mixture may contain, per 100 parts by weight, the compound represented by chemical formula 3 in amounts of 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, and 1 part by weight or more and 2 parts by weight or less.

[0161] If the monomer mixture contains less than 0.1 parts by weight of the compound represented by chemical formula 3 per 100 parts by weight, the surface hydrophilicity will be insufficient, resulting in poor dispersibility in the culture vessel. If it contains more than 5 parts by weight, the particle density will increase, leading to technical problems where the low-density properties are lost.

[0162] On the other hand, according to one embodiment of the invention, the step of polymerizing and recovering polystyrene particles from a monomer mixture containing the compound represented by chemical formula 1 may include the step of reacting the monomer mixture containing the compound represented by chemical formula 1 with an ethylene-based unsaturated crosslinking agent to polymerize and recover polystyrene particles.

[0163] The ethylene-based unsaturated crosslinking agent includes all of the above-mentioned components.

[0164] In the method for producing the microcarriers for cell culture, the compound represented by chemical formula 2 may be included in an amount of 80 to 150 parts by weight per 100 parts by weight of the ethylene-based unsaturated crosslinking agent.

[0165] More specifically, the compound represented by chemical formula 2 may be included in amounts of 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, or 80 parts by weight or more and 150 parts by weight or less, 85 parts by weight or more and 150 parts by weight or less, 90 parts by weight or more and 150 parts by weight or less, 80 parts by weight or more and 125 parts by weight or less, 85 parts by weight or more and 125 parts by weight or less, 90 parts by weight or more and 125 parts by weight or less, 80 parts by weight or more and 100 parts by weight or less, 85 parts by weight or more and 100 parts by weight or less, 90 parts by weight or more and 100 parts by weight or less, 80 parts by weight or more and 99 parts by weight or less, 85 parts by weight or more and 99 parts by weight or less, and 90 parts by weight or more and 99 parts by weight or less per 100 parts by weight of the ethylene-based unsaturated crosslinking agent.

[0166] By including 80 to 150 parts by weight of the compound represented by chemical formula 2 with 100 parts by weight of the ethylene-based unsaturated crosslinking agent, the low density of the compound represented by chemical formula 2 can control the low density characteristics of the particles, enabling separation from cells due to the density difference, while simultaneously improving dispersibility in cell culture reactors or culture media.

[0167] If the compound represented by chemical formula 2 is included in an excessively small amount relative to 100 parts by weight of the ethylene-based unsaturated crosslinking agent, the particle density may increase, causing it to lose its low-density properties and resulting in sedimentation in the culture medium. If it is included in excessive amounts, the particle density may reach 0.99 g / cm³. 3 When the levels are below or very low, technical problems can arise where rapid agitation is required to disperse the cells in the culture medium, affecting their properties.

[0168] The monomer mixture may include the compound represented by chemical formula 1 and a styrene-based monomer mixture.

[0169] The styrene monomer mixture may contain 90 to 100 parts by weight of the compound represented by chemical formula 2 per 100 parts by weight.

[0170] Specifically, the styrene monomer mixture may contain 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 per 100 parts by weight of the compound represented by chemical formula 2.

[0171] By including the compound represented by chemical formula 2 in the above-mentioned amount per 100 parts by weight of the styrene monomer mixture, the density of the final cell culture microcarriers can be precisely controlled, enabling separation from cells due to density differences, while simultaneously improving dispersibility in the cell culture reactor or culture medium.

[0172] Furthermore, the compound represented by chemical formula 1 may be included in an amount of 1 to 15 parts by weight per 100 parts by weight of the compound represented by chemical formula 2.

[0173] Specifically, per 100 parts by weight of the compound represented by chemical formula 2, the compound represented by chemical formula 1 may be included in amounts of 1 part by weight or more, 1.5 parts by weight or more, 15 parts by weight or less, 12 parts by weight or less, 10 parts by weight or less, 6 parts by weight or less, 1 part by weight or more and 15 parts by weight or less, 1.5 parts by weight or more and 15 parts by weight or less, 1 part by weight or more and 12 parts by weight or less, 1.5 parts by weight or more and 12 parts by weight or less, 1 part by weight or more and 10 parts by weight or less, 1 part by weight or more and 6 parts by weight or less, and 1.5 parts by weight or more and 6 parts by weight or less.

[0174] If the compound represented by chemical formula 1 is present in an excessively small amount relative to 100 parts by weight of the compound represented by chemical formula 2, there may not be enough reaction sites for the cell-adhering ligand to be immobilized, resulting in poor cell adhesion. Conversely, if the compound is present in an excessive amount, technical problems may arise where the particle density increases, causing a loss of low-density properties.

