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

Abstract

The present invention relates to a microcarrier for cell culture and a cell culture composition using same, the microcarrier comprising: polystyrene-based particles; and a coating layer formed on the polystyrene-based particles and comprising polydopamine and poly(meth)acrylate.

Description

Microcarrier for cell culture, method for manufacturing a microcarrier for cell culture, and cell culture composition using the same

[0001] Cross-citation with related application(s)

[0002] This application claims the benefit of priority based on Korean Patent Application No. 10-2025-0024622 filed on February 25, 2025, and all contents disclosed in the document of said Korean patent application are incorporated herein as part of this specification.

[0003] The present invention relates to a microcarrier for cell culture, a method for manufacturing a microcarrier for cell culture, and a cell culture composition using the same.

[0004] As the fields of biopharmaceuticals and regenerative medicine expand, there is a growing demand for mass cell culture technology capable of efficiently producing cells, tissues, microorganisms, and more.

[0005] Adherent cells are cultured using microcarriers in a 3D bioreactor. Cells, culture medium, and microcarriers are placed inside the bioreactor, and the medium is stirred to bring the cells and microcarriers into contact, thereby causing the cells to attach to the surface of the microcarriers for culture. The microcarriers used in this process are suitable for large-scale cell culture because they provide a high surface area to volume ratio, which allows cells to attach and proliferate, compared to 2D culture.

[0006] Currently commercially available microcarriers have a density of approximately 1.1 to 1.3 g / cm³. 3 and the cell density is approximately 1.2 g / cm³ 3This is the extent of the problem. In this case, while it is advantageous for attaching cells during the initial stages of culture in the bioreactor, centrifugation is difficult for cell separation and recovery after culture, and filtering methods based on the size of microcarriers and cells must be used. However, this approach presents problems such as filter clogging, prolonged processing times, easy physical damage and contamination of cells, and potential cell loss.

[0007] To solve this problem, a density of 1.0 g / cm³ 3 Lower than or equal to 1.3 g / cm³ 3 Although microcarriers were manufactured using superior material properties, this method has the disadvantage that the achievable density range is limited and it is difficult to sufficiently secure a yield of microcarriers with perfect spherical shapes without damage or destruction.

[0008] The present invention relates to providing a microcarrier for cell culture that enables separation of cells by density control based on density difference, while simultaneously improving dispersibility in a cell culture reactor or medium and enhancing cell adhesion.

[0009] In addition, the present invention relates to a method for manufacturing the above-mentioned microcarrier for cell culture.

[0010] In addition, the present invention relates to a cell culture composition using the above-mentioned microcarrier for cell culture.

[0011] To solve the above problem, the present specification provides a microcarrier for cell culture comprising: polystyrene-based particles; and a coating layer formed on the polystyrene-based particles and comprising polydopamine and poly(meth)acrylate; wherein, in a spectrum measured by ATR-FTIR, the microcarrier for cell culture has a polydopamine coating rate calculated by the following Equation 1 of 70% or more and 98% or less, and an apparent density of 0.98 g / cm³ 3 Exceeding 1.01 g / cm³ 3Provides a microcarrier for cell culture that is less than [size].

[0012] [Mathematical Formula 1]

[0013] Polydopamine coating rate (%) = Area of ​​polydopamine present / Surface area of ​​microcarriers * 100.

[0014]

[0015] In this specification, the steps of polymerizing and recovering polystyrene-based particles are also included; and

[0016] A method for manufacturing a microcarrier for cell culture is provided, comprising the step of immersing recovered polystyrene-based particles in a solution containing a polydopamine precursor and poly(meth)acrylate and stirring at a temperature of 15°C or lower to form a coating layer containing polydopamine on the polystyrene-based particles.

[0017] In this specification, a cell culture composition comprising a cell and a microcarrier for cell culture is also provided.

[0018] A microcarrier for cell culture, a method for manufacturing a microcarrier for cell culture, and a cell culture composition using the same will be described in more detail below according to specific embodiments of the invention.

[0019]

[0020] Unless explicitly stated otherwise in this specification, technical terms are used merely to refer to specific embodiments and are not intended to limit the invention.

[0021] The singular forms used in this specification include plural forms unless the phrases clearly indicate otherwise.

[0022] As used in this specification, the meaning of 'includes' specifies certain characteristics, regions, integers, steps, actions, elements, and / or components, and does not exclude the existence or addition of other specific characteristics, regions, integers, steps, actions, elements, components, and / or groups.

[0023] Also, 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 said ordinal numbers. For example, within the scope of the present invention, the first component may also be named the second component, and similarly, the second component may be named the first component.

[0024] In this specification, the alkyl group may be a straight chain or a branched chain, and the number of carbon atoms is not particularly limited but is preferably 1 to 10. According to another embodiment, the number of carbon atoms of the alkyl group is 1 to 6. Specific examples of alkyl groups include methyl, ethyl, propyl, n-propyl, isopropyl, butyl, n-butyl, isobutyl, tert-butyl, sec-butyl, 1-methyl-butyl, 1-ethyl-butyl, pentyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, n-hexyl, 1-methylpentyl, 2-methylpentyl, 4-methyl-2-pentyl, 3,3-dimethylbutyl, 2-ethylbutyl, heptyl, n-heptyl, 1-methylhexyl, cyclopentylmethyl, cyclohectylmethyl, octyl, n-octyl, tert-octyl, 1-methylheptyl, 2-ethylhexyl, 2-propylpentyl, n-nonyl, 2,2-dimethylheptyl, 1-ethyl-propyl, 1,1-dimethyl-propyl, isohexyl, Examples include 2-methylpentyl, 4-methylhexyl, 5-methylhexyl, etc., but are not limited to these.

[0025] In this specification, the 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 number of carbon atoms of the cycloalkyl group is 3 to 10. Specifically, cyclopropyl, cyclobutyl, cyclopentyl, 3-methylcyclopentyl, 2,3-dimethylcyclopentyl, cyclohexyl, 3-methylcyclohexyl, 4-methylcyclohexyl, 2,3-dimethylcyclohexyl, 3,4,5-trimethylcyclohexyl, 4-tert-butylcyclohexyl, cycloheptyl, cyclooctyl, bicyclo[2,2,1]heptyl, etc., are included but are not limited thereto. The cycloalkyl group may be substituted or unsubstituted, and in the case of substitution, examples of substituents are as described above.

[0026] In the present specification, a heterocycloalkyl group refers to a cycloalkyl group comprising one or more non-carbon atoms or heteroatoms, and specifically, the heteroatoms may comprise one or more atoms selected from the group consisting of O, N, Se, and S, etc.

[0027] The present invention will be described in more detail below.

[0028]

[0029] According to one embodiment of the invention, a microcarrier for cell culture comprises: polystyrene-based particles; and a coating layer formed on the polystyrene-based particles and comprising polydopamine and poly(meth)acrylate, wherein the polydopamine coating rate calculated by the following Equation 1 is 70% or more and 98% or less, and the apparent density is 0.98 g / cm³ 3 Exceeding 1.01 g / cm³ 3 Microcarriers for cell culture with a size of less than 1 can be provided.

[0030] [Mathematical Formula 1]

[0031] Polydopamine coating rate (%) = Area of ​​polydopamine present / Surface area of ​​microcarriers * 100.

[0032]

[0033] The inventors confirmed through experiments that in the case of the microcarrier for cell culture of the above embodiment, by introducing poly(meth)acrylate together with polydopamine onto the surface of polystyrene-based particles, particle dispersibility and cell adhesion within the cell culture reactor can be improved due to the excellent hydrophilicity of poly(meth)acrylate, and completed the invention.

[0034] In particular, as the cell culture micro satisfies a polydopamine coating rate of 70% or more and 98% or less calculated by the above mathematical formula 1, the density difference between the cell and the micro carrier is small, allowing for centrifugation when separating and recovering cells after culture, while also improving particle dispersion and cell adhesion within the cell culture reactor due to excellent hydrophilicity.

[0035]

[0036] Specifically, the microcarrier for cell culture of the above embodiment may have a polydopamine coating rate of 70% or more and 98% or less, calculated by the following mathematical formula 1.

[0037] [Mathematical Formula 1]

[0038] Polydopamine coating rate (%) = Area of ​​polydopamine present / Surface area of ​​microcarriers * 100.

[0039]

[0040] More specifically, the microcarrier for cell culture of the above embodiment may have a polydopamine coating rate calculated by the following mathematical formula 1 of 70% or more, 75% or more, 98% or less, 95% or less, 70% or more and 98% or less, 70% or more and 95% or less, 75% or more and 98% or less, and 75% or more and 95% or less.

[0041] As the polydopamine coating rate calculated by the above mathematical formula 1 is 70% or more and 98% or less, particle dispersion and cell adhesion within the cell culture reactor can be improved due to excellent hydrophilicity.

[0042] If the polydopamine coating rate calculated by the above mathematical formula 1 is less than 70%, particle dispersion and cell adhesion within the cell culture reactor may be poor, and if cell adhesion is poor in the early stages of culture due to uneven coating between particles, it may have a negative effect on cell proliferation. In addition, due to uneven coating, there is a problem that the coating material may detach and enter the culture medium during cell culture.

[0043] If the polydopamine coating rate calculated by the above mathematical formula 1 exceeds 98%, the density of the microcarrier for cell culture increases, making it difficult to centrifuge when separating and recovering cells after culture.

[0044] The method for measuring the coating rate is not significantly limited, but, for example, it can be measured using PiFM (Photo-induced Force Microscopy).

[0045] Specifically, after impregnating the aforementioned cell culture microcarrier with polydimethylsiloxane (PDMS), etc., microtoming was performed to obtain a cross-sectional sample of the cell culture microcarrier, and the spectrum of polydopamine (1300 cm⁻¹) was analyzed using Photo-induced Force Microscopy (PiFM). -1 up to 1700 cm -1 ) The coating rate can be derived through the PiFM image.

