Microcarriers for cell culture, method for producing the same, and cell culture composition using the same

Abstract

The present invention relates to a microcarrier for cell culture, which comprises polystyrene particles containing at least one of a hydrocarbon oil having 12 or more carbon atoms or a void derived therefrom; and magnetic particles, a method for producing the same, and a cell culture composition using the same.

Description

Technical Field

[0001] Cross-reference to related applications This application claims the benefit of priority based on Korean Patent Application No. 10-2021-0128333, filed on September 28, 2021, and Korean Patent Application No. 10-2022-0112921, filed on September 6, 2022, and all the contents disclosed in the documents of the Korean patent applications are incorporated herein by reference in their entirety.

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

Background Art

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

[0004] Adherent cells are cultured using microcarriers in a 3D bioreactor. By placing cells, a culture solution, and microcarriers in the bioreactor and stirring the culture solution to bring the cells into contact with the microcarriers, the cells are attached to the surface of the microcarriers and cultured. The microcarriers used at this time provide a high surface area / volume ratio for cells to attach and grow, and thus are suitable for large-scale cell culture.

[0005] Currently commercially used microcarriers have a density of about 1.1 to 1.3 g / cm 3 and the density of cells is about 1.2 g / cm 3is the degree. In this case, it is advantageous for the cells in the initial stage of culture to adhere in the bioreactor. However, when separating and recovering the cells after culture, centrifugation is difficult, and a filtering method based on the size of the microcarriers and the cells must be used. However, in such cases, there are problems such as the filter clogging or the process time being long, and physical damage and contamination of the cells are likely to occur, and cell loss may occur.

[0006] Therefore, there is a need to develop a new microcarrier that has a low density so that cell culture is possible and can be separated more easily during the separation and recovery of the microcarriers and cells after cell culture.

Summary of the Invention

Problems to be Solved by the Invention

[0007] The present invention provides a microcarrier for cell culture that has a low density so that cell culture is possible and can be separated more easily during the separation and recovery of the microcarriers and cells after cell culture.

[0008] The present invention also provides a method for producing the microcarrier for cell culture.

[0009] The present invention also relates to a cell culture composition using the microcarrier for cell culture.

Means for Solving the Problems

[0010] In order to solve the above problems, the present specification provides a microcarrier for cell culture, which includes at least one or more of hydrocarbon oils having 12 or more carbon atoms, or voids derived therefrom; and magnetic particles; and polystyrene-based particles containing the same.

[0011] The present specification also provides a method for producing a microcarrier for cell culture, which includes a step of performing a suspension polymerization reaction of a monomer composition containing magnetic particles and styrene monomers in the presence of a hydrocarbon oil having 12 or more carbon atoms.

[0012] Also provided herein is a cell culture composition comprising cells and the microcarriers for cell culture.

[0013] Hereinafter, the microcarriers for cell culture, a method for producing the same, and a cell culture composition using the same according to specific embodiments of the invention will be described in more detail.

[0014] Unless expressly stated otherwise herein, the technical terms are merely for referring to specific examples and are not intended to limit the present invention.

[0015] As used herein, the singular forms also include the plural forms unless the context clearly dictates otherwise.

[0016] As used herein, the meaning of "comprising" does not exclude the presence or addition of other specific features, regions, integers, steps, operations, elements, and / or components, while specifying a particular feature, region, integer, step, operation, element, and / or component.

[0017] And, terms including ordinal numbers such as "first" and "second" herein are used for the purpose of distinguishing one component from another and are not limited by the ordinal numbers. For example, within the scope of the rights of the present invention, the first component can also be named the second component, and similarly, the second component can be named the first component.

[0018] Hereinafter, the present invention will be described in more detail.

[0019] According to one embodiment of the invention, a microcarrier for cell culture can be provided, which comprises polystyrene-based particles containing at least one or more of hydrocarbon oils having 12 or more carbon atoms or voids derived therefrom; and magnetic particles.

[0020] In the case of the microcarrier for cell culture of the above-described embodiment, the inventors have found that at least one of hydrocarbon oils having 12 or more carbon atoms or voids derived therefrom is contained inside the polystyrene-based particles, which can not only reduce the density of the microcarrier for cell culture but also ensure homogeneous spherical microcarriers in a high yield. The inventors completed the invention through experiments

[0021] In addition, in the case of the microcarrier for cell culture of the above-described embodiment, the inventors have found that magnetic particles are contained, and through experiments, it has been confirmed that during the separation and recovery of the microcarrier and cells after cell culture, separation and purification can be more easily performed using magnetism. The inventors completed the invention

[0022] Specifically, the microcarrier for cell culture may include polystyrene-based particles containing at least one of hydrocarbon oils having 12 or more carbon atoms or voids derived therefrom; and magnetic particles. That is, the polystyrene-based particles may contain one type of hydrocarbon oil having 12 or more carbon atoms, one type of void derived from a hydrocarbon oil having 12 or more carbon atoms, or a mixture of these two types

[0023] When the polystyrene-based particles contain one type of void derived from a hydrocarbon oil having 12 or more carbon atoms, or when they contain all of the hydrocarbon oil having 12 or more carbon atoms and the voids derived from the hydrocarbon oil having 12 or more carbon atoms, the polystyrene-based particles may correspond to porous polystyrene-based particles

[0024] The polystyrene-based particles are synthesized by suspension polymerization in the presence of a hydrocarbon oil as in the production method of other embodiments described later, and the hydrocarbon oil continues to remain trapped inside the polystyrene particles, or a part or all of the hydrocarbon oil trapped under high-speed suspension polymerization stirring conditions may escape to form voids inside the polystyrene particles

[0025] The void means the empty space inside the polystyrene-based particles and can be used to mean pores, hollows, holes, voids, etc.

[0026] The void may be derived from a hydrocarbon oil having 12 or more carbon atoms. Specifically, the void corresponds to a space formed while the hydrocarbon oil having 12 or more carbon atoms is phase-separated from polystyrene during suspension polymerization.

[0027] Therefore, the fact that the polystyrene-based particles contain only one kind of hydrocarbon oil having 12 or more carbon atoms means that the hydrocarbon oil was completely phase-separated during the polymerization process but did not disappear outside the particles during the polymerization and washing processes. The fact that the polystyrene-based particles contain only one kind of void derived from a hydrocarbon oil having 12 or more carbon atoms means that the hydrocarbon oil was completely phase-separated during the polymerization process but disappeared outside the particles during the polymerization and washing processes. Also, the fact that the polystyrene-based particles contain all of a hydrocarbon oil having 12 or more carbon atoms and voids derived from a hydrocarbon oil having 12 or more carbon atoms means a state in which the hydrocarbon oil was partially phase-separated and disappeared during the polymerization process and part of it remained.

[0028] Specifically, the diameter of the void may be 0.1 μm or more and 5 μm or less. More specifically, the diameter of the void may be 0.1 μm or more, 0.5 μm or more, 1 μm or more, and may also be 5 μm or less, 4 μm or less, 3 μm or less, and may be 0.1 μm or more and 5 μm or less, 0.5 μm or more and 5 μm or less, 1 μm or more and 5 μm or less, 0.1 μm or more and 4 μm or less, 0.5 μm or more and 4 μm or less, 1 μm or more and 4 μm or less, 0.1 μm or more and 3 μm or less, 0.5 μm or more and 3 μm or less, 1 μm or more and 3 μm or less.

[0029] The method for measuring the diameter of the void is not greatly restricted. For example, after embedding a microcarrier for cell culture in epoxy, a cross-section can be produced through ion milling, and then the shape of the particle cross-section can be confirmed by SEM to measure the diameter of the internal void of the particle.

[0030] As described above, since the voids are derived from a hydrocarbon oil having 12 or more carbon atoms, voids having a small size with a diameter of 0.1 μm or more and 5 μm or less can be realized, and thereby, the density of the microcarriers can be easily adjusted.

[0031] Conventionally, in order to reduce the density of polystyrene particles, a low-boiling foaming agent has been introduced during the polystyrene polymerization process, and then expanded polystyrene has been produced through an additional foaming process. In such a case, however, large voids having a size of several tens of μm are formed, making it difficult to adjust the density of the microcarriers.

[0032] The number of carbon atoms of the hydrocarbon oil may be 12 or more, or 12 or more and 50 or less, or 12 or more and 16 or less. If the number of carbon atoms of the hydrocarbon oil is excessively reduced to less than 12, there is a limit in producing a plurality of particles having a non-uniform and concave particle surface due to a decrease in the phase separation rate of the hydrocarbon oil and polystyrene during suspension polymerization.