[0175] Furthermore, if the monomer compound contains a compound represented by the following chemical formula 3, the compound represented by chemical formula 3 may be present in an amount of 0.1 parts by weight or more and 5 parts by weight or less per 100 parts by weight of the ethylene-based unsaturated crosslinking agent.

[0176] More specifically, the compound represented by chemical formula 3 may be included in amounts of 0.1 parts by weight or more, 0.5 parts by weight or more, 1 part by weight or more, 5 parts by weight or less, 3 parts by weight or less, 2 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, or 1 part by weight or more and 2 parts by weight or less per 100 parts by weight of the ethylene-based unsaturated crosslinking agent.

[0177] When the monomer compound contains the compound represented by the following chemical formula 3, by including 0.1 to 5 parts by weight of the compound represented by chemical formula 3 per 100 parts by weight of the ethylene-based unsaturated crosslinking agent, the substituents of the final cell culture microcarrier can be hydrolyzed, thereby improving the dispersibility in the cell culture reactor or culture medium.

[0178] When the compound represented by chemical formula 3 is included in an amount exceeding 5 parts by weight per 100 parts by weight of the ethylene-based unsaturated crosslinking agent, technical problems may occur where the particle density increases and the low-density properties are lost.

[0179] Furthermore, if the monomer compound includes a compound represented by the following chemical formula 3, the styrene monomer mixture may contain 0.1 parts by weight or more and 5 parts by weight or less of the compound represented by the chemical formula 3 per 100 parts by weight.

[0180] Specifically, the styrene monomer mixture may contain, per 100 parts by weight, the compound represented by chemical formula 3 in amounts of 0.1 parts by weight or more, 0.5 parts by weight or more, 1 part by weight or more, 5 parts by weight or less, 3 parts by weight or less, 2.5 parts by weight or less, 0.1 parts by weight or more and 5 parts by weight or less, 0.5 parts by weight or more and 5 parts by weight or less, 1 part by weight or more and 5 parts by weight or less, 0.1 parts by weight or more and 3 parts by weight or less, 0.5 parts by weight or more and 3 parts by weight or less, 1 part by weight or more and 3 parts by weight or less, 0.1 parts by weight or more and 2.5 parts by weight or less, 0.5 parts by weight or more and 2.5 parts by weight or less, and 1 part by weight or more and 2.5 parts by weight or less.

[0181] When the monomer compound includes a compound represented by the following chemical formula 3, by including 0.1 to 5 parts by weight of the compound represented by chemical formula 3 per 100 parts by weight of the styrene monomer mixture, the substituents of the final cell culture microcarrier can be hydrolyzed, thereby improving the dispersibility in the cell culture reactor or culture medium.

[0182] Furthermore, if the monomer compound contains a compound represented by the following chemical formula 3, the compound represented by chemical formula 1 may be included in an amount of 110 to 500 parts by weight per 100 parts by weight of the compound represented by chemical formula 3.

[0183] Specifically, per 100 parts by weight of the compound represented by chemical formula 3, the compound represented by chemical formula 1 may be included in amounts of 110 parts by weight or more, 150 parts by weight or more, 500 parts by weight or less, 400 parts by weight or less, 300 parts by weight or less, 200 parts by weight or less, or 110 parts by weight or more and 500 parts by weight or less, 110 parts by weight or more and 400 parts by weight or less, 110 parts by weight or more and 300 parts by weight or less, 110 parts by weight or more and 200 parts by weight or less, 150 parts by weight or more and 500 parts by weight or less, 150 parts by weight or more and 400 parts by weight or less, 150 parts by weight or more and 300 parts by weight or less, or 150 parts by weight or more and 200 parts by weight or less.

[0184] If the compound represented by chemical formula 1 is included in an amount of less than 110 parts by weight per 100 parts by weight of the compound represented by chemical formula 3, a problem may occur where the compound represented by chemical formula 1 is washed away before polymerization due to its relatively high water solubility and is not introduced to the particle surface. If it is included in an amount exceeding 500 parts by weight, a technical problem may occur where the particle density becomes high and the low-density properties are lost.

[0185] In the method for producing the microcarriers for cell culture, the step of polymerizing and recovering the polystyrene particles may include a step of carrying out a suspension polymerization reaction of the monomer composition and recovering the result of the suspension polymerization reaction.

[0186] More specifically, the suspension polymerization reaction of the monomer composition may include the steps of: mixing the monomer composition with an aqueous dispersion and applying shear force to homogenize the monomer composition in the aqueous dispersion into droplet form; and performing suspension polymerization of the homogenized monomer composition at a stirring speed of 300 rpm to 1000 rpm.

[0187] In the step of homogenizing the monomer composition into droplet form in an aqueous dispersion, stirring can be performed at a stirring speed of 300 rpm to 1000 rpm, or 400 rpm to 800 rpm.