[0046]

[0047] The above-mentioned microcarrier for cell culture has a spectrum of 3300 cm⁻¹ in the ATR-FTIR spectrum. -11720 cm for the height of the peak -1 The ratio of the peak height may be 0.33 or greater and 1.0 or less.

[0048] More specifically, the cell culture microcarrier has a spectrum of 3300 cm⁻¹ in the spectrum measured by ATR-FTIR. -1 1720 cm for the height of the peak -1 The ratio of the peak height may be 0.33 or more, 0.34 or more, 1.0 or less, 0.9 or less, 0.87 or less, or 0.33 or more, 1.0 or less, 0.33 or more, 0.9 or less, 0.33 or more, 0.87 or less, 0.34 or more, 1.0 or less, 0.34 or more, 0.9 or less, or 0.34 or more, 0.87 or less.

[0049] In the spectrum measured by the above ATR-FTIR, 3300 cm⁻¹ -1 1720 cm for the height of the peak -1 The ratio of the peak height can be achieved by controlling the composition of the coating layer manufacturing solution as described above.

[0050] 1720 cm⁻¹ in the spectrum measured by the above ATR-FTIR -1 The peak appears due to the aliphatic ether (CO) functional group contained in the poly(meth)acrylate, and at 3300 cm⁻¹ -1 The peak is caused by polydopamine, which allows us to indirectly infer the ratio of polydopamine to poly(meth)acrylate within the coating layer.

[0051] In the spectrum measured by the above ATR-FTIR, 3300 cm⁻¹ -1 Regarding the height of the peak 1720 cm -1If the ratio of peak heights decreases excessively, it may be difficult to sufficiently realize the enhancement of surface hydrophilicity by poly(meth)acrylate, leading to a technical problem where some of the microcarriers for cell culture fail to enter the medium and remain at the medium-air interface. Additionally, in the spectrum measured by the aforementioned ATR-FTIR, at 3300 cm⁻¹ -1 Regarding the height of the peak 1720 cm -1 If the ratio of peak height increases excessively, the density of the final microcarriers produced increases, which may prevent the realization of low-density characteristics.

[0052]

[0053] In addition, the microcarrier for cell culture of the above embodiment has an apparent density of 0.98 g / cm³ 3 Exceeding 1.01 g / cm³ 3 Less than, 0.98 g / cm³ 3 Exceeding 1.003 g / cm³ 3 Less than, 0.99 g / cm³ 3 Above 1.003 g / cm³ 3 Less than, 0.995 g / cm³ 3 Above 1.003 g / cm³ 3 Less than, 0.997 g / cm³ 3 Above 1.003 g / cm³ 3 Less than, 0.997 g / cm³ 3 Exceeding 1.003 g / cm³ 3 It may be less than. As it has the aforementioned low-density range, cells and microcarriers can be easily separated through the difference in sedimentation speed due to gravity when separating and recovering microcarriers and cells after cell culture.

[0054] If the density of the microcarriers for cell culture becomes excessively high, the density difference between the cells and the microcarriers is small, which may make centrifugation difficult when separating and recovering cells after culture; conversely, if the density becomes excessively low, the microcarriers may float only on the surface of the culture medium during the initial stages of culture, which may cause problems in attaching cells.

[0055] The above cells are adherent animal cells, although examples are not significantly limited, but may be, for example, fibroblasts, epithelial cells, osteoblasts, chondrocytes, hepatocytes, human-derived umbilical cord blood cells, human bone marrow-derived mesenchymal stem cells, CHO (Chinese hamster ovary) cells, kidney cells (HEK293, BHK21, MDCK, vero cells, etc.), or a mixture of two or more of these.

[0056] In addition, the density difference between the microcarrier for cell culture and the cell is 0.02 g / cm³ 3 Above 0.20 g / cm³ 3 It may be less than or equal to 0.02 g / cm³. 3 Above 0.20 g / cm³ 3 By satisfying the following, cells and microcarriers can be easily separated through the difference in sedimentation velocity due to gravity when separating and recovering microcarriers and cells after cell culture.

[0057]

[0058] The microcarrier for cell culture of the above embodiment may include polystyrene-based particles. The polystyrene-based particles may include a styrene-based monomer homopolymer, or a copolymer of a styrene-based monomer and other monomers and a crosslinking agent, and the styrene-based monomer may include a styrene monomer or a derivative thereof.

[0059]

[0060] Specifically, the polystyrene-based particles may include a monomer compound represented by the following chemical formula 1.

[0061] [Chemical Formula 1]

[0062]

[0063] In the above Chemical Formula 1, L0 is an arylene group having 6 or more carbon atoms or -(C=O)-, L1 and L2 are each independently an alkylene group having 1 or more carbon atoms, R1 is a reactive functional group capable of ring-opening reactions, and R 10 is hydrogen or an alkyl group with 1 or more carbon atoms, and n is an integer greater than or equal to 0.

[0064]

[0065] The inventors have completed the invention by confirming that, in the case of the cell culture microcarrier of the above embodiment, by including a compound represented by Chemical Formula 1 as a polystyrene-based monomer, the density of the final cell culture microcarrier can be controlled to enable separation due to the density difference with the cell, while simultaneously improving dispersibility in a cell culture reactor or medium and improving cell adhesion.

[0066]

[0067] Conventional microcarriers for cell culture controlled particle density by incorporating non-reactive low-density oil into the particles, but there was a technical problem in that the embedded low-density oil leaked out if the particles were physically damaged.

[0068] Accordingly, the inventors confirmed that by including the compound represented by Formula 1 as a monomer in a polystyrene-based polymer used for cell culture microcarriers, the particle density can be controlled, enabling separation based on the density difference with the cells, while simultaneously improving dispersibility in cell culture reactors or media. Furthermore, by including the compound represented by Formula 1 as a monomer in a polystyrene-based polymer, reaction sites are provided on the surface of the cell culture microcarrier where cell-adhesive ligands can be immobilized by chemical bonding, thereby improving cell adhesion.

[0069]

[0070] As the compound represented by Chemical Formula 1 above includes a reactive functional group capable of ring-opening reactions, a reaction site is provided on the surface of a microcarrier for cell culture where a cell-adhesive ligand can be fixed by chemical bonding, thereby improving cell adhesion.

[0071]

[0072] The above-mentioned reactive functional group capable of ring-opening reactions may refer to a reactive functional group capable of ring-opening reactions such as hydrolyzed ring opening.

[0073] Specifically, the reactive functional group capable of the ring-opening reaction may include a heterocycloalkyl group.

[0074] The above heterocycloalkyl group may refer to a cycloalkyl group comprising one or more non-carbon atoms or heteroatoms. The above heteroatoms may include one or more atoms selected from the group consisting of O, N, Se, and S, and preferably may include O.

[0075] The above heterocycloalkyl group is not significantly limited, but may include, for example, an epoxy group.

[0076]

[0077] Specifically, the polystyrene-based particles include a reaction product of a compound represented by Chemical Formula 1 and an ethylene-based unsaturated crosslinking agent, and may include at least 1 part by weight and no more than 10 parts by weight of the compound represented by Chemical Formula 1 per 100 parts by weight of the ethylene-based unsaturated crosslinking agent.

[0078] Specifically, the polystyrene-based particles include a reaction product of a compound represented by Chemical Formula 1 and an ethylene-based unsaturated crosslinking agent, and may include the compound represented by Chemical Formula 1 in an amount of 1 part by weight or more, 1.5 parts by weight or more, 10 parts by weight or less, 5 parts by weight or less, or 1 part by weight or more and 10 parts by weight or less, 1.5 parts by weight or more and 10 parts by weight or less, 1 part by weight or more and 5 parts by weight or less, or 1.5 parts by weight or more and 5 parts by weight or less, with respect to 100 parts by weight of the ethylene-based unsaturated crosslinking agent.

[0079]

[0080] If the compound represented by Chemical Formula 1 is included in an excessively small amount per 100 parts by weight of the above ethylene-based unsaturated crosslinking agent, sufficient reaction sites for the cell adhesion ligand to be immobilized are not provided, which may result in poor cell adhesion. If the compound represented by Chemical Formula 1 is included in an excessively large amount, the relative proportion of the high-density compound is increased, causing the particle density to become higher than that of the cell culture medium, which may result in a loss of low-density characteristics.

[0081] Specifically, the compound represented by the above chemical formula 1 may include any one of the compounds represented by the following chemical formula 1-1 to the compound represented by chemical formula 1-3.

[0082] [Chemical Formula 1-1]

[0083]

[0084] In the above chemical formula 1-1,

[0085] R 11is hydrogen or an alkyl group having 1 or more carbon atoms, and

[0086] [Chemical Formula 1-2]

[0087]

[0088] In the above chemical formula 1-2,

[0089]

[0090] R 12 is hydrogen or an alkyl group having 1 or more carbon atoms, and

[0091] [Chemical Formula 1-3]

[0092]

[0093] In the above chemical formula 1-3,

[0094] R 13 It is hydrogen or an alkyl group with 1 or more carbon atoms.

[0095]

[0096] By including any one of the compounds represented by Chemical Formula 1-1 to Chemical Formula 1-3 as a monomer in polystyrene-based particles, a reaction site is provided on the surface of a microcarrier for cell culture where a cell-adhesive ligand can be fixed by chemical bonding by controlling the weight ratio of the styrene-based monomer and the compounds represented by Chemical Formula 1-1 to Chemical Formula 1-3, thereby improving cell adhesion.

