Particle complex, cell culture composition using same, and method for producing cell therapeutic agent

Abstract

The present invention relates to a particle complex, a cell culture composition using same, and a method for preparing a cell therapeutic agent, wherein the particle complex comprises: spherical particles; and a protein immobilized on the surface of the particles.

Description

Particle complex, cell culture composition using the same, and method for producing cell therapeutic agent

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

[0002] This application claims the benefit of priority to Korean Patent Application No. 10-2023-0152073, filed November 6, 2023, Korean Patent Application No. 10-2024-0021286, filed February 14, 2024, and Korean Patent Application No. 10-2024-0154399, filed November 4, 2024, the entire contents of which are incorporated herein by reference.

[0003] The present invention relates to a particle complex, a cell culture composition using the same, and a method for producing a cell therapeutic agent.

[0004] As the fields of biopharmaceuticals and regenerative medicine expand, the demand for cell mass culture technologies that can efficiently produce cells, tissues, and microorganisms is increasing.

[0005] Meanwhile, existing anticancer cell therapy uses T cells (autologous cells) derived from each patient, so it takes a long time and costs money to increase the amount of cells, and since cancer patients in general have low immune cell function, there was a disadvantage in that it was not possible to guarantee quality that could be used for treatment.

[0006] To address this, attempts have been made to differentiate and utilize stem cells, such as induced pluripotent stem cells (iPSCs) derived from other cells. This differentiation requires protein stimulation, and when applied on a flat (2D) surface, it presents limitations in terms of mass production and automation.

[0007] Accordingly, along with the 3D conversion of particle-utilizing processes, there is a need for the development of a technology that allows particles to be completely separated from cells after culture.

[0008] The present invention relates to a technology for securing the stability of a differentiation platform by covalently fixing a protein necessary for cell differentiation, and providing a particle complex that can be completely separated after cell culture to secure the stability of a cell therapy product.

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

[0010] In addition, the present invention relates to a method for manufacturing a cell therapeutic agent using the particle complex.

[0011] To solve the above problem, the present specification provides a particle complex comprising a spherical particle; and a protein fixed to the surface of the particle.

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

[0013] The present specification also provides a method for producing a cell therapeutic agent, comprising: a step of culturing the cell culture composition; and a step of removing a particle complex from the culture result.

[0014] Hereinafter, a particle complex according to a specific embodiment of the invention, a cell culture composition using the same, and a method for manufacturing a cell therapeutic agent will be described in more detail.

[0015]

[0016] Unless explicitly stated otherwise in this specification, terminology is used only to describe specific embodiments and is not intended to limit the invention.

[0017] As used herein, the singular forms also include the plural forms unless the context clearly dictates otherwise.

[0018] As used herein, the term “including” means specifying a particular characteristic, region, integer, step, operation, element and / or component, but does not exclude the presence or addition of other particular characteristics, regions, integers, steps, operations, elements, components and / or groups.

[0019] In this specification, the term "substitution" means that another functional group is bonded instead of a hydrogen atom in a compound, and the position of substitution is not limited as long as it is a position where a hydrogen atom is substituted, i.e., a position where a substituent can be substituted, and when two or more are substituted, the two or more substituents may be the same or different from each other.

[0020] The term "substituted or unsubstituted" as used herein means a group unsubstituted or substituted with one or more substituents selected from the group consisting of deuterium; a halogen group; a cyano group; a nitro group; a hydroxy group; a carbonyl group; an ester group; an imide group; an amide group; a primary amino group; a carboxyl group; a sulfonic acid group; a sulfonamide group; a phosphine oxide group; an alkoxy group; an aryloxy group; an alkylthioxy group; an arylthioxy group; an alkylsulfoxy group; an arylsulfoxy group; a silyl group; a boron group; an alkyl group; a cycloalkyl group; an alkenyl group; an aryl group; an aralkyl group; an aralkenyl group; an alkylaryl group; an alkoxysilylalkyl group; an arylphosphine group; or a heterocyclic group containing one or more of N, O, and S atoms, or a substituted or unsubstituted group in which two or more of the above-mentioned substituents are linked. For example, a "substituent having two or more substituents connected" may be a biphenyl group. That is, the biphenyl group may be an aryl group, or may be interpreted as a substituent having two phenyl groups connected.

[0021] In this specification, , or means a bond that is connected to another substituent, and a direct bond means that there is no separate atom in the part indicated by L.

[0022] In the present specification, an alkyl group is a monovalent functional group derived from an alkane, which may be straight or branched, and the number of carbon atoms in the straight-chain alkyl group is not particularly limited, but is preferably 1 to 20. In addition, the number of carbon atoms in the branched-chain alkyl group is 3 to 20. 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, octyl, n-octyl, tert-octyl, 1-methylheptyl, 2-ethylhexyl, 2-propylpentyl, n-nonyl, 2,2-dimethylheptyl, 1-ethyl-propyl, 1,1-dimethyl-propyl, isohexyl, 2-methylpentyl, 4-methylhexyl, Examples thereof include, but are not limited to, 5-methylhexyl, 2,6-dimethylheptan-4-yl, etc. The alkyl group may be substituted or unsubstituted, and examples of the substituent when substituted are as described above.