[0188] In the step of suspension polymerization of the homogenized monomer composition at a stirring speed of 300 rpm to 1000 rpm or 400 rpm to 800 rpm, it is possible to produce microcarriers with a high proportion of perfectly spherical particles without damage or destruction, while lowering the density of microcarriers due to the structure of the compound represented by chemical formula 1 during the formation of the polystyrene particle structure.

[0189] In the step of suspension polymerization of the homogenized monomer composition at a stirring speed of 300 rpm to 1000 rpm, or 400 rpm to 800 rpm, the examples of the suspension polymerization conditions are not very limited, but for example, the process can be carried out at a temperature of 50°C to 100°C for 3 to 18 hours.

[0190] On the other hand, the method for producing the microcarriers for cell culture may further include washing and drying steps after the step of polymerizing and recovering polystyrene particles from a monomer mixture containing the compound represented by chemical formula 1.

[0191] Specifically, the washing step may include filtering the reaction product through a 30 μm to 100 μm sieve, followed by stirring it 5 to 7 times at room temperature in 100% ethanol.

[0192] The aforementioned drying step includes a step of vacuum drying at room temperature in a vacuum oven. However, it is not limited to this, and any commonly known drying method can be used without any particular restrictions.

[0193] On the other hand, in the present invention, cell-adhering substances can be introduced to the particle surface by chemical bonding. For example, the present invention may include a step of modifying the particles with a solution containing one or more selected from the group consisting of gelatin, collagen, fibronectin, chitosan, poly-L-lysine, vitronectin, peptides including RGD, lignin, cationic dextran, dihydroxyphenylalanine (DOPA), dopamine, norepinephrine, epinephrine, epigallocatechin, and derivatives thereof.

[0194] A solution containing one or more selected from the group consisting of gelatin, collagen, fibronectin, chitosan, poly-L-lysine, vitronectin, peptides including RGD, lignin, cationic dextran, dihydroxyphenylalanine (DOPA), dopamine, norepinephrine, epinephrine, epigallocatechin, and derivatives thereof can act as an adhesion factor that adheres cells to microcarriers, increasing the degree of adhesion between cells and microcarriers, and thus becoming suitable for large-scale cell culture.

[0195] Specifically, the step of introducing the cell adhesion induction layer may include immersing the surface of the polystyrene particles in a solution containing one or more selected from the group consisting of gelatin, collagen, fibronectin, chitosan, poly-L-lysine, vitronectin, peptides including RGD, lignin, cationic dextran, dihydroxyphenylalanine (DOPA), dopamine, norepinephrine, epinephrine, epigallocatechin, and derivatives thereof for 10 to 20 hours, or 15 to 20 hours, or 17 to 19 hours to react.

[0196] The cell culture microcarriers produced by the method for producing cell culture microcarriers described above may have an epoxy content of 40 μmol / g or more and 100 μmol / g or less.

[0197] Specifically, the microcarrier for cell culture may have an epoxy content of 40 μmol / g or more, 45 μmol / g or more, 49 μmol / g or more, 49.5 μmol / g or more, 40 μmol / g or more and 100 μmol / g or less, 45 μmol / g or more and 100 μmol / g or less, 49 μmol / g or more and 100 μmol / g or less, or 49.5 μmol / g or more and 100 μmol / g or less.

[0198] The epoxy content can be calculated by adding the cell culture microcarrier to an acidic solution, then adding an indicator solution, and titrating with NaOH, using the following formula.

[0199] [Mathematical formula] Epoxy content (μmol / g) = [V0(ml) - V(ml)] × C NaOH (mol / L)

[0200] In the above formula, V0 is the volume of NaOH added to the control group which contains deionized water instead of particles, V is the amount of NaOH added to the actual sample, and C NaOH This indicates the concentration of NaOH used in the titration.

[0201] By satisfying the epoxy content requirement, the aforementioned microcarriers for cell culture can achieve excellent cell adhesion. If the epoxy content is excessively reduced, technical problems may arise such as a decrease in particle dispersion and a reduction in the efficiency of introducing surface modifiers.

[0202] According to another embodiment of the invention, a cell culture composition comprising cells and the cell culture microcarrier of the first embodiment can be provided. The cell culture microcarrier includes all of the contents described above in the first embodiment.

[0203] The aforementioned cells are not limited to adherent animal cells, but may include, for example, fibroblasts, epithelial cells, osteoblasts, chondrocytes, hepatocytes, human umbilical cord blood cells, human bone marrow-derived mesenchymal stem cells, CHO (Chinese hamster ovary) cells, kidney cells (HEK293, BHK21, MDCK, vero cells, etc.), or mixtures of two or more of these.