[0097]

[0098] Meanwhile, the above polystyrene-based particles may further include a compound represented by the following chemical formula 2 as a monomer compound, in addition to the compound represented by chemical formula 1.

[0099] [Chemical Formula 2]

[0100]

[0101] In the above Chemical Formula 2, R2 to R6 are each independently hydrogen or an alkyl group having one or more carbon atoms, and at least one of R2 to R6 is It is an alkyl group with 1 or more carbon atoms.

[0102] By including a compound represented by Chemical Formula 2 as a polystyrene-based monomer, the density of the final microcarrier for cell culture can be precisely controlled by adjusting the content of the compound represented by Chemical Formula 2, thereby enabling separation due to the density difference with the cells, and at the same time, dispersibility in the cell culture reactor or medium can be improved.

[0103] Specifically, the compound represented by the above chemical formula 2 may include one or more compounds selected from the group consisting of compounds represented by the following chemical formula 2-1 to compounds represented by the following chemical formula 2-3.

[0104] [Chemical Formula 2-1]

[0105]

[0106] [Chemical Formula 2-2]

[0107]

[0108] [Chemical Formula 2-3]

[0109]

[0110] In the above chemical formulas 2-1 to 2-3, R 21 to R 26 Each is independently an alkyl group having 1 or more carbon atoms.

[0111]

[0112] By including 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 as polystyrene monomers, the density of the final microcarrier for cell culture can be precisely controlled by controlling the content of the compound represented by Chemical Formula 2, thereby enabling separation due to the density difference with the cells, and at the same time, dispersibility in the cell culture reactor or medium can be improved.

[0113]

[0114] More specifically, the compound represented by the above chemical formula 2 may include one or more compounds selected from the group consisting of compounds represented by the following chemical formula 2-4 to compounds represented by the following chemical formula 2-6.

[0115] [Chemical Formula 2-4]

[0116]

[0117] [Chemical Formula 2-5]

[0118]

[0119] [Chemical Formula 2-6]

[0120]

[0121] In the above chemical formulas 2-4 to 2-6, R 21 to R 26 Each is independently an alkyl group having 1 or more carbon atoms.

[0122]

[0123] Meanwhile, the compound represented by the above chemical formula 2 has a density of 0.92 g / cm³ 3 It may be less than.

[0124] Specifically, the compound represented by the above chemical formula 2 has a density of 0.92 g / cm³ 3 Below, 0.91 g / cm³ 3 Below, 0.906 g / cm³ 3 Below, 0.9 g / cm³ 3 Below, 0.89 g / cm³3 Below, 0.5 g / cm³ 3 Above, 0.6 g / cm³ 3 Above, 0.7 g / cm³ 3 Above, 0.8 g / cm³ 3 Above, or 0.5 g / cm³ 3 Above 0.92 g / cm³ 3 Below, 0.6 g / cm³ 3 Above 0.92 g / cm³ 3 Below, 0.7 g / cm³ 3 Above 0.92 g / cm³ 3 Below, 0.8 g / cm³ 3 Above 0.92 g / cm³ 3 Below, 0.5 g / cm³ 3 Above 0.91 g / cm³ 3 Below, 0.6 g / cm³ 3 Above 0.91 g / cm³ 3 Below, 0.7 g / cm³ 3 Above 0.91 g / cm³ 3 Below, 0.8 g / cm³ 3 Above 0.91 g / cm³ 3 Below, 0.5 g / cm³ 3 Above 0.906 g / cm³ 3 Below, 0.6 g / cm³ 3 Above 0.906 g / cm³ 3 Below, 0.7 g / cm³ 3 Above 0.906 g / cm³ 3 Below, 0.8 g / cm³ 3 Above 0.906 g / cm³ 3 Below, 0.5 g / cm³ 3 Above 0.9 g / cm³ 3 Below, 0.6 g / cm³ 3 Above 0.9 g / cm³ 3 Below, 0.7 g / cm³ 3 Above 0.9 g / cm³ 3 Below, 0.8 g / cm³ 3 Above 0.9 g / cm³ 3 Below, 0.5 g / cm³ 3 Above 0.89 g / cm³ 3 Below, 0.6 g / cm³ 3 Above 0.89 g / cm³3 Below, 0.7 g / cm³ 3 Above 0.89 g / cm³ 3 Below, 0.8 g / cm³ 3 Above 0.89 g / cm³ 3 It may be less than.

[0125]

[0126] The density of the compound represented by the above chemical formula 2 is 0.92 g / cm³ 3 By satisfying the following, the density of the final microcarrier for cell culture can be precisely controlled by controlling the content of the compound represented by Chemical Formula 2, thereby enabling separation based on the density difference with the cell, and at the same time, dispersibility in the cell culture reactor or medium can be improved.

[0127] For example, the compound represented by the above chemical formula 2 may include 4-methylstyrene, trimethylstyrene, 4-ethenyl-2-methyl-1-(2-methylpropyl)benzene, 1-ethenyl-2-methyl-4-(1-methylethyl)benzene, 1-ethenyl-2,3-dimethylbenzene, 4-(1,1-dimethylethyl)-2-ethenyl-1-methylbenzene, 2-ethenyl-4-methyl-1-(1-methylethyl)benzene, 1-ethenyl-2-(1-methylethyl)benzene, 2-tert-butylstyrene, 4-tert-butylstyrene, and 1-ethenyl-3-(1-methylethyl)benzene.

[0128]

[0129] Meanwhile, the above polystyrene-based particles may further include a compound represented by the following chemical formula 3 as a monomer compound, in addition to the compound represented by chemical formula 1.

[0130] [Chemical Formula 3]

[0131]

[0132] In the above chemical formula 3, L 30 is -O(C=O)- or -(C=O)O- and R 30 is a directly bonded or an alkylene group having 1 or more carbon atoms, and R 31 is an alkyl group with 1 or more carbon atoms.

[0133]

[0134] That is, the polystyrene-based particles may include a monomer compound represented by Chemical Formula 1, or include a compound represented by Chemical Formula 1 and a compound represented by Chemical Formula 2, or include a compound represented by Chemical Formula 1 and a compound represented by Chemical Formula 3, or include a compound represented by Chemical Formula 1, a compound represented by Chemical Formula 2, and a compound represented by Chemical Formula 3.

[0135]

[0136] By including a compound represented by Chemical Formula 3 as a polystyrene-based monomer, the substituents of the final-prepared microcarrier for cell culture can be hydrolyzed, thereby improving dispersibility in a cell culture reactor or medium.

[0137]

[0138] For example, the compound represented by the above chemical formula 3 may include acetoxystyrene.

[0139]

[0140] The above polystyrene-based particles may include the reaction product of a monomer mixture and an ethylene-based unsaturated crosslinking agent.

[0141] As described above, the monomer mixture may include a compound represented by Chemical Formula 1. Additionally, the monomer mixture may include a compound represented by Chemical Formula 1 and a compound represented by Chemical Formula 2. Additionally, the monomer mixture may include a compound represented by Chemical Formula 1 and a compound represented by Chemical Formula 3. Additionally, the monomer mixture may include compounds represented by Chemical Formula 1 to Chemical Formula 3.

[0142] That is, the polystyrene-based particles include the reaction product of a compound represented by Chemical Formula 1, a styrene-based monomer mixture, and an ethylene-based unsaturated crosslinking agent, and the styrene-based monomer mixture may include a compound represented by Chemical Formula 2 or a compound represented by Chemical Formula 3.

[0143]

[0144] Specifically, the compound represented by Chemical Formula 2 may be included in an amount of 80 parts by weight or more and 99 parts by weight or less per 100 parts by weight of the ethylene-based unsaturated crosslinking agent.

[0145] More specifically, with respect to 100 parts by weight of the ethylene-based unsaturated crosslinking agent, the compound represented by Formula 2 may be included in an amount of 80 parts by weight or more, 85 parts by weight or more, 90 parts by weight or more, 99 parts by weight or less, 95 parts by weight or less, or 80 parts by weight or more and 99 parts by weight or less, 85 parts by weight or more and 99 parts by weight or less, 90 parts by weight or more and 99 parts by weight or less, 80 parts by weight or more and 95 parts by weight or less, 85 parts by weight or more and 95 parts by weight or less, or 90 parts by weight or more and 95 parts by weight or less.

[0146] By including 80 parts by weight or more and 99 parts by weight or less of the compound represented by Chemical Formula 2 with respect to 100 parts by weight of the above ethylene-based unsaturated crosslinking agent, the low density characteristics of the particles can be controlled due to the low density of the compound represented by Chemical Formula 2, thereby enabling separation by density difference with the cells, and at the same time, dispersibility in a cell culture reactor or medium can be improved.

[0147] If the compound represented by Chemical Formula 2 is included in an excessively small amount per 100 parts by weight of the above ethylene-based unsaturated crosslinking agent, the particle density increases, causing a loss of low-density characteristics and potentially leading to sedimentation within the medium; if it is included in an excessively large amount, the particle density becomes 0.99 g / cm³ 3As the level becomes very low, high-speed stirring to disperse it within the medium may cause technical problems that affect cell characteristics.

[0148]

[0149] In addition, the compound represented by Chemical Formula 2 may be included in an amount of 90 parts by weight or more and 100 parts by weight or less per 100 parts by weight of the styrene-based monomer mixture.

[0150] Specifically, the compound represented by Formula 2 may be included in an amount of 90 parts by weight or more, 91 parts by weight or more, 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, with respect to 100 parts by weight of the styrene-based monomer mixture.

[0151] By including 90 parts by weight or more and 100 parts by weight or less of the compound represented by Chemical Formula 2 with respect to 100 parts by weight of the above styrene-based monomer mixture, the density of the final microcarrier for cell culture can be precisely controlled, enabling separation based on the density difference with the cell, and at the same time, dispersibility in the cell culture reactor or medium can be improved.

