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

Abstract

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

Description

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

[0001] This application claims the benefit of priority to Korean Patent Application No. 10-2024-0067363, filed May 23, 2024, and Korean Patent Application No. 10-2025-0066732, filed May 22, 2025, the entire contents of which are incorporated herein by reference.

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

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

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

[0005] Currently commercially available microcarriers have densities of about 1.05 to 1.3 g / cm. 3 , and the cell density is about 1.05 to 1.2 g / cm 3This is a degree of difficulty. While this is advantageous for initial cell attachment within the bioreactor, centrifugation is difficult for cell separation and recovery after culture, requiring the use of microcarriers and a filtration method based on cell size. However, this method poses problems such as filter clogging, prolonged processing times, potential physical damage and contamination of cells, and potential cell loss.

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

[0007] The present invention relates to providing a cell culture microcarrier capable of separating cells by density difference by controlling density, while improving dispersibility in a cell culture reactor or medium.

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

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

[0010] In order to solve the above problem, the present specification provides a polystyrene-based particle comprising a compound represented by the following chemical formula 1 as a monomer compound; and the apparent density of the cell culture microcarrier is 0.99 g / cm. 3 Above 1.04 g / cm 3 Below, a micro carrier for cell culture is provided.

[0011] [Chemical Formula 1]

[0012]

[0013] In the above chemical formula 1,

[0014] R1 to R5 are each independently hydrogen or an alkyl group having 1 or more carbon atoms,

[0015] At least one of the above R1 to R5 It is an alkyl group with 1 or more carbon atoms.

[0016]

[0017] The present specification also includes a step of polymerizing and recovering polystyrene particles from a styrene monomer mixture including a compound represented by the following chemical formula 1; and the apparent density of the cell culture microcarrier is 0.99 g / cm. 3 Above 1.04 g / cm 3 Below, a method for manufacturing a micro carrier for cell culture is provided.

[0018] [Chemical Formula 1]

[0019]

[0020] In the above chemical formula 1,

[0021] R1 to R5 are each independently hydrogen or an alkyl group having 1 or more carbon atoms,

[0022] At least one of the above R1 to R5 It is an alkyl group with 1 or more carbon atoms.

[0023]

[0024] Also provided herein is a cell culture composition comprising cells and microcarriers for cell culture.

[0025] Hereinafter, a cell culture microcarrier according to a specific embodiment of the invention, a method for manufacturing a cell culture microcarrier, and a cell culture composition using the same will be described in more detail.

[0026]

[0027] Unless explicitly stated otherwise in this specification, terminology is used only to describe specific embodiments and is not intended to limit the invention.

[0028] As used herein, the singular forms also include the plural forms unless the context clearly dictates otherwise.

[0029] As used herein, the term “including” means specifying a particular characteristic, region, integer, step, operation, element and / or component, but does not exclude the presence or addition of other particular characteristics, regions, integers, steps, operations, elements, components and / or groups.

[0030] In addition, terms including ordinal numbers, such as "first" and "second," in this specification are used to distinguish one component from another and are not limited by the ordinal numbers. For example, within the scope of the present invention, the first component may also be referred to as the second component, and similarly, the second component may be referred to as the first component.

[0031] Hereinafter, the present invention will be described in more detail.

[0032]

[0033] According to one embodiment of the invention, a polystyrene particle comprising a compound represented by the above chemical formula 1 as a monomer compound; and an apparent density of the cell culture microcarrier is 0.99 g / cm 3 Above 1.04 g / cm 3 Below, a micro carrier for cell culture can be provided.

[0034] The inventors of the present invention have confirmed that, in the case of the cell culture microcarrier of the above embodiment, by including a compound represented by the above chemical formula 1 as a polystyrene monomer, the density of the final cell culture microcarrier can be controlled, thereby enabling separation by density difference with cells, and improving dispersibility in a cell culture reactor or medium, thereby completing the invention.

[0035] Conventional cell culture microcarriers controlled the density of particles by encapsulating non-reactive low-density oil inside the particles, but there was a technical problem in that the low-density oil encapsulated inside leaked out when the particles were physically damaged.

[0036] Accordingly, the present inventors have developed a polystyrene monomer having a density of 0.9 g / cm used in microcarriers for cell culture. 3 By including a compound represented by the above chemical formula 1 satisfying the following, the density of particles can be controlled, allowing separation by density difference with cells, and at the same time, dispersibility in a cell culture reactor or medium can be improved, thereby completing the invention.

[0037]

[0038] Specifically, in the case of the cell culture microcarrier of the above embodiment, it may include polystyrene particles including a compound represented by the following chemical formula 1 as a monomer compound.

[0039] [Chemical Formula 1]

[0040]

[0041] 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 It is an alkyl group with 1 or more carbon atoms.

[0042]

[0043] By including the compound represented by the above chemical formula 1 as a polystyrene monomer, the density of the final cell culture microcarrier can be precisely controlled by adjusting the content of the compound represented by the above chemical formula 1, thereby enabling separation by density difference with cells, and at the same time, dispersibility in a cell culture reactor or medium can be improved.

[0044]

[0045] Specifically, the compound represented by the above chemical formula 1 may include one or more compounds selected from the group consisting of compounds represented by the following chemical formula 1-1 to compounds represented by the following chemical formula 1-3.

[0046] [Chemical Formula 1-1]

[0047]

[0048] [Chemical Formula 1-2]

[0049]

[0050] [Chemical Formula 1-3]

[0051]

[0052] In the above chemical formulas 1-1 to 1-3, R6 to R 11 are each independently an alkyl group having 1 or more carbon atoms.

[0053] By including at least one compound selected from the group consisting of a compound represented by the above chemical formula 1-1 to a compound represented by the above chemical formula 1-3 as a polystyrene monomer, the density of the final cell culture microcarrier can be precisely controlled by adjusting the content of the compound represented by the above chemical formula 1, thereby enabling separation by density difference with cells, and at the same time improving dispersibility in a cell culture reactor or medium.

[0054]

[0055] More specifically, the compound represented by the above chemical formula 1 may include at least one compound selected from the group consisting of a compound represented by the following chemical formula 1-4 to a compound represented by the following chemical formula 1-6.

[0056] [Chemical Formula 1-4]

[0057]

[0058] [Chemical Formula 1-5]

[0059]

[0060] [Chemical Formula 1-6]

[0061]

[0062] 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.