[0204] Furthermore, the density difference between the cell culture microcarriers and the cells is 0.20 g / cm³. 3 The following may also apply: The density difference between the cell culture microcarrier and the cells is 0.20 g / cm³. 3 By satisfying the following conditions, when separating and recovering microcarriers from cells after cell culture, the difference in sedimentation rates due to gravity allows for easy separation of cells and microcarriers, while simultaneously preventing the problem in the early stages of culture where microcarriers float only on the surface of the culture medium, making it difficult for cells to adhere to them.

[0205] The cell culture composition may further include a culture medium solution. The culture medium solution can contain various additives for sufficiently satisfying the environmental conditions such as nutrients, pH, temperature, and osmotic pressure that are close to the biological conditions based on body fluids such as plasma and lymph fluid. Various substances widely known in the technical field related to cell culture can be used without limitation for this.

[0206] As an example, the microcarrier for cell culture of the above-described embodiment has a density smaller than that of the culture medium solution, is injected into the culture medium solution, and floats inside the culture medium solution under stirring conditions. Subsequently, as the number of cells adhering to the surface of the low-density microcarrier increases, the density of the microcarrier to which the cells are attached (hereinafter referred to as "microcarrier-cell conjugate") gradually increases and gradually sinks in the culture medium solution.

[0207] Therefore, after adding a cell detachment enzyme to the microcarrier to which cells are attached (microcarrier-cell conjugate) and separating it by centrifugation to separate the cells from the microcarrier-cell conjugate, the cultured cells can be easily obtained.

Advantages of the Invention

[0208] According to the present invention, there can be provided a microcarrier for cell culture having high surface hydrophilicity and improved cell adhesiveness and having improved dispersibility in a cell culture reactor or a culture medium, a method for producing the microcarrier for cell culture, and a cell culture method using the same.

Brief Description of the Drawings

[0209] [Figure 1] It is a SEM image of the surface shape of the microcarrier for cell culture of Example 1.

Modes for Carrying Out the Invention

[0210] The invention will be described in more detail with the following examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following examples.

[0211] Example 1 Polyvinyl alcohol (molecular weight 85-124K, 87-89% hydrolysis) was dissolved in distilled water at a concentration of 2% to prepare an aqueous dispersion, which was then stirred at room temperature for 20 minutes.

[0212] A monomer composition was prepared by mixing styrene, glycidyl methacrylate, and t-butylstyrene monomers with divinylbenzene, a crosslinking agent, in a weight ratio of 0.05:0.05:0.9:1, dissolving them thoroughly in 25 g of the mixture, and then adding 2% by weight of V-65 initiator (amount of initiator added: based on the total amount of monomers and crosslinking agent) and stirring for an additional 5 minutes.

[0213] 600 g of aqueous dispersion was added to a 1 L reactor, and the monomer composition was added. A shear force was applied to the aqueous dispersion and monomer composition at a speed of 400 rpm at room temperature, and the monomer composition was dispersed in the aqueous dispersion into fine droplets and homogenized.

[0214] The homogenized mixture was reacted at 85°C for 6 hours under nitrogen purging while being stirred at a stirring speed of 400 rpm to produce polystyrene particles. The particles were washed twice with distilled water at 60°C and five times with ethanol, and then dried in an oven at 80°C to recover. The recovered polystyrene particles were used as microcarriers for cell culture.

[0215] The physical properties of the aforementioned polystyrene particles are as follows:

[0216] Average diameter: 178 μm (measured using a PSA instrument, based on D50 standard) Apparent density: 0.99~1.04 g / cm³ 3

[0217] Example 2 A microcarrier for cell culture was produced in the same manner as in Example 1, except that the weight ratio of the monomers styrene, glycidyl methacrylate, and t-butylstyrene to the crosslinking agent divinylbenzene was adjusted to 0.07:0.03:0.9:1.

[0218] Example 3 Microcarriers for cell culture were produced in the same manner as in Example 1, except that the weight ratio of the monomers styrene, glycidyl methacrylate, and t-butylstyrene to the crosslinking agent divinylbenzene was adjusted to 0.085:0.015:0.9:1.

[0219] Example 4 In Example 1, the monomers styrene, glycidyl methacrylate, t-butylstyrene, and acetoxystyrene (density: 1.06 g / cm³) were used. 3 A microcarrier for cell culture was manufactured in the same manner as in Example 1, except that the weight ratio of the crosslinking agent, divinylbenzene, was adjusted to 0.06:0.02:0.91:0.01:1.

[0220] Example 5 In Example 1, the monomers styrene, glycidyl methacrylate, t-butylstyrene, and acetoxystyrene (density: 1.06 g / cm³) were used. 3 A microcarrier for cell culture was manufactured in the same manner as in Example 1, except that the weight ratio of the crosslinking agent, divinylbenzene, was adjusted to 0.05:0.03:0.90:0.02:1.

