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. 10-2024-0067363 dated May 23, 2024, and Korean Patent Application No. 10-2025-0066732 dated May 22, 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 1.05–1.3 g / cm³. 3 The cell density is approximately 1.05-1.2 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, and has improved dispersibility in a cell culture reactor or culture medium.

[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 address the above issues, this specification provides for polystyrene particles containing a compound represented by the following chemical formula 1 as a monomer compound, wherein the apparent density of the microcarrier for cell culture is 0.99 g / cm³. 3 More than 1.04g / cm 3 The following microcarriers for cell culture are provided.

[0011] [ka]

[0012] In the above chemical formula 1, R1 to R5 are each independently hydrogen or an alkyl group having 1 or more carbon atoms. At least one of the R1 to R5 is an alkyl group having 1 or more carbon atoms.

[0013] This specification also includes the step of polymerizing and recovering polystyrene particles from a styrene monomer mixture containing a compound represented by the following chemical formula 1, wherein the apparent density of the microcarriers for cell culture is 0.99 g / cm³. 3 More than 1.04g / cm 3 The following method for producing microcarriers for cell culture is provided.

[0014] [ka]

[0015] In the above chemical formula 1, R1 to R5 are each independently hydrogen or an alkyl group having 1 or more carbon atoms. At least one of the R1 to R5 is an alkyl group having 1 or more carbon atoms.

[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] And in this specification, terms including ordinal numbers such as "first" and "second" are used for the purpose of distinguishing one component from other components and are not limited by said 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] Hereinafter, the present invention will be described in more detail.

[0023] According to an embodiment of the invention, a polystyrene-based particle containing a compound represented by the chemical formula 1 as a monomer compound; is included, and the apparent density of the microcarrier for cell culture is 0.99 g / cm 3 to 1.04 g / cm 3 below, and a microcarrier for cell culture can be provided.

[0024] The inventors of the present invention, in the case of the microcarrier for cell culture of the above embodiment, by including the compound represented by the chemical formula 1 as a polystyrene-based monomer, controlled the density of the finally produced microcarrier for cell culture so that separation due to the density difference from cells was possible and the dispersibility in a cell culture reactor or a medium could be improved, and thus completed the invention.

[0025] Conventional microcarriers for cell culture included non-reactive low-density oil inside the particles to control the density of the particles, but there was a technical problem that when the particles were physically damaged, the low-density oil included inside flowed out.

[0026] Therefore, the inventors of the present invention, by including the compound represented by the chemical formula 1 that satisfies the following as a polystyrene-based monomer used for the microcarrier for cell culture, were able to control the density of the particles so that separation due to the density difference from cells was possible and at the same time the dispersibility in a cell culture reactor or a medium could be improved, and thus completed the invention. 3 to satisfy the following, and completed the invention.

[0027] 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.

[0028] [ka]

[0029] In the above chemical formula 1, R1 to R5 are each independently hydrogen or an alkyl group having 1 or more carbon atoms, and at least one of R1 to R5 is an alkyl group having 1 or more carbon atoms.

[0030] By including the compound represented by chemical formula 1 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 1, enabling separation from cells due to density differences, and simultaneously improving dispersibility in the cell culture reactor or culture medium.

[0031] Specifically, the compound represented by chemical formula 1 may include one or more compounds selected from the group consisting of compounds represented by chemical formulas 1-1 to 3 below.

[0032] [ka]

[0033] In the chemical formulas 1-1 to 1-3, R6 to R 11 Each of these is independently an alkyl group having one or more carbon atoms.

[0034] By including one or more compounds selected from the group consisting of compounds represented by chemical formulas 1-1 to 1-3 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 1, enabling separation from cells due to density differences, and simultaneously improving dispersibility in the cell culture reactor or culture medium.

[0035] More specifically, the compound represented by chemical formula 1 may include one or more compounds selected from the group consisting of compounds represented by chemical formulas 1-4 to 6.

[0036] [ka]

[0037] In the chemical formulas 1-4 to 1-6, R6 to R 11 Each of these is independently an alkyl group having one or more carbon atoms.

[0038] On the other hand, the compound represented by chemical formula 1 has a density of 0.92 g / cm³. 3 The following is also acceptable.

[0039] Specifically, the compound represented by chemical formula 1 has a density of 0.92 g / cm³. 3 Below, 0.91g / cm 3 Below, 0.906g / cm 3 Below, 0.9g / cm 3 Below, 0.89g / cm 3 Below, 0.5g / cm 3 More than 0.6g / cm 3 More than 0.7g / cm 3 More than 0.8g / cm 3 or more, or 0.5 g / cm³ 3 More than 0.92g / cm 3 Below, 0.6g / cm 3 More than 0.92g / cm 3 Below, 0.7g / cm 3 More than 0.92g / cm 3 Below, 0.8g / cm 3 More than 0.92g / cm 3 Below, 0.5g / cm 3 More than 0.91g / cm 3 Below, 0.6g / cm 3 More than 0.91g / cm 3 Below, 0.7g / cm 3 More than 0.91g / cm3 Below, 0.8g / cm 3 More than 0.91g / cm 3 Below, 0.5g / cm 3 More than 0.906g / cm 3 Below, 0.6g / cm 3 More than 0.906g / cm 3 Below, 0.7g / cm 3 More than 0.906g / cm 3 Below, 0.8g / cm 3 More than 0.906g / cm 3 Below, 0.5g / cm 3 More than 0.9g / cm 3 Below, 0.6g / cm 3 More than 0.9g / cm 3 Below, 0.7g / cm 3 More than 0.9g / cm 3 Below, 0.8g / cm 3 More than 0.9g / cm 3 Below, 0.5g / cm 3 More 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.