[0063]

[0064] Meanwhile, the compound represented by the above chemical formula 1 has a density of 0.92 g / cm 3 It could be as follows:

[0065] Specifically, the compound represented by the above chemical formula 1 has a density of 0.92 g / cm 3 Below, 0.91 g / cm 3 Below, 0.906 g / cm 3 Below 0.9 g / cm 3 Below, 0.89 g / cm 3 Below 0.5 g / cm 3 Above, 0.6 g / cm 3 Above, 0.7 g / cm 3 Above, 0.8 g / cm 3 or more than 0.5 g / cm 3 Above 0.92 g / cm 3 Below 0.6 g / cm 3 Above 0.92 g / cm 3 Below 0.7 g / cm 3 Above 0.92 g / cm 3 Below 0.8 g / cm3 Above 0.92 g / cm 3 Below 0.5 g / cm 3 Above 0.91 g / cm 3 Below 0.6 g / cm 3 Above 0.91 g / cm 3 Below 0.7 g / cm 3 Above 0.91 g / cm 3 Below 0.8 g / cm 3 Above 0.91 g / cm 3 Below 0.5 g / cm 3 Above 0.906 g / cm 3 Below 0.6 g / cm 3 Above 0.906 g / cm 3 Below 0.7 g / cm 3 Above 0.906 g / cm 3 Below 0.8 g / cm 3 Above 0.906 g / cm 3 Below 0.5 g / cm 3 More than 0.9 g / cm 3 Below 0.6 g / cm 3 More than 0.9 g / cm 3 Below 0.7 g / cm 3 More than 0.9 g / cm 3 Below 0.8 g / cm 3 More than 0.9 g / cm 3 Below 0.5 g / cm 3 Above 0.89 g / cm 3 Below 0.6 g / cm 3 Above 0.89 g / cm 3 Below 0.7 g / cm 3 Above 0.89 g / cm 3 Below 0.8 g / cm 3 Above 0.89 g / cm 3 It could be as follows:

[0066]

[0067] The density of the compound represented by the above chemical formula 1 is 0.92 g / cm 3By satisfying the following, the density of the final cell culture microcarrier can be precisely controlled by adjusting the content of the compound represented by the above chemical formula 1, thereby enabling separation by density difference with cells, and at the same time, dispersibility in a cell culture reactor or medium can be improved.

[0068] For example, the compound represented by the above 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, 1-ethenyl-3-(1-methylethyl)benzene.

[0069]

[0070] Meanwhile, the cell culture microcarrier may include polystyrene particles. The polystyrene particles may include a styrene monomer homopolymer, or a copolymer of a styrene monomer with other monomers or a crosslinking agent, and the styrene monomer may include a styrene monomer or a derivative thereof.

[0071] Preferably, the microcarrier for cell culture may be made of polystyrene particles.

[0072] Specifically, the apparent density of the polystyrene particles is 0.99 g / cm 3 Above 1.04 g / cm 3 It may be as follows. As it has the low density range described above, when separating and recovering microcarriers and cells after cell culture, cells and microcarriers can be easily separated through the difference in sedimentation rate due to gravity.

[0073] The density of the above polystyrene particles is 1.04 g / cm 3If it exceeds 0.99 g / cm, the difference in density between cells and microcarriers is small, making centrifugation difficult when separating and recovering cells after culture. 3 If it is less than this, a problem may occur in the early stage of culture, where the microcarriers float only on the surface of the culture medium, making it difficult for cells to attach.

[0074] The above cells are not particularly limited to adherent animal cells, but may be, for example, fibroblasts, epithelial cells, osteoblasts, chondrocytes, hepatocytes, human-derived umbilical cord blood cells, human bone marrow-derived mesenchymal stem cells, CHO (Chinese hamster ovary) cells, kidney cells (HEK293, BHK21, MDCK, vero cells, etc.), or a mixture of two or more thereof.

[0075] The density of the above cells is 1.02 g / cm 3 More than 1.1 g / cm 3 It may be less than.

[0076] In addition, the density difference between the cell culture microcarrier and the cell is 0.02 g / cm 3 Above 0.20 g / cm 3 The difference in density between the cell culture microcarrier and the cell may be 0.02 g / cm 3 Above 0.20 g / cm 3 By satisfying the following, when separating and recovering microcarriers and cells after cell culture, cells and microcarriers can be easily separated through the difference in sedimentation rate due to gravity.

[0077]

[0078] The above polystyrene particles may include a reaction product of a styrene monomer mixture and an ethylenically unsaturated crosslinking agent.

[0079] Specifically, the reaction product of the styrene monomer mixture and the ethylenically unsaturated crosslinking agent may be included in an amount of 90 parts by weight or more, based on 100 parts by weight of the total polystyrene particles. More specifically, the reaction product of the styrene monomer mixture and the ethylenically unsaturated crosslinking agent may be included in an amount of 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, based on 100 parts by weight of the total polystyrene particles.

[0080]

[0081] In addition, the polystyrene particles may contain 51 parts by weight or more of the compound represented by the chemical formula 1 based on 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 the chemical formula 1 based on 100 parts by weight of the styrene monomer mixture.

[0082] By including the compound represented by the above chemical formula 1 in the above-described amount with respect to 100 parts by weight of a styrene-based monomer mixture, the density of the final cell culture microcarrier can be precisely controlled, enabling separation by density difference with cells, and at the same time improving dispersibility in a cell culture reactor or medium.

[0083] If the compound represented by the above chemical formula 1 is included in an excessively small amount relative to 100 parts by weight of the styrene monomer mixture, the overall density of the polystyrene particles may be higher than the target level.

[0084] In addition, the polystyrene particles include a reaction product of a styrene monomer mixture and an ethylenically unsaturated crosslinking agent, and may include 60 parts by weight or more and 200 parts by weight or less of the ethylenically unsaturated crosslinking agent with respect to 100 parts by weight of the styrene monomer mixture.

[0085] 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, or 60 parts by weight or more and 150 parts by weight or less, per 100 parts by weight of the styrene-based monomer mixture.

[0086] When the ethylene-based unsaturated crosslinking agent is included in an excessively small amount relative to 100 parts by weight of the above styrene-based monomer mixture, there is a limitation in that it is difficult for the shape of the particles to stably maintain a spherical shape as the crosslinking density of the polystyrene-based polymer decreases.

[0087] 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, it is difficult for the particle shape to stably maintain a spherical shape, and the overall density of the polystyrene-based particles may become higher than the target level.

[0088]

[0089] In addition, the polystyrene particles may contain 51 parts by weight or more and 200 parts by weight or less of the monomer represented by the chemical formula 1 based on 100 parts by weight of the ethylene-based unsaturated crosslinking agent.

[0090] Specifically, the polystyrene particles may contain the monomer represented by the chemical formula 1 in an amount of 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, or 150 parts by weight or less, 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, or 65 parts by weight or more and 150 parts by weight or less.

[0091] If the monomer represented by the above chemical formula 1 is included in an excessively small amount relative to 100 parts by weight of the above ethylene-based unsaturated crosslinking agent, there is a limitation that it is difficult for the particle shape to stably maintain a spherical shape and it is difficult for the overall density of the polystyrene particles to be reduced to the target level.

[0092] In addition, when the compound represented by the above chemical formula 1 is included in an excessive amount relative to 100 parts by weight of the above ethylene-based unsaturated crosslinking agent, the crosslinking density of the polystyrene-based polymer decreases, making it difficult for the particle shape to stably maintain a spherical shape, and there is a limitation that the overall density of the polystyrene-based particles decreases below the target level.

[0093]

[0094] An example of the above ethylenically unsaturated crosslinking agent is divinylbenzene.

[0095]

[0096] Conventionally, foamed styrene has been manufactured 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 became excessively wide, making it difficult to secure a yield within a range applicable as a microcarrier.