[0152]

[0153] In addition, for every 100 parts by weight of the compound represented by Chemical Formula 2, the compound represented by Chemical Formula 1 may be included in an amount of 1 part by weight or more and 10 parts by weight or less.

[0154] Specifically, for 100 parts by weight of the compound represented by Chemical Formula 2, the compound represented by Chemical Formula 1 may be included in an amount of 1 part by weight or more, 1.5 parts by weight or more, 10 parts by weight or less, 1 part by weight or more and 10 parts by weight or less, or 1.5 parts by weight or more and 10 parts by weight or less.

[0155] If the compound represented by Chemical Formula 1 is included in an excessively small amount per 100 parts by weight of the compound represented by Chemical Formula 2, sufficient reaction sites for the cell-adhesive ligand to be immobilized are not provided, which may result in poor cell adhesion, and if the compound is included in an excessively large amount, a technical problem may occur in which the density of the particles increases and the low-density characteristics are lost.

[0156]

[0157] In addition, when the monomer compound includes a compound represented by the following chemical formula 3, the compound represented by the chemical formula 3 may be included 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.

[0158] More specifically, with respect to 100 parts by weight of the ethylene-based unsaturated crosslinking agent, the compound represented by Chemical Formula 3 may be included in an amount of 0.1 parts by weight or more, 0.5 parts by weight or more, 1 part by weight or more, 5 parts by weight or less, 3 parts by weight or less, 2 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, and 1 part by weight or more and 2 parts by weight or less.

[0159] When the monomer compound includes a compound represented by the following chemical formula 3, the compound represented by the chemical formula 3 is included in an amount of 0.1 to 5 parts by weight per 100 parts by weight of the ethylene-based unsaturated crosslinking agent, thereby hydrolyzing the substituents of the finally prepared microcarrier for cell culture, so that the dispersibility in the cell culture reactor or medium can be improved.

[0160] If the compound represented by Chemical Formula 3 is included in an excessive amount for every 100 parts by weight of the above ethylene-based unsaturated crosslinking agent, a technical problem may occur in which the density of the particles increases and the low-density characteristics are lost.

[0161]

[0162] In addition, when the monomer compound includes a compound represented by the following chemical formula 3, the compound represented by the chemical formula 3 may be included 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 styrene-based monomer mixture.

[0163] Specifically, with respect to 100 parts by weight of the styrene-based monomer mixture, the compound represented by Chemical Formula 3 may be included in an amount of 0.1 parts by weight or more, 0.5 parts by weight or more, 1 part by weight or more, 5 parts by weight or less, 3 parts by weight or less, 2.5 parts by weight or less, 0.1 parts by weight or more and 5 parts by weight or less, 0.5 parts by weight or more and 5 parts by weight or less, 1 part by weight or more and 5 parts by weight or less, 0.1 parts by weight or more and 3 parts by weight or less, 0.5 parts by weight or more and 3 parts by weight or less, 1 part by weight or more and 3 parts by weight or less, 0.1 parts by weight or more and 2.5 parts by weight or less, 0.5 parts by weight or more and 2.5 parts by weight or less, and 1 part by weight or more and 2.5 parts by weight or less.

[0164] When the monomer compound includes a compound represented by the following chemical formula 3, the compound represented by the chemical formula 3 is included in an amount of 0.1 to 5 parts by weight per 100 parts by weight of the styrene-based monomer mixture, thereby providing sufficient reaction sites where cell-adhesive ligands can be immobilized while maintaining low-density characteristics, so that an effect of easy cell adhesion can be realized.

[0165]

[0166] In addition, when the monomer compound includes a compound represented by the following chemical formula 3, the compound represented by the chemical formula 1 may be included in an amount of 110 parts by weight or more and 500 parts by weight or less per 100 parts by weight of the compound represented by the chemical formula 3.

[0167] Specifically, for 100 parts by weight of the compound represented by Chemical Formula 3, the compound represented by Chemical Formula 1 may be included in an amount of 110 parts by weight or more, 150 parts by weight or more, 500 parts by weight or less, 400 parts by weight or less, 300 parts by weight or less, 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.

[0168] When the monomer compound includes a compound represented by the following chemical formula 3, if the compound represented by the chemical formula 1 is included in an excessively small amount per 100 parts by weight of the compound represented by the chemical formula 3, a problem may occur in which the compound represented by the chemical formula 1 is lost before polymerization due to its relatively high water solubility and is not introduced onto the particle surface, and if it is included in an excessively large amount, a technical problem may occur in which the density of the particles increases and the low-density characteristics are lost.

[0169]

[0170] In addition, the polystyrene-based particles may contain 60 parts by weight or more and 200 parts by weight or less of the ethylene-based unsaturated crosslinking agent per 100 parts by weight of the styrene-based monomer mixture.

[0171] Specifically, the ethylene-based unsaturated crosslinking agent may be included in an amount of 60 parts by weight or more and 200 parts by weight or less, 60 parts by weight or more and 150 parts by weight or less, 60 parts by weight or more and 130 parts by weight or less, 100 parts by weight or more and 200 parts by weight or less, 100 parts by weight or more and 150 parts by weight or less, 100 parts by weight or more and 130 parts by weight or less, 101 parts by weight or more and 200 parts by weight or less, 101 parts by weight or more and 150 parts by weight or less, and 101 parts by weight or more and 130 parts by weight or less, based on 100 parts by weight of the styrene-based monomer mixture.

[0172] If the ethylene-based unsaturated crosslinking agent is included in an excessively small amount per 100 parts by weight of the above styrene-based monomer mixture, the crosslinking density of the polystyrene-based polymer decreases, making it difficult to stably maintain a spherical shape for the particles.

[0173] On the other hand, if the ethylene-based unsaturated crosslinking agent is included in an excessive amount relative to 100 parts by weight of the styrene-based monomer mixture, there is a limitation in that it is difficult to lower the particle density to the target level.

[0174]

[0175] Examples of the above-mentioned ethylene-based unsaturated crosslinking agents include divinylbenzene.

[0176]

[0177] The diameter of the D50 particles of the microcarrier for cell culture may be 150 μm to 300 μm, 150 μm to 250 μm, 150 μm to 240 μm, or 155 μm to 240 μm. When the average diameter of the polystyrene-based particles satisfies the above-described range, the cell adhesion and culture performance is excellent. Meanwhile, if the average diameter of the microcarrier for cell culture becomes excessively small, there is a concern that the surface area available for cell culture will be small, leading to a decrease in culture efficiency; conversely, if it becomes excessively large, the interaction between adhered cells will be reduced, and the cell density within the culture vessel will decrease, which may lead to a decrease in cell culture efficiency.

[0178] The diameter of the cell culture microcarrier above refers to the distance between two points where a straight line passing through the center of gravity of the cell culture microcarrier meets the outermost surface of the cell culture microcarrier, and the average diameter of the cell culture microcarrier can be obtained by confirming the diameter of the entire cell culture microcarrier included in the cell culture microcarrier through an optical microscope.

[0179] The above-mentioned microcarrier for cell culture may be a group of individual particles having an average diameter of 150 μm to 300 μm, 150 μm to 250 μm, 150 μm to 240 μm, or 155 μm to 240 μm, and the individual particles included in this group may have an average diameter of 150 μm to 300 μm, 150 μm to 250 μm, 150 μm to 240 μm, or 155 μm to 240 μm. More specifically, 95% or 99% of the individual particles included in the group may have a diameter of 150 μm to 300 μm, 150 μm to 250 μm, 150 μm to 240 μm, or 155 μm to 240 μm.

[0180] In addition, the polystyrene-based particles may have a ratio of perfectly spherical particles without damage or destruction according to the following mathematical formula of greater than 90% and less than or equal to 100%, or greater than or equal to 92% and less than or equal to 100%, or greater than or equal to 95% and less than or equal to 100%, or greater than or equal to 96% and less than or equal to 99%.

[0181] [Mathematical Formula]

[0182] Percentage of perfectly spherical particles without damage or destruction (%) = (Number of polystyrene-based particles having a perfectly spherical shape without damage or destruction / Total number of polystyrene-based particles) x 100.

[0183] The ratio of perfectly spherical particles without damage or destruction according to the above mathematical formula can be obtained by measuring the number of particles having a perfectly spherical shape without damage or destruction among the total particles using SEM for the polystyrene-based particles, and calculating the percentage ratio of the number of perfectly spherical particles without damage or destruction relative to the total particles.

[0184] That is, the microcarrier for cell culture may contain a plurality of polystyrene-based particles, and whether any of these plurality of polystyrene-based particles have a perfectly spherical shape without damage or destruction can be visually determined through SEM.

[0185] If the ratio of perfectly spherical particles decreases to 90% or less without damage or destruction according to the above mathematical formula, there is a concern that the number of amorphous particles with uneven and pitted surfaces will increase, and that amorphous particles will float in the cell culture medium and cause physical impact to the cells being cultured, resulting in a decrease in cell culture efficiency to the point where cell culture becomes impossible.

[0186]

[0187] Meanwhile, it may include a coating layer formed on the above-mentioned polystyrene-based particles and comprising polydopamine and poly(meth)acrylate. The coating layer is formed on the surface of the polystyrene-based particles and modifies the surface of the microcarriers to be hydrophilic, thereby increasing the water dispersibility of the particles in the medium and controlling the buoyancy of the microcarriers in the culture medium. It also serves to provide sites for transmembrane proteins of cells to bind, thereby enabling adherent cells to be stably attached, dispersed, and cultured on the particle surface.