[0221] Example 6 In Example 1, instead of the monomer glycidyl methacrylate, 2-(2-oxyranylmethoxy)ethyl methacrylate (density: 1.1 g / cm³) was used. 3A microcarrier for cell culture was produced in the same manner as in Example 1, except that 2-(2-oxiranylmethoxy)ethyl methacrylate was used.

[0222] Example 7 In Example 1, without adding the monomer styrene and using 4-vinylphenyl glycidyl ether instead of glycidyl methacrylate, while using t-butylstyrene and 4-vinylphenyl glycidyl ether (density: 1.1 g / cm 3 A microcarrier for cell culture was produced in the same manner as in Example 1, except that the weight ratio of 4-vinylphenyl glycidyl ether (4-vinylphenylglycidyl ether) to divinylbenzene as a crosslinking agent was adjusted to 0.9:0.1:1.

[0223] Example 8 A microcarrier for cell culture was produced in the same manner as in Example 1, except that the weight ratio of the monomers styrene, glycidyl methacrylate, t-butylstyrene to divinylbenzene as a crosslinking agent was adjusted to 0.1:0.1:0.8:1.

[0224] Example 9 A microcarrier for cell culture was produced in the same manner as in Example 1, except that the weight ratio of the monomers styrene, glycidyl methacrylate, t-butylstyrene to divinylbenzene as a crosslinking agent was adjusted to 0.15:0.05:0.8:1.

[0225] Example 10 A microcarrier for cell culture was produced in the same manner as in Example 1, except that the weight ratio of the monomers styrene, glycidyl methacrylate, t-butylstyrene to divinylbenzene as a crosslinking agent was adjusted to 0.075:0.075:0.85:1.

[0226] Example 11 Microcarriers for cell culture were produced in the same manner as in Example 1, except that the weight ratio of the monomers styrene, glycidyl methacrylate, and t-butylstyrene to the crosslinking agent divinylbenzene was adjusted to 0.1:0.05:0.85:1.

[0227] Example 12 Microcarriers for cell culture were produced in the same manner as in Example 1, except that the weight ratio of the monomers styrene, glycidyl methacrylate, and t-butylstyrene to the crosslinking agent divinylbenzene was adjusted to 0.02:0.08:0.9:1.

[0228] Example 13 A microcarrier for cell culture was produced in the same manner as in Example 1, except that the weight ratio of the monomers styrene, glycidyl methacrylate, and t-butylstyrene to the crosslinking agent divinylbenzene was adjusted to 0.9:0.1:0:1.

[0229] Comparative Example 1 In the same manner as in Example 1, a microcarrier for cell culture was produced, except that the weight ratio of the monomers styrene, glycidyl methacrylate, and t-butylstyrene to the crosslinking agent divinylbenzene was adjusted to 0.1:0:0.9:1.

[0230] <Experimental Example: Measurement of Physical Properties of Microcarriers for Cell Culture> The physical properties of the cell culture microcarriers obtained in the above examples and comparative examples were measured using the following method, and the results are shown in Tables 1 and 2.

[0231] Experiment 1. Average particle size (unit: μm) The cell culture microcarriers obtained in the above examples and comparative examples were dispersed in ethanol at a 10% by weight level, and then the D50 (particle size value corresponding to the cumulative distribution percentage reaching 50%) particle diameter was measured using a PSA (Particle Size Analysis) instrument.

[0232] Experiment 2. Analysis of epoxy content 0.1 g of the cell culture microcarriers prepared in the above examples and comparative examples were dispersed in an HCl / acetone (volume ratio = 1:80) solution and then sonicated for 4 minutes.

[0233] 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) were added, and the solution was titrated with 0.1 M NaOH.

[0234] The epoxy content (μmol / g) was calculated using the following formula.

[0235] [Mathematical formula] Epoxy content (μmol / g) = [V0(ml) - V(ml)] × C NaOH (mol / L)

[0236] In the above formula, V0 is the volume of NaOH added to the control group containing 0.1g of DIW instead of particles, V is the amount of NaOH added to the actual sample, and C NaOH This indicates the concentration of NaOH used in the titration.

[0237] Experiment 3. Particle dispersion in the incubator After dispersing 1 g of the aforementioned cell culture microcarrier in 4 ml of 1x PBS, 10 mg of GRGDSK peptide was added and the mixture was reacted for 18 hours. The mixture was then washed three times with 1x PBS to produce cell culture microcarriers to which the peptide had adhered.