[0097] The average diameter of the polystyrene particles may be 50 ㎛ to 400 ㎛, or 60 ㎛ to 390 ㎛, or 80 ㎛ to 350 ㎛, or 90 ㎛ to 300 ㎛. When the average diameter of the polystyrene particles satisfies the above-described range, cell attachment and culture performance are excellent. On the other hand, when the average diameter of the polystyrene particles is less than 50 ㎛, there is a concern that the surface area available for cell culture may be small, thereby lowering the culture efficiency, and when it exceeds 400 ㎛, the interaction between attached cells may be reduced, the cell density in the incubator may be lowered, and thus the cell culture efficiency may be lowered.

[0098] The diameter of the polystyrene particles refers to the distance between two points where a straight line passing through the center of gravity of the polystyrene particles meets the outermost surface of the polystyrene particles, and the average diameter of the polystyrene particles can be obtained by checking the diameter of all polystyrene particles included in the cell culture microcarrier using an optical microscope.

[0099] The polystyrene particles may be a group of individual particles having an average diameter of 50 μm to 400 μm, or 60 μm to 390 μm, and the individual particles included in the group may have an average diameter of 50 μm to 400 μm, or 60 μm to 390 μm. More specifically, 95%, or 99% of the individual particles included in the group may have a diameter of 50 μm to 400 μm, or 60 μm to 390 μm.

[0100] In addition, the polystyrene particles may have a perfect spherical particle ratio of more than 90% and less than 100%, or more than 92% and less than 100%, or more than 95% and less than 100%, or more than 96% and less than 99% without damage or destruction according to the following mathematical formula.

[0101] [Mathematical formula]

[0102] Percentage of perfectly spherical particles without damage or destruction (%) = (Number of polystyrene particles with perfectly spherical shapes without damage or destruction / Number of total polystyrene particles) x 100.

[0103] The percentage of perfectly spherical particles without damage or destruction by the above mathematical formula can be obtained by measuring the number of particles having a perfect spherical shape without damage or destruction among all particles of the polystyrene particles using an SEM or optical microscope, and calculating the percentage ratio of the number of perfectly spherical particles without damage or destruction compared to all particles.

[0104] That is, the above cell culture microcarrier may contain a plurality of polystyrene particles, and whether or not the plurality of polystyrene particles have a perfectly spherical shape without damage or destruction can be determined with the naked eye through an SEM or optical microscope.

[0105] If the ratio of perfectly spherical particles without damage or destruction according to the above mathematical formula decreases to less than 90%, there is a concern that the number of irregular particles with uneven and sunken particle surfaces will increase, and the irregular particles will float in the cell culture medium, causing physical impact on the cells being cultured, thereby lowering the cell culture efficiency to the point where cell culture becomes impossible.

[0106]

[0107] Specifically, the D50 particle diameter of the cell culture microcarrier may be 100 ㎛ to 300 ㎛, or 100 ㎛ to 250 ㎛, or 120 ㎛ to 250 ㎛, or 130 ㎛ to 250 ㎛. When the average diameter of the cell culture microcarrier satisfies the above-described range, cell attachment and culture performance are excellent. On the other hand, when the D50 particle diameter of the cell culture microcarrier is less than 100 ㎛, there is a concern that the surface area available for cell culture may be small, thereby lowering the culture efficiency, and when it exceeds 300 ㎛, the interaction between attached cells may be reduced, the cell density in the incubator may be lowered, thereby lowering the cell culture efficiency.

[0108]

[0109] Meanwhile, the cell culture microcarrier may include a cell adhesion inducing layer formed on the polystyrene particles.

[0110] The above cell adhesion inducing layer is composed of cell adhesive substances, which serve to provide a site where transmembrane proteins of cells can bind, thereby enabling adherent cells to stably attach, spread, and be cultured.

[0111] The polymer forming the cell adhesion-inducing layer may include, but is not particularly limited to, one or more selected from the group consisting of gelatin, collagen, fibronectin, chitosan, poly L-lysine, vitronectin, peptides including RGD, lignin, cationic dextran, and derivatives thereof.

[0112]

[0113] In addition, the cell culture microcarrier may optionally include a primer polymer layer formed between the polystyrene particles and the cell adhesion inducing layer. That is, 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 inducing layer formed on the surface of the primer polymer layer.

[0114] The above primer polymer layer acts as an adhesive layer that can introduce a functional polymer to a polystyrene surface without functional groups, thereby effectively introducing a polymer layer for cell attachment to the microcarrier surface and allowing it to be stably maintained during culture.

[0115] The above primer polymer layer may include, but is not particularly limited to, at least one selected from the group consisting of L-dihydroxyphenylalanine (L-DOPA), dopamine, norepinephrine, epinephrine, epigallocatechin, and derivatives thereof as catechol derivatives capable of inducing aqueous adhesion.

[0116]

[0117]

[0118] Meanwhile, according to another embodiment of the invention, a step of polymerizing and recovering polystyrene particles from a styrene monomer mixture including a compound represented by the following chemical formula 1; and an apparent density of the cell culture microcarrier is 0.99 g / cm 3 Above 1.04 g / cm 3 Below, a method for manufacturing a micro carrier for cell culture can be provided.

[0119]

[0120] In the method for manufacturing a cell culture microcarrier of the above embodiment, the contents of the polystyrene particles and the compound represented by the chemical formula 1 include all of the contents described above.

[0121]

[0122] In the method for manufacturing the above cell culture microcarrier, the step of polymerizing and recovering the polystyrene particles may include the step of performing a suspension polymerization reaction of a monomer composition containing a styrene monomer and recovering the suspension polymerization reaction result.

[0123]

[0124] Specifically, the compound represented by the above chemical formula 1 may include one or more compounds selected from the group consisting of compounds represented by the following chemical formula 1-1 to compounds represented by the following chemical formula 1-3.

[0125] [Chemical Formula 1-1]

[0126]

[0127] [Chemical Formula 1-2]

[0128]

[0129] [Chemical Formula 1-3]

[0130]

[0131] In the above chemical formulas 1-1 to 1-3, R6 to R 11 are each independently an alkyl group having 1 or more carbon atoms.

[0132] By including at least one compound selected from the group consisting of a compound represented by the above chemical formula 1-1 to a compound represented by the above chemical formula 1-3 as a polystyrene monomer, the density of the final cell culture microcarrier can be precisely controlled by adjusting the content of the compound represented by the above chemical formula 1, thereby enabling separation by density difference with cells, and at the same time improving dispersibility in a cell culture reactor or medium.

[0133]

[0134] More specifically, the compound represented by the above chemical formula 1 may include at least one compound selected from the group consisting of a compound represented by the following chemical formula 1-4 to a compound represented by the following chemical formula 1-6.

[0135] [Chemical Formula 1-4]

[0136]

[0137] [Chemical Formula 1-5]

[0138]

[0139] [Chemical Formula 1-6]

[0140]

[0141] 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.