[0023] In this specification, (meth)acrylate is meant to include both acrylate and methacrylate.

[0024] In this specification, “dispersed phase composition” means a composition capable of forming a dispersed phase (or droplets) after being mixed with a continuous phase composition.

[0025] In this specification, “continuous phase composition” means a composition that can form a continuous phase after being mixed with a dispersed phase composition.

[0026] In this specification, unless otherwise specifically defined or explained, the temperature at which the manufacturing process is performed (or each manufacturing step) or the temperature at which the numerical properties of the manufactured particles are calculated or measured may be room temperature. Specifically, "room temperature" in this specification may mean a temperature in a state where no heating or cooling is performed, for example, a temperature in the range of 15 to 30°C.

[0027] Hereinafter, the present invention will be described in more detail.

[0028]

[0029] 1. Particle complex

[0030] According to one embodiment of the invention, a particle complex may be provided, comprising: a spherical particle; and a protein fixed to the surface of the particle.

[0031] The inventors of the present invention have experimentally confirmed that, in the case of the particle complex of the above embodiment, the structural stability of the particle complex can be secured by fixing the protein to the surface of a spherical particle through a physical bond such as a chemical covalent bond or adsorption, and the stability of the cell therapy product can be secured by completely separating the particle complex from the cell culture medium using a cell strainer, thereby completing the invention.

[0032] Specifically, the particle complex of the above embodiment may comprise particles. The particle may be spherical in shape. The spherical particles not only have excellent durability and are not damaged or destroyed by external impacts, but also have a uniform particle surface and a large surface area, which can enhance protein fixation efficiency.

[0033] Whether the above particles have a spherical shape can be visually determined through SEM. The above spherical particles may theoretically include spherical particles, which are perfectly spherical, or particles that are not spherical but have a shape close to a spherical shape.

[0034] More specifically, the circularity of the spherical particles related to the present embodiment is typically 0.88 or more, or 0.90 or more, or 0.91 or more, or 0.92 or more, or 1 or less, or 0.98 or less, or 0.97 or less, or 0.88 to 1, or 0.90 to 1, or 0.91 to 1, or 0.92 to 1.

[0035] When the circularity of the above spherical particles is within the above range, there is a tendency to suppress the deterioration of particle durability and protein fixation performance. In addition, circularity is defined by the following equation, and when the circularity is 1, the particle is theoretically spherical. Conversely, the circularity value decreases as the particle shape moves away from a sphere.

[0036] Circularity = (perimeter of equivalent circle with same area as particle projection shape) / (actual perimeter of particle projection shape)

[0037] Specifically, the examples of the method for measuring the circularity are not particularly limited, but for example, it can be measured from a cross-sectional SEM image. Specifically, the particle area S [㎛] obtained from the cross-sectional SEM image 2 ], and is calculated from the following equation using the circumferential length L [㎛].

[0038] Circularity = 4πS / (L) 2

[0039] There are no specific restrictions on the method for determining particle boundaries, and commercially available analysis software can be used either automatically or manually. Polygonal approximations are preferred, with a polygonal approximation of 15 or more sides being more preferable. If the polygonal approximation is less than 15 sides, the background portion may be treated as part of the particle when approximating the curved portion.

[0040] The particle cross-section image is obtained using a reflection electron image acquired at an acceleration voltage of 5 kV using a scanning electron microscope (SEM). The method for preparing a sample for SEM observation to obtain a particle cross-section image is not particularly limited; after cutting the particle cross-section to prepare a sample, the particle cross-section image is obtained using an SEM. In addition, the imaging magnification is typically 10 times or more, or 50 times or more, or 100 times or more, or 1000 times or less.

[0041] However, when using conventional, widely used culture plates instead of the aforementioned spherical particles, protein immobilization relies on simple adsorption rather than covalent bonding, making it difficult to control the amount and uniformity of protein. Furthermore, protein coating on flat plates is difficult to mass-produce and automate, making protein immobilization on particles essential for mass culture.

[0042] In addition, the particles may have a D50 diameter of more than 20 μm, or less than 180 μm, or more than 20 μm and less than 180 μm, or from 30 μm to 175 μm, or from 50 μm to 160 μm, or from 50 μm to 130 μm, or from 50 μm to 80 μm, or from 80 μm to 120 μm, or from 120 μm to 160 μm. When the D50 diameter of the particles satisfies the above-described range, cell differentiation and culture performance are excellent.

[0043] The diameter of the particle refers to the distance between two points where a straight line passing through the center of gravity of the particle meets the outermost surface of the particle, and the D50 diameter of the particle refers to the size of the 50th particle when the total number of particles is converted to 100.

