Polyacrylic microparticle, and particle composite, cell culture composition, and method for preparing cell therapeutic agent using same

Abstract

The present invention relates to a polyacrylic microparticle suitable for cell culture and cell differentiation, a particle composite, and a cell culture composition and a method for preparing a cell therapeutic agent using same.

Description

Polyacrylic microparticles, particle composites using the same, cell culture compositions, and methods for manufacturing cell therapeutic agents

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

[0002] This application claims the benefit of priority based on Korean Patent Application No. 10-2025-0003482 filed January 9, 2025 and Korean Patent Application No. 10-2025-0022483 filed February 20, 2025, and all contents disclosed in the documents of said Korean patent applications are incorporated herein as part of this specification.

[0003] The present invention relates to polyacrylic microparticles, a particle composite using the same, a cell culture composition, and a method for manufacturing a cell therapeutic agent.

[0004]

[0005] As the fields of biopharmaceuticals and regenerative medicine expand, there is a growing demand for mass cell culture and differentiation technologies capable of efficiently producing cells, tissues, and microorganisms.

[0006]

[0007] Meanwhile, existing anticancer cell therapies have the disadvantage of requiring a long time and cost to proliferate cells because they use T cells (autologous cells) derived from each patient, and the quality suitable for treatment cannot be guaranteed since typical cancer patients have impaired immune cell function.

[0008] To address this, there have been attempts to differentiate and use stem cells, such as allogeneic induced pluripotent stem cells (iPSCs). This differentiation requires stimulation using proteins; however, with conventional adsorption-type fixation surfaces, protein concentration control is impossible, making stable fixation impossible. Furthermore, since an excessive amount of protein must be injected, economic efficiency is low. Additionally, when protein stimulation is applied on a flat plate (2D), there were limitations in mass production and automation.

[0009] Accordingly, there is a need for research on microparticles suitable for cell culture and differentiation that possess appropriate density and hydrophilicity to achieve excellent dispersibility within the culture medium while being easy to modify on the surface. Furthermore, there is a need to develop a technology that enables stable protein immobilization and control of protein binding concentration, thereby ensuring excellent differentiation efficiency.

[0010]

[0011] The present invention relates to providing polyacrylic microparticles suitable for cell culture and cell differentiation, which have appropriate density and hydrophilicity to achieve excellent dispersibility in a culture medium while being easy to modify on the surface.

[0012] In addition, the present invention relates to a technology for providing a particle complex capable of controlling the optimal concentration of proteins and ensuring the stability of a differentiation platform by fixing proteins necessary for cell differentiation through covalent bonds.

[0013] In addition, the present invention relates to a cell culture composition using the polyacrylic microparticles or particle complexes.

[0014] In addition, the present invention relates to a method for manufacturing a cell therapeutic agent, comprising the steps of: culturing the cell culture composition; and removing polyacrylic microparticles or particle complexes from the culture product.

[0015] To solve the above problem, the present specification provides polyacrylic microparticles comprising a (meth)acrylate copolymer containing two or more different repeating units, wherein the epoxy content is 50 μmol / g or more and 2000 μmol / g or less, and the acrylate copolymer containing two or more different repeating units comprises a (meth)acrylate repeating unit containing a reactive functional group and a (meth)acrylate repeating unit containing an aliphatic functional group.

[0016]

[0017] The present specification also provides a particle complex comprising: the polyacrylic microparticles; and a protein immobilized on the surface of the particles.

[0018]

[0019] In addition to the above, a cell culture composition comprising a cell and the polyacrylic microparticles or particle complex is provided.

[0020]

[0021] The present specification also provides a method for manufacturing a cell therapeutic agent, comprising the steps of: culturing a cell culture composition; and removing polyacrylic microparticles or particle complexes from the culture product.

[0022]

[0023] The following describes in more detail the polyacrylic microparticles according to specific embodiments of the invention, the particle composites using the same, the cell culture composition, and the method for manufacturing a cell therapeutic agent.

[0024]

[0025] Unless explicitly stated otherwise in this specification, technical terms are used merely to refer to specific embodiments and are not intended to limit the invention.

[0026] The singular forms used in this specification include plural forms unless the phrases clearly indicate otherwise.

[0027] As used in this specification, the meaning of 'includes' specifies certain characteristics, regions, integers, steps, actions, elements, and / or components, and does not exclude the existence or addition of other specific characteristics, regions, integers, steps, actions, elements, components, and / or groups.

[0028] Also, in this specification, terms including ordinal numbers such as 'first' and 'second' are used for the purpose of distinguishing one component from another and are not limited by said ordinal numbers. For example, within the scope of the present invention, the first component may also be named the second component, and similarly, the second component may be named the first component.

[0029] In this specification, the term "substitution" means that another functional group is bonded in place of a hydrogen atom in a compound, and the substitution site is not limited to the site where the hydrogen atom is substituted, that is, the site where the substituent can be substituted, and in the case of two or more substitutions, the two or more substituents may be the same or different from each other.

[0030] In this specification, the term “substituted or unsubstituted” means substituted or unsubstituted with one or more substituents selected from the group consisting of deuterium; halogen group; cyano group; nitro group; hydroxyl group; carbonyl group; ester group; imide group; amide group; primary amino group; carboxyl group; sulfonic acid group; sulfonamide group; phosphine oxide group; alkoxy group; aryloxy group; alkylthioxy group; arylthioxy group; alkyl sulfoxy group; aryl sulfoxy group; silyl group; boron group; alkyl group; cycloalkyl group; alkenyl group; aryl group; aralkyl group; aralkenyl group; alkylaryl group; alkoxysilylalkyl group; arylphosphine group; or heterocyclic groups comprising one or more of N, O, and S atoms, or substituted or unsubstituted with two or more of the exemplified substituents connected. For example, “substituents connected with two or more substituents” may be a biphenyl group. That is, the biphenyl group can be an aryl group, or it can be interpreted as a substituent consisting of two connected phenyl groups.

[0031] In this specification, , or means a bond connected to another substituent, and a direct bond means a case where there is no separate atom in the part indicated by L.

[0032] In this specification, the alkyl group may be a straight chain or a branched chain, and the number of carbon atoms is not particularly limited. According to another embodiment, the number of carbon atoms of the alkyl group is 1 to 6. Specific examples of alkyl groups include methyl, ethyl, propyl, n-propyl, isopropyl, butyl, n-butyl, isobutyl, tert-butyl, sec-butyl, 1-methyl-butyl, 1-ethyl-butyl, pentyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, n-hexyl, 1-methylpentyl, 2-methylpentyl, 4-methyl-2-pentyl, 3,3-dimethylbutyl, 2-ethylbutyl, heptyl, n-heptyl, 1-methylhexyl, cyclopentylmethyl, cyclohectylmethyl, octyl, n-octyl, tert-octyl, 1-methylheptyl, 2-ethylhexyl, 2-propylpentyl, n-nonyl, 2,2-dimethylheptyl, 1-ethyl-propyl, 1,1-dimethyl-propyl, isohexyl, Examples include 2-methylpentyl, 4-methylhexyl, 5-methylhexyl, etc., but are not limited to these.

[0033] In this specification, the cycloalkyl group is a monovalent functional group derived from a cycloalkane, and may be monocyclic or polycyclic, and is not particularly limited, but has 3 to 20 carbon atoms. According to another embodiment, the number of carbon atoms of the cycloalkyl group is 3 to 10. Specifically, cyclopropyl, cyclobutyl, cyclopentyl, 3-methylcyclopentyl, 2,3-dimethylcyclopentyl, cyclohexyl, 3-methylcyclohexyl, 4-methylcyclohexyl, 2,3-dimethylcyclohexyl, 3,4,5-trimethylcyclohexyl, 4-tert-butylcyclohexyl, cycloheptyl, cyclooctyl, bicyclo[2,2,1]heptyl, etc., are included but are not limited thereto. The cycloalkyl group may be substituted or unsubstituted, and in the case of substitution, examples of substituents are as described above.

[0034] In the present specification, a heterocycloalkyl group refers to a cycloalkyl group comprising one or more non-carbon atoms or heteroatoms, and specifically, the heteroatoms may comprise one or more atoms selected from the group consisting of O, N, Se, and S, etc.

[0035] In this specification, (meth)acrylate means including both acrylate and methacrylate.

[0036] The present invention will be described in more detail below.

[0037]

[0038] According to one embodiment of the invention, polyacrylic microparticles may be provided, comprising a (meth)acrylate copolymer containing two or more different repeating units, wherein the epoxy content is 50 μmol / g or more and 2000 μmol / g or less, and the acrylate copolymer containing two or more different repeating units comprises a (meth)acrylate repeating unit containing a reactive functional group and a (meth)acrylate repeating unit containing an aliphatic functional group.

[0039] The inventors confirmed that in the case of the polyacrylic microparticles of the above-mentioned embodiment, as the epoxy content satisfies the above range, they possess appropriate density and hydrophilicity, thereby achieving excellent dispersibility in a culture medium, while also being easy to modify the surface, making them suitable for cell culture and cell differentiation, and thus completed the invention.

[0040]

[0041] As the acrylate copolymer containing two or more different repeating units simultaneously contains a (meth)acrylate repeating unit containing a reactive functional group and a (meth)acrylate repeating unit containing an aliphatic functional group, the finally produced polyacrylate microparticles have appropriate density and hydrophilicity, thereby achieving excellent dispersibility in a culture medium, while also being easy to modify the surface, making them suitable for cell culture and cell differentiation.

[0042]

[0043] Meanwhile, the (meth)acrylate repeating unit containing the above-mentioned reactive functional group may include a repeating unit derived from a compound represented by the following chemical formula 1.

[0044] [Chemical Formula 1]

[0045]

[0046] In the above chemical formula 1, L1 and L2 are each independently alkylene groups with 1 or more carbon atoms, R1 is a reactive functional group capable of ring-opening reactions, and n is an integer greater than or equal to 0.

[0047] As the acrylate copolymer containing two or more different repeating units includes a (meth)acrylate repeating unit containing the reactive functional group, ligand attachment through chemical reaction is facilitated and dispersibility in a cell culture reactor or medium can be improved.

[0048] Specifically, as the (meth)acrylate repeating unit containing the reactive functional group comprises a repeating unit derived from a compound represented by Chemical Formula 1, ligand attachment through chemical reaction is facilitated and dispersibility in a cell culture reactor or medium can be improved.

[0049]

[0050] In addition, conventional microparticles had a problem in that the binding force between particles and ligands was weak because they relied on physical adsorption rather than covalent bonding due to the absence of functional groups for ligand attachment.

[0051]

[0052] Accordingly, the inventors confirmed that the acrylate copolymer containing two or more different repeating units comprises a (meth)acrylate repeating unit containing a reactive functional group, and that the (meth)acrylate repeating unit containing a reactive functional group comprises a repeating unit derived from a compound represented by Chemical Formula 1, thereby providing a reaction site on the surface of the microparticle where a ligand can be fixed by chemical bonding, which can improve the ligand attachment efficiency and attachment strength.

[0053]

[0054] The above-mentioned reactive functional group capable of ring-opening reactions may refer to a reactive functional group capable of ring-opening reactions such as hydrolyzed ring opening.

[0055] Specifically, the reactive functional group capable of the ring-opening reaction may include a heterocycloalkyl group.