[0040] The density of the compound represented by the above chemical formula 1 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 1, enabling separation from cells due to density differences, and simultaneously improving dispersibility in the cell culture reactor or culture medium.

[0041] For example, the compound represented by chemical formula 1 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.

[0042] On the other hand, the cell culture microcarrier may include polystyrene particles. The polystyrene particles may include styrene monomer homopolymers, or copolymers of styrene monomers with other monomers and crosslinking agents, and the styrene monomer may include styrene monomers or derivatives thereof.

[0043] Preferably, the cell culture microcarrier may consist of polystyrene particles.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] The density of the aforementioned cells is 1.02 g / cm³. 3 More than 1.1g / cm 3 It is acceptable to be less than [a certain value].

[0048] Furthermore, the density difference between the cell culture microcarriers and the cells is 0.02 g / cm³. 3 More than 0.20g / cm 3 The following may also apply: The density difference between the cell culture microcarrier and the cells is 0.02 g / cm³. 3 More than 0.20g / cm 3 By satisfying the following conditions, cells and microcarriers can be easily separated and recovered after cell culture due to the difference in sedimentation rates caused by gravity.

[0049] The polystyrene particles may include the reaction product of a styrene monomer mixture and an ethylene-based unsaturated crosslinking agent.

[0050] Specifically, the mixture may contain 90 parts by weight or more of the reaction product of the styrene monomer mixture and the ethylene unsaturated crosslinking agent per 100 parts by weight of polystyrene particles. More specifically, the mixture may contain 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, or 95 parts by weight or more and 100 parts by weight or less of the reaction product of the styrene monomer mixture and the ethylene unsaturated crosslinking agent per 100 parts by weight of polystyrene particles.

[0051] Furthermore, the polystyrene particles may contain 51 parts by weight or more of the compound represented by chemical formula 1 per 100 parts by weight of the styrene monomer mixture. Specifically, the polystyrene particles may contain 51 parts by weight or more and 100 parts by weight or less, 55 parts by weight or more and 100 parts by weight or less, or 60 parts by weight or more and 100 parts by weight or less of the compound represented by chemical formula 1 per 100 parts by weight of the styrene monomer mixture.

[0052] By including the compound represented by chemical formula 1 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.

[0053] If an excessively small amount of the compound represented by chemical formula 1 is included in 100 parts by weight of the styrene monomer mixture, the overall density of the polystyrene particles may exceed the target level.

[0054] Furthermore, the polystyrene particles include the reaction product of a styrene monomer mixture and an ethylene-based unsaturated crosslinking agent, and the ethylene-based unsaturated crosslinking agent may be present in an amount of 60 to 200 parts by weight per 100 parts by weight of the styrene monomer mixture.

[0055] Specifically, the styrene monomer mixture may contain 60 to 200 parts by weight, or 60 to 150 parts by weight, of the ethylene-based unsaturated crosslinking agent per 100 parts by weight.

[0056] When the ethylene-based unsaturated crosslinking agent is present in an excessively small amount relative to 100 parts by weight of the styrene monomer mixture, the crosslinking density of the polystyrene polymer decreases, which limits the stability of the particle shape, making it difficult to maintain a spherical form.

[0057] In contrast, if the ethylene-based unsaturated crosslinking agent is excessively present in 100 parts by weight of the styrene monomer mixture, the particles may not stably maintain a spherical shape, and the overall density of the polystyrene particles may exceed the target level.

[0058] Furthermore, the polystyrene particles may contain 51 to 200 parts by weight of the monomer represented by chemical formula 1 per 100 parts by weight of the ethylene-based unsaturated crosslinking agent.

[0059] Specifically, the polystyrene particles may contain, per 100 parts by weight of the ethylene-based unsaturated crosslinking agent, 51 parts by weight or more, 60 parts by weight or more, 65 parts by weight or more, 200 parts by weight or less, 180 parts by weight or less, 150 parts by weight or less of the monomer represented by chemical formula 1, or 51 parts by weight or more and 200 parts by weight or less, 60 parts by weight or more and 200 parts by weight or less, 65 parts by weight or more and 200 parts by weight or less, 51 parts by weight or more and 180 parts by weight or less, 60 parts by weight or more and 180 parts by weight or less, 65 parts by weight or more and 180 parts by weight or less, 51 parts by weight or more and 150 parts by weight or less, 60 parts by weight or more and 150 parts by weight or less, and 65 parts by weight or more and 150 parts by weight or less.

[0060] When an excessively small amount of the monomer represented by chemical formula 1 is included with 100 parts by weight of the ethylene-based unsaturated crosslinking agent, there is a limit to how stably the particle morphology can be maintained in a spherical shape, and how easily the overall density of the polystyrene particles can be reduced to the target level.

[0061] Furthermore, if the compound represented by chemical formula 1 is excessively present in proportion to 100 parts by weight of the ethylene-based unsaturated crosslinking agent, the crosslinking density of the polystyrene polymer decreases, making it difficult for the particles to stably maintain a spherical shape, and there is a limit to how low the overall density of the polystyrene particles can fall below the target level.