[0044] The method for measuring the D50 diameter of the above particles is not particularly limited, and various conventionally known methods for measuring the D50 diameter of particles can be applied without limitation. However, as an example, the measurement can be made by analyzing a SEM (scanning electron microscopy) image.

[0045] If the D50 diameter of the above particles increases excessively beyond the above-described range, the surface area to volume decreases, which may result in a problem of reduced cell differentiation efficiency due to reduced interaction between cells and particles.

[0046] On the other hand, if the D50 diameter of the particles is excessively reduced beyond the above-described range, it is difficult to completely separate the particle complex from the cell culture medium using a cell strainer, which may cause problems with the safety of the final cell therapy product.

[0047] The above particle may comprise a single particle or a particle group composed of multiple single particles. The single particle refers to one particle, and the particle group refers to a plurality of particle matrices in which two or more particles are mixed.

[0048] At this time, the single particle may include a protein immobilized on its surface. That is, a protein immobilized on the surface of a single particle may be included. Furthermore, in a particle group comprising two or more particles, a protein immobilized on the surface of each single particle may be included.

[0049] The above particles may be suspension polymerization particles. Suspension polymerization particles refer to particles obtained through a step of mixing a continuous phase composition and a disperse phase composition and performing polymerization in a suspension state.

[0050] The above-mentioned dispersed phase composition means a composition capable of forming a dispersed phase (or droplet) after being mixed with a continuous phase composition, and the above-mentioned continuous phase composition means a composition capable of forming a continuous phase after being mixed with a dispersed phase composition.

[0051] In one example, the dispersed phase composition may comprise a polymerizable monomer.

[0052] In one example of the present application, the polymerizable monomer is a monomer having an unsaturated bond between carbon atoms, and may be a monomer further including 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 above-described group may be referred to as a hydrophilic group.

[0053] In one example, the polymerizable monomer may be a (meth)acrylate monomer. In this case, the (meth)acrylate monomer having a hydrophilic group may include one or more of the hydrophilic groups described above.

[0054] Although not particularly limited, examples of (meth)acrylate monomers having an epoxy group as a hydrophilic group include glycidyl (meth)acrylate, 4,5-epoxybutyl (meth)acrylate, or 9,10-epoxy stearyl (meth)acrylate.

[0055] Although not particularly limited, (meth)acrylate monomers having an amide group as a hydrophilic group include, for example, (meth)acrylamide or N-methylol (meth)acrylamide.

[0056] Although not particularly limited, examples of (meth)acrylate monomers having a carboxyl group as a hydrophilic group include acrylic acid, methacrylic acid, maleic acid, itaconic acid, etc.

[0057] Although not particularly limited, the (meth)acrylate monomer having an alkoxy group as a hydrophilic group may have a methoxy group or an ethoxy group. For example, methoxyethyl (meth)acrylate may be used.

[0058] Although not particularly limited, the (meth)acrylate monomer having a hydroxyl group as a hydrophilic group may be hydroxyethyl (meth)acrylate, hydroxypropyl (meth)acrylate, or hydroxybutyl (meth)acrylate.

[0059] One or more of the monomers listed above may be used as a (meth)acrylate monomer having a hydrophilic group. In a specific example of the present application, a monomer having an unsaturated bond between carbon atoms and an epoxy group may be used as the polymerizable monomer.

[0060] In one example, the above-described dispersed composition may further comprise a cross-linking agent. The usable cross-linking agent is not particularly limited, but may include, for example, a multifunctional (meth)acrylate such as ethylene glycol di(meth)acrylate, trimethylolpropane tri(meth)acrylate, pentaerythritol tri(meth)acrylate, dipentaerythritol hexa(meth)acrylate, dipentaerythritol hexamethacrylate, or trimethylpropane trimethacrylate. Alternatively, one or more of the cross-linking agents listed above may be used together. Considering the formation of a hydrophilic surface of the particles, etc., it may be preferable to use ethylene glycol di(meth)acrylate.

[0061] In one example, the dispersed composition may contain the cross-linking agent in an amount of 50 to 500 parts by weight relative to 100 parts by weight of the polymerizable monomer. Specifically, the content of the cross-linking agent may be 100 parts by weight or more, 150 parts by weight or more, 200 parts by weight or more, 250 parts by weight or more, or 300 parts by weight or more. In addition, the upper limit may be, for example, 450 parts by weight or less, 400 parts by weight or less, or 350 parts by weight or less. When the above-described content is satisfied, it is advantageous in stably forming an emulsion and securing particle strength and morphological characteristics through a desired level of cross-linking.

[0062] In one example, the above-described dispersed composition may further include an initiator. The type of initiator is not particularly limited as long as it does not hinder securing particle characteristics according to the manufacturing method of the present application. For example, initiators such as organic peroxide initiators or azo initiators may be used. Specifically, compounds such as 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)hexyne-3, or di-cumyl peroxide, and mixtures thereof may be used, but are not limited thereto.