[0056] The above heterocycloalkyl group may refer to a cycloalkyl group comprising one or more non-carbon atoms or heteroatoms. The above heteroatoms may include one or more atoms selected from the group consisting of O, N, Se, and S, and preferably may include O.

[0057] The above heterocycloalkyl group is not significantly limited, but may include, for example, an epoxy group.

[0058]

[0059] Meanwhile, the (meth)acrylate repeating unit containing the above-mentioned aliphatic functional group may include a (meth)acrylate repeating unit containing an aliphatic functional group having 6 or more carbon atoms, 8 or more carbon atoms, 6 or more and 15 or fewer carbon atoms, 6 or more and 12 or fewer carbon atoms, 8 or more and 15 or fewer carbon atoms, or 8 or more and 12 or fewer carbon atoms.

[0060] When using (meth)acrylate repeating units containing aliphatic functional groups with fewer than 6 carbon atoms, excessive polymerization occurs, resulting in a density higher than necessary, which may lead to reduced dispersibility in cell culture reactors or media, or reduced differentiation and culture efficiency during differentiation and culture.

[0061]

[0062] Specifically, the (meth)acrylate repeating unit containing the aliphatic functional group may include a repeating unit derived from a compound represented by the following chemical formula 2.

[0063] [Chemical Formula 2]

[0064]

[0065] In the above chemical formula 2, R2 is an alkyl group having 6 or more carbon atoms.

[0066] Specifically, in the above chemical formula 2, R2 may be an alkyl group having 6 or more carbon atoms, 8 or more carbon atoms, 6 or more and 15 or fewer carbon atoms, 6 or more and 12 or fewer carbon atoms, 8 or more and 15 or fewer carbon atoms, or 8 or more and 12 or fewer carbon atoms.

[0067] In the case of the microparticles of the above embodiment, the acrylate copolymer containing two or more different repeating units includes (meth)acrylate repeating units containing aliphatic functional groups, so the density of the particles can be controlled, thereby improving dispersibility in a cell culture reactor or medium, which increases contact with cells during differentiation and culture, and thus, improvement in differentiation and culture efficiency can be expected.

[0068] In the case where R2 in the above chemical formula 2 is an alkyl group with fewer than 6 carbon atoms, excessive polymerization occurs, causing the density to become higher than necessary, which may reduce dispersibility in cell culture reactors or media, or reduce differentiation and culture efficiency during differentiation and culture.

[0069]

[0070] Specifically, the (meth)acrylate repeating unit containing the aliphatic functional group includes a repeating unit derived from the compound represented by Chemical Formula 2, thereby allowing for control of particle density and improved dispersibility in cell culture reactors or media, which increases contact with cells during differentiation and culture, and thus allows for the expectation of improved differentiation and culture efficiency.

[0071] The compound represented by the above chemical formula 2 may include, for example, 2-ethylhexyl methacrylate or dodecyl methacrylate.

[0072]

[0073] In addition, the acrylate copolymer containing two or more different repeating units may contain 40 parts by weight or more and 750 parts by weight or less of the aliphatic functional group (meth)acrylate repeating unit with respect to 100 parts by weight of the reactive functional group (meth)acrylate repeating unit.

[0074] Specifically, the acrylate copolymer comprising two or more different repeating units comprises, with respect to 100 parts by weight of the (meth)acrylate repeating unit comprising the reactive functional group, the (meth)acrylate repeating unit comprising the aliphatic functional group in an amount of 40 parts by weight or more, 100 parts by weight or more, 101 parts by weight or more, 110 parts by weight or more, 120 parts by weight or more, 140 parts by weight or more, 750 parts by weight or less, 500 parts by weight or less, 450 parts by weight or less, 400 parts by weight or less, 350 parts by weight or less, 40 parts by weight or more and 750 parts by weight or less, 40 parts by weight or more and 500 parts by weight or less, 40 parts by weight or more and 450 parts by weight or less, 40 parts by weight or more and 400 parts by weight or less, 40 parts by weight or more and 350 parts by weight or less, 100 parts by weight or more and 750 parts by weight or less, 100 parts by weight or more and 500 parts by weight or less, 100 parts by weight or more and 450 parts by weight or less, 100 parts by weight or more and 400 parts by weight or less, 100 parts by weight or more and 350 parts by weight or less, 101 parts by weight or more and 750 parts by weight or less, 101 parts by weight or more and 500 parts by weight or less, 101 parts by weight or more and 450 parts by weight or less, 101 parts by weight or more and 400 parts by weight or less, 101 parts by weight or more and 350 parts by weight or less, 110 parts by weight or more and 750 parts by weight or less, 110 parts by weight or more and 500 parts by weight or less, 110 parts by weight or more and 450 parts by weight or less, 110 parts by weight or more and 400 parts by weight or less, 110 parts by weight or more and 350 parts by weight or less, 120 parts by weight or more and 750 parts by weight or less, 120 parts by weight or more and 500 parts by weight or less, 120 parts by weight or more and 450 parts by weight or less, 120 parts by weight or more It may contain 400 parts by weight or less, 120 parts by weight or more and 350 parts by weight or less, 140 parts by weight or more and 750 parts by weight or less, 140 parts by weight or more and 500 parts by weight or less, 140 parts by weight or more and 450 parts by weight or less, 140 parts by weight or more and 400 parts by weight or less, and 140 parts by weight or more and 350 parts by weight or less.

[0075] If the (meth)acrylate repeating unit containing the aliphatic functional group is included in an excessively small amount for every 100 parts by weight of the (meth)acrylate repeating unit containing the reactive functional group, the particle density may increase, which may reduce dispersibility in the culture medium, and if the amount is included in an excessive amount, problems may arise such as reduced particle strength and increased hydrophobicity.

[0076]

[0077] In addition, the acrylate copolymer containing the above two or more different repeating units may contain 10 parts by weight or more and less than 50 parts by weight of (meth)acrylate repeating units containing the reactive functional group, based on 100 parts by weight of the total repeating units.

[0078] Specifically, the acrylate copolymer comprising two or more different repeating units comprises, with respect to 100 parts by weight of total repeating units, (meth)acrylate repeating units containing reactive functional groups in an amount of 10 parts by weight or more, 15 parts by weight or more, 20 parts by weight or more, less than 50 parts by weight, 49 parts by weight or less, 45 parts by weight or less, 42 parts by weight or less, 10 parts by weight or more and less than 50 parts by weight, 10 parts by weight or more and 49 parts by weight or less, 10 parts by weight or more and 45 parts by weight or less, 10 parts by weight or more and 42 parts by weight or less, 15 parts by weight or more and less than 50 parts by weight, 15 parts by weight or more and 49 parts by weight or less, 15 parts by weight or more and 45 parts by weight or less, 15 parts by weight or more and 42 parts by weight or less, 20 parts by weight or more and less than 50 parts by weight, 20 parts by weight or more and 49 parts by weight or less, 20 parts by weight or more and 45 parts by weight or less, and 20 parts by weight or more and 42 parts by weight or less. It can be included.

[0079] If the acrylate copolymer containing two or more different repeating units contains an excessively small amount of (meth)acrylate repeating units containing the reactive functional group per 100 parts by weight of the total repeating units, surface modification becomes difficult and ligand attachment may be impossible, and if it contains an excessive amount, problems may arise such as increased particle density and reduced strength.

[0080]

[0081] Specifically, the acrylate copolymer containing two or more different repeating units may contain at least 20 parts by weight and less than 75 parts by weight of (meth)acrylate repeating units containing aliphatic functional groups, based on 100 parts by weight of total repeating units.

[0082] More specifically, the acrylate copolymer comprising two or more different repeating units comprises, with respect to 100 parts by weight of total repeating units, (meth)acrylate repeating units containing aliphatic functional groups in an amount of 20 parts by weight or more, 50 parts by weight or more, 51 parts by weight or more, 55 parts by weight or more, 58 parts by weight or more, less than 75 parts by weight, 74.9 parts by weight or less, 74 parts by weight or less, 73 parts by weight or less, 20 parts by weight or more and less than 75 parts by weight, 50 parts by weight or more and less than 75 parts by weight, 51 parts by weight or more and less than 75 parts by weight, 55 parts by weight or more and less than 75 parts by weight, 58 parts by weight or more and less than 75 parts by weight, 20 parts by weight or more and less than 74.9 parts by weight, 50 parts by weight or more and less than 74.9 parts by weight, 51 parts by weight or more and less than 74.9 parts by weight, 55 parts by weight or more and less than 74.9 parts by weight, and 58 parts by weight or more and 74.9 parts by weight. It may include 20 parts by weight or more and 74 parts by weight or less, 50 parts by weight or more and 74 parts by weight or less, 51 parts by weight or more and 74 parts by weight or less, 55 parts by weight or more and 74 parts by weight or less, 58 parts by weight or more and 74 parts by weight or less, 20 parts by weight or more and 73 parts by weight or less, 50 parts by weight or more and 73 parts by weight or less, 51 parts by weight or more and 73 parts by weight or less, 55 parts by weight or more and 73 parts by weight or less, and 58 parts by weight or more and 73 parts by weight or less.

[0083] If the acrylate copolymer containing two or more different repeating units contains an excessively small amount of (meth)acrylate repeating units containing aliphatic functional groups per 100 parts by weight of the total repeating units, the particle density may increase, which may reduce dispersibility in the culture medium, and if it contains an excessive amount, problems may arise such as reduced particle strength and increased hydrophobicity.

[0084]

[0085] Specifically, the compound represented by the above chemical formula 1 may include any one of the compounds represented by the following chemical formula 1-1 to the compound represented by chemical formula 1-3.

[0086] [Chemical Formula 1-1]

[0087]

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

[0089] R 11 is hydrogen or an alkyl group having 1 or more carbon atoms, and

[0090] [Chemical Formula 1-2]

[0091]

[0092] In the above chemical formula 1-2,

[0093] R 12 is hydrogen or an alkyl group having 1 or more carbon atoms, and

[0094] [Chemical Formula 1-3]

[0095]

[0096] In the above chemical formula 1-3,

[0097] R 13 It is hydrogen or an alkyl group with 1 or more carbon atoms.

[0098]

[0099] Meanwhile, the polyacrylic microparticles may include a reaction product of a monomer compound comprising a compound represented by Chemical Formula 1 and a compound represented by Chemical Formula 2, and a polyfunctional acrylic crosslinking agent.

[0100] In the above embodiment, the polyacrylic microparticles can secure structural stability of the particle complex by immobilizing proteins on the surface of the polyacrylic microparticles, which include the compound represented by Chemical Formula 1 and the compound represented by Chemical Formula 2 as monomer compounds, through physical bonding such as chemical covalent bonding or adsorption, and can achieve excellent differentiation performance as the appropriate concentration of the protein can be controlled.

[0101]

[0102] At this time, the polyfunctional acrylic crosslinking agent may include an acrylic compound with two or more functions. The polyfunctional acrylic crosslinking agent is not significantly limited as long as it is an acrylic compound with two or more functions, but may include one or more polyfunctional acrylic crosslinking agents selected from the group consisting of, for example, ethylene glycol dimethacrylate or trimethylolpropane trimethacrylate. Preferably, the polyfunctional acrylic crosslinking agent may include ethylene glycol dimethacrylate and trimethylolpropane trimethacrylate.