[0033] Specifically, the hydrocarbon oil may contain a linear or branched saturated hydrocarbon compound having 12 or more and 50 or less carbon atoms. The linear or branched saturated hydrocarbon compound having 12 or more and 50 or less carbon atoms can be used alone or in combination, and examples of the linear or branched saturated hydrocarbon compound having 12 or more and 50 or less carbon atoms include normal alkanes having 12 or more and 16 or less carbon atoms, isoalkanes having 12 or more and 16 or less carbon atoms, or mixtures thereof.

[0034] More specifically, as the hydrocarbon oil, dodecane having 12 carbon atoms, hexadecane having 16 carbon atoms, or Isopar M (a mixture of isoalkanes having 12 or more and 14 or less carbon atoms and isoalkanes having 13 or more and 16 or less carbon atoms) can be used.

[0035] The hydrocarbon oil can be contained in an amount of 30% by weight or less, or 10% by weight or more and 30% by weight or less, based on the total weight (100% by weight) of the dispersion phase composition containing the styrene monomer. Specifically, the lower limit of the content of the hydrocarbon oil is 10% by weight or more, or 11% by weight or more, or 12% by weight or more, or 13% by weight or more, or 14% by weight or more, and the upper limit thereof may be, for example, 30% by weight or less, or 25% by weight or less, or 21% by weight or less.

[0036] More specifically, the hydrocarbon oil may be contained in an amount of 10% by weight or more and 30% by weight or less, 11% by weight or more and 30% by weight or less, 12% by weight or more and 30% by weight or less, 13% by weight or more and 30% by weight or less, 14% by weight or more and 30% by weight or less, 10% by weight or more and 25% by weight or less, 11% by weight or more and 25% by weight or less, 12% by weight or more and 25% by weight or less, 13% by weight or more and 25% by weight or less, 14% by weight or more and 25% by weight or less, 10% by weight or more and 20% by weight or less, 11% by weight or more and 20% by weight or less, 12% by weight or more and 20% by weight or less, 13% by weight or more and 20% by weight or less, 14% by weight or more and 20% by weight or less, based on the total weight (100% by weight) of the dispersion phase composition containing the styrene monomer.

[0037] When the content of the hydrocarbon oil is used less than the content range, it is difficult to ensure carrier particles with low density characteristics. When the content range is excessively exceeded, the shape of the polystyrene-based particles hardly has a spherical shape and the uniformity of the particles produced decreases.

[0038] The density of the hydrocarbon oil is 0.75 g / cm 3 or more and 0.80 g / cm 3 or less, 0.75 g / cm 3 or more and 0.795 g / cm 3 or less, or 0.75 g / cm 3 or more and 0.791 g / cm 3 or less. Since the density of the hydrocarbon oil is within the above range and is very low, the density of the polystyrene-based particles can be significantly reduced thereby.

[0039] However, if the density of the hydrocarbon oil decreases excessively to less than 0.75 g / cm 3 there is a limit to the production of a large number of particles with a non-uniform and concave surface due to a decrease in the phase separation rate of the hydrocarbon oil and polystyrene during suspension polymerization.

[0040] The apparent density of the microcarrier may be 0.95 g / cm 3 or more and less than 1.05 g / cm 3 Specifically, the apparent density of the microcarrier may be 0.95 g / cm 3 or more, 0.99 g / cm 3 or more, 0.995 g / cm 3 or more, 0.996 g / cm 3 or more, and may be less than 1.05 g / cm 3 , less than 1.049 g / cm 3 , less than 1.04 g / cm 3 , less than 1.03 g / cm 3 , and may be less than 0.95 g / cm 3 or more and less than 1.05 g / cm 3 , less than 0.99 g / cm 3 or more and less than 1.05 g / cm 3 , less than 0.995 g / cm 3 or more and less than 1.05 g / cm 3 , less than 0.996 g / cm 3 or more and less than 1.05 g / cm 3 , less than 0.95 g / cm 3 or more and less than 1.049 g / cm 3 , less than 0.99 g / cm 3 or more and less than 1.049 g / cm 3 , less than 0.995 g / cm 3 or more and less than 1.049 g / cm 3 , less than 0.996 g / cm 3 or more and less than 1.049 g / cm 3 , less than 0.95 g / cm 3 or more and less than 1.04 g / cm 3 , less than 0.99 g / cm 3 or more and less than 1.04 g / cm 3 , less than 0.995 g / cm 3 or more and less than 1.04 g / cm 3 , less than 0.996 g / cm3 Above 1.04 g / cm 3 Below, 0.95 g / cm 3 Above 1.03 g / cm 3 Below, 0.99 g / cm 3 Above 1.03 g / cm 3 Below, 0.995 g / cm 3 Above 1.03 g / cm 3 Below, 0.996 g / cm 3 Above 1.03 g / cm 3 It may also be below.

[0041] By having the above-mentioned low-density range, when separating and recovering the microcarriers and cells after cell culture, the cells and microcarriers can be easily separated through the difference in sedimentation rates due to gravity.

[0042] The method for measuring the apparent density of the microcarriers is not greatly restricted. For example, a microcarrier sample under the condition where the drying process is completed can be added to an ethanol aqueous solution with adjusted density and distilled water respectively, and it can be confirmed whether the particles float or sediment to measure the apparent density.

[0043] More specifically, a microcarrier sample under the condition where the drying process is completed under normal temperature (25 °C) and normal pressure (1 atm) conditions is added to an ethanol aqueous solution with a density of 0.95 g / cm 3 , 0.97 g / cm 3 , 0.98 g / cm 3 or 0.995 g / cm 3 and distilled water (DIW) with a density of 1 g / cm 3 respectively, and it can be confirmed whether the particles float or sediment to evaluate the apparent density.

[0044] For example, when floating in an ethanol aqueous solution with a density of 0.95 g / cm 3 , the apparent density of the microcarrier is less than 0.95 g / cm 3 , and when sedimenting in distilled water (DIW) with a density of 1 g / cm 3 , the apparent density of the microcarrier is 1 g / cm 3is exceeded and the density is 0.95 g / cm 3 and sedimented in an aqueous ethanol solution, and suspended in distilled water (DIW) with a density of 1 g / cm 3 , the apparent density of the microcarrier is 0.95 g / cm 3 or more and less than 1 g / cm 3 is exceeded and the density is 0.95 g / cm 3 and sedimented in an aqueous ethanol solution, and suspended in an aqueous ethanol solution with a density of 0.97 g / cm 3 , the apparent density of the microcarrier is 0.95 g / cm 3 or more and less than 0.97 g / cm 3 is exceeded and the density is 0.98 g / cm 3 and sedimented in an aqueous ethanol solution, and suspended in an aqueous ethanol solution with a density of 0.995 g / cm 3 , the apparent density of the microcarrier is 0.98 g / cm 3 or more and less than 0.995 g / cm 3 and can be evaluated as less than that.

[0045] When the density of the microcarrier exceeds 1.05 g / cm 3 , the density difference between the cells and the microcarrier may be small and centrifugation may be difficult during cell separation and recovery after culture. When it is less than 0.95 g / cm 3 , there may be a problem that the microcarrier floats only on the surface of the culture solution at the initial stage of culture and it is difficult for cells to adhere.

[0046] The cells to which the microcarrier for cell culture of the above embodiment is applied are adherent animal cells, and the examples are not greatly limited. 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 a mixture of two or more of these may be used.

[0047] The density of the cells is 1.02 g / cm 3 or more and 1.1 g / cm 3 or less.

[0048] Also, the density difference between the microcarrier for cell culture and the cells may be 0.02 g / cm 3 or more and 0.20 g / cm 3 or less. The density difference between the microcarrier for cell culture and the cells may be 0.02 g / cm 3 or more and 0.20 g / cm 3 or less, and even if the density difference is not large, the cells and the microcarrier can be easily separated by including the magnetic particles.

[0049] On the other hand, the magnetic particles may include magnetic particles surface-treated with a hydrophobic functional group. The magnetic particles surface-treated with the hydrophobic functional group may mean magnetic particles having a hydrophobic functional group bonded to the surface of the magnetic particles.

[0050] Specifically, surface treatment means reacting a magnetic particle with a compound containing a hydrophobic functional group to bond the hydrophobic functional group to the surface of the magnetic particle in order to prevent aggregation of the magnetic particles and improve dispersibility.

[0051] In the above embodiment, the magnetic particles mean particles exhibiting magnetic properties. All substances interact with a magnetic field to generate an attractive force or a repulsive force. That is, when a magnetic field is applied to a substance, it is magnetized, and depending on the manner in which the object is magnetized, it is classified into a ferromagnetic material, a paramagnetic material, a diamagnetic material, a ferrimagnetic material, etc.