[0063] The content of the initiator is not particularly limited. An appropriate amount of the initiator may be used as long as it does not hinder securing the characteristics of the desired particles. For example, the dispersed phase composition may contain 0.1 part by weight or more, specifically 0.2 part by weight or more, 0.3 part by weight or more, 0.4 part by weight or more, 0.5 part by weight or more, 0.6 part by weight or more, 0.7 part by weight or more, 0.8 part by weight or more, 0.9 part by weight or more, or 1.0 part by weight or more of the initiator, based on 100 parts by weight of the combined weight of the polymerizable monomer and the crosslinking agent. In addition, the upper limit of the content of the initiator may be, for example, 5.0 parts by weight or less, specifically, 4.5 parts by weight or less, 4.0 parts by weight or less, 3.5 parts by weight or less, 3.0 parts by weight or less, 2.5 parts by weight or less, 2.0 parts by weight or less, or 1.5 parts by weight or less.

[0064] In one example, the continuous phase composition may comprise water and a polymeric surfactant.

[0065] Although not specifically limited, the water may be distilled or deionized water.

[0066] In one example, the continuous phase composition may be a mixture of water and a polymeric surfactant. That is, the continuous phase composition may consist solely of water and a polymeric surfactant.

[0067] In a specific example of the present application, the polymer surfactant has a weight average molecular weight (M) in a predetermined range. W ) and / or may have a hydration degree (hydrolysis degree) of a certain range.

[0068] In one example, the weight average molecular weight of the polymer surfactant may be 60,000 or more. Specifically, the lower limit of the weight average molecular weight may be, for example, 65,000 or more, 70,000 or more, 80,000 or more, or 85,000 or more. And, the upper limit of the weight average molecular weight may be, for example, 190,000 or less, 185,000 or less, 180,000 or less, 175,000 or less, 170,000 or less, 165,000 or less, 160,000 or less, 155,000 or less, 150,000 or less, 145,000 or less, 140,000 or less, 135,000 or less, 130,000 or less, 125,000 or less, or 124,000 or less. The weight average molecular weight can be measured using GPC.

[0069] In one example, the polymeric surfactant may have a degree of hydration in the range of 80 to 99%. More specifically, the lower limit of the degree of hydration may be, for example, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, or 87% or more, and the upper limit may be, for example, 98% or less, 97% or less, 96% or less, 95% or less, 94% or less, 93% or less, 92% or less, 91% or less, 90% or less, or 89% or less. As a surfactant having the degree of hydration as described above, polyvinyl alcohol may be used, for example. The degree of hydration may be measured using 1H-NMR.

[0070] Examples of usable polymeric surfactants include poly(vinyl alchol) (PVA) or polyvinylpyrrolidone.

[0071] In one example, in the continuous phase composition, the concentration of the polymer surfactant may be 1.0% or more. For example, when a continuous phase composition that is a mixture of water and a polymer surfactant is used, the content of the polymer surfactant may be 1.0% by weight or more based on the total weight (100% by weight) of the continuous phase composition. Specifically, the lower limit of the concentration of the polymer surfactant in the continuous phase composition may be 1.5% by weight or more, or 2.0% by weight or more. And, the upper limit of the concentration of the polymer surfactant may be less than 4.0%, specifically, for example, 3.5% or less, 3.0% or less, or 2.5% or less. When a polymer surfactant satisfying the above concentration is used, uniform and stable droplets can be formed when the continuous phase and the powder phase are mixed, and it is advantageous in obtaining the shape of the final particles targeted in the present application.

[0072] In one example, a mixture comprising the dispersed phase and the continuous phase (or a mixture of the dispersed phase and the continuous phase) can satisfy a viscosity in the range of 1.0 to 2.5 cP. Within this viscosity range, collisions and breakage between liquid crystals or particles can be reduced, and the shape of the manufactured particles can be made more uniform. The viscosity can be measured according to the shear rate. The viscosity can be measured using a rotational viscometer, LVDV2T (Brookfield), at a shear rate range of 66 to 264 1 / s and at room temperature (15 to 30°C).

[0073] In one example, the mixture comprising the dispersed phase and the continuous phase has a density of 0.99 to 1.05 g / cm 3 It can satisfy the density of the range.

[0074] The above suspension polymerization can be carried out under conditions that do not hinder securing particle characteristics according to the manufacturing method of the present application. For example, the suspension polymerization reaction can be carried out at a temperature of 100°C or lower, more specifically, at a temperature of 40°C to 95°C. Furthermore, during the polymerization reaction at the above temperature, stirring can be performed at a speed of 600 rpm to 1,000 rpm.

[0075] In one example, the method may further include a washing step after the suspension polymerization reaction is completed. Washing can remove impurities unrelated to the particles in the suspension polymer. The washing method is not particularly limited, and any known washing method can be used. For example, the washing may be performed by placing the suspension polymer in a washing solution containing alcohol (e.g., alcohol including ethanol) and / or water (e.g., distilled water) and stirring the solution. Although not particularly limited, this washing process may be repeated, for example, two or more times.