[0103]

[0104] Specifically, the polyacrylic microparticles may contain at least 110 parts by weight and no more than 1,000 parts by weight of the monomer compound per 100 parts by weight of the polyfunctional acrylic crosslinking agent.

[0105] More specifically, the polyacrylic microparticles comprise, with respect to 100 parts by weight of the polyfunctional acrylic crosslinking agent, 110 parts by weight or more, 200 parts by weight or more, 250 parts by weight or more, 300 parts by weight or more, 1000 parts by weight or less, 500 parts by weight or less, 400 parts by weight or less, 110 parts by weight or more and 1000 parts by weight or less, 200 parts by weight or more and 1000 parts by weight or less, 250 parts by weight or more and 1000 parts by weight or less, 300 parts by weight or more and 1000 parts by weight or less, 110 parts by weight or more and 500 parts by weight or less, 200 parts by weight or more and 500 parts by weight or less, 250 parts by weight or more and 500 parts by weight or less, 110 parts by weight or more and 400 parts by weight or less, 200 parts by weight or more and 400 parts by weight or less, 250 It may contain at least 400 parts by weight and at least 300 parts by weight and at least 400 parts by weight.

[0106] If the monomer compound is included in an excessively small amount per 100 parts by weight of the polyfunctional acrylic crosslinking agent, the particle density may become excessively high, and if it is included in an excessively large amount, a problem may occur in which the particle strength becomes weak.

[0107]

[0108] In addition, the polyacrylic microparticles may contain at least 25 parts by weight and less than 750 parts by weight of the compound represented by Formula 2 per 100 parts by weight of the polyfunctional acrylic crosslinking agent.

[0109] Specifically, the polyacrylic microparticles comprise, with respect to 100 parts by weight of the polyfunctional acrylic crosslinking agent, a compound represented by Formula 2 in an amount of 25 parts by weight or more, 100 parts by weight or more, 110 parts by weight or more, 150 parts by weight or more, 200 parts by weight or more, 230 parts by weight or more, less than 750 parts by weight, less than 500 parts by weight, less than 300 parts by weight, 299 parts by weight or less, 295 parts by weight or less, or 25 parts by weight or more and less than 750 parts by weight, 100 parts by weight or more and less than 750 parts by weight, 110 parts by weight or more and less than 750 parts by weight, 150 parts by weight or more and less than 750 parts by weight, 200 parts by weight or more and less than 750 parts by weight, 230 parts by weight or more and less than 750 parts by weight, 25 parts by weight or more and less than 500 parts by weight, 100 parts by weight or more and less than 500 parts by weight, or 110 parts by weight or more Less than 500 parts by weight, 150 parts by weight or more and less than 500 parts by weight, 200 parts by weight or more and less than 500 parts by weight, 230 parts by weight or more and less than 500 parts by weight, 25 parts by weight or more and less than 300 parts by weight, 100 parts by weight or more and less than 300 parts by weight, 110 parts by weight or more and less than 300 parts by weight, 150 parts by weight or more and less than 300 parts by weight, 200 parts by weight or more and less than 300 parts by weight, 230 parts by weight or more and less than 300 parts by weight, 25 parts by weight or more and 299 parts by weight or less, 100 parts by weight or more and 299 parts by weight or less, 110 parts by weight or more and 299 parts by weight or less, 150 parts by weight or more and 299 parts by weight or less, 200 parts by weight and 299 parts by weight or less, 230 parts by weight or more and 299 parts by weight or less, 25 parts by weight or more and 295 parts by weight or less, It may contain 100 parts by weight or more and 295 parts by weight or less, 110 parts by weight or more and 295 parts by weight or less, 150 parts by weight or more and 295 parts by weight or less, 200 parts by weight or more and 295 parts by weight or less, and 230 parts by weight or more and 295 parts by weight or less.

[0110] If the compound represented by Chemical Formula 2 is included in an excessively small amount per 100 parts by weight of the polyfunctional acrylic crosslinking agent, the particle density may become excessively large, and if it is included in an excessively large amount, the particle strength may decrease and hydrophobicity may increase.

[0111]

[0112] Meanwhile, the acrylate copolymer containing two or more different repeating units may further include repeating units derived from a compound represented by the following chemical formula 3, in addition to (meth)acrylate repeating units containing reactive functional groups and (meth)acrylate repeating units containing aliphatic functional groups.

[0113] [Chemical Formula 3]

[0114]

[0115] In the above chemical formula 3, L3 is an alkylene group having 1 or more carbon atoms.

[0116]

[0117] Specifically, in the above chemical formula 3, L3 may be an alkylene group having 1 or more carbon atoms, an alkylene group having 2 or more carbon atoms, and, for example, an ethylene group.

[0118] By including repeating units derived from the compound represented by the above chemical formula 3, the hydrophilicity of the particle can be improved through the introduction of hydroxyl groups.

[0119]

[0120] The acrylate copolymer containing two or more different repeating units may contain at least 1 part by weight and no more than 20 parts by weight of repeating units derived from the compound represented by Chemical Formula 3, based on 100 parts by weight of total repeating units.

[0121] Specifically, the acrylate copolymer containing two or more different repeating units may contain, with respect to 100 parts by weight of total repeating units, repeating units derived from the compound represented by Chemical Formula 3 in an amount of 1 part by weight or more, 5 parts by weight or more, 6 parts by weight or more, 20 parts by weight or less, 15 parts by weight or less, 10 parts by weight or less, 1 part by weight or more and 20 parts by weight or less, 5 parts by weight or more and 20 parts by weight or less, 6 parts by weight or more and 20 parts by weight or less, 1 part by weight or more and 15 parts by weight or less, 5 parts by weight or more and 15 parts by weight or less, 6 parts by weight or more and 15 parts by weight or less, 1 part by weight or more and 10 parts by weight or less, 5 parts by weight or more and 10 parts by weight or less, and 6 parts by weight or more and 10 parts by weight or less.

[0122] By including repeating units derived from the compound represented by Chemical Formula 3 to satisfy the above range, the epoxy content of the final microparticles is appropriately controlled, and surface modification of the particles can be facilitated.

[0123] The particle strength of the acrylate copolymer containing two or more different repeating units may be weakened if it contains an excessively small amount of repeating units derived from the compound represented by Chemical Formula 3 per 100 parts by weight of the total repeating units.

[0124]

[0125] In addition, the acrylate copolymer containing two or more different repeating units may contain, for every 100 parts by weight of the (meth)acrylate repeating unit containing the aliphatic functional group, a repeating unit derived from the compound represented by Chemical Formula 3 in an amount of 1 part by weight or more and 20 parts by weight or less.

[0126] Specifically, the acrylate copolymer containing two or more different repeating units may contain, with respect to 100 parts by weight of the (meth)acrylate repeating unit containing the aliphatic functional group, repeating units derived from the compound represented by Chemical Formula 3 in an amount of 1 part by weight or more, 5 parts by weight or more, 6 parts by weight or more, 20 parts by weight or less, 15 parts by weight or less, 1 part by weight or more and 20 parts by weight or less, 5 parts by weight or more and 20 parts by weight or less, 6 parts by weight or more and 20 parts by weight or less, 1 part by weight or more and 15 parts by weight or less, 5 parts by weight or more and 15 parts by weight or less, 6 parts by weight or more and 15 parts by weight or less, 1 part by weight or more and 10 parts by weight or less, 5 parts by weight or more and 10 parts by weight or less, and 6 parts by weight or more and 10 parts by weight or less.

[0127] By including repeating units derived from the compound represented by Chemical Formula 3 to satisfy the above range, the epoxy content of the final microparticles is appropriately controlled, and surface modification of the particles can be facilitated.

[0128] The particle strength of an acrylate copolymer containing two or more different repeating units may be weakened if it contains an excessively small amount of repeating units derived from a compound represented by Chemical Formula 3 per 100 parts by weight of (meth)acrylate repeating units containing aliphatic functional groups.

[0129]

[0130] Meanwhile, the above-mentioned polyacrylic microparticles may include polyacrylic microparticles. Preferably, the above-mentioned polyacrylic microparticles may be composed of polyacrylic microparticles.

[0131]

[0132] According to one embodiment of the invention, the polyacrylic microparticles may have an epoxy content of 50 μmol / g or more and 2000 μmol / g or less.

[0133] Specifically, the polyacrylic microparticles have an epoxy content of 50 μmol / g or more, 300 μmol / g or more, 400 μmol / g or more, 2000 μmol / g or less, 1000 μmol / g or less, 800 μmol / g or less, 499 μmol / g or less, 450 μmol / g or less, 50 μmol / g or more and 2000 μmol / g or less, 300 μmol / g or more and 2000 μmol / g or less, 400 μmol / g or more and 2000 μmol / g or less, 50 μmol / g or more and 1000 μmol / g or less, 300 μmol / g or more and 1000 μmol / g or less, 400 μmol / g or more and 1000 μmol / g or less, 50 μmol / g or more and 800 μmol / g or less, and 300 μmol / g or more and 800 It may be μmol / g or less, 400 μmol / g or more and 800 μmol / g or less, 50 μmol / g or more and 499 μmol / g or less, 300 μmol / g or more and 499 μmol / g or less, 400 μmol / g or more and 499 μmol / g or less, 50 μmol / g or more and 450 μmol / g or less, 300 μmol / g or more and 450 μmol / g or less, or 400 μmol / g or more and 450 μmol / g or less.

[0134] As the epoxy content of the above polyacrylic microparticles satisfies the above range, surface modification of the particles becomes easier.

[0135] If the epoxy content of the above polyacrylic microparticles is reduced excessively, it becomes difficult to achieve surface modification with sufficient density, and if it is increased excessively, there is a possibility that the particle strength will be weakened.

[0136] The method for measuring the epoxy content is not significantly limited, but it can be calculated by the following mathematical formula after dispersing the polyacrylic microparticles in an acidic solution and then titrating with a basic solution.

[0137] [Mathematical Formula]

[0138] Epoxy content (μmol / g) = [V0(ml)-V(ml)]*C (mol / L)

[0139] In the above mathematical formula, V0 is the volume of basic solution added to the control group containing 0.1g of DIW instead of particles, V is the volume of basic solution added to the actual sample, and C represents the concentration of the basic solution used in the titration.

[0140]

[0141] According to one embodiment of the invention, the apparent density of the polyacrylic microparticles is 1.15 g / cm³ 3 It may be less than.

[0142] Specifically, the apparent density of the polyacrylic microparticles is 0.99 g / cm³ 3 Above, 1.00 g / cm³ 3 Above, 1.05 g / cm³ 3 Above, 1.08 g / cm³ 3 Above, 1.15 g / cm³ 3 Below, 1.11 g / cm³ 3 Less than or equal to 0.99 g / cm³ 3 Above 1.15 g / cm³ 3 Below, 1.00 g / cm³ 3 Above 1.15 g / cm³ 3 Below, 1.05 g / cm³ 3 Above 1.15 g / cm³ 3 Below, 1.08 g / cm³ 3 Above 1.15 g / cm³ 3 Below, 0.99 g / cm³ 3 Above 1.11 g / cm³ 3 Below, 1.00 g / cm³ 3 Above 1.11 g / cm³ 3 Below, 1.05 g / cm³ 3 Above 1.11 g / cm³ 3 Below, 1.08 g / cm³ 3 Above 1.11 g / cm³ 3 It may be less than.