[0062] Divinylbenzene is an example of the ethylene-based unsaturated crosslinking agent mentioned above.

[0063] Conventionally, foamed styrene has been produced by adding a blowing agent to lower the density of polystyrene particles. However, in this case, the distribution of the diameter range and density range of the polystyrene particles becomes excessively wide, making it difficult to ensure a yield within a range suitable for use as a microcarrier.

[0064] The average diameter of the polystyrene particles may be 50 μm to 400 μm, 60 μm to 390 μm, 80 μm to 350 μm, or 90 μm to 300 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.

[0065] 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.

[0066] 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.

[0067] 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%.

[0068] [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.

[0069] 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 or optical microscope, and calculating the percentage ratio of the number of perfectly spherical particles without damage or destruction to the total number of particles.

[0070] In other words, the cell culture microcarrier can contain multiple polystyrene particles, and whether or not these multiple polystyrene particles have a perfectly spherical shape without damage or destruction can be determined visually using a scanning electron microscope or an optical microscope.

[0071] 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.

[0072] 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.

[0073] On the other hand, the cell culture microcarrier may include a cell adhesion induction layer formed on the polystyrene particles.

[0074] 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.

[0075] 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, poly-L-lysine, vitronectin, peptides containing RGD, lignin, cationic dextran, and derivatives thereof.

[0076] Furthermore, the cell culture microcarrier may selectively include a primer polymer layer formed between the polystyrene particles and the cell adhesion induction layer. In other words, the cell culture microcarrier of the above embodiment may include the polystyrene particles; a primer polymer layer formed on the surface of the polystyrene particles; and a cell adhesion induction layer formed on the surface of the primer polymer layer.

[0077] The aforementioned primer polymer layer acts as an adhesive layer that allows functional polymers to be introduced to the polystyrene surface which lacks functional groups. This effectively introduces a polymer layer for cell adhesion to the surface of the microcarrier, and ensures that it is stably maintained even during culture.

[0078] The aforementioned primer polymer layer is not limited in its examples, but it may contain one or more catechol derivatives selected from the group consisting of L-dihydroxyphenylalanine (L-DOPA), dopamine, norepinephrine, epinephrine, epigallocatechin, and derivatives thereof, which can induce aqueous phase adhesion.

[0079] On the other hand, according to another embodiment of the invention, the step of polymerizing and recovering polystyrene particles from a styrene monomer mixture containing a compound represented by the following chemical formula 1, wherein the apparent density of the microcarriers for cell culture is 0.99 g / cm³. 3 More than 1.04g / cm 3 The following method for producing microcarriers for cell culture can be provided.

[0080] 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.

[0081] 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 a monomer composition containing styrene monomers and recovering the result of the suspension polymerization reaction.

[0082] Specifically, the compound represented by chemical formula 1 may include one or more compounds selected from the group consisting of compounds represented by chemical formulas 1-1 to 3 below.

[0083] [ka]

[0084] In the chemical formulas 1-1 to 1-3, R6 to R 11 Each of these is independently an alkyl group having one or more carbon atoms.

[0085] By containing one or more compounds selected from the group consisting of the compound represented by the chemical formula 1-1 and the compound represented by the chemical formula 1-3 as a polystyrene monomer, by adjusting the content of the compound represented by the chemical formula 1, the density of the microcarrier for final cell culture can be precisely controlled, separation due to the density difference from cells is possible, and at the same time, the dispersibility in a cell culture reactor or a culture medium can be improved.

[0086] More specifically, the compound represented by the chemical formula 1 may contain one or more compounds selected from the group consisting of the compound represented by the following chemical formula 1-4 and the compound represented by the following chemical formula 1-6.

[0087]

Chemical formula

[0088] In the above chemical formulas 1-4 to 1-6, R6 to R 11 are each independently an alkyl group having 1 or more carbon atoms.

[0089] On the other hand, the compound represented by the chemical formula 1 may have a density of 0.9 g / cm 3 or less.

[0090] Specifically, the compound represented by the chemical formula 1 has a density of 0.92 g / cm 3 or less, 0.91 g / cm 3 or less, 0.906 g / cm 3 or less, 0.9 g / cm 3 or less, 0.89 g / cm 3 or less, 0.5 g / cm 3 or more, 0.6 g / cm 3 or more, 0.7 g / cm 3 or more, 0.8 g / cm 3 or more, or 0.5 g / cm 3 or more and 0.92 g / cm 3 or less, 0.6 g / cm 3 or more and 0.92 g / cm 3 or less, 0.7 g / cm3 Above 0.92 g / cm 3 Below, 0.8 g / cm 3 Above 0.92 g / cm 3 Below, 0.5 g / cm 3 Above 0.91 g / cm 3 Below, 0.6 g / cm 3 Above 0.91 g / cm 3 Below, 0.7 g / cm 3 Above 0.91 g / cm 3 Below, 0.8 g / cm 3 Above 0.91 g / cm 3 Below, 0.5 g / cm 3 Above 0.906 g / cm 3 Below, 0.6 g / cm 3 Above 0.906 g / cm 3 Below, 0.7 g / cm 3 Above 0.906 g / cm 3 Below, 0.8 g / cm 3 Above 0.906 g / cm 3 Below, 0.5 g / cm 3 Above 0.9 g / cm 3 Below, 0.6 g / cm 3 Above 0.9 g / cm 3 Below, 0.7 g / cm 3 Above 0.9 g / cm 3 Below, 0.8 g / cm 3 Above 0.9 g / cm 3 Below, 0.5 g / cm 3 Above 0.89 g / cm 3 Below, 0.6 g / cm 3 Above 0.89 g / cm 3 Below, 0.7 g / cm 3 Above 0.89 g / cm 3 Below, 0.8 g / cm 3 Above 0.89 g / cm 3 It may also be below. By satisfying that the density of the compound represented by the chemical formula 1 is 0.92 g / cm 3 Below, by adjusting the content of the compound represented by the chemical formula 1, the density of the microcarrier for final cell culture can be precisely controlled, separation due to the density difference from cells becomes possible, and at the same time, the dispersibility in the cell culture reactor or the medium can be improved.