[0238] Before culturing, the necessary amount of the cell culture microcarriers was dispersed in a 20 mL glass vial with the culture medium and wetted for approximately 10-18 hours. After filtering the dispersed cell culture microcarriers with a cell strainer, they were added to a 100 mL 3D bioreactor along with 60 mL of culture medium, and mesenchymal stem cells (density: 1.05 g / cm³) were cultured. 3 The culture medium containing ) was filled and incubated for 24 hours. (Culture conditions: 37°C, 5% CO2 incubator, bioreactor 25 rpm stirring operation)

[0239] After 24 hours of incubation, particles that were not dispersed inside the incubator but floating at the interface of the culture medium were filtered and removed. The particles dispersed inside the incubator were thoroughly washed with DPBS or water, dried, and weighed. The degree of dispersion was calculated by determining the percentage ratio of the weight of dispersed particles to the total weight, and evaluated under the following criteria.

[0240] Best: Dispersion of 90% or more and 100% or less Top: Dispersion of 80% or more and 90% or less Medium: Dispersion of 60% or more and less than 80% Bottom: Dispersion less than 60%

[0241] Experiment 4. Initial degree of cell adhesion After dispersing 1 g of the aforementioned cell culture microcarrier in 4 ml of 1x PBS, 10 mg of GRGDSK peptide was added and the mixture was reacted for 18 hours. The mixture was then washed three times with 1x PBS to produce cell culture microcarriers to which the peptide had adhered.

[0242] Before culturing, 1 g of the aforementioned cell culture microcarriers, the amount required for cell culture, was pre-dispersed in a 20 mL glass vial with the culture medium and wetted for approximately 10-18 hours. After filtering the pre-dispersed cell culture microcarriers with a cell strainer, they were added to a 100 mL 3D bioreactor along with 60 mL of culture medium, and mesenchymal stem cells (density: 1.05 g / cm³) were cultured. 3The cells were incubated for 24 hours in a culture medium containing 1,800,000 cells. (Culture conditions: 37°C, 5% CO2 incubator, bioreactor 25 rpm stirring)

[0243] After 24 hours of incubation, 1 mL of the particle dispersion was collected from the bioreactor vessel. Using a Nucleocounter NC-200 (Chemometec) instrument, the percentage ratio of cells attached to the cell culture microcarrier to the total number of cells was determined and evaluated under the following criteria.

[0244] Best: Percentage ratio of cells attached to microcarriers relative to the total number of cells: 93% or higher and 100% or lower. Top: Percentage ratio of cells attached to microcarriers relative to the total number of cells: 85% or more and less than 93%. Medium: Percentage ratio of cells attached to microcarriers relative to the total number of cells: 60% or more and less than 85% Bottom: Percentage of cells attached to microcarriers relative to the total number of cells is less than 60%.

[0245] [Table 1]

[0246] As shown in Table 1 above, the microcarriers for cell culture in the examples were found to exhibit excellent cell adhesion due to improved particle dispersion under cell culture conditions and the ability to perform ligand peptide reactions for cell adhesion.

[0247] In Comparative Example 1, it was confirmed that the degree of dispersion and initial cell adhesion were poorer compared to the example.

[0248] Experiment 5. Apparent Density (unit: g / cm³) 3 ) The cell culture microcarriers produced in the above examples and comparative examples were subjected to a density of 0.99 g / cm³ at room temperature (25°C) and atmospheric pressure (1 atm). 3Distilled water, or water with a density of 1.01 g / cm³ 3 The apparent density was evaluated by adding each of the following to the cell culture medium and observing whether the particles floated or settled. The cell culture medium was prepared with 94.9% by weight Advanced MEM, 5% by weight Fetal bovine serum, and 1% by weight Gentamicin.

[0249] The cell culture microcarriers produced in the above examples and comparative examples were subjected to a density of 0.985 g / cm³ at room temperature (25°C) and atmospheric pressure (1 atm). 3 0.99 g / cm³ 3 , 0.997 g / cm³ 3 An aqueous solution of ethanol with a density of 1.02 g / cm³ 3 , 1.04 g / cm³ 3 The particles were added to each glycerol aqueous solution, and it was confirmed whether they floated or settled. The apparent density was then evaluated under the following criteria.

[0250] 1) 0.985 g / cm³ 3 <d<0.99g / cm 3 (0.985g / cm 3 Excess 0.99g / cm 3 less than) Density is 0.985 g / cm³ 3 It precipitates in an ethanol aqueous solution and has a density of 0.99 g / cm³. 3 Floating in an ethanol aqueous solution 2) 0.99 g / cm³ 3 <1.02 g / cm³ 3 (0.99g / cm 3 Excess 1.02g / cm 3 less than) Density is 0.99 g / cm³ 3 It precipitates in an ethanol aqueous solution and has a density of 1.02 g / cm³. 3 Floating in a glycerol aqueous solution 3) 0.99 g / cm³ 3 <1.04 g / cm³ 3 (0.99g / cm 3 Excess 1.04g / cm 3 less than) Density is 0.99 g / cm³ 3It precipitates in an ethanol aqueous solution and has a density of 1.04 g / cm³. 3 Floating in a glycerol aqueous solution 4) 0.99 g / cm³ 3 <d<1.01g / cm 3 (0.99g / cm 3 Excess 1.01g / cm 3 less than) Density is 0.99 g / cm³ 3 It settles in distilled water and has a density of 1.01 g / cm³. 3 Suspended in the cell culture medium 5) d > 1.04 g / cm³ 3 (1.04 g / cm³) 3 excess) Density is 1.04 g / cm³ 3 Precipitation in glycerol aqueous solution