[0143] With the above-mentioned density range, not only is dispersibility in the cell culture reactor or medium improved, but contact with cells during differentiation and culture is increased, so improvement in differentiation and culture efficiency can be expected.

[0144]

[0145] According to one embodiment of the invention, the D50 particle diameter of the microparticles may be 45 μm to 55 μm. When the average diameter of the microparticles satisfies the above-described range, cell differentiation and culture performance is excellent.

[0146] Meanwhile, if the average diameter of the microparticles is less than 45 μm, the surface area available for cell culture is small, resulting in lower culture efficiency. It is also difficult to completely separate the microparticles from the cell culture medium using a cell strainer, which may cause safety issues with the final cell therapy product. If the diameter exceeds 55 μm, the surface area relative to volume decreases, which reduces the interaction between cells and particles and thus may lead to a problem of lower cell differentiation efficiency.

[0147] The diameter of the microparticle refers to the distance between two points where a straight line passing through the center of gravity of the microparticle meets the outermost surface of the microparticle, and the average diameter of the microparticle can be obtained by confirming the diameter of the microparticle using an optical microscope.

[0148] The above microparticles may be a group of individual particles having an average diameter of 45 μm to 55 μm in D50 particle diameter, and the individual particles included in this group may have an average diameter of 45 μm to 55 μm. More specifically, 95% or 99% of the individual particles included in the group may have a diameter of 45 μm to 55 μm.

[0149]

[0150] The above particles may include a single particle or a group of particles composed of multiple single particles. The single particle refers to one particle, and the group of particles refers to a matrix of multiple particles in which two or more particles are mixed.

[0151] At this time, the single particle may include a protein for immune cell differentiation fixed to its surface by a covalent bond. That is, a protein for immune cell differentiation fixed to its surface by a covalent bond may be included in a single particle, which is a single particle. In addition, a protein for immune cell differentiation fixed to its surface by a covalent bond may be included in a particle group in which two or more particles are mixed, on the surface of each single particle.

[0152] The shape of the above polyacrylic microparticles may be spherical. The polyacrylic spherical particles have excellent durability so they are not damaged or destroyed by external impact, and furthermore, the particle surface is uniform and has a large surface area, which can increase the protein immobilization efficiency.

[0153] Whether the above particles have a spherical shape can be determined visually using SEM. The spherical particles may include both theoretically perfect spherical particles and particles that are not perfect spheres but have a shape close to a perfect sphere.

[0154]

[0155] The above polyacrylic microparticles may be suspension polymer particles. Suspension polymer particles refer to particles obtained through a step of mixing a continuous phase composition and a dispersed phase composition and proceeding with polymerization in a suspension state.

[0156] Specifically, the polyacrylic microparticles may be microfluidic chip particles. The microfluidic chip particles may be particles obtained through a method for manufacturing particles comprising: a) a step of injecting a dispersed phase composition containing a polymerizable monomer into a continuous phase composition through a microchannel to generate a droplet composed of the dispersed phase composition within the continuous phase composition; and b) a step of photopolymerizing the droplet.

[0157] In the step of generating droplets a) above, droplets of uniform size and shape can be manufactured through a specific method.

[0158] In the above step a) of generating droplets, droplets of uniform size can be produced by injecting a dispersed phase composition that forms droplets into a continuous phase composition through a microchannel to generate droplets.

[0159] Specifically, in the step a) of generating droplets, a microfluidic device including microchannels may be used.

[0160] For example, the microfluidic device may include a first supply unit to which a dispersed phase composition is supplied; a first flow path through which the dispersed phase composition supplied from the first supply unit can flow; a second supply unit to which a continuous phase composition is supplied; a second flow path through which the continuous phase composition supplied from the second supply unit can flow; and a plurality of microflow paths connecting the sides of the first and second flow paths to each other. The side of the flow path refers to a direction other than the direction of fluid flow within the flow path.

[0161] The first and second channels can be formed spaced apart from each other by the length of the microchannels of the desired length. The height and length of the microchannels are not particularly limited and can be appropriately adjusted according to the size of the target droplet.

[0162] In the step a) of generating droplets above, a dispersed phase composition may be supplied to a first supply unit of a microfluidic device and a continuous phase composition may be supplied to a second supply unit. The dispersed phase composition and the continuous phase composition may be injected into the first and second supply units, respectively, through a pump, but are not limited thereto.

[0163] The dispersed phase composition supplied to the first supply unit flows along the first flow path, and the continuous phase composition supplied to the second supply unit flows along the second flow path. At this time, the speed of the dispersed phase composition can be controlled to 1 μl / min to 100 ml / min. The speed of the continuous phase composition can be controlled to 10 μl / min to 500 ml / min.

[0164] The dispersed phase composition flowing through the first flow path flows into the second flow path through a plurality of microchannels and meets the continuous phase composition flowing through the second flow path.

[0165] The dispersed phase composition flowing from the first channel to the second channel through the plurality of microchannels generates droplets at the boundary between the microchannel and the second channel, and the generated droplets flow through the second channel together with the continuous phase composition.

[0166] The microfluidic device may further include a discharge section through which a droplet formed from a dispersed phase composition can be discharged. At this time, the microfluidic device may further include a third flow path connecting the second flow path and the discharge section.

[0167] The above dispersed phase composition is a precursor composition for forming particles and may be an oil phase insoluble in the continuous phase composition which is a water phase.

[0168] Specifically, the dispersed phase composition may include a polymerizable monomer, a crosslinking agent, and a photoinitiator.

[0169] The above-mentioned dispersed phase composition refers to a composition capable of forming a dispersed phase (or droplets) after being mixed with a continuous phase composition, and the above-mentioned continuous phase composition refers to a composition capable of forming a continuous phase after being mixed with a dispersed phase composition.

[0170]

[0171] In one example, the dispersed phase composition may include a polymerizable monomer.

[0172] In one example of the present application, the polymerizable monomer is a monomer having unsaturated carbon-carbon bonds and may be a monomer further comprising an epoxy group, an amide group, a carboxyl group, an alkoxy group, a sulfonate group, a thiol group, an amine group, or a hydroxyl group. The aforementioned groups may be referred to as hydrophilic groups.

[0173] In one example, the dispersed phase composition may further include a photoinitiator.

[0174] The type of photoinitiator mentioned above is not particularly limited, and various initiators known in the technical field to which the present invention belongs may be used as long as they do not hinder securing the particle characteristics of the particles described above.

[0175] As the above photoinitiator, for example, initiators such as ketone initiators, organic peroxide initiators, and azo initiators may be used. Specifically, compounds such as 2,2-dimethoxy-2-phenylacetophenone, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, 1-hydroxycyclohexylphenyl ketone, benzoyl peroxide, di-t-amyl peroxide, t-butyl peroxybenzoate, 2,5-dimethyl-2,5-di-(t-butylperoxy)hexane, 2,5-dimethyl-2,5-di-(t-butylperoxy)hexine-3, or di-cumyl peroxide, and mixtures thereof may be used, but are not limited thereto.

[0176] The above photoinitiator may be included in an amount of 0.1 parts by weight or more, 1 part by weight or more, 3 parts by weight or more, 5 parts by weight or more, 10 parts by weight or more, or 15 parts by weight or more, and 30 parts by weight or less, 25 parts by weight or less, 20 parts by weight or less, or 18 parts by weight or less, based on 100 parts by weight of the polymerizable monomer.

[0177] Within this range, an appropriate polymerization rate can be exhibited to manufacture microparticles with desired characteristics.

[0178] The above-described dispersed phase composition may include various additives known in the art to which the present invention belongs, in addition to the components described above, to the extent that they do not impair the purpose of the present invention.

[0179] Meanwhile, the above continuous phase composition may be an aqueous phase. Specifically, the above continuous phase composition may be an aqueous solution containing a surfactant and water.

[0180] The above water may be distilled water or deionized water, although it is not specifically limited.

[0181] The above surfactant may be an ionic surfactant or a nonionic surfactant. For example, the ionic surfactant may be sodium dodecyl sulfate (SDS), and the nonionic surfactant may be Tween 20, Tween 40, Tween 60, Tween 80, Triton X-100, polyvinyl alcohol (PVA), polyethylene glycol (PEG), etc.

[0182] The above continuous phase composition may contain the surfactant in an amount of 0.01 wt% or more, 0.05 wt% or more, 0.1 wt% or more, 0.2 wt% or more, or 0.3 wt% or more, and 5 wt% or less, 3 wt% or less, or 1 wt% or less, based on the total continuous phase composition. It is advantageous to manufacture microparticles with desired characteristics within this range.

[0183] Meanwhile, the droplet produced in step a) of generating the droplet may be introduced into step b) of photopolymerizing the droplet. At this time, the droplet may be mixed with an additional continuous phase composition before being introduced into the photopolymerization process.

[0184] For example, a suspension containing droplets discharged from the outlet of a microfluidic device can be mixed with an additional continuous phase composition and then introduced into a photopolymerization process. Through this process, the shape of the microparticles can be uniformly controlled.

[0185] The additional continuous phase composition mentioned above may have the same composition as or a different composition from the continuous phase composition used in the step of generating droplets a). As an example, in the step of generating droplets a), an aqueous solution of sodium dodecyl sulfate may be used as the continuous phase composition, and the suspension containing the droplets may be mixed with an aqueous solution of polyvinyl alcohol. However, it is not limited thereto, and the droplets generated in the step of generating droplets a) may be transferred directly to a photopolymerization process.

[0186] In step b) of photopolymerization above, particles are manufactured by photopolymerizing the droplets generated in step a). In step b) of photopolymerization above, photopolymerization of the droplets can be induced by irradiating the droplets with UV light using a UV spot curing device. The specific conditions for photopolymerization are not particularly limited, and various examples and conditions widely used in the field of conventional particle manufacturing can be applied without restriction.

[0187] A washing step may be further included after the above b) photopolymerization step. Impurities unrelated to the particles can be removed by washing. The washing method is not particularly limited, and known washing methods may be used. For example, the washing may be performed by introducing the particles into an alcohol such as ethanol and stirring. Although not particularly limited, such washing may be repeated, for example, three or more times.

[0188] After the washing described above, a drying step may be further included. Residual solvents, etc., can be removed through drying. The drying method is not particularly limited, and known drying methods may be used. For example, the drying may be performed using an oven or under room temperature conditions. Additionally, although not particularly limited, the drying may be performed under a vacuum.

[0189] The above method for manufacturing particles can produce particles having a uniform size and shape. In the case of conventional particle manufacturing methods, the production yield was low because, after particle formation, a process of separating particles with desired shapes and properties from those with undesirable shapes or properties was required; however, the above method for manufacturing particles can obtain particles with desired shapes and properties without undergoing such a separation process, thereby demonstrating a high production yield.

[0190] In one example, the mixture comprising the dispersed phase and the continuous phase is 0.99 to 1.2 g / cm³ 3 It can satisfy the density of the range.

[0191]

[0192] According to another embodiment of the invention, a particle complex comprising polyacrylic microparticles of the above-described embodiment; and a protein immobilized on the surface of the particles may be provided.

[0193] The above-mentioned polyacrylic microparticles may include all of the above-described contents.