[0188] The fact that the coating layer is formed on polystyrene-based particles may include cases where the coating layer contacts the surface of the polystyrene-based particles, or where another layer is formed on the polystyrene-based particles and the coating layer contacts the surface of the other layer. In this case, the composition of the other layer is not significantly limited, and various materials forming a functional layer known in the past may be applied without limitation, and the number of layers of the other layer is not limited, and may be applied as, for example, from 1 to 10 layers.

[0189] Examples of methods for forming a coating layer on the above-mentioned polystyrene-based particles are not significantly limited, and the coating layer can be manufactured by first producing the polystyrene-based particles and then coating the surface of the produced polystyrene-based particles with a solution containing polydopamine and poly(meth)acrylate. The specific coating method is not significantly limited, and various methods widely known for solution coating on particle surfaces can be applied without restriction.

[0190] The coating layer formed on the above-mentioned polystyrene-based particles may include polydopamine and poly(meth)acrylate. The coating layer contains both polydopamine and poly(meth)acrylate, and within the coating layer, polydopamine and poly(meth)acrylate may exist in a combined state.

[0191]

[0192] The above polydopamine is a polymer of dopamine monomers and acts as an adhesive layer capable of introducing a functional polymer onto the surface of polystyrene-based particles, thereby allowing a polymer layer for cell attachment to be effectively introduced onto the surface of a microcarrier and maintained stably even during culture.

[0193] The above-mentioned microcarrier for cell culture has a polydopamine content of 0.2 mg / cm² as measured by the BCA assay method. 2 to 1.0 mg / cm² 2 , or 0.25 mg / cm² 2to 1.0 mg / cm² 2 , or 0.5 mg / cm² 2 to 1.0 mg / cm² 2 It may be. The polydopamine content measured by the above BCA assay method is 0.2 mg / cm² 2 If it decreases excessively, such as to below, it may be difficult to sufficiently achieve cell adhesion induced by polydopamine. In addition, the polydopamine content measured by the above BCA assay method is 1.0 mg / cm² 2 If it increases excessively due to excess or other factors, polydopamine polymers (aggregates) exist in multiple layers on the particle surface, which can cause the coating material to detach within the culture medium.

[0194] The above BCA assay method can be carried out in the following manner.

[0195] 1) 1 mg of the cell culture microcarrier of the above embodiment is dispersed in 25 µl of 1X PBS.

[0196] 2) Add 500 µl of BCA working solution and react at 37°C for 30 minutes.

[0197] 3) Dispense 150 µL of reaction solution into a 96-well plate and measure the absorbance at 562 nm using a microplate reader.

[0198] 4) The polydopamine content on the surface is determined using a previously obtained standard curve.

[0199]

[0200] Meanwhile, as the above-mentioned poly(meth)acrylate is combined with polydopamine and incorporated into the coating layer, the dispersibility of particles in the culture medium and cell adhesion characteristics can be maximized due to the relatively higher hydrophilicity of the poly(meth)acrylate compared to polydopamine.

[0201] The above poly(meth)acrylate may include one or more functional groups containing one or more heteroatoms in addition to the (meth)acrylate group.

[0202] As the above poly(meth)acrylate contains one or more functional groups including one or more heteroatoms in addition to the (meth)acrylate group, the polarity of the coating layer is increased, thereby maximizing hydrophilicity and cell adhesion characteristics.

[0203] The above heteroatom may refer to one or more atoms selected from the group consisting of O, N, Si, and S as heteroatoms other than carbon.

[0204] Examples of functional groups including one or more heteroatoms in addition to the above (meth)acrylate group are not significantly limited, but for example, the above poly(meth)acrylate may include one or more functional groups selected from the group consisting of hydroxyl groups, epoxy groups, amine groups, and betaine groups.

[0205] More specifically, the poly(meth)acrylate may comprise one or more poly(meth)acrylates selected from the group consisting of poly2-hydroethyl (meth)acrylate, polyglycidyl (meth)acrylate, poly2-(dimethylamino)ethyl (meth)acrylate, polysulfobetaine (meth)acrylate, polycarboxybetaine (meth)acrylate, and polyoligo(ethylene glycol) methyl ether (meth)acrylate.

[0206]

[0207] The above polydopamine may be prepared from a precursor of the above polydopamine. The above precursor of the above polydopamine refers to a dopamine monomer and, although not significantly limited, may include, for example, dopamine hydrochloride.

[0208] In the microcarrier for cell culture of the above embodiment, the solution used to prepare the coating layer may contain at least 101 parts by weight and no more than 2000 parts by weight of the poly(meth)acrylate per 100 parts by weight of the polydopamine precursor.

[0209] Specifically, the solution used to manufacture the coating layer comprises, with respect to 100 parts by weight of the polydopamine precursor, 101 parts by weight or more and 2000 parts by weight or less, 200 parts by weight or more and 2000 parts by weight or less, 300 parts by weight or more and 2000 parts by weight or less, 400 parts by weight or more and 2000 parts by weight or less, 500 parts by weight or more and 2000 parts by weight or less, 101 parts by weight or more and 1500 parts by weight or less, 200 parts by weight or more and 1500 parts by weight or less, 300 parts by weight or more and 1500 parts by weight or less, 400 parts by weight or more and 1500 parts by weight or less, 500 parts by weight or more and 1500 parts by weight or less, 101 parts by weight or more and 1000 parts by weight or less, 200 parts by weight or more and 1000 parts by weight or less, and 300 parts by weight or more and 1000 parts by weight or less. It may contain 400 parts by weight or more and 1000 parts by weight or less, or 500 parts by weight or more and 1000 parts by weight or less.

[0210] If the solution used to manufacture the coating layer contains an excessively small amount of the poly(meth)acrylate relative to 100 parts by weight of the polydopamine precursor, it may be difficult to sufficiently improve surface hydrophilicity due to the poly(meth)acrylate, resulting in a technical problem where some of the microcarriers for cell culture cannot enter the medium and remain at the interface between the medium and air during cell culture. Additionally, if the solution used to manufacture the coating layer contains an excessively large amount of the poly(meth)acrylate relative to 100 parts by weight of the polydopamine precursor, the density of the final microcarriers increases, which may prevent the realization of low-density characteristics.

[0211] Meanwhile, the thickness of the coating layer can be determined through the difference between the radius of the microcarrier for cell culture and the radius of the polystyrene-based particle to which the coating layer is not introduced, and the value may be 40 nm or more and 250 nm or less.

[0212] Specifically, the thickness of the coating layer may be 40 nm or more, 45 nm or more, 250 nm or less, 230 nm or less, 210 nm or less, 40 nm or more, 250 nm or less, 40 nm or more, 230 nm or less, 40 nm or more, 210 nm or less, 45 nm or more, 250 nm or less, 45 nm or more, 230 nm or less, 45 nm or more, 210 nm or less.

[0213] If the thickness of the coating layer becomes excessively thin, the coating layer is too thin relative to the polystyrene-based particles, so the effect of modifying the microcarrier surface to be hydrophilic is negligible. If the thickness of the coating layer becomes excessively thick, the coating layer becomes thick relative to the polystyrene-based particles, which may cause problems such as an increase in the diameter or density of the microcarrier for cell culture, leading to a decrease in cell culture efficiency. Additionally, as the coating material is stacked in a multi-layer structure, there is a problem that the coating material may detach and flow into the culture medium during cell culture.

[0214]

[0215] In addition, the ratio of the thickness of the inter-coating layer to the D50 particle diameter of the microcarrier for cell culture may be 0.0002 or more and 0.0009 or less.

[0216] Specifically, the ratio of the thickness of the inter-coating layer to the D50 particle diameter of the microcarrier for cell culture may be 0.0002 or more and 0.0009 or less, 0.0002 or more and 0.00089 or less, or 0.0002 or more and 0.00088 or less.

[0217] If the thickness of the coating layer is excessively small, resulting in an excessively small ratio of the radius of the polystyrene-based particles to the thickness of the surface coating layer, the coating layer becomes too thin relative to the polystyrene-based particles, making the effect of modifying the microcarrier surface to be hydrophilic insignificant. Conversely, if the thickness of the coating layer is excessively large, resulting in an excessively large ratio of the radius of the polystyrene-based particles to the thickness of the surface coating layer, the coating layer becomes too thick relative to the polystyrene-based particles. This can lead to problems such as an increase in the diameter or density of the microcarriers for cell culture, which may result in a decrease in cell culture efficiency. Additionally, as the coating material is stacked in a multi-layer structure, there is a problem where the coating material may detach and enter the culture medium during cell culture.

[0218]

[0219] Meanwhile, according to another embodiment of the invention, a method for manufacturing a microcarrier for cell culture may be provided, comprising the steps of: polymerizing and recovering polystyrene-based particles; and immersing the recovered polystyrene-based particles in a solution containing a precursor of polydopamine and poly(meth)acrylate and stirring at a temperature of 15°C or lower to form a coating layer containing polydopamine and poly(meth)acrylate on the polystyrene-based particles.

[0220]

[0221] In the method for manufacturing a microcarrier for cell culture according to the above-mentioned embodiment, the details regarding the polystyrene-based particles, polydopamine, poly(meth)acrylate, and coating layer include all of the details described above.

[0222]

[0223] Specifically, the step of forming a coating layer comprising polydopamine and poly(meth)acrylate on the polystyrene-based particles may be carried out at 15°C or lower, 5°C or higher and 15°C or lower, or 7°C or higher and 13°C or lower.

[0224] As the step of forming a coating layer containing polydopamine and poly(meth)acrylate on the above-described polystyrene-based particles is carried out at a temperature lower than room temperature, the coating layer is uniformly formed, and the particle dispersibility and cell adhesion within the cell culture reactor can be improved due to the excellent hydrophilicity of the finally manufactured microcarrier for cell culture.