[0076] In one example, the method may further include a drying step after the washing. Drying can remove solvent residues and the like. The drying method is not particularly limited, and any known drying method can be used. For example, the drying may be performed using an oven or at room temperature. Furthermore, although not particularly limited, the drying may be performed in a vacuum.

[0077] In one example, the method may further include, after the suspension polymerization reaction is completed, the steps of centrifuging, washing, and drying the suspension polymer. The descriptions of washing and drying are the same as those described above.

[0078]

[0079] Additionally, the particle complex of the above embodiment may include particles having a coefficient of variation of particle diameter of 20% or less, or 10% or less, or 9% or less, or 8% or less, or 7% or less, or 5% or less, or 3% or less, or 2% or less, or 0.1% or more, or 0.1% to 20%, or 0.1% to 10%, or 0.1% to 9%, or 0.1% to 8%, or 0.1% to 7%, or 0.1% to 5%, or 0.1% to 3%, or 0.1% to 2%.

[0080] Examples of methods for measuring the coefficient of variation of the above particles are not limited, but can be obtained, for example, by the following mathematical formula 2.

[0081] [Mathematical Formula 2]

[0082] Coefficient of variation (%) = (Standard deviation of particle diameter / Average particle diameter) X 100

[0083] The standard deviation and average particle diameter of the particle diameter of the above mathematical formula 2 can be measured by analyzing the SEM (scanning electron microscopy) image.

[0084] As the coefficient of variation of the particle diameter decreases to the above-described range, the particles can be produced with a uniform size, thereby maximizing particle performance.

[0085] On the other hand, if the coefficient of variation of the particle diameter increases excessively, such as exceeding 20%, the particle diameter deviation is large and uneven, resulting in the generation of large particles, which reduces reproducibility in the production of cell therapy products and may cause a problem of reduced production efficiency due to the requirement of a separate classification process.

[0086] Meanwhile, the particles may be microfluidic chip particles. That is, particles having a coefficient of variation of the particle diameter of 20% or less, or 10% or less, or 9% or less, or 8% or less, or 7% or less, or 5% or less, or 3% or less, or 2% or less, or 0.1% or more, or 0.1% to 20%, or 0.1% to 10%, or 0.1% to 9%, or 0.1% to 8%, or 0.1% to 7%, or 0.1% to 5%, or 0.1% to 3%, or 0.1% to 2% may be microfluidic chip particles. On the other hand, when the particles are suspended polymer particles, the coefficient of variation of the particle diameter increases to more than 20%.

[0087] Specifically, the particle complex of the above embodiment may be a particle complex comprising a spherical particle; and a protein fixed to the surface of the particle; wherein the particle has a coefficient of variation of a particle diameter of 20% or less. In addition, the particle complex of the above embodiment may be a particle complex comprising a spherical particle; and a protein fixed to the surface of the particle; wherein the particle has a coefficient of variation of a particle diameter of 20% or less, and wherein the particle is a microfluidic chip particle.

[0088] More specifically, the microfluidic chip particles may be particles obtained through a method for producing particles, including the steps of: a) injecting a dispersed phase composition including a polymerizable monomer into a continuous phase composition through a microchannel to generate droplets composed of the dispersed phase composition within the continuous phase composition; and b) photopolymerizing the droplets.

[0089] In the step of generating the above a) droplets, droplets of uniform size and shape can be manufactured through a specific method.

[0090] In the step of generating droplets above a), a dispersed phase composition forming droplets is injected into a continuous phase composition through a microchannel to generate droplets, thereby producing droplets of a uniform size.

[0091] Specifically, in the step a) of generating the droplets, a microfluidic device including a microchannel may be used.

[0092] For example, the microfluidic device may include a first supply portion through which a dispersed phase composition is supplied; a first flow path through which the dispersed phase composition supplied from the first supply portion can flow; a second supply portion through which a continuous phase composition is supplied; a second flow path through which the continuous phase composition supplied from the second supply portion can flow; and a plurality of microflow paths connecting sides of the first and second flow paths. The side of the flow path refers to a direction other than the direction of fluid flow within the flow path.

[0093] More specifically, referring to FIG. 3, the microfluidic device may include a first supply unit (10) through which a dispersed phase composition is supplied; a first flow path (11) through which the dispersed phase composition supplied from the first supply unit can flow; a second supply unit (20) through which a continuous phase composition is supplied; a second flow path (21) through which the continuous phase composition supplied from the second supply unit can flow; and a plurality of microflow paths (12) connecting side surfaces of the first and second flow paths to each other. FIG. 1 is a drawing of a microfluidic device having a structure in which a first flow path (11) through which a dispersed phase composition flows is arranged between two second flow paths (21) through which a continuous phase composition flows, and both side surfaces of the first flow path (11) are connected to side surfaces of the two second flow paths (21) through a plurality of microflow paths, and is an example in which a plurality of microflow paths are arranged as densely as possible. The structure of the above microfluidic device is not limited to FIG. 3, and can be freely modified within a range that can achieve the purpose of the present invention with reference to FIG. 3.