[0091] For example, the compound represented by chemical formula 1 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.

[0092] The polystyrene particles may include the reaction product of a styrene monomer mixture and an ethylene-based unsaturated crosslinking agent.

[0093] Specifically, the mixture may contain 90 parts by weight or more of the reaction product of the styrene monomer mixture and the ethylene unsaturated crosslinking agent per 100 parts by weight of polystyrene particles. More specifically, the mixture may contain 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, or 95 parts by weight or more and 100 parts by weight or less of the reaction product of the styrene monomer mixture and the ethylene unsaturated crosslinking agent per 100 parts by weight of polystyrene particles.

[0094] Furthermore, the polystyrene particles may contain 51 parts by weight or more of the compound represented by chemical formula 1 per 100 parts by weight of the styrene monomer mixture. Specifically, the polystyrene particles may contain 51 parts by weight or more and 100 parts by weight or less, 55 parts by weight or more and 100 parts by weight or less, or 60 parts by weight or more and 100 parts by weight or less of the compound represented by chemical formula 1 per 100 parts by weight of the styrene monomer mixture.

[0095] By including the compound represented by chemical formula 1 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.

[0096] If an excessively small amount of the compound represented by chemical formula 1 is included in 100 parts by weight of the styrene monomer mixture, the overall density of the polystyrene particles may exceed the target level.

[0097] Furthermore, the polystyrene particles include the reaction product of a styrene monomer mixture and an ethylene-based unsaturated crosslinking agent, and the ethylene-based unsaturated crosslinking agent may be present in an amount of 60 to 200 parts by weight per 100 parts by weight of the styrene monomer mixture.

[0098] Specifically, the styrene monomer mixture may contain 60 to 200 parts by weight, or 60 to 150 parts by weight, of the ethylene-based unsaturated crosslinking agent per 100 parts by weight.

[0099] When the ethylene-based unsaturated crosslinking agent is present in an excessively small amount relative to 100 parts by weight of the styrene monomer mixture, the crosslinking density of the polystyrene polymer decreases, which limits the stability of the particle shape, making it difficult to maintain a spherical form.

[0100] In contrast, if the ethylene-based unsaturated crosslinking agent is excessively present in 100 parts by weight of the styrene monomer mixture, the particles may not stably maintain a spherical shape, and the overall density of the polystyrene particles may exceed the target level.

[0101] Furthermore, the polystyrene particles may contain 51 to 200 parts by weight of the compound represented by chemical formula 1 per 100 parts by weight of the ethylene-based unsaturated crosslinking agent.

[0102] Specifically, the polystyrene particles may contain, per 100 parts by weight of the ethylene-based unsaturated crosslinking agent, 51 parts by weight or more, 60 parts by weight or more, 65 parts by weight or more, 200 parts by weight or less, 180 parts by weight or less, 150 parts by weight or less of the compound represented by chemical formula 1, or 51 parts by weight or more and 200 parts by weight or less, 60 parts by weight or more and 200 parts by weight or less, 65 parts by weight or more and 200 parts by weight or less, 51 parts by weight or more and 180 parts by weight or less, 60 parts by weight or more and 180 parts by weight or less, 65 parts by weight or more and 180 parts by weight or less, 51 parts by weight or more and 150 parts by weight or less, 60 parts by weight or more and 150 parts by weight or less, 65 parts by weight or more and 150 parts by weight or less.

[0103] When the compound represented by chemical formula 1 is included in an excessively small amount relative to 100 parts by weight of the ethylene-based unsaturated crosslinking agent, there is a limit to how stably the particle morphology can be maintained in a spherical shape.

[0104] Furthermore, if the compound represented by chemical formula 1 is excessively present in proportion to 100 parts by weight of the ethylene-based unsaturated crosslinking agent, the crosslinking density of the polystyrene polymer decreases, making it difficult for the particles to stably maintain a spherical shape, and there is a limit to how low the overall density of the polystyrene particles can fall below the target level.

[0105] Divinylbenzene is an example of the ethylene-based unsaturated crosslinking agent mentioned above.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] 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.

[0110] On the other hand, the method for producing the cell culture microcarrier may further include a washing step and a drying step after the step of polymerizing and recovering polystyrene particles from a styrene monomer mixture containing the compound represented by chemical formula 1.

[0111] Specifically, the washing step may include filtering the reaction product through a sieve with a diameter of 30 μm to 100 μm, followed by stirring 3 to 5 times with distilled water at 50 to 75°C, and stirring 3 to 5 times with ethanol at room temperature.

[0112] The aforementioned drying step includes drying in a convection oven at room temperature or up to 80°C. However, it is not limited to this, and any drying method known to be commonly used may be used without any particular restrictions.