[0194] The inventors confirmed through experiments that the particle complex of the above-mentioned embodiment is economical as it satisfies the protein binding density and can achieve excellent differentiation performance as it enables control of the appropriate protein concentration, thereby completing the invention.

[0195] When using a conventionally widely used culture plate instead of the polyacrylic microparticles of the above embodiment, there is a limitation in that it is difficult to control the amount and uniformity of the protein because the protein is immobilized using simple adsorption rather than covalent bonding. Furthermore, since it is difficult to mass-produce and automate proteins coated on plates, it is essential to immobilize the protein onto particles for mass culture.

[0196]

[0197] The above particle complex has a protein binding density of 0.2 μg / cm² 2 Above 2.0 µg / cm² 2 It may be less than or equal to. Specifically, the particle complex has a protein binding density of 0.2 μg / cm² 2 Above, 0.25 µg / cm² 2 Above, 2.0 µg / cm² 2 Below, 1.8 µg / cm² 2 Below, 1.5 µg / cm² 2 Less than or equal to 0.2 µg / cm² 2 Above 2.0 µg / cm² 2 Below, 0.2 µg / cm² 2 Above 1.8 µg / cm² 2 Below, 0.2 µg / cm² 2 Above 1.5 µg / cm² 2 Below, 0.25 µg / cm² 2 Above 2.0 µg / cm² 2 Below, 0.25 µg / cm² 2 Above 1.8 µg / cm² 2 Below, 0.25 µg / cm² 2 Above 1.5 µg / cm² 2 It may be less than.

[0198] The protein binding density of the above particle complex can be achieved by controlling the type and weight ratio of monomers used in the manufacture of polyacrylic microparticles included in the particle complex, thereby securing the structural stability of the particle complex by fixing proteins to the surface of the polyacrylic microparticles through physical bonding such as chemical covalent bonding or adsorption, and enabling the control of the appropriate protein concentration.

[0199] The protein binding density of the above particle complex is 0.2 μg / cm² 2 If it is less than 2.0 µg / cm², it may be difficult for protein-induced stem cell differentiation to occur sufficiently, and 2.0 µg / cm² 2 If exceeded, a technical problem may arise where proteins fail to bind because the density of proteins becomes higher than the density of ligands to bind at the cell surface.

[0200] The method for measuring the protein binding density is not significantly limited, but, for example, a standard solution with a controlled concentration using unbound protein is prepared, and a BCA assay is performed to create a standard curve. The BCA assay is also performed in a solution containing the particle complex, and the absorbance is compared to inversely calculate the protein concentration of the particle complex; subsequently, the protein binding density can be calculated using the concentration and specific surface area of ​​each particle complex.

[0201]

[0202] The above protein may be a protein for immune cell differentiation. The above protein for immune cell differentiation may play a role in providing the necessary stimulation to differentiate stem cells, such as allogeneic induced pluripotent stem cells (iPSCs), into immune cells.

[0203] Specific examples of the above-mentioned proteins for immune cell differentiation are not particularly limited, and examples include GM-CSF, IL4, IL-1b, TNFa, PGE2, DLL1, DLL4, TGFb, or a mixture of two or more of these, and other commonly known proteins can be applied without limitation.

[0204] Since the above protein has the use of immune cell differentiation, when the particle complex of the above embodiment is cultured together with stem cells, the stem cells can be differentiated into immune cells to manufacture a cell therapy agent for anticancer treatment, etc. By introducing such particles, the technical limitations of surface protein adsorption used in existing immune cell differentiation platforms can be effectively overcome, enabling the economical production of a large amount of immune cells and providing high marketability as an off-the-shelf drug.

[0205]

[0206] The above protein can be fixed to the particle surface by covalent bonds or physical bonds. The particle complex of the above embodiment can secure structural stability by fixing the protein to the particle surface by chemical bonds (e.g., covalent bonds) or physical bonds (e.g., adsorption). In particular, if the protein is fixed to the particle surface by chemical bonds (e.g., covalent bonds), mass production and automation can be stably carried out through a 3D process compared to conventional technology, thereby increasing process efficiency.

[0207] The above covalent bond is a bond formed when atoms share electrons during a chemical bond, and the protein can be fixed to the surface by covalent bond while the particle is dispersed in water.

[0208] Specifically, the above covalent bond may include a bonding functional group represented by the following chemical formula 4.

[0209] [Chemical Formula 4]

[0210]

[0211] In the above chemical formula 4, R 10 It is hydrogen or alkyl.

[0212] That is, the above covalent bond may have a structure in which a particle and a protein are bonded through a binding functional group represented by Chemical Formula 4. More specifically, a particle may be bonded to one end of the binding functional group represented by Chemical Formula 4, and a protein may be bonded to the other end of the binding functional group represented by Chemical Formula 4.

[0213] For example, the above covalent bond may include a bond represented by the following chemical formula 4-1.

[0214] [Chemical Formula 4-1]

[0215]

[0216] In the above chemical formula 4-1, R 10 is hydrogen or alkyl, X is a polyacrylic microparticle, and Y is a protein.

[0217]

[0218] The covalent bond represented by Chemical Formula 4-1 above can be formed by the reaction between the epoxy group on the particle surface and the amine group contained in the protein. The reaction conditions between the epoxy group on the particle surface and the amine group contained in the protein are not significantly limited, and various conventionally known covalent bond conditions can be applied without restriction. However, for example, ammonium sulfate can be added to a solution in which particles and protein are mixed in PBS (Phosphate buffered saline), and the reaction can be carried out for 16 hours or more while shaking at a speed of 250 rpm or more at room temperature.

[0219] In addition, the above covalent bond may include a bonding functional group represented by the following chemical formula 5.

[0220] [Chemical Formula 5]

[0221]

[0222] That is, the above covalent bond may have a structure in which a particle and a protein are bonded through a binding functional group represented by Chemical Formula 5. More specifically, a particle may be bound to one end of the binding functional group represented by Chemical Formula 5, and a protein may be bound to the other end of the binding functional group represented by Chemical Formula 5.

[0223] For example, the above covalent bond may include a bond represented by the following chemical formula 6.

[0224] [Chemical Formula 6]

[0225]

[0226] In the above chemical formula 6, X is a polyacrylic microparticle and Y is a protein.

[0227]

[0228] The covalent bond represented by the above chemical formula 6 can be formed by the reaction between the epoxy group on the particle surface and the thiol group contained in the protein. The reaction conditions between the epoxy group on the particle surface and the thiol group contained in the protein are not significantly limited, and various conventionally known covalent bond conditions can be applied without restriction. However, for example, ammonium sulfate can be added to a solution in which particles and protein are mixed in PBS (Phosphate buffered saline), and the reaction can be carried out for 16 hours or more while shaking at a speed of 250 rpm or more at room temperature.

[0229] In addition, the above covalent bond may include a bonding functional group represented by the following chemical formula 7.

[0230] [Chemical Formula 7]

[0231]

[0232] In the above chemical formula 7, R 20 is hydrogen or alkyl.

[0233] That is, the above covalent bond may have a structure in which a particle and a protein are bonded through a binding functional group represented by the above chemical formula 7. More specifically, a particle may be bound to one end of the binding functional group represented by the above chemical formula 7, and a protein may be bound to the other end of the binding functional group represented by the above chemical formula 7.

[0234] For example, the above covalent bond may include a bond represented by the following chemical formula 8.

[0235] [Chemical Formula 8]

[0236]

[0237] In the above chemical formula 8, R 20 is hydrogen or alkyl, X is a polyacrylic microparticle, and Y is a protein.

[0238]

[0239] The covalent bond represented by Chemical Formula 8 above can be formed by the reaction between the carboxyl group on the particle surface and the amine group contained in the protein. The reaction conditions between the carboxyl group on the particle surface and the amine group contained in the protein are not significantly limited, and various conventionally known covalent bond conditions can be applied without restriction. However, for example, ammonium sulfate can be added to a solution in which particles and proteins are mixed in PBS (Phosphate buffered saline), and the reaction can be carried out for 16 hours or more while shaking at a speed of 250 rpm or more at room temperature.

[0240]

[0241] Meanwhile, since the particle complex of the above-described embodiment has the use of immune cell differentiation, when the particle complex of the above-described embodiment is cultured together with stem cells, the stem cells can be differentiated into immune cells to manufacture a cell therapy agent for anticancer treatment, etc. By introducing such particles, the technical limitations of surface protein adsorption used in existing immune cell differentiation platforms can be effectively overcome, enabling the economical production of a large amount of immune cells and providing high marketability as an off-the-shelf drug.

[0242] The particle composite of the above embodiment may have a free protein ratio of 0.1% or less, or 0.0001% to 0.1% according to the following mathematical formula 1.

[0243] [Mathematical Formula 1]

[0244] Free protein percentage (%) = (W1 / W2) * 100

[0245] In the above Equation 1, W1 is the mass of protein released from the particle complex after shaking culture and centrifugation of the solution containing the particle complex, and W2 is the mass of protein fixed to the particle complex before shaking culture and centrifugation of the solution containing the particle complex.

[0246] This appears to be due to the particle complex of the above-mentioned embodiment securing structural stability by fixing proteins to the particle surface through covalent bonds. Accordingly, compared to conventional technology, mass production and automation can be stably carried out through a 3D process, thereby increasing process efficiency.

[0247] On the other hand, if the ratio of free protein according to the above mathematical formula 1 increases excessively to more than 0.1%, the protein cannot be stably fixed from the particle complex, which may cause a problem where the efficiency of the process is significantly reduced when mass-producing and automating through a 3D process.

[0248] The solution containing the particle complex is a mixture of the particle complex and a solvent, and examples of the solvent are not significantly limited, and various conventionally known solvents can be applied without limitation. However, for example, PBS (Phosphate buffered saline) may be used. The particle complex may be added to the solution containing the particle complex so that the protein concentration satisfies 1 µg / mL.

[0249] Examples of the above shaking culture conditions are not significantly limited, and various conventionally known shaking culture conditions can be applied without restriction. However, for example, the reaction can be carried out for 7 hours or more while shaking at a speed of 250 rpm or more in a shaking culture at room temperature.

[0250] Examples of the above centrifugation conditions are not significantly limited, and various conventionally known centrifugation conditions can be applied without restriction. However, for example, centrifugation can be performed at 10,000 xg for 10 minutes.

[0251]

[0252] Examples of methods for measuring the mass of protein released from the above particle complex are not significantly limited, and various conventionally known quantitative analysis methods can be applied without limitation. However, as an example, ELISA (Enzyme-Linked Immunosorbent Assay) can be cited.

[0253] Meanwhile, the particle composite of the above-described embodiment may consist of the polyacrylic microparticles and a protein immobilized on the surface of the particles. The details regarding the polyacrylic microparticles and the protein immobilized on the surface of the particles are the same as those described above. If the particle composite includes other components (e.g., magnetic particles) in addition to the polyacrylic microparticles and the protein immobilized on the surface of the particles, there is a limitation in that it is difficult to maintain quality because they are difficult to remove from the particle composite, and process efficiency deteriorates as a separate removal process is required for reuse.

[0254]

[0255] According to another embodiment of the invention, a cell culture composition comprising a cell and the polyacrylic microparticles of the first embodiment or the particle complex of the first embodiment may be provided. The details regarding the polyacrylic microparticles and the particle complex include all the details described above in the first embodiment.