[0052] The magnetic particles can be manufactured by solution synthesis, coprecipitation, sol-gel method, high-energy pulverization, hydrothermal synthesis, microemulsion synthesis, synthesis by thermal decomposition, or sonochemical synthesis, but are not limited thereto.

[0053] The diameter of the magnetic particles surface-treated with the hydrophobic functional group may be 0.1 nm or more and 1000 nm or less. That is, the magnetic particles surface-treated with the hydrophobic functional group may be magnetic nanoparticles surface-treated with the hydrophobic functional group.

[0054] The nanoparticles mean particles having a size of nanometers (nm). The size of nanometers is obtained by reducing the size of micrometers (10 -6 ) to 1 / 1000, and when the size of the substance becomes smaller at the nanometer level, various and specific physical, chemical, mechanical, and electronic characteristics are exhibited.

[0055] When the size of the magnetic substance becomes smaller at the nanometer level, each particle forms a magnetic single domain. In the colloidal solution of such particles, the direction of the magnetic dipole is oriented in an unspecified direction due to the thermal fluctuation of each particle, and the net magnetic force shown externally is shown as "0". However, when a magnetic field larger than the internal thermal energy is applied from the outside, the magnetic dipoles of the particles are aligned in one direction and turn into a magnet.

[0056] The types of the magnetic particles are not greatly limited, and metal nanoparticles can be used. For example, it can contain one or more metals selected from the group consisting of gold (Au), silver (Ag), cobalt (Co), copper (Cu), iron (Fe), chromium (Cr), nickel (Ni), palladium (Pd), platinum (Pt), and tin (Sn), or oxides thereof. Specifically, the magnetic particles can contain iron (Fe) particles.

[0057] The magnetic particles can contain a hydrophobic ligand containing a hydrophobic functional group on the surface of the magnetic particles. That is, the magnetic particles surface-treated with the hydrophobic functional group can contain a hydrophobic ligand containing a hydrophobic functional group on the surface of the magnetic particles.

[0058] By including magnetic particles whose microcarriers are surface-treated with hydrophobic functional groups, compared with microcarriers containing magnetic particles that are not surface-treated, not only can metal particles be independently dispersed to prevent particle aggregation, but also the oxidation stability can be improved.

[0059] The ligand means a molecule or ion that binds around the central metal ion of a compound to which it is coordinately bonded, and can mean an atom or atomic group that forms a coordinate bond while providing an electron pair to the central metal atom in a complex compound.

[0060] That is, the hydrophobic ligand can mean a hydrophobic molecule or hydrophobic ion that binds around the central magnetic particle ion of magnetic particles surface-treated with hydrophobic functional groups.

[0061] Specifically, the hydrophobic ligand can include one or more ligands selected from the group consisting of fatty acids having 2 to 20 carbon atoms or their derivatives and fatty acid amines having 2 to 20 carbon atoms or their derivatives.

[0062] The fatty acid derivative can include fatty acid ions, and the fatty acid amine derivative can include fatty acid amine ions.

[0063] The fatty acid is a compound containing a hydrocarbon chain and a carboxyl group (-COOH) at its end. The hydrocarbon chain has hydrophobicity, but can interact with hydrophilic substances through the carboxyl group at the end. The fatty acid may be an unsaturated fatty acid or a saturated fatty acid.

[0064] The unsaturated fatty acid is a fatty acid having one or more double bonds, and -COOH or -COO at the end - is exposed on the surface of the magnetic particles and can form hydrogen bonds, ionic bonds, covalent bonds, etc. with hydrophilic groups and / or unsaturated coordination sites of the magnetic particles.

[0065] Since the unsaturated fatty acid contains a double bond, a bent hydrocarbon chain of the unsaturated fatty acid can be oriented outside the magnetic particles while forming a hydrophobic channel.

[0066] That is, the hydrophobic functional group described above can include a hydrocarbon chain derived from a fatty acid having 2 to 20 carbon atoms or a derivative thereof, or a fatty acid amine having 2 to 20 carbon atoms, or a derivative thereof. More specifically, the hydrophobic functional group can include an alkyl group having 2 to 50 carbon atoms or an alkenyl group having 2 to 50 carbon atoms.

[0067] Thereby, the hydrophobic ligand can include the hydrophobic functional group and -COOH or -COO bonded to the end thereof. - can be included.

[0068] The type of the fatty acid having 2 to 20 carbon atoms is not greatly limited. For example, it can include any one of oleic acid, caproic acid, and stearic acid.

[0069] Also, the type of the fatty acid amine having 2 to 20 carbon atoms is not greatly limited. For example, it can include any one of oleylamine, butylamine, and octylamine.

[0070] For example, the magnetic particles surface-treated with the hydrophobic functional group can have a bonding structure as shown in FIG. 1.

[0071] When manufacturing the magnetic particles surface-treated with the hydrophobic functional group, a fatty acid having 2 to 20 carbon atoms is added to a basic aqueous solution to form fatty acid ions having 2 to 20 carbon atoms, and then reacted with magnetic nanoparticles, whereby -COO contained at the end of the fatty acid having 2 to 20 carbon atoms. - and the coordination bond between the magnetic nanoparticles can be formed as shown in FIG. 1.

[0072] On the other hand, the density of the magnetic particles is 5 g / cm 3 or more and 6 g / cm 3The following may be applicable. That is, the density of the magnetic particles surface-treated with the hydrophobic functional group may be 5 g / cm 3 or more and 6 g / cm 3 or less.

[0073] Specifically, the density of the magnetic particles may be 5 g / cm 3 or more, 5.1 g / cm 3 or more, 5.15 g / cm 3 or more, or 6 g / cm 3 or less, 5.8 g / cm 3 or less, 5.5 g / cm 3 or less, 5.3 g / cm 3 or less, or 5 g / cm 3 or more and 6 g / cm 3 or less, 5 g / cm 3 or more and 5.8 g / cm 3 or less, 5 g / cm 3 or more and 5.5 g / cm 3 or less, 5 g / cm 3 or more and 5.3 g / cm 3 or less, 5.1 g / cm 3 or more and 6 g / cm 3 or less, 5.1 g / cm 3 or more and 5.8 g / cm 3 or less, 5.1 g / cm 3 or more and 5.5 g / cm 3 or less, 5.1 g / cm 3 or more and 5.3 g / cm 3 or less, 5.15 g / cm 3 or more and 5.8 g / cm 3 or less, 5.15 g / cm 3 or more and 5.5 g / cm 3 or less, 5.15 g / cm 3 or more and 5.3 g / cm 3 or less.

[0074] As described above, the density of the magnetic particles is 5 g / cm 3 or more and 6 g / cm 3When, for 100 parts by weight of a hydrocarbon oil having 12 or more carbon atoms, magnetic particles are excessively added in an amount exceeding 5 parts by weight, the overall density of the particles increases due to the excessive inclusion of magnetic particles with a high density, resulting in not only the particles not floating smoothly in the incubator but also showing cytotoxicity in some cases.

[0075] Also, the diameter of the magnetic particles surface-treated with the hydrophobic functional group may be 100 nm or more and 2000 nm or less. The method for measuring the diameter of the magnetic particles is not greatly restricted. For example, it can be measured using the dynamic light scattering method (DLS).

[0076] Specifically, the diameter of the magnetic particles surface-treated with the hydrophobic functional group may be 100 nm or more, 300 nm or more, 500 nm or more, or 2000 nm or less, 1500 nm or less, 1000 nm or less, 800 nm or less, 600 nm or less, or 100 nm or more and 2000 nm or less, 100 nm or more and 1500 nm or less, 100 nm or more and 1000 nm or less, 100 nm or more and 800 nm or less, 100 nm or more and 600 nm or less, 300 nm or more and 2000 nm or less, 300 nm or more and 1500 nm or less, 300 nm or more and 1000 nm or less, 300 nm or more and 800 nm or less, 300 nm or more and 600 nm or less, or 500 nm or more and 2000 nm or less, 500 nm or more and 1500 nm or less, 500 nm or more and 1000 nm or less, 500 nm or more and 800 nm or less, 500 nm or more and 600 nm or less.

[0077] The polystyrene-based particles can contain a polystyrene-based polymer in which at least one or more of a hydrocarbon oil having 12 or more carbon atoms or voids derived therefrom; and magnetic particles; are dispersed inside.

[0078] That is, the polystyrene-based particles can contain a polystyrene-based polymer matrix and at least one or more of a hydrocarbon oil having 12 or more carbon atoms or voids derived therefrom; and magnetic particles; dispersed inside the polystyrene-based polymer matrix.

[0079] Specifically, the microcarrier for cell culture may exist in a state where magnetic particles are dispersed in at least one or more of the hydrocarbon oils having 12 or more carbon atoms or the voids derived therefrom.