[0113] On the other hand, the method for producing the cell culture microcarriers may further include a step of washing and a step of drying; followed by a step of applying a cell adhesion-inducing layer onto the polystyrene particles.

[0114] Selectively, the method for producing the cell culture microcarriers may further include, after a washing step and a drying step, a step of applying a primer polymer layer to the surface of the polystyrene particles, and a step of applying a cell adhesion induction layer to the surface of the primer polymer layer.

[0115] Specifically, the step of applying a primer polymer layer to the surface of the polystyrene particles may include the step of immersing the polystyrene particles in the primer polymer solution for 1 to 10 hours, 2 to 6 hours, or 3 to 5 hours.

[0116] The aforementioned primer polymer layer is not limited in its examples, but it may contain one or more catechol derivatives selected from the group consisting of L-dihydroxyphenylalanine (L-DOPA), dopamine, norepinephrine, epinephrine, epigallocatechin, and derivatives thereof, which can induce aqueous phase adhesion.

[0117] After recovering and drying the manufactured polystyrene particles, a primer polymer layer is applied to modify the surface of the microcarriers to be hydrophilic, thereby allowing for water dispersion. This enables the stable introduction of a cell adhesion induction layer, allowing for adjustment of the buoyancy of the microcarriers in the culture medium, and thus providing the effect of stably adhering and culturing cells.

[0118] In this case, the ratio of the radius of the polystyrene particles to the thickness of the primer polymer layer may be 1:0.00001 to 1:0.01, or 1:0.0001 to 1:0.001.

[0119] On the other hand, the method for manufacturing microcarriers according to the above embodiment may further include the step of applying a cell adhesion induction layer to the surface of the primer polymer layer after the step of applying a primer polymer layer to the surface of the polystyrene-based particles.

[0120] The step of applying a cell adhesion-inducing layer to the surface of the primer polymer layer may include coating 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, and derivatives thereof.

[0121] A solution containing one or more selected from the group consisting of gelatin, collagen, fibronectin, chitosan, poly-L-lysine, vitronectin, peptides containing RGD, lignin, cationic dextran, and derivatives thereof can act as an adhesion factor to bond cells to microcarriers. When a primer polymer layer is coated with a solution containing gelatin, the degree of adhesion between cells and microcarriers can be increased, making it suitable for large-scale cell culture.

[0122] Specifically, the step of applying a cell adhesion induction layer to the surface of the primer polymer layer may include a step of immersing the substance obtained in the step of applying the primer polymer layer to 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, and derivatives thereof, for 10 to 20 hours, or 15 to 20 hours, or 17 to 19 hours.

[0123] 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.

[0124] 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.

[0125] The density of the aforementioned cells is 1.02 g / cm³. 3 More than 1.1g / cm 3 It is acceptable to be less than [a certain value].

[0126] Furthermore, the density difference between the cell culture microcarriers and the cells is 0.02 g / cm³. 3 More than 0.20g / cm 3 The following may also apply: The density difference between the cell culture microcarrier and the cells is 0.02 g / cm³. 3 More than 0.20g / cm 3 By satisfying the following conditions, cells and microcarriers can be easily separated and recovered after cell culture due to the difference in sedimentation rates caused by gravity.

[0127] The cell culture composition may further include a culture medium solution. The culture medium solution may contain nutrients and various additives to adequately satisfy environmental conditions such as pH, temperature, and osmotic pressure, which are close to those of living organisms based on body fluids such as plasma or lymph. A wide variety of substances widely known in the field of cell culture technology can be used without limitation.

[0128] As an example, the cell culture microcarrier of the above embodiment has a density lower than that of the culture medium and, when injected into the culture medium, floats within the culture medium under agitation conditions. Subsequently, as the number of cells adhering to the surface of the low-density microcarrier increases, the density of the cell-attached microcarrier (hereinafter referred to as "microcarrier-cell conjugate") gradually increases, causing it to gradually sink in the culture medium.

[0129] Therefore, cultured cells can be easily obtained by separating the cells from the microcarriers (microcarrier-cell conjugates) to which cells are attached, after adding a cell desorption enzyme, and then separating them by centrifugation. [Effects of the Invention]

[0130] According to the present invention, it is possible to provide a microcarrier for cell culture that has high surface hydrophilicity and improved cell adhesion, as well as improved dispersibility in a cell culture reactor or culture medium, a method for producing a microcarrier for cell culture, and a cell culture method using the same. [Modes for carrying out the invention]

[0131] The invention will be described in more detail by the following embodiments. However, the following embodiments are merely illustrative of the present invention, and the content of the present invention is not limited to the following embodiments.

[0132] Example 1 Polyvinyl alcohol (molecular weight 85-124K, 87-89% hydrolysis) was dissolved in distilled water at a concentration of 2%, and 2% by weight of sodium chloride additive (based on the weight of distilled water) was added to the aqueous dispersion. The mixture was then stirred at room temperature for 20 minutes.

[0133] The monomers are styrene (Sigma-Aldrich) and methylstyrene (Sigma-Aldrich, density: 0.89-0.92 g / cm³). 3A monomer composition was prepared by mixing 60 g of a mixture of ) and divinylbenzene (TCI), a crosslinking agent, in a weight ratio of 2:3:5 and thoroughly dissolving it, then adding 2% by weight of V-65 initiator (amount of initiator added: based on the sum of monomer and crosslinking agent) and stirring for an additional 5 minutes.