[0256] In addition, the density difference between the microparticles and the cells is 0 g / cm³ 3 Above 0.10 g / cm³ 3 It may be less than or equal to 0 g / cm³. The density difference between the microparticles and the cells is 0 g / cm³ 3 Above 0.10 g / cm³ 3 By satisfying the following, contact between cells and particles increases during culture, and improvement in differentiation and culture efficiency can be expected.

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

[0258]

[0259] According to another embodiment of the invention, a method for manufacturing a cell therapeutic agent may be provided, comprising the steps of: culturing a cell culture composition of the other embodiment; and removing polyacrylic microparticles or particle complexes from the culture product. The cell culture composition includes all the details described above in the other embodiment.

[0260] In the step of culturing the cell culture composition of the other embodiment above, the cells contained in the cell culture composition can be differentiated into immune cells. The specific culture conditions are not particularly limited, and any culture conditions capable of differentiating stem cells into immune cells can be applied without limitation.

[0261] Meanwhile, in the step of removing polyacrylic microparticles or particle complexes from the culture product, the safety of the cell therapeutic agent can be ensured by separating and removing the polyacrylic microparticles or particle complexes from the product of the cell culture step. The method of removing particle complexes from the culture product is not particularly limited, and various conventionally known methods and conditions can be applied without restriction. However, as an example, the polyacrylic microparticles or particle complexes can be separated and removed using a cell strainer.

[0262]

[0263] According to the present invention, microparticles suitable for cell culture and cell differentiation that have appropriate density and hydrophilicity to achieve excellent dispersibility in a culture medium while being easy to modify the surface, particle composites that secure the stability of a differentiation platform by fixing proteins required for cell differentiation through covalent bonds and enable control of the appropriate concentration of proteins, and cell culture compositions using the same, and a method for manufacturing a cell therapeutic agent may be provided.

[0264]

[0265] The invention is described in more detail in the following examples. However, the following examples are merely illustrative of the invention, and the scope of the invention is not limited by the following examples.

[0266]

[0267] Polyacrylic Microparticles

[0268] Example 1

[0269] Polyvinyl alcohol (molecular weight 85-124K, 87-89% hydrolysis) was dissolved in distilled water at a concentration of 2% to prepare an aqueous dispersion, and then stirred at room temperature for 20 minutes.

[0270] A monomer composition was prepared by stirring glycidyl methacrylate (GMA) and 2-ethylhexyl methacrylate (EHMA) as monomers, and ethylene glycol dimethacrylate (EGDMA) and trimethylolpropane trimethacrylate (TMPTMA) as crosslinking agents in the weight ratios shown in Table 1, and then adding 2 wt% of V-65 initiator (initiator amount: based on the total sum of monomers and crosslinking agents) to 25 g of the mixture and stirring for an additional 5 minutes.

[0271] 600g of an aqueous dispersion was added to a 1L reactor, and the monomer composition was added. A shear force was applied to the aqueous dispersion and the monomer composition at a speed of 400 rpm at room temperature to disperse the monomer composition into the aqueous dispersion in the form of fine droplets and homogenize it.

[0272] Microparticles were prepared by reacting the homogenized mixture under nitrogen purging at 85°C for 6 hours while stirring at a stirring speed of 400 rpm, and after washing three times with distilled water at 60°C and five times with ethanol, the particles were filtered through a 70 µm sieve and recovered by drying in an oven at 70°C.

[0273]

[0274] The physical properties of the above microparticles are as follows.

[0275] Average diameter: 50 µm

[0276] Apparent density: 1.04 g / cm³ 3 ~ 1.2 g / cm 3

[0277]

[0278] Examples 2 and 3

[0279] Microparticles were prepared in the same manner as in Example 1, except that the weight ratio of the monomer and the crosslinking agent in Example 1 was adjusted to satisfy Table 1 below.

[0280]

[0281] Comparative Example 1

[0282] Corning, product name: Synthermax II was used as microparticles.

[0283]

[0284] Comparative Example 2

[0285] Microparticles were prepared in the same manner as in Example 1, except that the weight ratio of the monomer and the crosslinking agent in Example 1 was adjusted to satisfy Table 1 below.

[0286]

[0287] Comparative Example 3

[0288] Microparticles were prepared in the same manner as in Example 1, except that the weight ratio of the monomer and the crosslinking agent in Example 1 was adjusted to satisfy Table 1 below.

[0289] In the case of the polyacrylic microparticles of Comparative Example 3, it was confirmed that the aliphatic methacrylate content was excessively low, resulting in increased apparent density and reduced dispersibility.

[0290]

[0291] Comparative Example 4

[0292] Microparticles were prepared in the same manner as in Example 1, except that the weight ratio of the monomer and the crosslinking agent in Example 1 was adjusted to satisfy Table 1 below.

[0293] In the case of the polyacrylic microparticles of Comparative Example 4, the aliphatic methacrylate content was excessively high, which resulted in lower particle strength and increased hydrophobicity, making them unsuitable for cell culture use.

[0294]

[0295] Monomer (weight%) Crosslinking agent (weight%) GMAEHMAHEMADMAEGDMATMPTMA Example 1 33.346.71010 Example 2 33.346.71010 Example 3 16.758.351010 Comparative Example 1 Comparative Example 2 5050 Comparative Example 3 60201010 Comparative Example 4 8.571.51010

[0296] The content of each component in Table 1 above refers to the weight % based on the total sum of monomers and crosslinking agents.

[0297] GMA: Glycidyl methacrylate

[0298] EHMA: 2-Ethylhexyl methacrylate

[0299] HEMA: 2-Hydroxyethyl methacrylate

[0300] DMA: Dodecyl methacrylate

[0301] EGDMA: Ethylene glycol dimethacrylate

[0302] TMPTMA: Trimethylolpropane trimethacrylate

[0303]

[0304] <Experimental Example: Measurement of Physical Properties of Microparticles>

[0305] For the microparticles obtained in the above examples and comparative examples, physical properties were measured by the following method, and the results are shown in Table 2.

[0306]

[0307] Experiment 1. Average particle size (Unit: μm)

[0308] For the particles obtained in the above examples and comparative examples, after observing them under a microscope, the D50 (particle size value corresponding to the cumulative distribution percentage reaching 50%) particle diameter was measured using image analysis software.

[0309]

[0310] Experiment 2. Apparent Density (Unit: g / cm³) 3 )

[0311] For the microparticles prepared in the above examples and comparative examples, various densities (1.02 g / cm³) were obtained using OptiPrep Density Gradient Medium (Sigma-Aldrich) under conditions of room temperature (25 ℃) and atmospheric pressure (1 atm). 3 , 1.04 g / cm 3 , 1.06 g / cm 3 , 1.08 g / cm 3 , 1.1 g / cm 3 , 1.12 g / cm 3 , 1.14 g / cm 3 , 1.16 g / cm 3 , 1.18 g / cm 3 , 1.20 g / cm 3 , 1.22 g / cm 3 Solutions of ) were prepared. The density of the particles is evaluated by dispersing them in each solution and observing.

[0312] 1) Particle density = Solution density

[0313] After 5 minutes have passed since the particles were dispersed in the solution, if it is observed that the particles are not settling or floating in the solution but are dispersed throughout, it is determined that the density of the solution and the density of the particles are the same.

[0314] 2) Particle density > Solution density

[0315] After dispersing particles in a solution, if it is observed that most of the particles settle in the solution after 5 minutes, it is determined that the particle density is higher than the density of the solution.

[0316] 3) Particle density < Solution density

[0317] After dispersing particles in a solution, if it is observed that most of the particles are suspended in the solution after 5 minutes, it is determined that the particle density is lower than the density of the solution.

[0318]

[0319] Experiment 3. Epoxy Content Analysis

[0320] 0.1 g of microparticles prepared in the above examples and comparative examples were dispersed in an HCl / acetone (volume ratio = 1:80) solution and then sonicated for 4 minutes.

[0321] After adding 2 drops of indicator solution (0.1 wt% cresol red + 0.1 wt% thymol blue, volume ratio 1:3, pH 7, 0.01 M NaOH), the solution was titrated with 0.1 M NaOH.

[0322] The epoxy content (μmol / g) was calculated using the following formula.

[0323] [Mathematical Formula]

[0324] Epoxy content (μmol / g) = [V0(ml)-V(ml)]*C NaOH (mol / L)

[0325] In the above mathematical formula, V0 is the volume of NaOH added to the control group containing 0.1g of DIW instead of particles, V is the amount of NaOH added to the actual sample, and C NaOH represents the concentration of NaOH used in the titration.

[0326]

[0327] Experiment 4. Coefficient of Variation (CV)

[0328] For 100 microparticles prepared in the above examples and comparative examples, the average particle diameter was obtained by analyzing scanning electron microscopy (SEM) images or microscope images and then calculated using the following mathematical formula 1.

[0329] [Mathematical Formula 1]

[0330] Coefficient of variation (%) = (Standard deviation of particle diameter / Average particle diameter) X 100.

[0331]

[0332] Experiment 5. Hydrophilicity

[0333] After dispersing the microparticles prepared in the above examples and comparative examples in DI water at a high concentration of 50 w%, 100 uL of the dispersion is drawn using a 1 mL pipette and tip, and then dispensed after about 10 seconds, the amount of particles adhering to the wall of the pipette tip is observed. In the case of particles with normal or higher hydrophilicity, particles remain in only less than 10% of the area where the solution came into contact; in the case of particles with low hydrophilicity, particles remain in an area of ​​10% or more but less than 30%; and in the case of particles with very low hydrophilicity, particles remain in an area of ​​30% or more.

[0334]

[0335] Average particle size (㎛, D50) CV (%) Epoxy content (μmol / g) Apparent density (g / cm³) 3 Hydrophilic Example 1 48.4 3.5 800 1.11 Ordinary Example 2 48.4 3.5 800 1.09 Ordinary Example 3 54.9 5.5 400 1.08 High Comparative Example 1 1.05 Low Comparative Example 2 53.1 7.7 1 200 1.2 Ordinary

[0336] As shown in Table 2 above, the microparticles of the example are 1.11 g / cm³ 3 It was confirmed that the low density below is suitable for cell culture, which improves particle dispersion under cell culture conditions and exhibits excellent hydrophilicity, suggesting that high differentiation efficiency can be expected during cell differentiation experiments.

[0337] On the other hand, it was confirmed that the microparticles of Comparative Example 1 had poor hydrophilicity as they did not contain surface functional groups, unlike the microparticles of the Example.

[0338] The microparticles of Comparative Example 2 were made using conventional polyacrylic microparticles, and it was confirmed that they were unsuitable for cell culture because their density was excessively high and they quickly settled in the culture medium during cell culture or differentiation.

[0339]

[0340] Particle Complex

[0341] Examples 4 to 6

[0342] (1) Preparation of particles

[0343] Glycidyl methacrylate (GMA) and 2-ethylhexyl methacrylate (EHMA) as monomers, and ethylene glycol dimethacrylate (EGDMA) and trimethylolpropane trimethacrylate (TMPTMA) as crosslinking agents were stirred in the weight ratios shown in Table 3, after which irgacure 651 was added as a photoinitiator at 1% by weight relative to the weight of the stirred solution, and the mixture was stirred at room temperature for about 5 minutes to prepare a dispersed phase composition.