[0080] As described above, the microcarrier for cell culture contains the suspension polymerization reaction product of a monomer composition containing a hydrocarbon oil having 12 or more carbon atoms, magnetic particles, and a styrene monomer, and may contain voids which are spaces formed while the hydrocarbon oil having 12 or more carbon atoms is phase-separated from polystyrene during suspension polymerization.

[0081] Therefore, due to the inclusion of magnetic particles in the monomer composition, magnetic particles may exist in a dispersed state in at least one or more of the hydrocarbon oils having 12 or more carbon atoms or the voids derived therefrom.

[0082] Specifically, the polystyrene-based particles may contain the suspension polymerization reaction product of a monomer composition containing a hydrocarbon oil having 12 or more carbon atoms, magnetic particles surface-treated with a hydrophobic functional group, and a styrene monomer.

[0083] The monomer composition may contain 0.01 part by weight or more and 5 parts by weight or less of magnetic particles with respect to 100 parts by weight of the hydrocarbon oil having 12 or more carbon atoms. Specifically, the monomer composition may contain 0.01 part by weight or more, 0.02 part by weight or more, 0.04 part by weight or more of magnetic particles with respect to 100 parts by weight of the hydrocarbon oil having 12 or more carbon atoms, and may contain 5 parts by weight or less, 4 parts by weight or less, and may contain 0.01 part by weight or more and 5 parts by weight or less, 0.02 part by weight or more and 5 parts by weight or less, 0.02 part by weight or more and 4 parts by weight or less, 0.04 part by weight or more and 4 parts by weight or less.

[0084] When the amount of magnetic particles is excessively reduced to less than 0.01 part by weight with respect to 100 parts by weight of a hydrocarbon oil having 12 or more carbon atoms, the magnetism of the microcarriers is not sufficiently realized. When separating and recovering the microcarriers and cells after cell culture, not only is the separation and purification not easy, but there is also a limit in that an additional recovery process is involved and the process becomes complicated.

[0085] On the contrary, when the amount of magnetic particles is excessively added to exceed 5 parts by weight with respect to 100 parts by weight of a hydrocarbon oil having 12 or more carbon atoms, the overall density of the particles increases due to the excessive inclusion of magnetic particles with a high density, and not only do the particles not float smoothly in the incubator, but they may also exhibit cytotoxicity.

[0086] Further, the monomer composition can contain an ethylenically unsaturated crosslinking agent in an amount exceeding 0.033 part by weight and less than 3 parts by weight with respect to 1 part by weight of the styrene monomer.

[0087] Specifically, when the amount of the ethylenically unsaturated crosslinking agent is excessively reduced to less than 0.033 part by weight with respect to 1 part by weight of the styrene monomer, the crosslinking density of the polystyrene-based polymer decreases, making it difficult to stably produce the voids formed by the hydrocarbon oil and there is a limit in that it is difficult to sufficiently lower the density of the polystyrene-based particles.

[0088] On the contrary, when the amount of the ethylenically unsaturated crosslinking agent is excessively increased to 3 parts by weight or more with respect to 1 part by weight of the styrene monomer, while the crosslinking density of the polystyrene-based polymer increases, there is a limit in that it is difficult for the overall density of the polystyrene-based particles to drop to the targeted level.

[0089] Further, the content of the hydrocarbon oil may be 10% by weight or more and 30% by weight or less, or 14% by weight or more and 21% by weight or less with respect to the total weight of the monomer composition. When the content of the hydrocarbon oil is excessively reduced, the amount of the hydrocarbon oil impregnated into the particles decreases, and it is difficult for the density of the polystyrene-based particles to sufficiently decrease. Also, when the content of the hydrocarbon oil is excessively increased, it becomes difficult for the shape of the polystyrene-based particles to form a sphere, and the uniformity of the produced particles decreases.

[0090] Examples of the ethylenically unsaturated crosslinking agent include divinylbenzene.

[0091] Based on the total surface area of the polystyrene-based particles, the ratio of the surface area of the polystyrene-based particles in contact with the micropores present on the surface of the polystyrene-based particles may be less than 0.01%. The total surface area of the polystyrene-based particles means the sum of the surface areas exposed to the air in the outermost shell of the polystyrene-based particles, and the surface area of the polystyrene-based particles in contact with the micropores present on the surface of the polystyrene-based particles means the sum of the surface areas where the micropores present on the surface of the polystyrene-based particles contact the outermost shell surface of the polystyrene-based particles. Further, the micropores are voids having a maximum diameter in the micrometer size range, for example, voids having a maximum diameter of 1 μm or more and 500 μm or less.

[0092] More specifically, based on the total surface area of the polystyrene-based particles, the ratio of the surface area of the polystyrene-based particles in contact with the micropores present on the surface of the polystyrene-based particles can be obtained through the following formula (1).

[0093] [Formula (1)] Ratio (%) of the surface area of the polystyrene-based particles in contact with the micropores present on the surface of the polystyrene-based particles based on the total surface area of the polystyrene-based particles = [(Surface area of the polystyrene-based particles in contact with the micropores present on the surface of the polystyrene-based particles) / (Total surface area of the polystyrene-based particles)] × 100

[0094] That the ratio of the surface area of the polystyrene-based particles in contact with the micropores present on the surface of the polystyrene-based particles is less than 0.01% based on the total surface area of the polystyrene-based particles means that there are almost no or extremely few micro-sized pores present on the surface of the polystyrene-based particles. That is, there may be no micropores on the surface of the polystyrene-based particles.

[0095] Conventionally, in order to reduce the density of polystyrene particles, a foaming agent has been added to produce expanded polystyrene. However, in this case, due to the excessively wide distribution of the diameter range and density range of the polystyrene particles, it has been difficult to ensure a yield within a range applicable as a microcarrier for cell culture.

[0096] On the other hand, since the ratio of the surface area of the polystyrene-based particles in contact with the micropores present on the surface of the polystyrene-based particles is less than 0.01% based on the total surface area of the polystyrene-based particles, unlike conventional expanded polystyrene, there is no foaming process using a foaming agent at all, and there is an advantage that the distribution of the diameter range and density range of the polystyrene particles can be adjusted more precisely.

[0097] The average diameter of the microcarrier may be 50 μm or more and 400 μm or less.

[0098] Specifically, the average diameter of the microcarrier may be 50 μm or more, 75 μm or more, 100 μm or more, 120 μm or more, or 400 μm or less, 300 μm or less, 250 μm or less, 200 μm or less, or 50 μm or more and 400 μm or less, 50 μm or more and 300 μm or less, 50 μm or more and 250 μm or less, 50 μm or more and 200 μm or less, 50 μm or more and 400 μm or less, 75 μm or more and 300 μm or less, 75 μm or more and 250 μm or less, 75 μm or more and 200 μm or less, 100 μm or more and 400 μm or less, 100 μm or more and 300 μm or less, 100 μm or more and 250 μm or less, 100 μm or more and 200 μm or less, 120 μm or more and 400 μm or less, 120 μm or more and 300 μm or less, 50 μm or more and 120 μm or less, 50 μm or more and 120 μm or less. When the average diameter of the microcarrier satisfies the above range, it is excellent in cell adhesion and culture performance. On the other hand, when the average diameter of the microcarrier is less than 50 μm, there may be a problem that the surface area available for cell culture is small and the culture efficiency decreases. When it exceeds 400 μm, the interaction between attached cells decreases, the cell density in the incubator decreases, and the problem of decreased cell culture efficiency may occur.

[0099] The diameter of the microcarrier means the distance between two points where a straight line passing through the center of gravity of the microcarrier contacts the outermost surface of the microcarrier, and the average diameter of the microcarrier can be determined by checking the diameters of all the particles contained in the microcarrier for cell culture through an optical microscope. Also, the average particle size of the microcarrier can be confirmed through the diameters of all the microcarriers obtained in the manufacturing process of the microcarrier and their average diameters.

[0100] The microcarrier may be a group of individual particles having an average diameter of 50 μm or more and 400 μm or less, and the individual microparticles contained in such a group can have an average diameter of 50 μm or more and 400 μm or less. More specifically, 95% or 99% of the individual microparticles contained in the group can have a diameter of 50 μm or more and 400 μm or less.

[0101] Also, the microcarrier may have a specific surface area of 200 cm 2 / g or more and 1000 cm 2 / g or less. Specifically, the microcarrier may have a specific surface area of 200 cm 2 / g or more, 300 cm 2 / g or more, 330 cm 2 / g or more, 350 cm 2 / g or more, and may also be 1000 cm 2 / g or less, 500 cm 2 / g or less, 450 cm 2 / g or less, and may also be 200 cm 2 / g or more and 1000 cm 2 / g or less, 300 cm 2 / g or more and 1000 cm 2 / g or less, 300 cm 2 / g or more and 500 cm 2 / g or less, 300 cm 2 / g or more and 450 cm 2 / g or less, 330 cm 2 / g or more and 450 cm 2 / g or less, 350 cm 2450 cm / g or more 2 It may be 450 cm / g or less.