[0094] The first and second channels can be formed spaced apart from each other by the length of the microchannel of the desired length. The height and length of the microchannel are not particularly limited and can be appropriately adjusted depending on the size of the desired droplet.

[0095] In the step a) of generating droplets, a dispersed phase composition may be supplied to a first supply unit of the 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, via a pump, but are not limited thereto.

[0096] 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 from 1 μL / min to 100 ml / min. The speed of the continuous phase composition can be controlled from 10 μL / min to 500 ml / min.

[0097] The dispersed phase composition flowing through the first flow path flows into the second flow path through a plurality of micro-flow paths and meets the continuous phase composition flowing through the second flow path.

[0098] The dispersed phase composition flowing from the first flow path to the second flow path through the above-described plurality of microchannels generates droplets at the boundary between the microchannels and the second flow path, and the generated droplets flow through the second flow path together with the continuous phase composition.

[0099] The above microfluidic device may further include a discharge port through which droplets formed from the dispersed composition may be discharged. In this case, the microfluidic device may further include a third flow path (31) connecting the second flow path (21) and the discharge port (40), as shown in FIG. 3.

[0100] The above-mentioned dispersed composition is a precursor composition for forming particles, and may be an oil phase that is insoluble in a continuous phase composition that is a water phase.

[0101] Specifically, the dispersed composition may include a polymerizable monomer, a crosslinking agent, and a photoinitiator.

[0102] The polymerizable monomer may be a monomer having one or more unsaturated bonds. For example, the polymerizable monomer may be a (meth)acrylate monomer or a (meth)acrylamide monomer having a (meth)acryloyl group, a vinyl monomer having a vinyl group, or a mixture thereof.

[0103] Specifically, as monomers containing an epoxy group, glycidyl methacrylate, glycidyl acrylate, and glycidyl acrylamide can be used alone or in a mixture with monomers not containing an epoxy group.

[0104] The above epoxy group-free monomers include methyl methacrylate, methyl acrylate, ethyl acrylate, ethyl hexyl acrylate, butyl acrylate, hydroxyethyl methacrylate, trimethylolpropane triacrylate, N-isopropylacrylamide, N-butyl acrylate, etc.

[0105] The cross-linking agent can form a cross-linking structure with a polymerizable monomer or a prepolymer formed therefrom, thereby allowing the particles to maintain a spherical shape. When the particles have a spherical shape, they can have excellent cell culture efficiency due to a large specific surface area. For example, an ethylenically unsaturated cross-linking agent having two or more unsaturated bonds can be used as the cross-linking agent. More specifically, the cross-linking agent may be a multifunctional (meth)acrylate such as ethylene glycol dimethyl(meth)acrylate, ethylene glycol di(meth)acrylate, trimethylolpropane tri(meth)acrylate, pentaerythritol tri(meth)acrylate, or dipentaerythritol hexa(meth)acrylate. Alternatively, one or more of the cross-linking agents listed above may be used together. Considering the formation of a hydrophilic surface of the particles, etc., it may be preferable to use ethylene glycol di(meth)acrylate.

[0106] The content ratio of the crosslinking agent to 1 part by weight of the above monomer may be 0.1 to 10 parts by weight, or 0.5 to 2 parts by weight. When the above content is satisfied, it is advantageous to stably form an emulsion and secure particle strength and morphological characteristics through a desired level of crosslinking.

[0107] In one example, the dispersion may be a solution in which glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EGDMA) are mixed in a mass ratio of 5:1 to 1:5, or 4:1 to 1:4, or 3:1 to 1:3, or 2:1 to 1:2.

[0108] The type of the photoinitiator is not particularly limited, and various initiators known in the technical field to which the present invention belongs can be used as long as they do not hinder securing the particle characteristics of the particles described above.

[0109] As the photoinitiator, an initiator such as a ketone initiator, an organic peroxide initiator, or an azo initiator can be used, for example. Specifically, compounds such as 2,2-dimethoxy-2-phenylacetophenone, 2-Hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, 1-Hydroxycyclohexyl phenyl 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)hexyne-3 or di-cumyl peroxide, and mixtures thereof can be used, but are not limited thereto.

[0110] The 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, but not more than 30 parts by weight, not more than 25 parts by weight, not more than 20 parts by weight, or not more than 18 parts by weight, based on 100 parts by weight of the polymerizable monomer.

[0111] Within this range, microparticles with desired characteristics can be manufactured by exhibiting an appropriate polymerization rate.

[0112] The above-described dispersion composition may contain various additives known in the technical field to which the present invention pertains, in addition to the components described above, within a range that does not interfere with the purpose of the present invention.