[0344] A continuous phase composition was prepared by dissolving SDS (sodium dodecyl sulfate) in deionized water at a concentration of 0.5 wt%.

[0345] The dispersed phase composition (injection rate of 100 µl / min) and the continuous phase composition (injection rate of 300 µl / min), prepared prior to the first and second supply sections of the microfluidic device (step emulsification chip), were respectively injected through a pump. The dispersed phase composition injected into the first supply section formed droplets within the continuous phase composition. The suspension containing the droplets was discharged through the discharge section.

[0346] Subsequently, the suspension containing the above droplets was collected in an aqueous solution of PVA (molecular weight 85,000 to 125,000) to disperse the droplets in a final 2% by weight of PVA. After shaking to ensure the droplets were evenly dispersed, photopolymerization was carried out using a UV spot curing device.

[0347] The polymerized particles were recovered and washed twice with distilled water and five times with ethanol. Afterwards, the particles were dried in an oven at approximately 80°C to obtain the particles.

[0348] (2) Preparation of particle composites

[0349] The particles obtained in (1) above were recovered, and 0.1-10 g of the particles and 5-100 mL of solvent (type: borate buffer pH 9, Diglyme, DMSO, etc.) were placed in a glass vial. EDA (Ethylenediamine) was added in an amount of at least 2 equivalents relative to the epoxy amount of the particles, a magnetic bar was inserted, and the lid was closed. The glass vial was placed in an oil bath at 30-80 ℃ and reacted for at least 24 hours. Afterward, the particles were washed 5 times with the solvent and 5 times with DW, then dried in an oven at about 80 ℃ to obtain the particles.

[0350] 0.1-10 g of the obtained particles and 5-100 mL of solvent (type: borate buffer pH 9, Diglyme, DMSO, etc.) were placed in a glass vial, and at least 2 equivalents of glycidol (2,3-Epoxypropanol) based on amine were added, followed by the insertion of a magnetic bar and the closing of the lid. The glass vial was placed in an oil bath at 30-80 ℃ and reacted for at least 24 hours. Afterward, the particles were washed 5 times with the solvent and 5 times with distilled water, then dried in an oven at approximately 80 ℃ to obtain the particles.

[0351] 0.1-10 g of the obtained particles and 5-100 mL of solvent (type: DMF) were placed in a glass vial, succinic anhydride at least 10 equivalents based on the hydroxyl group was added, and DMAP at least 1 equivalent was added as a catalyst, after which a magnetic bar was inserted and the lid was closed. The glass vial was placed in an oil bath at 30-80°C and reacted for 24 hours. Afterward, the particles were washed 5 times with the solvent and 5 times with DW, then dried in an oven at approximately 80°C to obtain the particles.

[0352] 20 mg of the sequentially modified particles were reacted with 0.001-0.02 mmol of EDC and 0.001-0.02 mmol of Sulfo-NHS in a tube while inverting at 37°C for at least 4 hours. After the EDC / NHS activation was completed, the particles were washed three times (using centrifugation), and the amount of protein was adjusted and added according to the calculations in Table 4 to ensure protein labeling. The resulting covalently bonded protein particles were reacted at 37°C for 24 hours while inverting the tube, and these were used as the particle complexes for Examples 4 to 6. The prepared particle complexes were placed in PBS and stored at 4°C.

[0353]

[0354] Example 7

[0355] Particles and particle composites were prepared in the same manner as in Example 4, except that the composition of the monomer was adjusted as shown in Table 3.

[0356]

[0357] Comparative Example 5

[0358] (1) Preparation of particles

[0359] Polyvinyl alcohol (molecular weight 85-124K, 87-89% hydrolysis) was dissolved in distilled water at a concentration of 2% to prepare an aqueous dispersion, and then stirred at room temperature for 20 minutes.

[0360] A monomer composition was prepared by adding 2% by weight of V-65 initiator (initiator input amount: based on the total sum of monomer and crosslinking agent) to 25 g of a mixture sufficiently dissolved by mixing monomers styrene and t-butylstyrene and a crosslinking agent divinylbenzene in a weight ratio of 0.1:0.9:1 and stirring for an additional 5 minutes.

[0361] 600g of an aqueous dispersion was added to a 1L reactor, and the monomer composition was added. A shear force was applied to the aqueous dispersion and the monomer composition at a speed of 400 rpm at room temperature to disperse the monomer composition into the aqueous dispersion in the form of fine droplets and homogenize it.

[0362] Polystyrene particles were prepared by reacting the homogenized mixture under nitrogen purging at 85°C for 6 hours while stirring at a stirring speed of 400 rpm, and after washing three times with distilled water at 60°C and five times with ethanol, the particles were filtered through a 70 µm sieve and recovered by drying in an oven at 70°C.

[0363]

[0364] (2) Preparation of particle composites

[0365] After mixing the particles obtained in (1) above with a PBS solution containing a protein for immune cell differentiation, the mixture was reacted in a shaking culture at room temperature at a speed of 250 rpm or more for 16 hours or more to allow the protein to be adsorbed on the surface of the particles, thereby producing the particle complex of Comparative Example 5.

[0366]

[0367] Comparative Examples 6 to 9

[0368] (1) Preparation of particles

[0369] Glycidyl methacrylate (GMA) as a monomer and ethylene glycol dimethacrylate (EGDMA) as a crosslinking agent were stirred in the weight ratios shown in Table 3, and then irgacure 651 was added as a photoinitiator at 1% by weight relative to the weight of the stirred solution, and the mixture was stirred at room temperature for about 5 minutes to prepare a dispersed phase composition.

[0370] A continuous phase composition was prepared by dissolving SDS (sodium dodecyl sulfate) in deionized water at a concentration of 0.5 wt%.

[0371] The dispersed phase composition (injection rate of 20 μl / min) and the continuous phase composition (injection rate of 200 μl / min), prepared prior to the first supply section and the second supply section of the microfluidic device (190 μm flow focusing chip), were each injected through a pump. The dispersed phase composition injected into the first supply section formed droplets within the continuous phase composition. The suspension containing the droplets was discharged through the discharge section.

[0372] Subsequently, the suspension containing the above droplets was collected in an aqueous solution of PVA (molecular weight 85,000 to 125,000) to disperse the droplets in a final 2% by weight of PVA. After shaking to ensure the droplets were evenly dispersed, photopolymerization was carried out using a UV spot curing device.

[0373] The polymerized particles were recovered and washed twice with distilled water and five times with ethanol. Afterwards, the particles were dried in an oven at approximately 80°C to obtain the particles.

[0374]

[0375] (2) Preparation of particle composites

[0376] The particles obtained in (1) above were washed three times with PBS (Phosphate buffered saline) by centrifuging at 10,000 xg for 10 minutes. Afterwards, immune cell differentiation proteins (GM-CSF, IL4, IL-1b, TNFa, PGE2, DLL1, DLL4, TGFb, etc. were each applied independently) were dissolved in PBS, and the amount of protein was adjusted and added so that the protein was labeled on the surface of the particles as described in Table 4.

[0377] 3M ammonium sulfate of the same volume as the PBS solution mixed with the particle obtained in (1) above and the protein for immune cell differentiation was added so that the final solution became 1.5M ammonium sulfate. Subsequently, the reaction was carried out for more than 16 hours at room temperature in a shaking incubator while shaking at a speed of 250 rpm or more, thereby covalently bonding the epoxy group exposed on the surface of the particle obtained in (1) above with the amine group or thiol group of the protein.

[0378] Subsequently, the protein-covalently bonded particles were separated from the mixed solution by centrifugation at 10,000 xg for 10 minutes, the supernatant was removed, pH 12 Tris buffer was added, and vortexing was performed. After removing the buffer by centrifugation, pH 4 Acetate buffer was added and vortexing was performed. Subsequently, the protein-covalently bonded particles obtained by washing three more times with Tween 20 0.5 wt% PBS were used as the particle complexes of Comparative Examples 6 to 9. The particle complexes were placed in PBS and stored at 4°C.

[0379]

[0380] Monomer (weight%) Crosslinking Agent (weight%) GMAEHMAHEMADMAEGDMATMPTMA Example 4 33.346.71010 Example 5 33.346.71010 Example 6 33.346.71010 Example 7 16.758.351010 Comparative Example 5------Comparative Example 6 33.35016.7 Comparative Example 7 33.35016.7 Comparative Example 8 33.35016.7 Comparative Example 9 33.35016.7

[0381] The content of each component in Table 3 above refers to the weight % based on the total sum of monomers and crosslinking agents.

[0382] GMA: Glycidyl methacrylate

[0383] EHMA: 2-Ethylhexyl methacrylate

[0384] HEMA: 2-Hydroxyethyl methacrylate

[0385] DMA: Dodecyl methacrylate

[0386] EGDMA: Ethylene glycol dimethacrylate

[0387] TMPTMA: Trimethylolpropane trimethacrylate

[0388]

[0389] <Experimental Example: Measurement of Physical Properties of Particle Composites>

[0390] For the microparticles and particle composites obtained in the above examples and comparative examples, physical properties were measured by the following method, and the results are shown in Table 4.

[0391]

[0392] Experiment 1. Average particle size (Unit: μm)

[0393] For the microparticles obtained in the above examples and comparative examples, the D50 (particle size value corresponding to the cumulative distribution percentage reaching 50%) particle diameter was measured using image analysis software after observation with a microscope.

[0394]

[0395] Experiment 2. Apparent Density (Unit: g / cm³) 3 )

[0396] For the microparticles prepared in the above examples and comparative examples, various densities (1.02 g / cm³) were obtained using OptiPrep Density Gradient Medium (Sigma-Aldrich) under conditions of room temperature (25 ℃) and atmospheric pressure (1 atm). 3 , 1.04 g / cm 3 , 1.06 g / cm 3 , 1.08 g / cm 3 , 1.1 g / cm 3 , 1.12 g / cm 3 , 1.14 g / cm 3 , 1.16 g / cm 3 , 1.18 g / cm 3 , 1.20 g / cm 3 , 1.22 g / cm 3 Solutions of ) were prepared. The density of the particles is evaluated by dispersing them in each solution and observing.

[0397] 1) Particle density = Solution density

[0398] After 5 minutes have passed since the particles were dispersed in the solution, if it is observed that the particles are not settling or floating in the solution but are dispersed throughout, it is determined that the density of the solution and the density of the particles are the same.

[0399] 2) Particle density > Solution density

[0400] After dispersing particles in a solution, if it is observed that most of the particles settle in the solution after 5 minutes, it is determined that the particle density is higher than the density of the solution.

[0401] 3) Particle density < Solution density

[0402] After dispersing particles in a solution, if it is observed that most of the particles are suspended in the solution after 5 minutes, it is determined that the particle density is lower than the density of the solution.

[0403]

[0404] Experiment 3. Epoxy Content Analysis

[0405] 0.1 g of microparticles prepared in the above examples and comparative examples were dispersed in an HCl / acetone (volume ratio = 1:80) solution and then sonicated for 4 minutes.

[0406] After adding 2 drops of indicator solution (0.1 wt% cresol red + 0.1 wt% thymol blue, volume ratio 1:3, pH 7, 0.01 M NaOH), the solution was titrated with 0.1 M NaOH.