[0102] The method for measuring the specific surface area is not greatly limited. For example, the specific surface area of the microcarrier can be calculated from the apparent density using the following formula.

[0103] [Formula] Specific surface area of microcarrier (cm 2 / g) = 3 / (r × ρ)

[0104] In the above formula, r represents the average radius of the microcarrier, and ρ represents the apparent density of the microcarrier.

[0105] The surface of the polystyrene-based particles may further include a primer polymer layer, a cell adhesion induction layer, or a combination layer thereof. That is, the surface of the polystyrene-based particles may further include one type of primer polymer layer, one type of cell adhesion induction layer, or a mixed layer of one type of primer polymer layer and one type of cell adhesion induction layer. In the mixed layer of one type of primer polymer layer and one type of cell adhesion induction layer, the lamination order thereof is not particularly limited, and all structures in which the cell adhesion induction layer is laminated on the primer polymer layer or the primer polymer layer is laminated on the cell adhesion induction layer are applicable.

[0106] On the other hand, the primer polymer layer serves as an adhesive layer capable of introducing a functional polymer onto the surface of the polystyrene-based particles having no functional group, whereby a polymer layer for cell adhesion is effectively introduced onto the surface of the microcarrier and stably maintained during culturing.

[0107] The primer polymer layer is not limited to a large extent, but can include any one or more selected from the group consisting of catechol derivatives capable of inducing aqueous adhesion, such as L-dihydroxyphenylalanine (L-DOPA), dopamine, polydopamine, norepinephrine, epinephrine, epigallocatechin, and derivatives thereof.

[0108] On the other hand, the cell adhesion-inducing layer is composed of cell adhesion substances, which serve to provide a site where transmembrane proteins of cells can bind, enabling adherent cells to adhere, spread, and be cultured stably.

[0109] The polymer forming the cell adhesion-inducing layer is not limited to a large extent, but can include any one or more selected from the group consisting of gelatin, collagen, fibronectin, chitosan, polydopamine, poly-L-lysine, vitronectin, peptides containing RGD, acrylic polymers containing RGD, lignin, cationic dextran, and derivatives thereof.

[0110] As an example, the microcarrier includes a primer polymer layer formed on the surface of polystyrene-based particles, is dispersed in water by modifying the surface of the microcarrier to be hydrophilic, and by introducing a cell adhesion-inducing layer on the surface of the primer polymer layer, the floating degree of the microcarrier in the culture solution can be adjusted, and the effect of stably adhering and culturing cells can be achieved.

[0111] On the other hand, the ratio of the radius of the polystyrene-based 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.

[0112] When the ratio of the radius of the polystyrene-based particles to the thickness of the surface coating layer is less than 1:0.00001, the primer polymer layer is excessively thin compared to the polystyrene-based particles, and the effect of modifying the microcarrier surface to be hydrophilic is negligible. When it exceeds 1:0.01, the primer polymer layer becomes thicker compared to the polystyrene-based particles, and there is a risk that the degree of adhesion between the cells and the microcarriers will decrease during cell culture.

[0113] According to another embodiment of the invention, there is provided a method for manufacturing a microcarrier for cell culture, including the step of performing a suspension polymerization reaction of a monomer composition containing magnetic particles and styrene monomer in the presence of a hydrocarbon oil having 12 or more carbon atoms.

[0114] The content regarding the hydrocarbon oil, magnetic particles, and styrene monomer includes all the content described above in the one embodiment. That is, the magnetic particles can include magnetic particles surface-treated with a hydrophobic functional group.

[0115] The magnetic particles surface-treated with the hydrophobic functional group can mean magnetic particles having a hydrophobic functional group bonded to the surface of the magnetic particles.

[0116] Specifically, surface treatment means reacting a magnetic particle with a compound containing a hydrophobic functional group to bond a hydrophobic functional group to the surface of the magnetic particle in order to prevent aggregation of the magnetic particles and improve dispersibility.

[0117] In the above embodiment, the magnetic particles mean particles exhibiting magnetic properties. All substances interact with a magnetic field to generate an attractive force or a repulsive force. That is, when a magnetic field is applied to a substance, it is magnetized, and depending on the manner in which the object is magnetized, it is classified into ferromagnetic materials, paramagnetic materials, diamagnetic materials, ferrimagnetic materials, etc.

[0118] The magnetic particles can be produced by, but are not limited to, solution synthesis, co-precipitation, sol-gel method, high-energy milling, hydrothermal synthesis, microemulsion synthesis, synthesis by thermal decomposition, or sonochemical synthesis.

[0119] The diameter of the magnetic particles surface-treated with the hydrophobic functional group may be 0.1 nm or more and 1000 nm or less. That is, the magnetic particles surface-treated with the hydrophobic functional group may be magnetic nanoparticles surface-treated with the hydrophobic functional group.

[0120] The nanoparticles mean particles having a size of nanometers. The size of nanometers is obtained by reducing the size of micrometers (10 -6 ) to 1 / 1000, and when the size of a substance becomes small at the nanometer level, it exhibits various and specific physical, chemical, mechanical, and electronic properties.

[0121] When the size of a magnetic substance becomes small at the nanometer level, each particle comes to form a magnetic single domain. In a colloidal solution of such particles, the direction of the magnetic dipole is oriented in an unspecified direction due to the thermal fluctuation of each particle, and the net magnetic force that appears externally is "0". However, when a magnetic field larger than the internal thermal energy is applied from the outside, the magnetic dipoles of the particles are aligned in one direction and turn into a magnetic body.

[0122] The type of the magnetic particles is not greatly limited, and metal nanoparticles can be used. For example, it can contain one or more metals selected from the group consisting of gold (Au), silver (Ag), cobalt (Co), copper (Cu), iron (Fe), chromium (Cr), nickel (Ni), palladium (Pd), platinum (Pt), and tin (Sn), or an oxide thereof. Specifically, the magnetic particles can contain iron (Fe) particles.

[0123] The magnetic particles can include a hydrophobic ligand containing a hydrophobic functional group on the surface of the magnetic particles. That is, the magnetic particles surface-treated with the hydrophobic functional group can include a hydrophobic ligand containing a hydrophobic functional group on the surface of the magnetic particles.

[0124] Since the method for manufacturing the microcarrier includes magnetic particles surface-treated with a hydrophobic functional group, compared with the case of including non-surface-treated magnetic particles, not only can metal particles be independently dispersed on the finally manufactured microcarrier to prevent particle aggregation, but also the oxidation stability can be improved.

[0125] The ligand means a molecule or ion bonded around the central metal ion of a compound in which coordination bonds are formed, and can mean an atom or atomic group that forms a coordination bond while providing an electron pair to the central metal atom in a complex compound.

[0126] That is, the hydrophobic ligand can mean a hydrophobic molecule or hydrophobic ion bonded around the central magnetic particle ion of the magnetic particles surface-treated with a hydrophobic functional group.

[0127] Specifically, the hydrophobic ligand can include a fatty acid having 2 to 20 carbon atoms or a derivative thereof, or a fatty acid amine having 2 to 20 carbon atoms, or a derivative thereof.

[0128] The fatty acid derivative can include a fatty acid ion, and the fatty acid amine derivative can include a fatty acid amine ion.

[0129] The fatty acid is a compound containing a hydrocarbon chain and a carboxyl group (-COOH) at its terminal. The hydrocarbon chain has hydrophobicity but can interact with a hydrophilic substance through the terminal carboxyl group. The fatty acid may be an unsaturated fatty acid or a saturated fatty acid.

[0130] The unsaturated fatty acid is a fatty acid having one or more double bonds, and -COOH or -COO at the terminal -It can form hydrogen bonds, ionic bonds, covalent bonds, etc. with hydrophilic groups exposed on the surface of magnetic particles and / or unsaturated coordination sites of magnetic particles.

[0131] Since the unsaturated fatty acid contains a double bond, a hydrophobic channel can be formed while the bent hydrocarbon chain of the unsaturated fatty acid is oriented outside the magnetic particles.

[0132] That is, the aforementioned hydrophobic functional group can include a hydrocarbon chain derived from a fatty acid having 2 to 20 carbon atoms or its derivative, or a fatty acid amine having 2 to 20 carbon atoms, or its derivative. More specifically, the hydrophobic functional group can include an alkyl group having 2 to 50 carbon atoms, or an alkenyl group having 2 to 50 carbon atoms.