[0142]

[0143] Meanwhile, the compound represented by the above chemical formula 1 has a density of 0.9 g / cm 3 It could be as follows:

[0144] Specifically, the compound represented by the above chemical formula 1 has a density of 0.92 g / cm 3 Below, 0.91 g / cm 3 Below, 0.906 g / cm 3 Below 0.9 g / cm 3 Below, 0.89 g / cm 3Below 0.5 g / cm 3 Above, 0.6 g / cm 3 Above, 0.7 g / cm 3 Above, 0.8 g / cm 3 or more than 0.5 g / cm 3 Above 0.92 g / cm 3 Below 0.6 g / cm 3 Above 0.92 g / cm 3 Below 0.7 g / cm 3 Above 0.92 g / cm 3 Below 0.8 g / cm 3 Above 0.92 g / cm 3 Below 0.5 g / cm 3 Above 0.91 g / cm 3 Below 0.6 g / cm 3 Above 0.91 g / cm 3 Below 0.7 g / cm 3 Above 0.91 g / cm 3 Below 0.8 g / cm 3 Above 0.91 g / cm 3 Below 0.5 g / cm 3 Above 0.906 g / cm 3 Below 0.6 g / cm 3 Above 0.906 g / cm 3 Below 0.7 g / cm 3 Above 0.906 g / cm 3 Below 0.8 g / cm 3 Above 0.906 g / cm 3 Below 0.5 g / cm 3 More than 0.9 g / cm 3 Below 0.6 g / cm 3 More than 0.9 g / cm 3 Below 0.7 g / cm 3 More than 0.9 g / cm 3 Below 0.8 g / cm 3 More than 0.9 g / cm 3 Below 0.5 g / cm 3 Above 0.89 g / cm 3 Below 0.6 g / cm 3 Above 0.89 g / cm 3Below 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 be as follows. The density of the compound represented by the above chemical formula 1 is 0.92 g / cm 3 By satisfying the following, the density of the final cell culture microcarrier can be precisely controlled by adjusting the content of the compound represented by the above chemical formula 1, thereby enabling separation by density difference with cells, and at the same time, dispersibility in a cell culture reactor or medium can be improved.

[0145] For example, the compound represented by the above 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, 1-ethenyl-3-(1-methylethyl)benzene.

[0146]

[0147] The above polystyrene particles may include a reaction product of a styrene monomer mixture and an ethylenically unsaturated crosslinking agent.

[0148] Specifically, the reaction product of the styrene monomer mixture and the ethylenically unsaturated crosslinking agent may be included in an amount of 90 parts by weight or more, based on 100 parts by weight of the total polystyrene particles. More specifically, the reaction product of the styrene monomer mixture and the ethylenically unsaturated crosslinking agent may be included in an amount of 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, based on 100 parts by weight of the total polystyrene particles.

[0149]

[0150] In addition, the polystyrene particles may contain 51 parts by weight or more of the compound represented by the chemical formula 1 based on 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 the chemical formula 1 based on 100 parts by weight of the styrene monomer mixture.

[0151] By including the compound represented by the above chemical formula 1 in the above-described amount with respect to 100 parts by weight of a styrene-based monomer mixture, the density of the final cell culture microcarrier can be precisely controlled, enabling separation by density difference with cells, and at the same time improving dispersibility in a cell culture reactor or medium.

[0152] If the compound represented by the above chemical formula 1 is included in an excessively small amount relative to 100 parts by weight of the styrene monomer mixture, the overall density of the polystyrene particles may be higher than the target level.

[0153]

[0154] In addition, the polystyrene particles include a reaction product of a styrene monomer mixture and an ethylenically unsaturated crosslinking agent, and may include 60 parts by weight or more and 200 parts by weight or less of the ethylenically unsaturated crosslinking agent with respect to 100 parts by weight of the styrene monomer mixture.

[0155] 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, or 60 parts by weight or more and 150 parts by weight or less, per 100 parts by weight of the styrene-based monomer mixture.

[0156] When the ethylene-based unsaturated crosslinking agent is included in an excessively small amount relative to 100 parts by weight of the above styrene-based monomer mixture, there is a limitation in that it is difficult for the shape of the particles to stably maintain a spherical shape as the crosslinking density of the polystyrene-based polymer decreases.

[0157] 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, it is difficult for the particle shape to stably maintain a spherical shape, and the overall density of the polystyrene-based particles may become higher than the target level.

[0158]

[0159]

[0160] In addition, the polystyrene particles may contain 51 parts by weight or more and 200 parts by weight or less of the compound represented by the chemical formula 1 based on 100 parts by weight of the ethylene-based unsaturated crosslinking agent.

[0161] Specifically, the polystyrene particles may contain the compound represented by the chemical formula 1 in an amount of 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, or 150 parts by weight or less, 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, or 65 parts by weight or more and 150 parts by weight or less.

[0162] If the compound represented by the above chemical formula 1 is included in an excessively small amount relative to 100 parts by weight of the above ethylene-based unsaturated crosslinking agent, there is a limitation that it is difficult for the particle shape to stably maintain a spherical shape.

[0163] In addition, when the compound represented by the above chemical formula 1 is included in an excessive amount relative to 100 parts by weight of the above ethylene-based unsaturated crosslinking agent, the crosslinking density of the polystyrene-based polymer decreases, making it difficult for the particle shape to stably maintain a spherical shape, and there is a limitation that the overall density of the polystyrene-based particles decreases below the target level.

[0164]

[0165] An example of the above ethylenically unsaturated crosslinking agent is divinylbenzene.

[0166]

[0167] More specifically, the suspension polymerization reaction of the monomer composition may include a step of mixing the monomer composition into an aqueous dispersion and applying a shear force to homogenize the monomer composition into droplets in the aqueous dispersion; and a step of suspension polymerizing the homogenized monomer composition at a stirring speed of 300 rpm to 1000 rpm.

[0168] In the step of homogenizing the above monomer composition into a droplet form in an aqueous dispersion, stirring may be performed at a stirring speed of 300 rpm to 1000 rpm, or 400 rpm to 800 rpm.

[0169] In the step of suspension polymerizing the homogenized monomer composition at a stirring speed of 300 rpm to 1000 rpm, or 400 rpm to 800 rpm, the density of the microcarrier can be further reduced by the structure of the compound represented by the chemical formula 1 during the formation of the particle structure of polystyrene, while producing a microcarrier with a high percentage of perfectly spherical particles without damage or destruction.

[0170] In the step of suspension polymerizing the homogenized monomer composition at a stirring speed of 300 rpm to 1000 rpm, or 400 rpm to 800 rpm, examples of the suspension polymerization conditions are not particularly limited, but for example, it may be performed at a temperature of 50°C to 100°C for 3 to 18 hours.

[0171]

[0172] Meanwhile, the method for manufacturing a microcarrier for cell culture 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 a compound represented by the chemical formula 1.

[0173] Specifically, the washing step may include a step of filtering the reaction product through a sieve of 30 μm to 100 μm, and then stirring it 3 to 5 times in distilled water at 50 to 75°C, and a step of stirring it 3 to 5 times in ethanol at room temperature.