[0225] If the step of forming a coating layer containing polydopamine and poly(meth)acrylate on the above polystyrene-based particles is carried out under temperature conditions exceeding 15°C, the coating layer is not formed uniformly, and the coating rate of the final microcarrier for cell culture is less than 70%, which may result in poor particle dispersibility and cell adhesion within the cell culture reactor.

[0226]

[0227] In addition, the step of immersing the recovered polystyrene-based particles in a solution containing a polydopamine precursor and poly(meth)acrylate and stirring at a temperature of 15°C or lower to form a coating layer containing polydopamine and poly(meth)acrylate on the polystyrene-based particles may specifically be carried out for 10 hours or more and 30 hours or less, 10 hours or more and 25 hours or less, 10 hours or more and 20 hours or less, 15 hours or more and 30 hours or less, 15 hours or more and 25 hours or less, and 15 hours or more and 20 hours or less.

[0228]

[0229] Specifically, the cell culture microcarrier finally manufactured by the method for manufacturing a cell culture microcarrier of the above-mentioned embodiment may have a polydopamine coating rate of 70% or more and 98% or less, calculated by the following mathematical formula 1.

[0230] [Mathematical Formula 1]

[0231] Polydopamine coating rate (%) = Area of ​​polydopamine present / Surface area of ​​microcarriers * 100.

[0232]

[0233] More specifically, the cell culture microcarrier finally manufactured by the method for manufacturing a cell culture microcarrier of the above embodiment may have a polydopamine coating rate calculated by the following mathematical formula 1 of 70% or more, 75% or more, 98% or less, 95% or less, 70% or more and 98% or less, 70% or more and 95% or less, 75% or more and 98% or less, and 75% or more and 95% or less.

[0234] As the polydopamine coating rate calculated by the above mathematical formula 1 is 70% or more and 98% or less, particle dispersion and cell adhesion within the cell culture reactor can be improved due to excellent hydrophilicity.

[0235] If the polydopamine coating rate calculated by the above mathematical formula 1 is less than 70%, particle dispersion and cell adhesion within the cell culture reactor may be poor, and if cell adhesion is poor in the early stages of culture due to uneven coating between particles, it may have a negative effect on cell proliferation. In addition, due to uneven coating, there is a problem that the coating material may detach and enter the culture medium during cell culture.

[0236] The method for measuring the coating rate is not significantly limited, but, for example, it can be measured using PiFM (Photo-induced Force Microscopy).

[0237] Specifically, after impregnating the aforementioned cell culture microcarrier with polydimethylsiloxane (PDMS), etc., microtoming was performed to obtain a cross-sectional sample of the cell culture microcarrier, and the spectrum of polydopamine (1300 cm⁻¹) was analyzed using Photo-induced Force Microscopy (PiFM). -1 up to 1700 cm-1 ) The coating rate can be derived through the PiFM image.

[0238]

[0239] A step of immersing the recovered polystyrene-based particles in a solution containing a polydopamine precursor and poly(meth)acrylate and stirring at a temperature of 15°C or lower to form a coating layer containing polydopamine and poly(meth)acrylate on the polystyrene-based particles; wherein the concentration of the polydopamine precursor in the solution may be 1.0 mg / ml or more and 10.0 mg / ml or less.

[0240] Specifically, the concentration of the polydopamine precursor in the solution containing the polydopamine precursor may be 1.0 mg / ml or more and 10.0 mg / ml or less, 2.0 mg / ml or more and 10.0 mg / ml or less, 3.0 mg / ml or more and 10.0 mg / ml or less, 4.0 mg / ml or more and 10.0 mg / ml or less, 1.0 mg / ml or more and 8.0 mg / ml or less, 2.0 mg / ml or more and 8.0 mg / ml or less, 3.0 mg / ml or more and 8.0 mg / ml or less, 4.0 mg / ml or more and 8.0 mg / ml or less, 1.0 mg / ml or more and 5.0 mg / ml or less, 2.0 mg / ml or more and 5.0 mg / ml or less, 3.0 mg / ml or more and 5.0 mg / ml or less, and 4.0 mg / ml or more and 5.0 mg / ml or less.

[0241] As the concentration of the polydopamine precursor in the solution containing the above-mentioned polydopamine precursor is 1.0 mg / ml or more and 10.0 mg / ml or less, the final microcarrier for cell culture can have improved particle dispersion and cell adhesion within the cell culture reactor due to its excellent hydrophilicity.

[0242]

[0243] If the concentration of the polydopamine precursor in the solution containing the above-mentioned polydopamine precursor is excessively low to less than 1.0 mg / ml, the coating layer is not formed uniformly, and the coating rate of the final microcarrier for cell culture is less than 70%, and consequently, particle dispersibility and cell adhesion within the cell culture reactor may be poor.

[0244] If the concentration of the polydopamine precursor in the solution containing the above-mentioned polydopamine precursor becomes excessively high, exceeding 10.0 mg / ml, the dopamine precursor may aggregate between the molecule and the polydopamine polymer, resulting in uneven coating thickness and increased surface roughness. Furthermore, polydopamine aggregates stacked in multiple layers on the surface of the coating substrate may easily detach due to low coating stability.

[0245] In addition, a step of immersing the recovered polystyrene-based particles in a solution containing a polydopamine precursor and poly(meth)acrylate and stirring at a temperature of 15°C or lower to form a coating layer containing polydopamine and poly(meth)acrylate on the polystyrene-based particles; wherein the concentration of the polydopamine precursor relative to the surface area of ​​the polystyrene-based particles is 0.3 mg / cm² 2 ≥ 1.0 mg / cm² 2 It may be less than.

[0246]

[0247] Specifically, the concentration of the polydopamine precursor relative to the surface area of ​​the polystyrene-based particle is 0.3 mg / cm² 2 ≥ 1.0 mg / cm² 2 Less than or equal to 0.5 mg / cm² 2 ≥ 1.0 mg / cm² 2 It may be less than.

[0248]

[0249] Meanwhile, the above-mentioned recovered polystyrene-based particles are immersed in a solution containing a polydopamine precursor and poly(meth)acrylate and stirred at a temperature of 15°C or lower to form a coating layer containing polydopamine and poly(meth)acrylate on the polystyrene-based particles; subsequently, the steps of washing and drying may be further included.

[0250] Specifically, the washing step may include filtering the reaction product through a sieve of 30 μm to 100 μm and then stirring at room temperature 5 to 7 times in 100% ethanol.

[0251] The above drying step includes placing the material in a vacuum oven and vacuum drying it at room temperature. However, it is not limited thereto, and any drying method known to be commonly used may be used without any particular restrictions.

[0252]

[0253] According to another embodiment of the invention, a cell culture composition comprising a cell and a microcarrier for cell culture of the first embodiment may be provided. The details regarding the microcarrier for cell culture include all the details described above in the first embodiment.

[0254] The above cells are adherent animal cells, although examples are not significantly limited, but may be, for example, fibroblasts, epithelial cells, osteoblasts, chondrocytes, hepatocytes, human-derived umbilical cord blood cells, human bone marrow-derived mesenchymal stem cells, CHO (Chinese hamster ovary) cells, kidney cells (HEK293, BHK21, MDCK, vero cells, etc.), or a mixture of two or more of these.

[0255]

[0256] In addition, the density difference between the microcarrier for cell culture and the cell is 0.02 g / cm³ 3 Above 0.20 g / cm³ 3 It may be less than or equal to 0.02 g / cm³. 3 Above 0.20 g / cm³ 3 By satisfying the following, cells and microcarriers can be easily separated through the difference in sedimentation velocity due to gravity when separating and recovering microcarriers and cells after cell culture.

[0257] The above cell culture composition may further include a culture medium solution. The culture medium solution may include various additives to sufficiently satisfy environmental conditions such as pH, temperature, and osmotic pressure, as well as nutrients based on body fluids such as plasma or lymph fluid. Various substances widely known in the field of cell culture technology may be used without limitation.

[0258] For example, the microcarrier for cell culture of the above embodiment has a density lower than that of the medium solution and is injected into the medium solution and floats within the medium solution under stirring conditions. Subsequently, as the number of cells attached to the surface of the low-density microcarrier increases, the density of the microcarrier with attached cells (hereinafter referred to as the 'microcarrier-cell complex') gradually increases and gradually sinks within the medium solution.

[0259] Accordingly, cultured cells can be easily obtained by separating the cells from the microcarrier-cell complex by treating the microcarrier (microcarrier-cell complex) to which the cells are attached with a cell detachment enzyme and then separating it through centrifugation.

[0260]

[0261] According to the present invention, a microcarrier for cell culture that enables separation of cells by density difference by controlling density, has improved dispersibility in a cell culture reactor or medium, and has improved cell adhesion, a method for manufacturing a microcarrier for cell culture, and a cell culture method using the same may be provided.

[0262] The invention is described in more detail in the following examples. However, the following examples are merely illustrative of the invention, and the scope of the invention is not limited by the following examples.

[0263]

[0264] <Preparation Example 1: Preparation of Polystyrene Particles>

[0265] Polyvinyl alcohol (molecular weight 85-124K, 87-89% hydrolysis) was dissolved in distilled water at a concentration of 2% to prepare an aqueous dispersion, and then stirred at room temperature for 20 minutes.

[0266] A monomer composition was prepared by adding 2% by weight of V-65 initiator (initiator input amount: based on the total sum of monomers and crosslinking agent) to 25 g of a mixture sufficiently dissolved by mixing monomers styrene, glycidyl methacrylate, t-butylstyrene and crosslinking agent divinylbenzene in a weight ratio of 0.05:0.05:0.9:1 and stirring for an additional 5 minutes.