[0134] 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.

[0135] 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 copolymer particles. The particles were washed three times with distilled water at 60°C and five times with ethanol, and then dried in an oven at 80°C to be recovered.

[0136] Average diameter: 183 μm (measured using a PSA instrument, based on D50 standard) Apparent density: 0.99~1.04 g / cm³ 3

[0137] Example 2 Microcarriers were produced in the same manner as in Example 1, except that the ratio of monomers to crosslinking agents was adjusted to a weight ratio of styrene:methylstyrene:divinylbenzene 1:4:5.

[0138] Average diameter: 196 μm (measured using a PSA instrument, based on D50 standard) Apparent density: 0.99~1.02 g / cm³ 3

[0139] Example 3 In Example 1, microcarriers were produced in the same manner as in Example 1, except that the ratio of monomers to crosslinking agents was adjusted to a weight ratio of styrene:methylstyrene:divinylbenzene 1:5:4 and the amount of initiator added was increased by 3%.

[0140] Average diameter: 183 μm (measured using a PSA instrument, based on D50 standard) Apparent density: 0.99~1.02 g / cm³ 3

[0141] Example 4 Microcarriers were produced in the same manner as in Example 1, except that the ratio of monomers excluding styrene and the crosslinking agent was adjusted to a weight ratio of methylstyrene:divinylbenzene 4:6.

[0142] Average diameter: 185 μm (measured using a PSA instrument, based on D50 standard) Apparent density: 0.99~1.02 g / cm³ 3

[0143] Example 5 In Example 1, the ratio of monomer to crosslinking agent was set to styrene:trimethylstyrene (Sigma-Aldrich, density: 0.906 g / cm³). 3 Microcarriers were manufactured in the same manner as in Example 1, except that the weight ratio of divinylbenzene was adjusted to 2:3:5.

[0144] Average diameter: 213 μm (measured using a PSA instrument, based on D50 standard) Apparent density: 0.99~1.04 g / cm³ 3

[0145] Example 6 Microcarriers were produced in the same manner as in Example 5, except that the ratio of monomers to crosslinking agents was adjusted to a weight ratio of styrene:trimethylstyrene:divinylbenzene 1:4:5.

[0146] Average diameter: 180 μm (measured using a PSA instrument, based on D50 standard) Apparent density: 0.99~1.04 g / cm³ 3

[0147] Example 7 Microcarriers were produced in the same manner as in Example 4, except that the ratio of monomer to crosslinking agent was adjusted to a weight ratio of trimethylstyrene:divinylbenzene 4:6.

[0148] Average diameter: 198 μm (measured using a PSA instrument, based on D50 standard) Apparent density: 0.99~1.04 g / cm³ 3

[0149] Example 8 Microcarriers were produced in the same manner as in Example 7, except that the ratio of monomer to crosslinking agent was adjusted to a weight ratio of trimethylstyrene:divinylbenzene 5:5.

[0150] Average diameter: 205 μm (measured using a PSA instrument, based on D50 standard) Apparent density: 0.99~1.02 g / cm³ 3

[0151] Example 9 In Example 8, the ratio of monomer to crosslinking agent was set to 4-tert-butylstyrene (Sigma-Aldrich, density: 0.875 g / cm³). 3 Microcarriers were manufactured in the same manner as in Example 8, except that the weight ratio of divinylbenzene was adjusted to 5:5.

[0152] Average diameter: 221 μm (measured using a PSA instrument, based on D50 standard) Apparent density: 0.99~1.04 g / cm³ 3

[0153] Example 10 Microcarriers were produced in the same manner as in Example 1, except that the ratio of monomers to crosslinking agents was adjusted to a weight ratio of styrene:4-tert-butylstyrene:divinylbenzene 2:3:5.

[0154] Average diameter: 231 μm (measured using a PSA instrument, based on D50 standard) Apparent density: 0.99~1.04 g / cm³ 3

[0155] Example 11 In Example 10, the microcarrier was produced in the same manner as in Example 10, except that the ratio of monomer to crosslinking agent was adjusted to a weight ratio of styrene:4-tert-butylstyrene:divinylbenzene 1:5:4 and the amount of initiator added was increased by 3%.

[0156] Average diameter: 183 μm (measured using a PSA instrument, based on D50 standard) Apparent density: 0.99~1.04 g / cm³ 3

[0157] Example 12 In Example 2, the ratio of monomer to crosslinking agent was set to 2,4-dimethylstyrene (Sigmal-Aldrich, density: 0.906 g / cm³). 3 Microcarriers were manufactured in the same manner as in Example 2, except that the weight ratio of methylstyrene:divinylbenzene was adjusted to 1:4:5.

[0158] Average diameter: 205 μm (measured using a PSA instrument, based on D50 standard) Apparent density: 0.99~1.04 g / cm³ 3

[0159] Example 13 Microcarriers were produced in the same manner as in Example 12, except that the ratio of monomers and crosslinking agents was adjusted to a weight ratio of 2,4-dimethylstyrene (Sigmal-Aldrich):methylstyrene:divinylbenzene 1:5:4 and the amount of initiator was increased to 3%.

[0160] Average diameter: 198 μm (measured using a PSA instrument, based on D50 standard) Apparent density: 0.99~1.02 g / cm³ 3

[0161] Example 14 Microcarriers were produced in the same manner as in Example 12, except that the ratio of monomer to crosslinking agent was adjusted to a weight ratio of 2,4-dimethylstyrene:trimethylstyrene:divinylbenzene of 1:4:5.