[0133] Thereby, the hydrophobic ligand can include the hydrophobic functional group and -COOH or -COO bonded to its end. - can be included.

[0134] The type of the fatty acid having 2 to 20 carbon atoms is not greatly restricted. For example, it can include any one of oleic acid, caproic acid, and stearic acid.

[0135] Also, the type of the fatty acid amine having 2 to 20 carbon atoms is not greatly restricted. For example, it can include any one of oleylamine, butylamine, and octylamine.

[0136] For example, the magnetic particles surface-treated with the hydrophobic functional group can have a bonding structure as shown in FIG. 1.

[0137] When manufacturing the magnetic particles surface-treated with the hydrophobic functional group, a fatty acid having 2 to 20 carbon atoms is added to a basic aqueous solution to form fatty acid ions having 2 to 20 carbon atoms, and then reacted with magnetic nanoparticles, so that -COO contained at the end of the fatty acid having 2 to 20 carbon atoms - and the coordination bond between the magnetic nanoparticles can be formed as shown in FIG. 1.

[0138] On the one hand, the density of the magnetic particles may be 5 g / cm 3 or more and 6 g / cm 3 or less. That is, the density of the magnetic particles surface-treated with the hydrophobic functional group may be 5 g / cm 3 or more and 6 g / cm 3 or less.

[0139] Specifically, the density of the magnetic particles may be 5 g / cm 3 or more, 5.1 g / cm 3 or more, 5.15 g / cm 3 or more, or 6 g / cm 3 or less, 5.8 g / cm 3 or less, 5.5 g / cm 3 or less, 5.3 g / cm 3 or less, or 5 g / cm 3 or more and 6 g / cm 3 or less, 5 g / cm 3 or more and 5.8 g / cm 3 or less, 5.1 g / cm 3 or more and 5.8 g / cm 3 or less, 5.1 g / cm 3 or more and 5.5 g / cm 3 or less, 5.1 g / cm 3 or more and 5.3 g / cm 3 or less, 5.15 g / cm 3 or more and 5.3 g / cm 3 or less.

[0140] As described above, when the density of the magnetic particles is 5 g / cm 3 or more and 6 g / cm 3 or less, when more than 5 parts by weight of the magnetic particles surface-treated with the hydrophobic functional group are excessively added to 100 parts by weight of the hydrocarbon oil having 12 or more carbon atoms, the overall density of the particles increases due to the excessive inclusion of the magnetic particles with a large density, and not only the particles do not float smoothly in the incubator but also may exhibit cytotoxicity.

[0141] Also, the diameter of the magnetic particles surface-treated with the hydrophobic functional group may be 100 nm or more and 2000 nm or less.

[0142] Specifically, the diameter of the magnetic particles surface-treated with the hydrophobic functional group may be 100 nm or more, 300 nm or more, 500 nm or more, or 2000 nm or less, 1500 nm or less, 1000 nm or less, 800 nm or less, 600 nm or less, or 100 nm or more and 2000 nm or less, 100 nm or more and 1500 nm or less, 100 nm or more and 1000 nm or less, 100 nm or more and 800 nm or less, 100 nm or more and 600 nm or less, 300 nm or more and 2000 nm or less, 300 nm or more and 1500 nm or less, 300 nm or more and 1000 nm or less, 300 nm or more and 800 nm or less, 300 nm or more and 600 nm or less, or 500 nm or more and 2000 nm or less, 500 nm or more and 1500 nm or less, 500 nm or more and 1000 nm or less, 500 nm or more and 800 nm or less, 500 nm or more and 600 nm or less.

[0143] The monomer composition can contain 0.01 to 5 parts by weight of magnetic particles with respect to 100 parts by weight of a hydrocarbon oil having 12 or more carbon atoms. Specifically, the monomer composition can contain 0.01 part by weight or more, 0.02 part by weight or more, 0.04 part by weight or more of magnetic particles with respect to 100 parts by weight of a hydrocarbon oil having 12 or more carbon atoms, and can contain 5 parts by weight or less, 4 parts by weight or less, and can contain 0.01 to 5 parts by weight, 0.02 to 5 parts by weight, 0.02 to 4 parts by weight, 0.04 to 4 parts by weight.

[0144] When the amount of magnetic particles is excessively reduced to less than 0.01 part by weight with respect to 100 parts by weight of a hydrocarbon oil having 12 or more carbon atoms, the magnetism of the microcarrier is not sufficiently realized, and when separating and recovering the microcarrier and cells after cell culture, not only is the separation and purification not easy, but there is a limit that an additional recovery step is involved and the process becomes complicated.

[0145] On the contrary, when the amount of magnetic particles is excessively added to exceed 5 parts by weight with respect to 100 parts by weight of a hydrocarbon oil having 12 or more carbon atoms, the overall density of the particles increases due to the excessive inclusion of high-density magnetic particles, and not only do the particles not float smoothly in the incubator, but they may also exhibit cytotoxicity.

[0146] Further, the monomer composition can contain an ethylenically unsaturated crosslinking agent in an amount of more than 0.033 parts by weight and less than 3 parts by weight with respect to 1 part by weight of the styrene monomer.

[0147] Specifically, when the amount of the ethylenically unsaturated crosslinking agent is excessively reduced to less than 0.033 parts by weight with respect to 1 part by weight of the styrene monomer, the crosslinking density of the polystyrene-based polymer decreases, making it difficult to stably produce voids generated by the hydrocarbon oil, and there is a limit to sufficiently reducing the density of the polystyrene-based particles.

[0148] On the other hand, when the amount of the ethylenically unsaturated crosslinking agent is excessively increased to 3 parts by weight or more with respect to 1 part by weight of the styrene monomer, while the crosslinking density of the polystyrene-based polymer increases, there is a limit to the difficulty of reducing the overall density of the polystyrene-based particles to the target level.

[0149] Further, the content of the hydrocarbon oil may be 10% by weight or more and 30% by weight or less, or 14% by weight or more and 21% by weight or less with respect to the total weight of the monomer composition. When the content of the hydrocarbon oil is excessively reduced, the amount of the hydrocarbon oil impregnated into the particles decreases, making it difficult to sufficiently reduce the density of the polystyrene-based particles. Also, when the content of the hydrocarbon oil is excessively increased, it becomes difficult for the polystyrene-based particles to have a spherical shape, and the uniformity of the particles produced decreases.

[0150] More specifically, the suspension polymerization reaction of the monomer composition can include a step of mixing the monomer composition with an aqueous dispersion and applying a shear force to homogenize the monomer composition into droplet form in the aqueous dispersion; and a step of subjecting the homogenized monomer composition to suspension polymerization at a stirring speed of 300 rpm or more and 1000 rpm or less.

[0151] In the step of suspension polymerization of the homogenized monomer composition at a stirring speed of 300 rpm or more and 1000 rpm or less, or 400 rpm or more and 800 rpm or less, while forming internal voids by phase separation of polystyrene and hydrocarbon oil during the formation of the particle structure of polystyrene and hydrocarbon oil, it is possible to lower the density of the microcarrier for cell culture while still being able to produce a microcarrier for cell culture with a high ratio of spherical particles.

[0152] In the step of suspension polymerization of the homogenized monomer composition at a stirring speed of 300 rpm or more and 1000 rpm or less, or 400 rpm or more and 800 rpm or less, although the examples of the suspension polymerization conditions are not greatly limited, for example, it can be carried out at a temperature of 50°C or more and 100°C for 3 hours or more and 18 hours or less.

[0153] On the other hand, the method for producing the microcarrier for cell culture can further include a step of washing and a step of drying the suspension polymerization reaction product after the step of performing the suspension polymerization reaction.

[0154] Specifically, the step of washing the suspension polymerization reaction product can include the steps of passing the suspension polymerization reaction product through a sieve of 10 μm or more and 100 μm or less, and then stirring at room temperature 5 - 7 times in 100% ethanol and / or distilled water at a high temperature of 50°C or more and 80°C or less.

[0155] The step of drying the suspension polymerization reaction product includes the step of putting it into a vacuum oven and drying it under vacuum at room temperature. However, it is not limited to this, and drying methods known to be commonly used can be used without other restrictions.

[0156] On the other hand, the method for producing the microcarrier for cell culture of the other embodiment can further include a step of applying a primer polymer layer, a cell adhesion induction layer, or a combined layer of these on the surface of the suspension polymerization reaction product after the step of performing the suspension polymerization reaction.

[0157] The content regarding the primer polymer layer and the cell adhesion induction layer includes all the content described above in the said embodiment.

[0158] According to another embodiment of the invention, a cell culture composition can be provided which includes cells and the microcarrier for cell culture of the said embodiment. The content regarding the microcarrier for cell culture includes all the content described above in the said embodiment.