[0113] Meanwhile, the continuous phase composition may be an aqueous solution. Specifically, the continuous phase composition may be an aqueous solution containing a surfactant and water.

[0114] The above water is not particularly limited, but may be distilled water or deionized water.

[0115] The surfactant may be an ionic surfactant or a non-ionic surfactant. For example, the ionic surfactant may be sodium dodecyl sulfate (SDS), and the non-ionic surfactant may be Tween 20, Tween 40, Tween 60, Tween 80, Triton X-100, polyvinyl alcohol (PVA), polyethylene glycol (PEG), etc.

[0116] The 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, but not more than 5 wt%, not more than 3 wt%, or not more than 1 wt%, based on the total continuous phase composition. It is advantageous to produce microparticles having desired characteristics within this range.

[0117] Meanwhile, the droplets produced in the step a) of generating droplets may be introduced into the step b) of photopolymerizing the droplets. In this case, the droplets may be mixed with an additional continuous phase composition before being introduced into the photopolymerization process.

[0118] 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. This process allows for uniform control of the shape of the microparticles.

[0119] The additional continuous phase composition described above may have the same composition as or a different composition from the continuous phase composition used in step a) of generating droplets. As an example, in step a) of generating droplets, 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, this is not limited thereto, and the droplets generated in step a) of generating droplets may be directly transferred to the photopolymerization process.

[0120] In the above b) photopolymerization step, the droplets generated in step a) are photopolymerized to produce particles. In the b) photopolymerization step, the droplets can be photopolymerized by irradiating UV light to the droplets using a UV spot curing device. The specific conditions for photopolymerization are not particularly limited, and various examples and conditions widely used in the conventional particle manufacturing field can be applied without limitation.

[0121] A washing step may be further included after the photopolymerization step (b). Impurities unrelated to the particles can be removed by washing. The washing method is not particularly limited, and any known washing method can be used. For example, the washing may be performed by placing the particles in an alcohol, such as ethanol, and stirring. Although not particularly limited, this washing process may be repeated, for example, three or more times.

[0122] After the above washing, a drying step may be further included. Residual solvents and the like can be removed through drying. The drying method is not particularly limited, and any known drying method can be used. For example, the drying may be performed using an oven or at room temperature. Furthermore, although not particularly limited, the drying may be performed in a vacuum.

[0123] The above particle manufacturing method can produce particles having a uniform size and shape. In the case of existing particle manufacturing methods, the production yield was low due to the process of sorting particles having the desired shape and properties from particles having an undesirable shape or undesirable properties after particle formation. However, the above particle manufacturing method can obtain particles having the desired shape and properties without this sorting process, thereby exhibiting a high production yield.

[0124]

[0125] Meanwhile, the particle complex of the above embodiment may include a protein fixed to the particle surface.

[0126] The above protein may be a protein for immune cell differentiation. The above immune cell differentiation protein may serve as a stimulus necessary for differentiating stem cells, such as allogeneic induced pluripotent stem cells (iPSCs), into immune cells.

[0127] Specific examples of the above-mentioned immune cell differentiation proteins are not particularly limited, and examples thereof include GM-CSF, IL4, IL-1b, TNFa, PGE2, DLL1, DLL4, TGFb, or a mixture of two or more thereof, and in addition, conventionally known proteins can be applied without limitation.

[0128] Since the above protein has a function of differentiating immune cells, when the particle complex of the above embodiment is cultured with stem cells, the stem cells can be differentiated into immune cells, thereby producing 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 large quantities of immune cells, and high marketability as an off-the-shelf drug can be achieved.

[0129] The protein content ratio per 1g of the above particles may be 0.05 mg to 10 mg. If the protein content ratio per 1g of the above particles is excessively reduced beyond the above numerical range, it may be difficult to sufficiently achieve stem cell differentiation by the protein. On the other hand, if the protein content ratio per 1g of the above particles is excessively increased beyond the above numerical range, a technical problem may arise in which the protein becomes more dense than the ligand to which it binds on the cell surface, resulting in the generation of proteins that are unable to bind.

[0130] The protein may be immobilized on the particle surface via covalent or physical bonding. The particle complex of the above embodiment can secure structural stability by immobilizing the protein on the particle surface via chemical bonding (e.g., covalent bonding) or physical bonding (e.g., adsorption). In particular, when the protein is immobilized on the particle surface via chemical bonding (e.g., covalent bonding), mass production and automation can be stably performed through a 3D process compared to conventional technologies, thereby increasing process efficiency.

[0131] The above covalent bond is a bond that is created when atoms share electrons during a chemical bond, and the protein can be fixed to the surface of the particle by a covalent bond while it is dispersed in water.

[0132] Specifically, the covalent bond may include a bonding functional group represented by the following chemical formula 1.

[0133] [Chemical Formula 1]

[0134]

[0135] In the above chemical formula 1, R1 is hydrogen or alkyl.