[0162] Average diameter: 187 μm (measured using a PSA instrument, based on D50 standard) Apparent density: 0.99~1.02 g / cm³ 3

[0163] Example 15 In Example 12, the microcarrier was produced in the same manner as in Example 12, except that the ratio of monomer to crosslinking agent was adjusted to a weight ratio of 2,4-dimethylstyrene:4-tert-butylstyrene:divinylbenzene 1:5:4 and the amount of initiator was increased to 3%.

[0164] Average diameter: 218 μm (measured using a PSA instrument, based on D50 standard) Apparent density: 0.99~1.04 g / cm³ 3

[0165] [Table 1]

[0166] Comparative Example 1 Polyvinyl alcohol (molecular weight 85-124K, 87-89% hydrolysis) was dissolved in distilled water at a concentration of 2%, and 2% by weight of sodium chloride additive (based on the weight of distilled water) was added to the aqueous dispersion. The mixture was then stirred at room temperature for 20 minutes.

[0167] The weight ratio of the monomer styrene to the crosslinking agent divinylbenzene is 1:1, and the amount of oil added (based on the total sum of monomer, crosslinking agent, and oil) is 10% by weight, using Isopar M (a mixture of isoalkanes with 12 to 14 carbon atoms and isoalkanes with 13 to 16 carbon atoms; density 0.79 g / cm³) as the oil. 3 A monomer composition was prepared by adding 2% by weight of V-65 initiator (amount of initiator added: based on the sum of monomer and crosslinking agent) to 120 g of a mixture obtained by mixing and thoroughly dissolving the ingredients, and stirring for an additional 5 minutes.

[0168] 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.

[0169] 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 three times 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.

[0170] The physical properties of the aforementioned polystyrene particles are as follows:

[0171] Average diameter: 194 μm (measured using a PSA instrument, based on D50 standard) Apparent density: 1.003 g / cm³ 3 excess Gap diameter: 0.05μm~4μm

[0172] Comparative Example 2 The amount of oil added (based on the sum of monomers, crosslinking agent, and oil) is 14% by weight. The oil used is Isopar M (a mixture of isoalkanes with 12 to 14 carbon atoms and isoalkanes with 13 to 16 carbon atoms; density 0.79 g / cm³). 3 Microcarriers were manufactured in the same manner as in Comparative Example 1, except that ) was mixed in.

[0173] The physical properties of the aforementioned polystyrene particles are as follows:

[0174] Average diameter: 190.6 μm (measured using a PSA instrument, based on D50 standard) Apparent density: 0.98-0.99 g / cm³ 3 Gap diameter: 0.05μm~4μm

[0175] Comparative Example 3 The amount of oil added (based on the sum of monomers, crosslinking agent, and oil) is 20% by weight, and the oil is used as Isopar M (a mixture of isoalkanes with 12 to 14 carbon atoms and isoalkanes with 13 to 16 carbon atoms; density 0.79 g / cm³). 3 Microcarriers were manufactured in the same manner as in Comparative Example 1, except that ) was mixed in.

[0176] The physical properties of the aforementioned polystyrene particles are as follows:

[0177] Average diameter: 188.07 μm (measured using a PSA instrument, based on D50 standard) Apparent density: 0.97~0.98 g / cm³ 3 Gap diameter: 0.05μm~4μm

[0178] Comparative Example 4 In the above-mentioned Example 1, microcarriers were manufactured in the same manner as in Example 1, except that styrene was used alone as the monomer and the weight ratio with divinylbenzene, which is a crosslinking agent, was adjusted to 1:1.

[0179] Average diameter: 198 μm (measured using a PSA instrument, based on D50 standard) Apparent density: >1.04 g / cm³ 3

[0180] Reference example 1 Microcarriers were manufactured in the same manner as in Example 1, except that the content of each monomer was adjusted as shown in Table 2.

[0181] Average diameter: 204.5 μm (measured using a PSA instrument, based on D50 standard) Apparent density: >1.04 g / cm³ 3

[0182] [Table 2]

[0183] <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 Table 3.

[0184] 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.

[0185] Experiment 2. Apparent density (unit: g / cm³) 3 ) The cell culture microcarriers produced in the above examples and comparative examples were subjected to a density of 0.985 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 / cm³3 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.

[0186] 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 3 It 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<1.003g / cm 3 (0.99g / cm 3 Excess 1.003g / cm 3 less than) Density is 0.99 g / cm³ 3 It settles in distilled water and has a density of 1.003 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

[0187] Experiment 3. Particle Dispersion Degree in the Incubator Before culturing, the required amount of the microcarriers for cell culture was pre-dispersed together with the culture medium in a 20 mL glass vial and wetted for about 10 to 18 hours. After filtering the pre-dispersed microcarriers for cell culture through a cell strainer, they were put into a 100 mL 3D bioreactor together with 60 mL of the culture medium and stirred for 24 hours under the actual cell culture conditions. (Culture conditions: 37 °C, 5% CO2 incubator, bioreactor stirred at 25 rpm)

[0188] After culturing for 24 hours, the particles floating on the interface at the upper end of the culture medium without being dispersed inside the incubator were filtered off. The particles dispersed inside the incubator were washed thoroughly with DPBS or water and then dried to measure the weight, and the percentage ratio of the weight of the dispersed particles to the total weight was calculated to calculate the dispersion degree, which was evaluated according to the following criteria.