[0174] The above drying step includes drying in a convection oven at room temperature or within 80°C. However, this is not limited to this, and any commonly known drying method can be used without any particular restrictions.

[0175]

[0176] Meanwhile, the method for manufacturing the microcarrier for cell culture may further include a washing step and a drying step; and then, a step of applying a cell adhesion inducing layer on the polystyrene particles.

[0177]

[0178] Optionally, the method for manufacturing the microcarrier for cell culture may further include a washing step and a drying step; thereafter, a step of applying a primer polymer layer to the surface of the polystyrene particles; and a step of applying a cell adhesion inducing layer to the surface of the primer polymer layer.

[0179] Specifically, the step of applying a primer polymer layer to the surface of the polystyrene particles may include a step of immersing the polystyrene particles in a primer polymer solution for 1 to 10 hours, or 2 to 6 hours, or 3 to 5 hours.

[0180] The above primer polymer layer may include, but is not particularly limited to, at least one selected from the group consisting of L-dihydroxyphenylalanine (L-DOPA), dopamine, norepinephrine, epinephrine, epigallocatechin, and derivatives thereof as catechol derivatives capable of inducing aqueous adhesion.

[0181] The polystyrene particles manufactured above are recovered, dried, and then a primer polymer layer is applied to modify the surface of the microcarrier to be hydrophilic, thereby dispersing the particles in water and stably introducing a cell adhesion inducing layer, thereby controlling the floating degree of the microcarrier in the culture medium and having the effect of stably culturing cells by attachment.

[0182] At this time, the ratio of the radius of the polystyrene particles and the thickness of the primer polymer layer may be 1:0.00001 to 1:0.01, or 1:0.0001 to 1:0.001.

[0183] Meanwhile, the method for manufacturing a microcarrier of the above embodiment may further include a step of applying a cell adhesion inducing layer to the surface of the primer polymer layer after the step of applying a primer polymer layer to the surface of the polystyrene particles.

[0184]

[0185] The step of applying a cell adhesion inducing layer to the surface of the primer polymer layer may include a step of coating with a solution containing at least one selected from the group consisting of gelatin, collagen, fibronectin, chitosan, poly L-lysine, vitronectin, peptides including RGD, lignin, cationic dextran, and derivatives thereof.

[0186] The solution containing at least one selected from the group consisting of the above gelatin, collagen, fibronectin, chitosan, poly L-lysine, vitronectin, peptides including RGD, lignin, cationic dextran, and derivatives thereof can act as an adhesive factor for adhering cells and microcarriers, so that when the primer polymer layer is coated with the solution containing the above gelatin or the like, the adhesion between cells and microcarriers can be increased, making it more suitable for mass culture of cells.

[0187] Specifically, the step of applying a cell adhesion inducing layer to the surface of the primer polymer layer may include a step of immersing the material obtained in the step of applying the primer polymer layer to the surface of the polystyrene particles in a solution containing at least one selected from the group consisting of gelatin, collagen, fibronectin, chitosan, poly L-lysine, vitronectin, peptides including RGD, lignin, cationic dextran, and derivatives thereof for 10 to 20 hours, or 15 to 20 hours, or 17 to 19 hours.

[0188]

[0189] According to another embodiment of the invention, a cell culture composition comprising cells and a cell culture microcarrier of the above embodiment may be provided. The contents of the cell culture microcarrier include all of the contents described above in the above embodiment.

[0190] The above cells are not particularly limited to adherent animal cells, but may be, for example, fibroblasts, epithelial cells, osteoblasts, chondrocytes, hepatocytes, human-derived umbilical cord blood cells, human bone marrow-derived mesenchymal stem cells, CHO (Chinese hamster ovary) cells, kidney cells (HEK293, BHK21, MDCK, vero cells, etc.), or a mixture of two or more thereof.

[0191] The density of the above cells is 1.02 g / cm 3 More than 1.1 g / cm 3 It may be less than.

[0192] In addition, the density difference between the cell culture microcarrier and the cell is 0.02 g / cm 3 Above 0.20 g / cm 3 The difference in density between the cell culture microcarrier and the cell may be 0.02 g / cm 3 Above 0.20 g / cm 3 By satisfying the following, when separating and recovering microcarriers and cells after cell culture, cells and microcarriers can be easily separated through the difference in sedimentation rate due to gravity.

[0193] The above cell culture composition may further include a medium solution. The medium solution may include various additives to sufficiently satisfy environmental conditions such as pH, temperature, and osmotic pressure, as well as nutrients similar to those of a living organism based on body fluids such as plasma or lymph. Various materials widely known in the art of cell culture may be used without limitation.

[0194] For example, the cell culture microcarrier of the above embodiment has a density lower than that of the medium solution, and is injected into the medium solution and floats within the medium solution under stirring conditions. Thereafter, as the number of cells attached to the surface of the low-density microcarrier increases, the density of the cell-attached microcarrier (hereinafter referred to as a "microcarrier-cell complex") gradually increases, and the microcarrier gradually sinks within the medium solution.

[0195] Accordingly, the cultured cells can be easily obtained by separating the cells from the microcarrier-cell complex by centrifuging the microcarrier (microcarrier-cell complex) to which the cells are attached after adding a cell detachment enzyme.

[0196]

[0197] According to the present invention, a cell culture microcarrier having high surface hydrophilicity and enhanced cell adhesion, and improved dispersibility in a cell culture reactor or medium, a method for producing a cell culture microcarrier, and a cell culture method using the same can be provided.

[0198] The invention is described in more detail in the following examples. However, the following examples are merely illustrative of the present invention, and the scope of the present invention is not limited by the following examples.

[0199]

[0200] Example 1

[0201] Polyvinyl alcohol (molecular weight 85-124K, 87-89% hydrolysis) was dissolved in distilled water at 2%, and 2 wt% (based on the weight of distilled water) of sodium chloride additive was added to the aqueous dispersion, and stirred at room temperature for 20 minutes.

[0202] Monomers styrene (Sigma-Aldrich), methylstyrene (Sigma-Aldrich, density: 0.89 - 0.92 g / cm 3 ) and the crosslinking agent divinylbenzene (TCI) were mixed in a weight ratio of 2:3:5, and 2 wt% of V-65 initiator (initiator input amount: based on the total amount of monomer and crosslinking agent) was added to 60 g of the sufficiently dissolved mixture, and the mixture was stirred for an additional 5 minutes to prepare a monomer composition.

[0203] 600 g of an aqueous dispersion was added to a 1 L reactor, and the monomer composition was added thereto. A shear force was applied to the aqueous dispersion and the monomer composition at a speed of 400 rpm at room temperature to disperse the monomer composition in the aqueous dispersion in the form of fine droplets and homogenize the dispersion.

[0204] The homogenized mixture was stirred at a stirring speed of 400 rpm and reacted at 85°C under nitrogen purging for 6 hours 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 recover the particles.

[0205] Average diameter: 183 ㎛ (based on D50 measured using PSA equipment)

[0206] Apparent density: 0.99 ~ 1.04 g / cm 3

[0207]

[0208] Example 2

[0209] A microcarrier was manufactured in the same manner as in Example 1, except that the monomer and crosslinking agent ratios in Example 1 were adjusted to have a weight ratio of styrene: methylstyrene: divinylbenzene of 1:4:5.