[0159] The said cells are adherent animal cells, and examples thereof are not greatly limited, 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 (such as HEK293, BHK21, MDCK, vero cells, etc.), or a mixture of two or more of these may also be used.

[0160] The density of the said cells may be 1.02 g / cm 3 or more and less than 1.1 g / cm 3

[0161] Also, the density difference between the microcarrier for cell culture and the said cells may be 0.02 g / cm 3 or more and 0.20 g / cm 3 or less. Even if the density difference between the microcarrier for cell culture and the said cells is 0.02 g / cm 3 or more and 0.20 g / cm 3 or less and the density difference does not become large, the cells and the microcarrier can be easily separated by including magnetic particles.

[0162] ​The cell culture composition can further contain a culture medium solution. The culture medium solution can contain various additives for sufficiently satisfying the environmental conditions such as nutrients, pH, temperature, and osmotic pressure close to the conditions of a living body based on body fluids such as blood plasma and lymph fluid, and various substances widely known in the technical field related to cell culture can be used without limitation.

[0163] As an example, the microcarrier for cell culture of the above-described embodiment has a density smaller than that of the culture medium solution, is injected into the culture medium solution, and floats inside the culture medium solution under stirring conditions. Thereafter, as the number of cells attached to the surface of the low-density microcarrier increases, the density of the microcarrier to which cells are attached (hereinafter referred to as "microcarrier-cell conjugate") gradually increases and gradually sinks in the culture medium solution.

[0164] Therefore, after subjecting the microcarrier to which cells are attached (microcarrier-cell conjugate) to a cell detachment enzyme addition treatment, it can be separated by centrifugation, and cells cultured by separating cells from the microcarrier-cell conjugate can be easily secured.

Advantages of the Invention

[0165] According to the present invention, it is possible to provide a microcarrier for cell culture that has a low density so as to enable cell culture and can be more easily separated than when separating and recovering the microcarrier and cells after cell culture, a method for producing the same, and a cell culture method using the same.

Brief Description of the Drawings

[0166]

Figure 1

Figure 2

Figure 3

Figure 4

Mode for Carrying Out the Invention

[0167] The invention will be described in more detail with the following examples. However, the following examples are merely illustrative of the invention, and the content of the invention is not limited by the following examples.

Examples

[0168] <Production Example: Production of Magnetic Particles> 130 ml of distilled water was added with Fe 3+ (Iron(III) chloride hexahydrate, 5.41 g) and Fe 2+ salt (Iron(II) chloride tetrahydrate, 1.98 g), and stirred at 400 - 600 rpm to produce an aqueous solution of magnetic particles.

[0169] 2 g of oleic acid and 13 ml of acetone were mixed and then added to the aqueous solution of magnetic particles.

[0170] After stirring for 30 minutes, 15 ml of aqueous ammonium hydroxide solution (solid content 25 wt%) was gradually added.

[0171] The temperature was raised to 80°C - 90°C and stirred for 1 hour or more, then the temperature was lowered to 65°C - 75°C, and 1 N HCl was added until the pH of the reactant reached 2.

[0172] After washing with distilled water until the pH of the final reactant reached 7, it was dried at room temperature for 2 days or more to produce surface-treated magnetic particles (particle diameter: 500 - 600 nm, particle density: 5.15 g / cm 3 ). The diameter of the magnetic particles was measured using the dynamic light scattering method (DLS).

[0173] The density (true density) of the magnetic particles was measured using a pycnometer.

[0174] <Example: Production of Microcarriers for Cell Culture> Example 1 (1) Production of the Dispersive Phase A mixture of styrene monomer (st), divinylbenzene (DVB) as a crosslinking agent, the magnetic particles of the above Production Example, and low-density oil (Isopar M, density 0.791 g / cm 3 ) was stirred in a 100 ml vial. Then, benzoyl peroxide (BPO, 2.1 wt%) and tert-butyl peroxybenzoate (t-BP, 0.3 wt%) as thermal initiators were additionally added to the vial and stirred at room temperature for 5 to 10 minutes. The content ratios between the components are as described in Table 1.

[0175] (2) Production of the Continuous Phase 6 g of PVA with a weight average molecular weight in the range of 85,000 to 125,000 and a hydrolysis rate of 87 to 89% was dissolved in 600 g of distilled water. The detailed content is as shown in Table 1.

[0176] (3) Particle Production by Suspension Polymerization 600 g of a 1% PVA aqueous solution was mixed with the dispersive phase and stirred in an oil bath until a homogeneous dispersion was obtained. Specifically, while stirring at 400 to 500 rpm at room temperature, the temperature of the oil bath was gradually increased, and suspension polymerization was carried out under the conditions of a temperature of 80°C to 90°C and a speed of 400 to 600 rpm. The polymerization was carried out under a nitrogen purge.

[0177] (4) Particle Acquisition After 4 hours of reaction, the particles produced were collected through a 45-μm sieve, washed three times with ethanol and three times with hot distilled water at 70°C or higher and 80°C or lower, and then dried in an 80°C oven.

[0178] (5) Particle surface treatment After polymerization, the particles dried at room temperature were immersed in a tris buffer (pH 8.0) in which dopamine hydrochloride was dissolved at 1 mg / mL, and stirred at room temperature for 1 hour or more to introduce a polydopamine layer on the particle surface. After the reaction, the particles were collected through a 45-μm sieve, washed three times with ethanol, and then dried in an 80°C oven.

[0179] Examples 2 to 4 Particles were obtained through the same process as in Example 1, except that the compositions of the dispersed phase and the continuous phase were adjusted as shown in Table 1 below.

[0180] Comparative Example 1 Particles were obtained through the same process as in Example 1, except that magnetic particles were not added.

[0181] Comparative Examples 2 and 3 Particles were obtained through the same process as in Example 1, except that the compositions of the dispersed phase and the continuous phase were adjusted as shown in Table 1 below and low-density oil was not added.

[0182] Reference Example 1 Particles were obtained through the same process as in Example 1, except that magnetic particles whose surfaces were not hydrophobically modified with oleic acid were added instead of the magnetic particles in the production example.

[0183]

Table 1

[0184] <Experimental Example: Physical Property Measurement of Microcarriers for Cell Culture> For the microcarriers for cell culture obtained from the above Examples, Comparative Examples, and Reference Examples, the physical properties were measured by the following method, and the results are shown in Table 2.

[0185] 1. Particle size For the microcarriers for cell culture obtained from the above Examples, Comparative Examples, and Reference Examples, 100 particle diameters were measured through an optical microscope, and the arithmetic mean value thereof was determined.

[0186] The SEM image of the microcarriers for cell culture obtained from Example 1 is shown in Figure 2.

[0187] 2. Particle internal structure and void diameter (1) Particle internal structure For the microcarriers for cell culture obtained from Example 1, the particle internal structure was confirmed through SEM. Specifically, after embedding the particles in epoxy, a cross-section was produced through ion milling, and then the shape of the particle cross-section was confirmed through SEM, which is shown in Figure 3.

[0188] (2) Void diameter After embedding the microcarriers for cell culture obtained from the above Examples and Comparative Examples in epoxy, a cross-section was produced through ion milling, and then the shape of the particle cross-section was confirmed by SEM to determine the minimum diameter and the maximum diameter of the internal voids of the particles.

[0189] 3. Apparent density For the microcarriers for cell culture obtained from the above Examples, Comparative Examples, and Reference Examples, a sample under the condition that the drying process was completed was prepared, and the sample was placed under normal temperature (25 °C) and normal pressure (1 atm) conditions with an ethanol aqueous solution having a density of 0.95 g / cm 3 , 0.97 g / cm 3 , 0.98 g / cm 3 or 0.995 g / cm 3 and an ethanol aqueous solution having a density of 1 g / cm 3Added to distilled water (DIW) respectively, and it was confirmed whether the particles floated or settled, and the apparent density was evaluated under the following criteria.

[0190] 1) Floating in an ethanol aqueous solution with a density of 0.95 g / cm 3 : less than 0.95 g / cm 3 2) Settling in distilled water (DIW) with a density of 1 g / cm 3 : greater than 1 g / cm 3 3) Settling in an ethanol aqueous solution with a density of 0.95 g / cm 3 and floating in distilled water (DIW) with a density of 1 g / cm 3 : 0.95 g / cm 3 or more and less than 1 g / cm 3 4) Settling in an ethanol aqueous solution with a density of 0.95 g / cm 3 and floating in an ethanol aqueous solution with a density of 0.97 g / cm 3 : 0.95 g / cm 3 or more and less than 0.97 g / cm 3 5) Settling in an ethanol aqueous solution with a density of 0.98 g / cm 3 and floating in an ethanol aqueous solution with a density of 0.995 g / cm 3 : 0.98 g / cm 3 or more and less than 0.995 g / cm 3

[0191] 4. Specific surface area From the obtained apparent density, the specific surface area of the microcarrier for cell culture was calculated using the following formula.