[0407] The epoxy content (μmol / g) was calculated using the following formula.

[0408] [Mathematical Formula]

[0409] Epoxy content (μmol / g) = [V0(ml)-V(ml)]*C NaOH (mol / L)

[0410] In the above mathematical formula, V0 is the volume of NaOH added to the control group containing 0.1g of DIW instead of particles, V is the amount of NaOH added to the actual sample, and C NaOH represents the concentration of NaOH used in the titration.

[0411]

[0412] Experiment 6. Protein Stability

[0413] The particle complex obtained in the above examples and comparative examples was dissolved in PBS at a protein concentration of 1 µg / mL in a 1 mL EP tube and reacted in a shaking incubator at room temperature for at least 7 hours while shaking at a speed of 250 rpm or higher. Afterward, the supernatant obtained by centrifugation at 10,000 xg for 10 minutes was collected. Then, an ELISA (Enzyme-Linked Immunosorbent Assay) was performed on the supernatant to measure the mass of protein released from the particle complex, the ratio of free protein was calculated according to the following Equation 1, and protein stability was evaluated under the following criteria.

[0414] [Mathematical Formula 1]

[0415] Free protein percentage (%) = (W1 / W2) * 100

[0416] In the above mathematical formula 1,

[0417] W1 is the mass of protein released from the particle complex after shaking culture and centrifugation of the solution containing the particle complex, and

[0418] W2 is the mass of protein immobilized on the particle complex before shaking culture and centrifugation of the solution containing the particle complex.

[0419] [Evaluation Criteria]

[0420] Top: Free protein ratio according to Mathematical Formula 1 is 0.1% or less

[0421] Medium: Free protein ratio according to Mathematical Formula 1 exceeds 0.1% and is 10% or less

[0422] H: Free protein ratio according to Mathematical Formula 1 exceeds 10%

[0423]

[0424] Experiment 7. Protein Binding Density

[0425] 80 µL of the solution containing the dispersed particle complexes obtained in the above examples and comparative examples was added to 540 µL of BCA reaction solution (reagent, Thermofisher Scientific) and reacted at 37°C at 250 rpm for 30 minutes. Eight types of solutions with unbound proteins ranging from 0 to 2000 µg / mL were prepared and reacted as described above to create standard solutions. 200 µL of these solutions were dispensed into 96-well plates, and the absorbance was measured at 560 nm. After constructing a standard curve based on the concentration-absorbance of the unbound proteins, the concentrations were inversely calculated using the absorbances of the samples. The protein binding density was calculated in µg / cm² using the concentration and specific surface area of ​​each particle complex. 2 Convert to.

[0426] [metewand]

[0427] Deficient particles: Protein binding density < 0.2 ug / cm³ 2

[0428] Optimal particle size: 0.2 < protein binding density < 2 ug / cm³ 2

[0429] Excess particles: Protein binding density > 2 µg / cm³ 2

[0430]

[0431] Experiment 8. Cell Differentiation Performance

[0432] The particle complexes obtained in the above examples and comparative examples are sterilized with 70% ethanol for 15 minutes, washed three times with PBS, and then placed into each well of a 24-well plate in quantities ranging from 1,000 to 400,000 as desired. 50,000 HSC cells are placed into each well of the 24-well plate along with 1 mL of cell culture medium and cultured with the particles for one week in a 37°C, 25% CO₂ incubator. After one week, the particles are removed using a cell strainer. After culturing for another two weeks in a 37°C, 25% CO₂ incubator, the total number of cells is measured, and the cell differentiation / proliferation rate is calculated by comparing it to the initial number of cells. This proliferation rate is compared to a control group coated with protein on a plate.

[0433] [metewand]

[0434] Upper: Proliferation rate of 80% or more compared to the control group

[0435] Medium: Growth rate of 40%-80% compared to the control group

[0436] H: Proliferation rate of 40% or less compared to the control group

[0437]

[0438] Average particle size (㎛, D50) Apparent density (g / cm³) 3 )Epoxy content (μmol / g) Protein stability Protein binding density (ug / cm²) 2 Cell differentiation performance Example 4 48.41.12800 Upper 0.37 Lower Example 5 48.41.12800 Upper 0.43 Lower Example 6 48.41.12800 Upper 0.29 Lower Example 7 49.01.08400 Upper 0.33 Upper Comparative Example 5 1551.00 - Lower 0.10 Lower Comparative Example 6 123.81.24 - Upper 0.04 Lower Comparative Example 7 123.81.24 - Upper 2.32 Lower Comparative Example 8 123.81.24 - Upper 2.82 Lower Comparative Example 9 123.81.24 - Upper 5.31 Lower

[0439] As shown in Table 4 above, the particle composite of the example exhibited excellent protein binding density under cell culture conditions compared to the comparative example, and it was confirmed that it had excellent cell differentiation performance.

Claims

1. 2 (meth)acrylate copolymer comprising different repeating units, and The epoxy content is 50 μmol / g or more and 2000 μmol / g or less, and The acrylate copolymer comprising two or more different repeating units is a polyacrylic microparticle comprising a (meth)acrylate repeating unit comprising a reactive functional group and a (meth)acrylate repeating unit comprising an aliphatic functional group.

2. In Paragraph 1, Polyacrylic microparticles comprising an acrylate copolymer containing two or more different repeating units, wherein the copolymer comprises 40 parts by weight or more and 750 parts by weight or less of an aliphatic functional group (meth)acrylate repeating unit per 100 parts by weight of a (meth)acrylate repeating unit containing a reactive functional group.

3. In Paragraph 1, The above polyacrylic microparticles have an apparent density of 1.15 g / cm³ 3 Polyacrylic microparticles.

4. In Paragraph 1, Polyacrylic microparticles comprising (meth)acrylate repeating units containing aliphatic functional groups, wherein the (meth)acrylate repeating units containing aliphatic functional groups having 6 or more carbon atoms.

5. In Paragraph 1, Polyacrylic microparticles comprising (meth)acrylate repeating units containing the above-mentioned reactive functional groups, wherein the (meth)acrylate repeating units comprises repeating units derived from a compound represented by the following chemical formula 1: [Chemical Formula 1] In the above chemical formula 1, L1 and L2 are each independently alkylene groups having 1 or more carbon atoms, and R1 is a reactive functional group capable of ring-opening reactions, and n is an integer greater than or equal to 0.

6. In Paragraph 1, Polyacrylic microparticles comprising (meth)acrylate repeating units containing the above-mentioned aliphatic functional groups, wherein the repeating units are derived from a compound represented by the following chemical formula 2: [Chemical Formula 2] In the above chemical formula 2 R2 is an alkyl group with 6 or more carbon atoms.

7. In Paragraph 1, Polyacrylic microparticles comprising an acrylate copolymer containing two or more different repeating units, wherein the (meth)acrylate repeating unit containing the reactive functional group is present in an amount of 10 parts by weight or more and less than 50 parts by weight per 100 parts by weight of the total repeating unit.

8. In Paragraph 1, Polyacrylic microparticles comprising an acrylate copolymer containing two or more different repeating units, wherein the copolymer comprises 20 parts by weight or more and less than 75 parts by weight of (meth)acrylate repeating units containing aliphatic functional groups per 100 parts by weight of total repeating units.

9. In Paragraph 5, Polyacrylic microparticles comprising any one of the compounds represented by the above chemical formula 1 to the compounds represented by the following chemical formula 1-1 to 1-3: [Chemical Formula 1-1] In the above chemical formula 1-1, R 11 is hydrogen or an alkyl group having 1 or more carbon atoms, and [Chemical Formula 1-2] In the above chemical formula 1-2, R 12 is hydrogen or an alkyl group having 1 or more carbon atoms, and [Chemical Formula 1-3] In the above chemical formula 1-3, R 13 It is hydrogen or an alkyl group with 1 or more carbon atoms.

10. In Paragraph 1, The acrylate copolymer comprising two or more different repeating units above is a polyacrylic microparticle comprising repeating units derived from a compound represented by the following chemical formula 3: [Chemical Formula 3] In the above chemical formula 3, L3 is an alkylene group with 1 or more carbon atoms.

11. In Paragraph 10, Polyacrylic microparticles comprising an acrylate copolymer containing two or more different repeating units, wherein the copolymer comprises 1 part by weight or more and 20 parts by weight or less of a repeating unit derived from a compound represented by Chemical Formula 3, based on 100 parts by weight of total repeating units.

12. In Paragraph 10, Polyacrylic microparticles comprising an acrylate copolymer containing two or more different repeating units, wherein the copolymer contains 1 part by weight or more and 20 parts by weight or less of a repeating unit derived from a compound represented by Formula 3, per 100 parts by weight of a (meth)acrylate repeating unit containing an aliphatic functional group.

13. In Paragraph 1, The above particles are polyacrylic microparticles comprising a single particle or a group of particles composed of multiple single particles.

14. In Paragraph 1, Polyacrylic microparticles having a D50 particle diameter of 45 μm to 55 μm.

15. Polyacrylic microparticles of claim 1; and A particle complex comprising a protein fixed to the surface of the above particle.

16. In Paragraph 15, The above particle complex has a protein binding density of 0.2 μg / cm² 2 Above 2.0 µg / cm² 2 Lee Ha-in, particle complex.

17. In Paragraph 15, A particle complex in which the above-mentioned protein is fixed to the particle surface by covalent or physical bonds.

18. In Paragraph 17, The above covalent bond comprises a bonding functional group represented by the following chemical formula 4, in a particle complex: [Chemical Formula 4] In the above chemical formula 4, R 10 It is hydrogen or alkyl.

19. In Paragraph 17, The above-mentioned covalent bond comprises a bond represented by the following chemical formula 4-1, in a particle complex: [Chemical Formula 4-1] In the above chemical formula 4-1, R 10 It is hydrogen or alkyl, and X is a polyacrylic microparticle, and Y is a protein.

20. In Paragraph 17, The above covalent bond comprises a bonding functional group represented by the following chemical formula 5, in a particle complex: [Chemical Formula 5] .

21. In Paragraph 17, The above-mentioned covalent bond comprises a bond represented by the following chemical formula 6, a particle complex: [Chemical Formula 6] In the above chemical formula 6, X is a polyacrylic microparticle, and Y is a protein.

22. In Paragraph 17, The above covalent bond comprises a bonding functional group represented by the following chemical formula 7, in a particle complex: [Chemical Formula 7] In the above chemical formula 7, R 20 is hydrogen or alkyl.

23. In Paragraph 17, The above-mentioned covalent bond comprises a bond represented by the following chemical formula 8, a particle complex: [Chemical Formula 8] In the above chemical formula 8, R 20 is hydrogen or alkyl, and X is a particle, and Y is a protein.

24. In Paragraph 17, A particle complex in which the above-mentioned covalent bond is formed by the reaction of an epoxy group or carboxyl group on the particle surface with an amine group or thiol group contained in a protein.

25. In Paragraph 15, The above protein is a particle complex that is a protein for immune cell differentiation.

26. Cells; and A cell culture composition comprising the polyacrylic microparticles of claim 1 or the particle complex of claim 15.

27. A step of culturing the cell culture composition of claim 26; and A method for manufacturing a cell therapeutic agent, comprising the step of removing the polyacrylic microparticles of claim 1 or the particle complex of claim 15 from the culture product.

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