[0251] Experiment 6. Feasibility of separation based on density difference. The cell culture microcarriers produced in the above examples and comparative examples were evaluated as follows by centrifuging a mixture of cell culture medium and microcarriers to determine whether separation of cells and the cell culture microcarriers was possible due to density differences under normal temperature (25°C) and atmospheric pressure (1 atm).

[0252] O: No particles settled at the bottom after centrifugation. X: Some particles settled at the bottom after centrifugation.

[0253] Experiment 7. Observation of surface shape The surface shape of the cell culture microcarriers produced in Example 1 was observed using a SEM (JEOL JSM7610F), and these are shown in Figure 1.

[0254] [Table 2]

[0255] As shown in Table 2 above, the microcarrier for cell culture in Examples 1-7 was 1.04 g / cm³. 3The following low densities are suitable for cell culture, confirming that microcarriers can be separated based on density differences under cell culture conditions.

[0256] In contrast, in Examples 8-13, it was confirmed that separation of microcarriers due to density differences was impossible because the particle density exceeded the density of the cell culture medium, thus preventing the acquisition of low-density characteristics.

[0257] In the case of Comparative Example 1, we were able to confirm that the apparent density becomes excessively low, which can lead to a problem where microcarriers float only on the surface of the culture medium during the initial stages of culture, making it difficult for cells to adhere.

Claims

1. Microcarriers for cell culture, comprising polystyrene particles containing a monomer compound represented by the following chemical formula 1; 【Chemistry 1】 In the aforementioned chemical formula 1, L ０ This is an arylene group with 6 or more carbon atoms or -(C=O)-, L １ and L ２ Each of these is independently an alkylene group having one or more carbon atoms. R １ It is a reactive functional group capable of ring-opening reactions, R １０ is hydrogen or an alkyl group having 1 or more carbon atoms, n is a non-negative integer.

2. The polystyrene particles include the reaction product of the compound represented by chemical formula 1 and an ethylene-based unsaturated crosslinking agent. The microcarrier for cell culture according to claim 1, comprising 1 to 15 parts by weight of the compound represented by chemical formula 1 per 100 parts by weight of the ethylene-based unsaturated crosslinking agent.

3. The cell culture microcarrier according to claim 1, wherein the compound represented by chemical formula 1 comprises one of the compounds represented by chemical formulas 1-1 to 1-3 below: 【Chemistry 2】 In the aforementioned chemical formula 1-1, R １１ is hydrogen or an alkyl group having 1 or more carbon atoms, 【Transformation 3】 In the above chemical formula 1-2, R １２ is hydrogen or an alkyl group having 1 or more carbon atoms, 【Chemistry 4】 In the above chemical formulas 1 and 3, R １３ is hydrogen or an alkyl group having one or more carbon atoms.

4. The aforementioned polystyrene particles are The microcarrier for cell culture according to claim 1, comprising a compound represented by the following chemical formula 2 as a monomeric compound: 【Transformation 5】 In the aforementioned chemical formula 2, R ２ to R ６ are each independently hydrogen or an alkyl group having 1 or more carbon atoms, The aforementioned R ２ ~R ６ At least one of them is an alkyl group having one or more carbon atoms.

5. The microcarrier for cell culture according to claim 4, wherein the compound represented by chemical formula 2 comprises one or more compounds selected from the group consisting of compounds represented by chemical formula 2-1 to compounds represented by chemical formula 2-3 below: 【Transformation 6】 In the aforementioned chemical formulas 2-1 to 2-3, R ２１ ~R ２６ Each of these is independently an alkyl group having one or more carbon atoms.

6. The compound represented by chemical formula 2 has a density of 0.92 g / cm³. ３ The following is a microcarrier for cell culture according to claim 4.

7. The polystyrene particles include the reaction product of the compound represented by chemical formula 1, a styrene monomer mixture, and an ethylene-based unsaturated crosslinking agent. The microcarrier for cell culture according to claim 4, comprising 80 to 150 parts by weight of the compound represented by chemical formula 2 per 100 parts by weight of the ethylene-based unsaturated crosslinking agent.