[0210] Average diameter: 196 ㎛ (based on D50 measured using PSA equipment)

[0211] Apparent density: 0.99 ~ 1.02 g / cm 3

[0212]

[0213] Example 3

[0214] A microcarrier was manufactured in the same manner as in Example 1, except that the monomer and crosslinking agent ratio in Example 1 was adjusted to have a weight ratio of styrene: methylstyrene: divinylbenzene of 1:5:4, and the initiator amount was increased by 3%.

[0215] Average diameter: 183 ㎛ (based on D50 measured using PSA equipment)

[0216] Apparent density: 0.99 ~ 1.02 g / cm 3

[0217]

[0218] Example 4

[0219] A microcarrier was manufactured in the same manner as in Example 1, except that the ratio of monomers and crosslinking agents other than styrene in Example 1 was adjusted to have a weight ratio of methylstyrene:divinylbenzene 4:6.

[0220] Average diameter: 185 ㎛ (based on D50 measured using PSA equipment)

[0221] Apparent density: 0.99 ~ 1.02 g / cm 3

[0222]

[0223] Example 5

[0224] In the above Example 1, the ratio of monomer and crosslinking agent was styrene: trimethylstyrene (Sigma-Aldrich, density: 0.906 g / cm 3) : A microcarrier was manufactured in the same manner as in Example 1, except that the weight ratio of divinylbenzene was adjusted to 2:3:5.

[0225] Average diameter: 213 ㎛ (based on D50 measured using PSA equipment)

[0226] Apparent density: 0.99 ~ 1.04 g / cm 3

[0227]

[0228] Example 6

[0229] A microcarrier was manufactured in the same manner as in Example 5, except that the ratio of monomers and crosslinking agents in Example 5 was adjusted to have a weight ratio of styrene:trimethylstyrene:divinylbenzene of 1:4:5.

[0230] Average diameter: 180 ㎛ (based on D50 measured using PSA equipment)

[0231] Apparent density: 0.99 ~ 1.04 g / cm 3

[0232]

[0233] Example 7

[0234] A microcarrier was manufactured in the same manner as in Example 4, except that the ratio of monomer and crosslinking agent in Example 4 was adjusted to have a weight ratio of trimethylstyrene:divinylbenzene 4:6.

[0235] Average diameter: 198 ㎛ (based on D50 measured using PSA equipment)

[0236] Apparent density: 0.99 ~ 1.04 g / cm 3

[0237]

[0238] Example 8

[0239] A microcarrier was manufactured in the same manner as in Example 7, except that the ratio of monomer and crosslinking agent in Example 7 was adjusted to have a weight ratio of trimethylstyrene:divinylbenzene 5:5.

[0240] Average diameter: 205 ㎛ (based on D50 measured using PSA equipment)

[0241] Apparent density: 0.99 ~ 1.02 g / cm 3

[0242]

[0243] Example 9

[0244] In the above Example 8, the ratio of monomer and crosslinking agent was 4-tert-butylstyrene (Sigma-Aldrich, density: 0.875 g / cm 3 ) : A microcarrier was manufactured in the same manner as in Example 8, except that the weight ratio of divinylbenzene was adjusted to 5:5.

[0245] Average diameter: 221 ㎛ (based on D50 measured using PSA equipment)

[0246] Apparent density: 0.99 ~ 1.04 g / cm 3

[0247]

[0248] Example 10

[0249] A microcarrier was manufactured in the same manner as in Example 1, except that the ratio of monomers and crosslinking agents in Example 1 was adjusted to have a weight ratio of styrene:4-tert-butylstyrene:divinylbenzene 2:3:5.

[0250] Average diameter: 231 ㎛ (based on D50 measured using PSA equipment)

[0251] Apparent density: 0.99 ~ 1.04 g / cm 3

[0252]

[0253] Example 11

[0254] A microcarrier was manufactured in the same manner as in Example 10, except that the ratio of monomers and crosslinking agents in Example 10 was adjusted to have a weight ratio of styrene:4-tert-butylstyrene:divinylbenzene of 1:5:4, and the amount of initiator added was increased by 3%.

[0255] Average diameter: 183 ㎛ (based on D50 measured using PSA equipment)

[0256] Apparent density: 0.99 ~ 1.04 g / cm 3

[0257]

[0258] Example 12

[0259] In the above Example 2, the ratio of monomer and crosslinking agent was 2,4-dimethylstyrene (Sigmal-Aldrich, density: 0.906 g / cm 3 ) : A microcarrier was manufactured in the same manner as in Example 2, except that the weight ratio of methylstyrene:divinylbenzene was adjusted to 1:4:5.

[0260] Average diameter: 205 ㎛ (based on D50 measured using PSA equipment)

[0261] Apparent density: 0.99 ~ 1.04 g / cm 3

[0262]

[0263] Example 13

[0264] A microcarrier was manufactured in the same manner as in Example 12, except that the ratio of monomer and crosslinking agent in Example 12 was adjusted to have a weight ratio of 2,4-dimethylstyrene (Sigmal-Aldrich): methylstyrene: divinylbenzene 1:5:4, and the amount of initiator was increased to 3%.

[0265] Average diameter: 198 ㎛ (based on D50 measured using PSA equipment)

[0266] Apparent density: 0.99 ~ 1.02 g / cm 3

[0267]

[0268] Example 14

[0269] A microcarrier was manufactured in the same manner as in Example 12, except that the ratio of monomers and crosslinking agents was adjusted to have a weight ratio of 2,4-dimethylstyrene:trimethylstyrene:divinylbenzene of 1:4:5.

[0270] Average diameter: 187 ㎛ (based on D50 measured using PSA equipment)

[0271] Apparent density: 0.99 ~ 1.02 g / cm 3

[0272]

[0273] Example 15

[0274] A microcarrier was manufactured in the same manner as in Example 12, except that the ratio of monomers and crosslinking agents in Example 12 was adjusted to have a weight ratio of 2,4-dimethylstyrene:4-tert-butylstyrene:divinylbenzene of 1:5:4, and the amount of initiator was increased to 3%.

[0275] Average diameter: 218 ㎛ (based on D50 measured using PSA equipment)

[0276] Apparent density: 0.99 ~ 1.04 g / cm 3

[0277]

[0278] Styrene (g) 4-methylstyrene (g) 2,4,6-trimethylstyrene (g) 4-tert-butylstyrene (g) 2,4-dimethylstyrene (g) DVB (g) V-65 (g) Example 11 218---301.2 Example 26 24---301.2 Example 36 30---241.8 Example 40 24---361.2 Example 51 2-18---301.2 Example 66 24---301.2 Example 70 24---361.2 Example 80 30---301.2 Example 90--30-301.2 Example 1012--18-301.2 Example 116--30-241.8 Example 12--24--6301.2 Example 13--30--6241.8 Example 14--24-6301.2 Example 15---306241.8

[0279] Comparative Example 1

[0280] Polyvinyl alcohol (molecular weight 85-124K, 87-89% hydrolysis) was dissolved in distilled water at 2%, and 2 wt% (based on the weight of distilled water) of sodium chloride additive was added to the aqueous dispersion, and stirred at room temperature for 20 minutes.