[0267] 600g of an aqueous dispersion was added to a 1L reactor, and the monomer composition was added. A shear force was applied to the aqueous dispersion and the monomer composition at a speed of 400 rpm at room temperature to disperse the monomer composition into the aqueous dispersion in the form of fine droplets and homogenize it.

[0268] Polystyrene particles were prepared by reacting the homogenized mixture under nitrogen purging at 85°C for 6 hours while stirring at a stirring speed of 400 rpm, and after washing three times with distilled water at 60°C and five times with ethanol, the particles were filtered through a 70 µm sieve and recovered by oven drying at 70°C. The recovered polystyrene particles were used as microcarriers for cell culture.

[0269] The physical properties of the above polystyrene particles are as follows.

[0270]

[0271] Average diameter: 240 µm (based on D50 measured using PSA equipment)

[0272] Apparent density: 0.997 ~ 1.003 g / cm³ 3

[0273]

[0274] <Preparation Example 2: Preparation of Polystyrene Particles>

[0275] Polystyrene particles were prepared in the same manner as in Preparation Example 1, except that the weight ratio of the monomers styrene, glycidyl methacrylate, and t-butylstyrene and the crosslinking agent divinylbenzene was adjusted to 0.07:0.03:0.9:1.

[0276]

[0277] <Preparation Example 3: Preparation of Polystyrene Particles>

[0278] Polystyrene particles were prepared in the same manner as in Preparation Example 1, except that the weight ratio of the monomers styrene, glycidyl methacrylate, and t-butylstyrene and the crosslinking agent divinylbenzene was adjusted to 0.085:0.015:0.9:1.

[0279]

[0280] <Comparative Manufacturing Example 1: Preparation of Polystyrene Particles>

[0281] Polystyrene particles were prepared using the same method as the above preparation example, except that the weight ratio of the monomer styrene and the crosslinking agent divinylbenzene was 3:1 and the amount of oil added (based on the total sum of monomer, crosslinking agent, and oil) was 20% by weight.

[0282] The physical properties of the above polystyrene particles are as follows.

[0283] Average diameter: 140 µm (based on D50 measured using PSA equipment)

[0284] Apparent density: 0.95~0.97 g / cm³ 3

[0285] Porosity: 7.3%

[0286] Pore ​​diameter: 0.05~4㎛

[0287]

[0288] <Comparative Manufacturing Example 2: Preparation of Polystyrene Particles>

[0289] Polystyrene particles were prepared in the same manner as in Preparation Example 1, except that the weight ratio of the monomers styrene, glycidyl methacrylate, and t-butylstyrene and the crosslinking agent divinylbenzene was adjusted to 0.095:0.005:0.9:1.

[0290]

[0291] <Comparative Manufacturing Example 3: Preparation of Polystyrene Particles>

[0292] Polystyrene particles were prepared in the same manner as in Preparation Example 1, except that the weight ratio of the monomers styrene, glycidyl methacrylate, and t-butylstyrene and the crosslinking agent divinylbenzene was adjusted to 0.005:0.095:0.9:1.

[0293]

[0294] <Examples 1-5 and Comparative Examples 1-5: Preparation of Microcarriers for Cell Culture>

[0295] Polydopamine and poly 2-hydroxyethyl methacrylate were coated onto the polystyrene particles of the above preparation example. Specifically, the polystyrene particles of the above preparation example were immersed in tris buffer (pH 8.0) in which dopamine hydrochloride and 2-hydroxyethyl methacrylate were dissolved at the concentrations indicated in Table 1 below, and coated with polydopamine and poly 2-hydroxyethyl methacrylate for 20 hours at the temperature indicated in Table 1 below under stirring conditions. The weight ratio of dopamine hydrochloride and 2-hydroxyethyl methacrylate, and the polydopamine concentration relative to the particle specific surface area at the time of coating, are as indicated in Table 1 below.

[0296]

[0297] Afterward, the particles were washed five times with ethanol, filtered through a 45 µm sieve, and dried in an 80 ℃ oven to be recovered. The surface-modified polystyrene particles recovered in this way were used as microcarriers for cell culture.

[0298]

[0299] Comparative Example 6

[0300] Microparticles were prepared in the same manner as in Example 1, except that the polystyrene particles of Comparative Example 2 were used.

[0301] In the case of the microcarrier of Comparative Example 6, it was confirmed that the cell adhesion was excessively poor, making it unsuitable for use as a microcarrier for cell culture.

[0302]

[0303] Comparative Example 7

[0304] Microparticles were prepared in the same manner as in Example 1, except that the polystyrene particles of Comparative Manufacturing Example 3 were used.

[0305] In the case of the microcarrier of Comparative Example 7, it was confirmed that the particle density was excessively high, making it impossible to achieve low-density characteristics.

[0306]

[0307]

[0308] Classification: Polystyrene particles, dopamine hydrochloride concentration relative to particle specific surface area (mg / cm²) 2 ) Coating Solution Concentration (mg / ml) Coating Temperature (°C) Dopamine Hydrochloride: HEMA Weight Ratio Example 1 Preparation Example 1 15101:5 Example 2 Preparation Example 1 15101:10 Example 3 Preparation Example 1 0.55101:10 Example 4 Preparation Example 2 15101:10 Example 5 Preparation Example 3 15101:10 Comparative Example 1 Preparation Example 1 15101:0 Comparative Example 2 Preparation Example 1 15101:25 Comparative Example 3 Preparation Example 1 15251:10 Comparative Example 4 Preparation Example 1 0.50.5251:10 Comparative Example 5 Preparation Example 1 15101:1

[0309] <Experimental Example: Measurement of Physical Properties of Microcarriers for Cell Culture> The physical properties of the microcarriers for cell culture obtained in the above examples and comparative examples were measured by the following method, and the results are shown in Table 2.

[0310]

[0311] Experiment 1. Average particle size (Unit: μm)

[0312] For the microcarriers for cell culture obtained in the above examples and comparative examples, after dispersing them in ethanol at a level of 10% by weight, the particle diameter of D50 (particle size value corresponding to the cumulative distribution percentage reaching 50%) was measured using a PSA (Particle size analysis) device.

[0313]

[0314] Experiment 2. Apparent Density (Unit: g / cm³) 3 )

[0315] For the microcarriers for cell culture prepared in the above examples and comparative examples, the density is 0.997 g / cm³ under conditions of room temperature (25 ℃) and atmospheric pressure (1 atm). 3 Distilled water, or with a density of 1.0013 g / cm³ 3 Apparent density was evaluated by adding each to a cell culture medium and checking whether the particles floated or settled. The cell culture medium was prepared with 94.9 wt% Advanced MEM, 5 wt% Fetal bovine serum, and 1 wt% Gentamicin.

[0316]

[0317] Experiment 3. Evaluation of Coating Properties

[0318] A cross-sectional sample of the cell culture microcarrier prepared in the above examples and comparative examples was prepared by impregnating the microcarrier for cell culture with polydimethylsiloxane (PDMS) and then microtoming it.

[0319] For the prepared sample, the 1650 cm⁻¹ IR spectrum of polydopamine that does not overlap with polydimethylsiloxane was obtained using Photo-induced Force Microscopy (PiFM). -1 Polydopamine PiFM images were obtained.

[0320] The coating layer thickness and coating rate were derived from the acquired PiFM images. The coating layer thickness represented the average of the measured values.

[0321]

[0322] Experiment 4. Particle Dispersion in the Incubator

[0323] 1 g of the above cell culture microcarrier was dispersed in 4 ml of 1x PBS, 10 mg of GRRGDSK peptide was added and reacted for 18 hours, and then washed 3 times with 1x PBS to prepare a cell culture microcarrier with attached peptide.

[0324] Prior to culture, the amount of the above-mentioned cell culture microcarrier required for cell culture was pre-dispersed in a 20 mL glass vial along with the medium and wetted for approximately 10 to 18 hours. After filtering the pre-dispersed cell culture microcarrier through a cell strainer, it was introduced into a 100 mL 3D bioreactor along with 60 mL of medium, and mesenchymal stem cells (density: 1.05 g / cm³) 3 The culture medium containing ) was filled and cultured for 24 hours. (Culture conditions: 37 ℃, 5% CO2 incubator, bioreactor stirring at 25 rpm)

[0325] After 24 hours of incubation, particles that were not dispersed inside the culture vessel and were floating on the upper interface of the medium were filtered out and removed. The particles dispersed inside the culture vessel were thoroughly washed with DPBS or water, dried, and weighed. The percentage ratio of the weight of the dispersed particles to the total weight was calculated to determine the degree of dispersion, and the results were evaluated according to the following criteria.

[0326] Best: Dispersion 90% or more and 100% or less

[0327] High: Variance of 80% or more and 90% or less

[0328] Medium: Variance of 60% or more and less than 80%

[0329] Lower: Variance less than 60%

[0330]

[0331] Experiment 5. Initial cell adhesion

[0332] 1 g of the above cell culture microcarrier was dispersed in 4 ml of 1x PBS, 10 mg of GRRGDSK peptide was added and reacted for 18 hours, and then washed 3 times with 1x PBS to prepare a cell culture microcarrier with attached peptide.

[0333] Prior to culture, 1 g of the above-mentioned cell culture microcarrier, the amount required for cell culture, was pre-dispersed in a 20 mL glass vial along with the medium and wetted for approximately 10 to 18 hours. After filtering the pre-dispersed cell culture microcarrier through a cell strainer, it was added to a 100 mL 3D bioreactor along with 60 mL of medium, and the vial was filled with a culture medium containing 1,800,000 mesenchymal stem cells (density: 1.05 g / cm³) and cultured for 24 hours. (Culture conditions: 37 ℃, 5% CO2 incubator, bioreactor stirring at 25 rpm)

[0334] After 24 hours of culture, 1 mL of the particle dispersion in the bioreactor vessel was recovered. Using a Nucleocounter NC-200 (Chemometec), the percentage ratio of the number of cells attached to the microcarrier for cell culture relative to the total number of cells was calculated and evaluated according to the following criteria.