[0189] Upper: Dispersion degree of 80% or more and 100% or less Medium: Dispersion degree of 60% or more and less than 80% Lower: Dispersion degree less than 60%

[0190] Experiment 4. Feasibility of Separation by Density Difference Regarding the microcarriers for cell culture produced in the above examples and comparative examples, under the conditions of normal temperature (25 °C) and normal pressure (1 atm), whether it was possible to separate the cells from the microcarriers for cell culture by density difference was evaluated as follows by centrifuging the mixture of the cell culture medium and the microcarriers.

[0191] O: No particles sank to the lower end after centrifugation. X: There were particles that sank to the lower end after centrifugation.

[0192] [Table 3]

[0193] As shown in Table 3 above, the microcarrier for cell culture in the examples was 1.04 g / cm³. 3 We were able to confirm that the following low densities are suitable for cell culture, which improves particle dispersion under cell culture conditions and enables the separation of microcarriers due to density differences.

[0194] In contrast, it was confirmed that separation of the microcarriers in Comparative Example 1 for cell culture was impossible due to density differences.

[0195] Furthermore, the cell culture microcarriers of Comparative Examples 2 to 3 contained low-density oil within the particles, which not only reduced particle dispersion under cell culture conditions but also made it possible for the low-density oil contained within to leak out due to damage to the cell culture microcarriers during culture.

[0196] Furthermore, in the case of Comparative Example 4, it was confirmed that not only was the particle dispersion poor under the cell culture conditions, but that separation of microcarriers due to density differences was impossible.

Claims

1. Polystyrene particles containing a monomer compound represented by the following chemical formula 1; The apparent density of the microcarriers for cell culture is 0.99 g / cm³. ３ 1.04g / cm or more ３ The following are microcarriers for cell culture: 【Chemistry 1】 In the aforementioned chemical formula 1, R １ ~R ５ Each of these is independently a hydrogen atom or an alkyl group having one or more carbon atoms. The aforementioned R １ ~R ５ At least one of them is an alkyl group having one or more carbon atoms.

2. The microcarrier for cell culture according to claim 1, wherein the compound represented by chemical formula 1 comprises one or more compounds selected from the group consisting of compounds represented by chemical formulas 1-1 to 3 below: 【Chemistry 2】 In the aforementioned chemical formulas 1-1 to 1-3, R ６ ~R １１ Each of these is independently an alkyl group having one or more carbon atoms.

3. The compound represented by the above Chemical Formula 1 has a density of 0.92 g / cm ３ The microcarrier for cell culture according to claim 1, which is as follows.

4. The polystyrene particles include a reaction product of a styrene monomer mixture and an ethylene-based unsaturated crosslinking agent. The microcarrier for cell culture according to claim 1, comprising 90 parts by weight or more of the reaction product of the styrene monomer mixture and the ethylene-based unsaturated crosslinking agent with respect to 100 parts by weight of the polystyrene particles.

5. 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.

6. The polystyrene particles include a reaction product of a styrene monomer mixture and an ethylene-based unsaturated crosslinking agent. The microcarrier for cell culture according to claim 1, comprising 51 parts by weight or more of the compound represented by chemical formula 1 per 100 parts by weight of the styrene monomer mixture.

7. The polystyrene particles include a reaction product of a styrene monomer mixture and an ethylene-based unsaturated crosslinking agent. The microcarrier for cell culture according to claim 1, comprising 51 to 200 parts by weight of the compound represented by chemical formula 1 per 100 parts by weight of the ethylene-based unsaturated crosslinking agent.

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

9. The step includes polymerizing and recovering polystyrene particles from a styrene monomer mixture containing a compound represented by the following chemical formula 1; The apparent density of the microcarriers for cell culture is 0.99 g / cm³. ３ 1.04g / cm or more ３ The method for producing microcarriers for cell culture is as follows: 【Transformation 3】 In the aforementioned chemical formula 1, R １ ~R ５ Each of these is independently a hydrogen atom or an alkyl group having one or more carbon atoms. The aforementioned R １ ~R ５ At least one of them is an alkyl group having one or more carbon atoms.

10. The method for producing a microcarrier for cell culture according to claim 9, wherein the compound represented by chemical formula 1 comprises one or more compounds selected from the group consisting of compounds represented by chemical formulas 1-1 to 3 below: 【Chemistry 4】 In the aforementioned chemical formulas 1-1 to 1-3, R ６ ~R １１ Each of these is independently an alkyl group having one or more carbon atoms.

11. The compound represented by the aforementioned chemical formula 1 has a density of 0.92 g / cm³. ３ The method for producing a microcarrier for cell culture according to claim 9 is as follows:

12. A method for producing a microcarrier for cell culture according to claim 9, comprising 51 parts by weight or more of the compound represented by chemical formula 1 per 100 parts by weight of the styrene monomer mixture.

13. A method for producing a microcarrier for cell culture according to claim 9, further comprising the step of coating a cell adhesion-inducing layer onto the polystyrene particles.

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

15. The cell culture composition according to claim 14, wherein the cells comprise 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.

16. The apparent density difference between the cell culture microcarriers and the cells is 0.02 g / cm³. ３ ~0.20 g / cm ３ The cell culture composition according to claim 14.

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