[0281] The weight ratio of styrene as a monomer and divinylbenzene as a crosslinking agent is 1:1, and the oil input amount (based on the total of monomer, crosslinking agent, and oil) is 10 wt%. Isopar M (a mixture of isoalkanes having 12 to 14 carbon atoms and isoalkanes having 13 to 16 carbon atoms; density 0.79 g / cm) is used as an oil. 3 ) was mixed and sufficiently dissolved, 2 wt% of V-65 initiator (initiator input: based on the total amount of monomer and crosslinking agent) was added to 120 g of the mixture, and the mixture was stirred for an additional 5 minutes to prepare a monomer composition.

[0282] 600 g of an aqueous dispersion was added to a 1 L reactor, and the monomer composition was added thereto. A shear force was applied to the aqueous dispersion and the monomer composition at a speed of 400 rpm at room temperature to disperse the monomer composition in the aqueous dispersion in the form of fine droplets and homogenize the dispersion.

[0283] The homogenized mixture was stirred at a stirring speed of 400 rpm and reacted at 85°C under nitrogen purging for 6 hours 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 particles. The recovered polystyrene particles were used as microcarriers.

[0284] The properties of the above polystyrene particles are as follows.

[0285]

[0286] Average diameter: 194 ㎛ (based on D50 measured using PSA equipment)

[0287] Apparent density: 1.003 g / cm 3 over

[0288] Pore ​​diameter: 0.05 ㎛ ~ 4 ㎛

[0289]

[0290] Comparative Example 2

[0291] Isopar M (a mixture of isoalkanes having 12 to 14 carbon atoms and isoalkanes having 13 to 16 carbon atoms; density 0.79 g / cm) as oil so that the oil input (based on the total sum of monomer, crosslinker, and oil) is 14 wt% 3 ) was mixed, a microcarrier was manufactured in the same manner as in Comparative Example 1.

[0292] The properties of the above polystyrene particles are as follows.

[0293]

[0294] Average diameter: 190.6 ㎛ (based on D50 measured using PSA equipment)

[0295] Apparent density: 0.98 ~ 0.99 g / cm 3

[0296] Pore ​​diameter: 0.05 ㎛ ~ 4 ㎛

[0297]

[0298] Comparative Example 3

[0299] Isopar M (a mixture of isoalkanes having 12 to 14 carbon atoms and isoalkanes having 13 to 16 carbon atoms; density 0.79 g / cm) as oil so that the oil input (based on the total sum of monomer, crosslinker, and oil) is 20 wt% 3 ) was mixed, a microcarrier was manufactured in the same manner as in Comparative Example 1.

[0300] The properties of the above polystyrene particles are as follows.

[0301]

[0302] Average diameter: 188.07 ㎛ (based on D50 measured using PSA equipment)

[0303] Apparent density: 0.97 ~ 0.98 g / cm 3

[0304] Pore ​​diameter: 0.05 ㎛ ~ 4 ㎛

[0305]

[0306] Comparative Example 4

[0307] A microcarrier was manufactured in the same manner as in Example 1, except that styrene was used alone as the monomer and the weight ratio with divinylbenzene, the crosslinking agent, was adjusted to be 1:1.

[0308] Average diameter: 198 ㎛ (based on D50 measured using PSA equipment)

[0309] Apparent density: > 1.04 g / cm 3

[0310]

[0311] Reference Example 1

[0312] In the above Example 1, a microcarrier was manufactured in the same manner as in the above Example 1, except that the content of each monomer was adjusted as shown in Table 2.

[0313] Average diameter: 204.5 ㎛ (based on D50 measured using PSA equipment)

[0314] Apparent density: > 1.04 g / cm 3

[0315]

[0316] Styrene (g) 4-methylstyrene (g) 2,4,6-trimethylstyrene (g) 4-tert-butylstyrene (g) 2,4-dimethylstyrene (g) DVB (g) IsoparM (g) V-65 (g) Comparative Example 127 - - - 276 1.2 Comparative Example 225.8 - - - 25.8 8.4 1.2 Comparative Example 324 - - - 241 21.2 Comparative Example 430 - - - 30 - 1.2 Reference Example 128.5 1.5 - --- 30 - 1.2

[0317] <Experimental Example: Measurement of Physical Properties of Microcarriers for Cell Culture>

[0318]

[0319] The physical properties of the cell culture microcarriers obtained in the above examples and comparative examples were measured using the following methods, and the results are shown in Table 3.

[0320]

[0321] Experiment 1. Average particle size (unit: ㎛)

[0322] For the cell culture microcarriers obtained in the above examples and comparative examples, they were dispersed in ethanol at a level of 10 wt%, and then the particle diameter D50 (particle size value corresponding to the cumulative distribution percentage reaching 50%) was measured using PSA (Particle size analysis) equipment.

[0323]

[0324] Experiment 2. Apparent density (unit: g / cm) 3 )

[0325] For the cell culture microcarriers manufactured in the above examples and comparative examples, the density was 0.985 g / cm under room temperature (25 ℃) and atmospheric pressure (1 atm). 3 , 0.99 g / cm 3 , 0.997 g / cm 3In ethanol aqueous solution and density 1.02 g / cm 3 , 1.04 g / cm 3 The apparent density was evaluated under the following criteria by adding each to an aqueous glycerol solution and checking whether the particles floated or settled.

[0326] 1) 0.985 g / cm 3 < d < 0.99 g / cm 3 (0.985 g / cm 3 Exceeding 0.99 g / cm 3 under)

[0327] Density is 0.985 g / cm 3 It precipitates in an ethanol solution and has a density of 0.99 g / cm. 3 Floating in aqueous ethanol solution

[0328] 2) 0.99 g / cm 3 < 1.02 g / cm 3 (0.99 g / cm 3 Exceeding 1.02 g / cm 3 less than )

[0329] Density is 0.99 g / cm 3 It precipitates in an ethanol solution and has a density of 1.02 g / cm. 3 Suspended in aqueous glycerol solution

[0330] 3) 0.99 g / cm 3 < 1.04 g / cm 3 (0.99 g / cm 3 Exceeding 1.04 g / cm 3 less than )

[0331] Density is 0.99 g / cm 3 It precipitates in an ethanol solution and has a density of 1.04 g / cm. 3 Suspended in aqueous glycerol solution

[0332] 4) 0.99 g / cm 3 < d < 1.003 g / cm 3 (0.99 g / cm 3 Exceeds 1.003 g / cm 3 under)

[0333] Density is 0.99 g / cm 3 It settles in distilled water and has a density of 1.003 g / cm 3 Suspended in human cell culture medium

[0334] 5) d > 1.04 g / cm 3 (1.04 g / cm 3 over)

[0335] Density is 1.04 g / cm 3 Sedimentation in aqueous glycerol solution

[0336]

[0337] Experiment 3. Particle dispersion in the incubator

[0338] The above cell culture microcarriers were pre-dispersed in the amount required for cell culture before culturing, along with the medium, in a 20 mL glass vial and wetting was performed for approximately 10 to 18 hours. The pre-dispersed cell culture microcarriers were then filtered through a cell strainer and placed in a 100 mL 3D bioreactor with 60 mL of medium, followed by stirring for 24 hours under actual cell culture conditions. (Culture conditions: 37°C, 5% CO2 incubator, bioreactor stirring at 25 rpm)

[0339] After 24 hours of culture, the particles that were not dispersed inside the incubator but were floating on the upper interface of the medium were filtered out and removed, the particles dispersed inside the incubator were thoroughly washed with DPBS or water, dried, and weighed. The percentage ratio of the weight of the dispersed particles to the total weight was calculated to calculate the degree of dispersion, and the evaluation was performed under the following criteria.