[0192] [Formula] Specific surface area of microcarrier (cm 2 / g) = 3 / (r × ρ)

[0193] In the above formula, r means the average radius of the microcarrier, and ρ means the apparent density of the microcarrier.

[0194] 5. Cell culture efficiency ​​​​​Fill a 100 mL bioreactor with a culture solution containing mesenchymal stem cells (density: 1.05 g / cm 3 ), add 1 g to 1.5 g of the microcarriers for cell culture obtained from the above examples and comparative examples, and stir at 25 rpm. At this time, the temperature of the culture solution was maintained at 37°C, and the cells were cultured for 5 days, and the cell culture efficiency of the microcarriers was evaluated according to the following criteria.

[0195] Above: The cell culture efficiency during cell culture is 80% or more Below: The cell culture efficiency during cell culture is less than 80%

[0196] 6. Magnetic separation efficiency A strong magnet was brought into contact with one side of a vial containing a solution in which the microcarriers for cell culture obtained from the above examples, comparative examples, and reference examples were dispersed, and it was visually confirmed whether the microcarriers for cell culture moved or not.

[0197] When the microcarriers for cell culture moved in the direction of the surface where the magnet was in contact by 90% or more, it was evaluated as "excellent", and when it moved by 90% or less, it was evaluated as "poor".

[0198] 7. Void component analysis For the microcarriers for cell culture obtained from the above examples, comparative examples, and reference examples, in order to analyze the components contained in the internal voids, the microcarriers for cell culture were cryogenically pulverized and dissolved in chloroform to extract unreacted residual compounds, and the detailed components were qualitatively and quantitatively analyzed through GC / FID (gas chromatography - flame ionization detector).

[0199] Specifically, standard samples of styrene (1 / 4000 mg / mL to 1 mg / mL), divinylbenzene (1 / 10000 mg / mL to 1 mg / mL), and Isopar M (1 / 400 mg / mL to 10 mg / mL) were prepared at different concentrations in a solvent mixture of chloroform and methanol at a volume ratio of 1:2. 1 μL of the standard samples was injected into a GC / FID instrument to create a calibration curve. 1 μL of the sample in Table 1 was injected, and the content was calculated using the calibration curve. GC / FID measurements were performed using a column on Rtx (styrene, Isopar M) with an inner diameter of 0.53 mm, a length of 30 m, and a film thickness of 5 μm or wax (divinylbenzene). The initial oven temperature was 50 °C, and the temperature was increased to 250 °C at a rate of 10 °C / min. The mobile phase gas used was helium gas at 15 mL / min.

[0200] At this time, when oil or components derived from it were detected, it was indicated as "O", and when oil or components derived from it were not detected, it was indicated as "×".

[0201]

Table 2

[0202] As shown in Table 2 above, the microcarriers for cell culture in the examples satisfied a low density of less than 1.05 g / cm³, and it was confirmed that carrier culture was effectively carried out under cell culture conditions and the magnetic separation efficiency was also excellent. 3 On the contrary, the microcarriers for cell culture in Comparative Example 1 did not contain magnetic particles and had a problem of reduced magnetic separation efficiency.

[0203] On the other hand, the microcarriers for cell culture in Comparative Example 2 had a particle density exceeding 1.05 g / cm³, and there was a problem that smooth stirring could not be performed in the incubator due to the density difference with the cells, resulting in a decrease in cell adhesion.

[0204] Also, the microcarriers for cell culture in Comparative Example 3 had a particle density of 1.05 g / cm³ 3 exceeding, and there was a problem that smooth stirring could not be performed in the incubator due to the density difference with the cells, resulting in a decrease in cell adhesion.

[0205] Also, the microcarriers for cell culture in Comparative Example 3 had a particle density of 1.05 g / cm³3 There was a problem that the cell culture efficiency decreased to such an extent that smooth stirring could not be performed in the incubator due to the density difference with the cells, resulting in a decrease in cell adhesion and making cell culture impossible.

[0206] In addition, since the microcarrier for cell culture in Reference Example 1 contains magnetic particles that are not surface-treated with a hydrophobic functional group, it was confirmed that the water-dispersed magnetic particles were not evenly dispersed in the dispersion phase containing the styrene monomer, and the water-dispersed magnetic particles aggregated with each other during polymerization. In addition, in the case of the particles produced in this way, there was a problem that the particle size distribution was large and the magnetic separation efficiency decreased.

Claims

1. comprising polystyrene-based particles containing at least one or more of hydrocarbon oils having 12 or more carbon atoms, or voids derived therefrom; and magnetic particles; the apparent density of the microcarriers being 0.95 g / cm3 or more and 1.05 g / cm3 or less, the polystyrene-based particles comprising the suspension polymerization reaction product of a monomer composition containing a hydrocarbon oil having 12 or more carbon atoms, magnetic particles, and a styrene monomer, the hydrocarbon oil being contained in an amount of 10% by weight or more and 30% by weight or less based on the total weight (100% by weight) of the dispersion phase composition containing the styrene monomer, a microcarrier for cell culture.

2. The microcarrier for cell culture according to claim 1, wherein the magnetic particles include magnetic particles surface-treated with a hydrophobic functional group.

3. the magnetic particles include a hydrophobic ligand containing a hydrophobic functional group on the magnetic particle surface, the hydrophobic ligand including one or more ligands selected from the group consisting of fatty acids having 2 to 20 carbon atoms or derivatives thereof, and fatty acid amines having 2 to 20 carbon atoms or derivatives thereof, the microcarrier for cell culture according to claim 1.

4. The microcarrier for cell culture according to claim 1, wherein the magnetic particles include one or more metals selected from the group consisting of gold (Au), silver (Ag), cobalt (Co), copper (Cu), iron (Fe), chromium (Cr), nickel (Ni), palladium (Pd), platinum (Pt), and tin (Sn), or oxides thereof.

5. The microcarrier for cell culture according to claim 1, wherein the magnetic particles are present in a dispersed state inside at least one or more of the hydrocarbon oils having 12 or more carbon atoms, or voids derived therefrom.

6. the average diameter of the microcarriers being 50 μm or more and 400 μm or less, The specific surface area of the microcarrier is 200 cm ２ / g or more and 1000 cm ２ / g or less, the microcarrier for cell culture according to claim 1.

7. The diameter of the voids is 0.1 μm or more and 5 μm or less, the microcarrier for cell culture according to claim 1.

8. The density of the hydrocarbon oil is 0.75 g / cm ３ or more and 0.80 g / cm ３ or less, the microcarrier for cell culture according to claim 1.

9. The density of the magnetic particles is 5 g / cm ３ or more and 6 g / cm ３ or less, the microcarrier for cell culture according to claim 1.

10. The hydrocarbon oil contains a linear or branched saturated hydrocarbon compound having 12 or more and 50 or less carbon atoms, the microcarrier for cell culture according to claim 1.

11. The monomer composition is based on 100 parts by weight of a hydrocarbon oil having 12 or more carbon atoms, containing 0.01 part by weight or more and 5 parts by weight or less of magnetic particles, the microcarrier for cell culture according to claim 1.

12. The monomer composition is based on 1 part by weight of a styrene monomer, containing more than 0.033 part by weight and less than 3 parts by weight of an ethylenically unsaturated crosslinking agent, the microcarrier for cell culture according to claim 1.

13. The surface of the polystyrene-based particles further contains a primer polymer layer, a cell adhesion induction layer, or a combination layer thereof, the microcarrier for cell culture according to claim 1.

14. including the step of performing a suspension polymerization reaction of a monomer composition containing magnetic particles and a styrene monomer in the presence of a hydrocarbon oil having 12 or more carbon atoms, The apparent density of the microcarrier is 0.95 g / cm3 or more and 1.05 g / cm3 or less, The method for producing a microcarrier for cell culture, wherein the hydrocarbon oil is contained in an amount of 10% by weight or more and 30% by weight or less based on the total weight (100% by weight) of the dispersion phase composition containing the styrene monomer.

15. The method for producing a microcarrier for cell culture according to claim 14, wherein the magnetic particles include magnetic particles surface-treated with a hydrophobic functional group.

16. A cell culture composition comprising cells and the microcarrier for cell culture according to any one of claims 1 to 13.

17. The cell culture composition according to claim 16, wherein the cells contain one or more compounds selected from the group consisting of fibroblasts, chondrocytes, mesenchymal stem cells, CHO, HEK 293, Vero cells, BHK21, and MDCK.

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