8. The polystyrene particles include the reaction product of the compound represented by chemical formula 1, a styrene monomer mixture, and an ethylene-based unsaturated crosslinking agent. The microcarrier for cell culture according to claim 4, comprising 90 to 100 parts by weight of the compound represented by chemical formula 2 per 100 parts by weight of the styrene monomer mixture.

9. The polystyrene particles include the reaction product of the compound represented by chemical formula 1, a styrene monomer mixture, and an ethylene-based unsaturated crosslinking agent. The microcarrier for cell culture according to claim 4, comprising 1 to 15 parts by weight of the compound represented by chemical formula 1 with respect to 100 parts by weight of the compound represented by chemical formula 2.

10. The cell culture microcarrier according to claim 1, comprising a compound represented by the following chemical formula 3 as a monomeric compound: 【Transformation 7】 In the aforementioned chemical formula 3, L ３０ is -O(C=O)- or -(C=O)O-, R ３０ These are directly bonded or alkylene groups having 1 or more carbon atoms. R ３１ It is an alkyl group having one or more carbon atoms.

11. The polystyrene particles include the reaction product of the compound represented by chemical formula 1, a styrene monomer mixture, and an ethylene-based unsaturated crosslinking agent. The microcarrier for cell culture according to claim 10, comprising 0.1 parts by weight or more and 5 parts by weight or less of the compound represented by chemical formula 3 per 100 parts by weight of the ethylene-based unsaturated crosslinking agent.

12. The polystyrene particles include the reaction product of the compound represented by chemical formula 1, a styrene monomer mixture, and an ethylene-based unsaturated crosslinking agent. The microcarrier for cell culture according to claim 10, comprising 0.1 parts by weight or more and 5 parts by weight or less of the compound represented by chemical formula 3 per 100 parts by weight of the styrene monomer mixture.

13. The polystyrene particles include the reaction product of the compound represented by chemical formula 1, a styrene monomer mixture, and an ethylene-based unsaturated crosslinking agent. The microcarrier for cell culture according to claim 10, comprising 110 to 500 parts by weight of the compound represented by chemical formula 1 with respect to 100 parts by weight of the compound represented by chemical formula 3.

14. The aforementioned microcarrier for cell culture is The cell culture microcarrier according to claim 1, comprising a cell adhesion-inducing layer formed on the polystyrene-based particles.

15. The apparent density of the microcarriers for cell culture is 0.99 g / cm³. ３ Excess: 1.04g / cm ３ A microcarrier for cell culture according to claim 1, which is less than [amount missing].

16. The cell culture microcarrier according to claim 1, wherein the D50 particle diameter of the cell culture microcarrier is 100 μm to 300 μm.

17. A method for producing microcarriers for cell culture, comprising the step of polymerizing and recovering polystyrene particles from a monomer mixture containing a compound represented by the following chemical formula 1: 【Transformation 8】 In the aforementioned chemical formula 1, L ０ This is an arylene group with 6 or more carbon atoms or -(C=O)-, L １ and L ２ Each of these is independently an alkylene group having one or more carbon atoms. R １ It is a reactive functional group capable of ring-opening reactions, R １０ is hydrogen or an alkyl group having 1 or more carbon atoms, n is a non-negative integer.

18. The monomer mixture comprises a compound represented by the following chemical formula 2, wherein the method for producing a microcarrier for cell culture according to claim 17: 【Chemistry 9】 In the aforementioned chemical formula 2, R ２ ~R ６ Each of these is independently a hydrogen atom or an alkyl group having one or more carbon atoms. The aforementioned R ２ ~R ６ At least one of them is an alkyl group having one or more carbon atoms.

19. The monomer mixture comprises a compound represented by the following chemical formula 3, wherein the method for producing a microcarrier for cell culture according to claim 17: 【Chemistry 10】 In the aforementioned chemical formula 3, L ３０ is -O(C=O)- or -(C=O)O-, R ３０ These are directly bonded or alkylene groups having 1 or more carbon atoms. R ３１ It is an alkyl group having one or more carbon atoms.

20. A method for producing a microcarrier for cell culture according to claim 17, further comprising the step of coating a cell adhesion-inducing layer onto the polystyrene particles.

21. A cell culture composition comprising cells and the cell culture microcarrier described in claim 1.

22. The cell culture composition according to claim 21, wherein the cells contain one or more compounds selected from the group consisting of fibroblasts, epithelial cells, osteoblasts, chondrocytes, hepatocytes, umbilical cord blood cells, mesenchymal stem cells, CHO cells, and kidney cells.

23. The apparent density difference between the cell culture microcarriers and the cells is 0.20 g / cm³. ３ The cell culture composition according to claim 21, which is as follows:

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