[0335] Superior: Percentage of cells attached to microcarriers relative to the total number of cells 93% or higher and 100% or lower

[0336] Top: Percentage of cells attached to microcarriers relative to the total number of cells 85% or more and less than 93%

[0337] Medium: Percentage of cells attached to microcarriers relative to the total number of cells, between 60% and less than 85%

[0338] H: Percentage of cells attached to microcarriers relative to the total number of cells is less than 60%

[0339]

[0340] Experiment 6. Possibility of Separation Based on Density Difference

[0341] For the microcarriers for cell culture prepared in the above examples and comparative examples, whether the cells and the microcarriers for cell culture can be separated by density difference under conditions of room temperature (25 ℃) and atmospheric pressure (1 atm) was evaluated as follows by centrifuging a mixture of cell culture medium and microcarriers.

[0342] O: No particles settled at the bottom after centrifugation.

[0343] X: Particles settled at the bottom after centrifugation.

[0344]

[0345] Experiment 7. IR Peak Ratio

[0346] The entire spectrum of the cell culture microcarriers prepared in the above examples, comparative examples, and reference examples was measured using an ATR-FTIR (Attenuated Total Reflection Fourier transform infrared spectroscopy) instrument.

[0347] Polydopamine peak (3300 cm⁻¹) in the above spectrum -1 Aliphatic ether (CO) peak for the height of ) (1720 cm⁻¹ -1 The ratio of the height was calculated.

[0348]

[0349] Classification Average particle size (㎛, D50) Apparent density (g / cm³) 3Dispersion (%) Initial Cell Adhesion (%) Density Difference Separability Coating Thickness (nm) Coating Rate (%) IR Peak Ratio Example 1 2400 0.997~1.003 Best Best O 138.9±4 294.05 0.342 Example 2 2400 0.997~1.003 Best Best O 149.6±6 090.28 0.867 Example 3 2400 0.997~1.003 Best Best O 128.8±8 086.5 0.440 Example Example 4 155 0.997~1.003 Best Best O13 1.1±5 688 8.3 0.845 Example 5 162 0.997~1.003 Best O14 2.8±6 579.4 0.738 Comparative Example 1 240 0.997~1.003 Best Best O16 2.4±3 498 20.142 Comparative Example 2 240 1.003 Exceeding Best Best X 187.9±5 291.6 1.295 Comparative Example 3 2400 0.997~1.003 Upper Medium O 174.5±5 955.5 0.435 Comparative Example 4 2400 0.997~1.003 Middle Lower O 159.8±1 2444.7 0.430 Comparative Example 5 2400 0.997~1.003 Upper Medium O 166.6±5 262.00.326

[0350] As shown in Table 1 above, the microcarrier for cell culture in the example is 1.003 g / cm³ 3 It was confirmed that the low density below is suitable for cell culture, which improves particle dispersion under cell culture conditions and enables the separation of microcarriers due to density differences. Furthermore, it was confirmed that excellent cell adhesion is exhibited as the cell adhesion ligand peptide reaction is possible.

Claims

Polystyrene-based particles; and A microcarrier for cell culture comprising a coating layer formed on the above-mentioned polystyrene-based particles and including polydopamine and poly(meth)acrylate, The polydopamine coating rate calculated by the following mathematical formula 1 is 70% or more and 98% or less, and Apparent density is 0.98 g / cm³ 3 Exceeding 1.01 g / cm³ 3 Microcarrier for cell culture, less than: [Mathematical Formula 1] Polydopamine coating rate (%) = Area of ​​polydopamine present / Surface area of ​​microcarriers * 100. In paragraph 1, The above-mentioned microcarrier for cell culture has a spectrum of 3300 cm⁻¹ in the ATR-FTIR spectrum. -1 1720 cm for the height of the peak -1 A microcarrier for cell culture having a peak height ratio of 0.33 or more and 1.0 or less. In paragraph 1, A microcarrier for cell culture, wherein the coating layer is prepared from a solution containing 101 parts by weight or more and 2000 parts by weight or less of the poly(meth)acrylate per 100 parts by weight of the polydopamine precursor. In paragraph 1, The above poly(meth)acrylate is a microcarrier for cell culture comprising one or more functional groups including one or more heteroatoms in addition to the (meth)acrylate group. In paragraph 1, The above poly(meth)acrylate comprises one or more functional groups selected from the group consisting of hydroxyl groups, epoxy groups, amine groups, and betaine groups, for use as a microcarrier for cell culture. In paragraph 1, A microcarrier for cell culture comprising one or more poly(meth)acrylates selected from the group consisting of poly2-hydroethyl (meth)acrylate, polyglycidyl (meth)acrylate, poly2-(dimethylamino)ethyl (meth)acrylate, polysulfobetaine (meth)acrylate, carboxybetaine (meth)acrylate, and polyoligo(ethylene glycol)methyl ether (meth)acrylate. In paragraph 1, A cell culture microcarrier having a D50 particle diameter of 150 μm to 300 μm. In paragraph 1, A microcarrier for cell culture having a coating layer thickness of 40 nm or more and 250 nm or less. In paragraph 1, A microcarrier for cell culture, wherein the ratio of the thickness of the coating layer to the D50 particle diameter of the microcarrier for cell culture is 0.0002 or more and 0.0009 or less. In paragraph 1, The above-mentioned polystyrene-based particles are microcarriers for cell culture comprising a monomer compound represented by the following chemical formula 1: [Chemical Formula 1] In the above chemical formula 1, L0 is an arylene group with 6 or more carbon atoms or -(C=O)-, and L1 and L2 are each independently alkylene groups having 1 or more carbon atoms, and R1 is a reactive functional group capable of ring-opening reactions, and R 10 is hydrogen or an alkyl group having 1 or more carbon atoms, and n is an integer greater than or equal to 0. In Paragraph 10, The above polystyrene-based particles comprise the reaction product of a compound represented by the above chemical formula 1 and an ethylene-based unsaturated crosslinking agent, and A microcarrier for cell culture comprising 1 part by weight or more and 10 parts by weight or less of a compound represented by Chemical Formula 1, per 100 parts by weight of the above ethylene-based unsaturated crosslinking agent. In Paragraph 10, A microcarrier for cell culture comprising any one of the compounds represented by the above chemical formula 1 to the compounds represented by the following chemical formula 1-1 to 1-3: [Chemical Formula 1-1] In the above chemical formula 1-1, R 11 is hydrogen or an alkyl group having 1 or more carbon atoms, and [Chemical Formula 1-2] In the above chemical formula 1-2, R 12 is hydrogen or an alkyl group having 1 or more carbon atoms, and [Chemical Formula 1-3] In the above chemical formula 1-4, R 13 It is hydrogen or an alkyl group with 1 or more carbon atoms. In paragraph 1, The above-mentioned polystyrene-based particles are, A microcarrier for cell culture comprising a compound represented by the following chemical formula 2 as a monomer compound: [Chemical Formula 2] In the above chemical formula 2, R2 to R6 are each independently hydrogen or an alkyl group having one or more carbon atoms, and At least one of the above R2 to R6 is It is an alkyl group with 1 or more carbon atoms. In Paragraph 13, A microcarrier for cell culture comprising one or more compounds selected from the group consisting of compounds represented by the following chemical formula 2 to compounds represented by the following chemical formula 2-3, wherein the compound represented by the above chemical formula 2 comprises: [Chemical Formula 2-1] [Chemical Formula 2-2] [Chemical Formula 2-3] In the above chemical formulas 2-1 to 2-3, R 21 to R 26 Each is independently an alkyl group having 1 or more carbon atoms. In Paragraph 13, The compound represented by the above chemical formula 2 has a density of 0.92 g / cm³ 3 Lee Ha-in, microcarrier for cell culture. In Paragraph 13, The above polystyrene-based particles comprise a reaction product of a compound represented by Chemical Formula 1, a styrene-based monomer mixture, and an ethylene-based unsaturated crosslinking agent, and A microcarrier for cell culture comprising 80 parts by weight or more and 99 parts by weight or less of a compound represented by Chemical Formula 2, based on 100 parts by weight of the above ethylene-based unsaturated crosslinking agent. In Paragraph 13, The above polystyrene-based particles comprise a reaction product of a compound represented by Chemical Formula 1, a styrene-based monomer mixture, and an ethylene-based unsaturated crosslinking agent, and A microcarrier for cell culture comprising 90 parts by weight or more and 100 parts by weight or less of a compound represented by Chemical Formula 2, based on 100 parts by weight of the above styrene-based monomer mixture. In Paragraph 13, The above polystyrene-based particles comprise a reaction product of a compound represented by Chemical Formula 1, a styrene-based monomer mixture, and an ethylene-based unsaturated crosslinking agent, and A microcarrier for cell culture comprising 1 part by weight or more and 10 parts by weight or less of a compound represented by Chemical Formula 1, per 100 parts by weight of a compound represented by Chemical Formula 2. A step of polymerizing and recovering polystyrene-based particles; and A method for manufacturing a microcarrier for cell culture, comprising the step of immersing recovered polystyrene-based particles in a solution containing a polydopamine precursor and poly(meth)acrylate and stirring at a temperature of 15°C or lower to form a coating layer containing polydopamine and poly(meth)acrylate on the polystyrene-based particles. A cell culture composition comprising cells and a microcarrier for cell culture according to claim 1. In paragraph 20, The apparent density difference between the cell culture microcarrier and the cell is 0.02 g / cm³ 3 Up to 0.20 g / cm 3 Phosphorus, cell culture composition.

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