[0340] Top: Dispersion 80% or more and 100% or less

[0341] Medium: Dispersion 60% or more and less than 80%

[0342] Ha: Dispersion less than 60%

[0343]

[0344] Experiment 4. Separation by density difference

[0345] Regarding the cell culture microcarriers manufactured in the above examples and comparative examples, whether separation of cells and the cell culture microcarriers by density difference was possible under room temperature (25°C) and atmospheric pressure (1 atm) conditions was evaluated by centrifuging a mixture of cell culture medium and microcarriers as follows.

[0346] O: No particles settled at the bottom after centrifugation.

[0347] X: There are particles that have settled at the bottom after centrifugation.

[0348]

[0349] Particle size (㎛, D50) Apparent density (g / cm) 3 )Dispersion density difference separation example 11830.99 - 1.04 medium example 21960.99 - 1.02 medium example 31830.99 - 1.02 medium example 41850.99 - 1.02 high example 52130.99 - 1.04 high example 61800.99 - 1.04 high example 71980.99 - 1.04 high example 82050.99 - 1.02 high example 92210.99 - 1.04 high example 102310.99 - 1.04 medium example 111830.99 - 1.04 high example 122050.99 - 1.04 Medium Example 131980.99 - 1.02 High Example 141870.99 - 1.04 Medium Example 152180.99 - 1.04 High Example 1194> 1.003 Medium Example 21910.98 - 0.99 Low Example 31910.97 - 0.98 Low Example 4198> 1.04 Low Example 1204.5> 1.04 Low Example

[0350] As shown in Table 3 above, the cell culture microcarrier of the example has a density of 1.04 g / cm 3It was confirmed that the microcarriers for cell culture at low densities below are suitable, and that this improves particle dispersion under cell culture conditions and enables separation of microcarriers by density differences. On the other hand, it was confirmed that the microcarriers for cell culture of Comparative Example 1 do not allow separation of microcarriers by density differences.

[0351] In addition, since the cell culture microcarriers of Comparative Examples 2 and 3 contained low-density oil within the particles, not only was the particle dispersion reduced under cell culture conditions, but there was also a possibility that the low-density oil incorporated therein would leak out due to damage to the cell culture microcarriers during culture.

[0352] In the case of Comparative Example 4, it was confirmed that not only was the particle dispersion poor under cell culture conditions, but also the separation of microcarriers due to density difference was impossible.

Claims

1. Polystyrene particles comprising a compound represented by the following chemical formula 1 as a monomer compound; The apparent density of the microcarrier for cell culture is 0.99 g / cm 3 Above 1.04 g / cm 3 Below, microcarriers for cell culture: [Chemical Formula 1] 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 above R1 to R5 It is an alkyl group with 1 or more carbon atoms.

2. In paragraph 1, A cell culture microcarrier comprising at least one compound selected from the group consisting of a compound represented by the chemical formula 1 below to a compound represented by the chemical formula 1-3 below: [Chemical Formula 1-1] [Chemical Formula 1-2] [Chemical Formula 1-3] In the above chemical formulas 1-1 to 1-3, R6 to R 11 are each independently an alkyl group having 1 or more carbon atoms.

3. In paragraph 1, The compound represented by the above chemical formula 1 has a density of 0.92 g / cm 3 Below, micro carriers for cell culture.

4. In paragraph 1, The above polystyrene particles include a reaction product of a styrene monomer mixture and an ethylenically unsaturated crosslinking agent, A cell culture microcarrier comprising 90 parts by weight or more of the reaction product of the styrene monomer mixture and the ethylenically unsaturated crosslinking agent relative to 100 parts by weight of the polystyrene particles.

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

6. In paragraph 1, The above polystyrene particles include a reaction product of a styrene monomer mixture and an ethylenically unsaturated crosslinking agent, A cell culture microcarrier comprising 51 parts by weight or more of a compound represented by the chemical formula 1 based on 100 parts by weight of the styrene-based monomer mixture.

7. In paragraph 1, The above polystyrene particles include a reaction product of a styrene monomer mixture and an ethylenically unsaturated crosslinking agent, A cell culture microcarrier comprising 51 to 200 parts by weight of a compound represented by the chemical formula 1 based on 100 parts by weight of the ethylenically unsaturated crosslinking agent.

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

9. A step of polymerizing and recovering polystyrene particles from a styrene monomer mixture containing a compound represented by the following chemical formula 1; including, The apparent density of the microcarrier for cell culture is 0.99 g / cm 3 Above 1.04 g / cm 3 Below, a method for manufacturing a microcarrier for cell culture: [Chemical Formula 1] 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 above R1 to R5 It is an alkyl group with 1 or more carbon atoms.

10. In paragraph 9, A method for producing a cell culture microcarrier, wherein the compound represented by the above chemical formula 1 comprises at least one compound selected from the group consisting of a compound represented by the following chemical formula 1-1 to a compound represented by the following chemical formula 1-3: [Chemical Formula 1-1] [Chemical Formula 1-2] [Chemical Formula 1-3] In the above chemical formulas 1-1 to 1-3, R6 to R 11 are each independently an alkyl group having 1 or more carbon atoms.

11. In paragraph 9, The compound represented by the above chemical formula 1 has a density of 0.92 g / cm 3 Below, a method for manufacturing a micro carrier for cell culture.

12. In paragraph 9, A method for producing a cell culture microcarrier, comprising 51 parts by weight or more of a compound represented by the chemical formula 1 based on 100 parts by weight of the styrene-based monomer mixture.

13. In paragraph 9, A method for manufacturing a cell culture microcarrier, further comprising the step of applying a cell adhesion inducing layer on the polystyrene particles.

14. A cell culture composition comprising cells and a cell culture microcarrier of the first clause.

15. In paragraph 14, A cell culture composition comprising at least one compound selected from the group consisting of fibroblasts, epithelial cells, osteoblasts, chondrocytes, hepatocytes, umbilical cord blood cells, mesenchymal stem cells, CHO cells, and kidney cells.

16. In paragraph 14, The difference in apparent density between the above cell culture microcarrier and the above cells is 0.02 g / cm 3 0.20 g / cm 3 Human, cell culture composition.

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