[0136] That is, the covalent bond may have a structure in which a particle and a protein are bonded via a bonding functional group represented by the above chemical formula 1. More specifically, a particle may be bonded to one end of the bonding functional group represented by the above chemical formula 1, and a protein may be bonded to the other end of the bonding functional group represented by the above chemical formula 1.

[0137] For example, the covalent bond may include a bond represented by the following chemical formula 2.

[0138] [Chemical Formula 2]

[0139]

[0140] In the above chemical formula 2, R1 is hydrogen or alkyl, X is a particle, and Y is a protein.

[0141] The covalent bond represented by the above chemical formula 2 can be formed by a reaction between an epoxy group on the particle surface and an 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 particularly limited, and various covalent bond conditions known in the art can be applied without limitation. However, as an example, ammonium sulfate can be added to a solution of particles and proteins mixed in PBS (phosphate buffered saline), and the mixture can be reacted for 16 hours or more while shaking at a speed of 250 rpm or higher at room temperature.

[0142] Additionally, the covalent bond may include a bonding functional group represented by the following chemical formula 3.

[0143] [Chemical Formula 3]

[0144]

[0145] That is, the covalent bond may have a structure in which a particle and a protein are bonded via a bonding functional group represented by the above chemical formula 3. More specifically, a particle may be bonded to one end of the bonding functional group represented by the above chemical formula 3, and a protein may be bonded to the other end of the bonding functional group represented by the above chemical formula 3.

[0146] For example, the covalent bond may include a bond represented by the following chemical formula 4.

[0147] [Chemical Formula 4]

[0148]

[0149] In the above chemical formula 4, X is a particle and Y is a protein.

[0150] The covalent bond represented by the above chemical formula 4 can be formed by a reaction between an epoxy group on the particle surface and a 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 particularly limited, and various covalent bond conditions known in the art can be applied without limitation. However, as an example, ammonium sulfate can be added to a solution of particles and proteins mixed in PBS (phosphate buffered saline), and the mixture can be reacted for 16 hours or more while shaking at room temperature at a speed of 250 rpm or higher.

[0151] Additionally, the covalent bond may include a bonding functional group represented by the following chemical formula 5.

[0152] [Chemical Formula 5]

[0153]

[0154] In the above chemical formula 5, R2 is hydrogen or alkyl.

[0155] That is, the covalent bond may have a structure in which a particle and a protein are bonded via a bonding functional group represented by the above chemical formula 5. More specifically, a particle may be bonded to one end of the bonding functional group represented by the above chemical formula 5, and a protein may be bonded to the other end of the bonding functional group represented by the above chemical formula 5.

[0156] For example, the covalent bond may include a bond represented by the following chemical formula 6.

[0157] [Chemical Formula 6]

[0158]

[0159] In the above chemical formula 6, R2 is hydrogen or alkyl, X is a particle, and Y is a protein.

[0160] The covalent bond represented by the above chemical formula 6 can be formed by a reaction between a carboxyl group on the particle surface and an 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 particularly limited, and various covalent bond conditions known in the art can be applied without limitation. However, as an example, ammonium sulfate can be added to a solution of particles and proteins mixed in PBS (phosphate buffered saline), and the mixture can be reacted for 16 hours or more while shaking at a speed of 250 rpm or higher at room temperature.

[0161]

[0162] Meanwhile, since the particle complex of the above embodiment has a purpose of differentiating immune cells, when the particle complex of the above embodiment is cultured together with stem cells, the stem cells can be differentiated into immune cells, thereby producing 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 large quantities of immune cells, and high marketability as an off-the-shelf drug can be achieved.

[0163] The particle complex of the above embodiment may have a glass protein ratio of 0.1% or less, or 0.0001% to 0.1%, according to the following mathematical formula 1.

[0164] [Mathematical Formula 1]

[0165] Free protein ratio (%) = (W1 / W2) * 100

[0166] In the above mathematical expression 1, W1 is the mass of protein released from the particle complex after shaking and centrifuging the solution containing the particle complex, and W2 is the mass of protein fixed to the particle complex before shaking and centrifuging the solution containing the particle complex.

[0167] This appears to be due to the particle complex of the above-mentioned embodiment securing structural stability by covalently anchoring the protein to the particle surface. Accordingly, compared to conventional technologies, mass production and automation can be achieved more reliably through 3D processing, thereby increasing process efficiency.

[0168] On the other hand, if the ratio of free protein according to the above mathematical formula 1 increases excessively to exceed 0.1%, the protein may not be stably fixed in the particle complex, which may cause a problem in that the efficiency of the process is greatly reduced when mass-produced or automated through a 3D process.

[0169] The solution containing the particle complex is a mixture of the particle complex and a solvent. The examples of the solvent are not particularly limited, and various solvents known in the art can be applied without limitation. However, as an example, PBS (phosphate buffered saline) can be used. The particle complex can be added so that the protein concentration in the solution containing the particle complex satisfies 1 ug / mL.

[0170] The above shaking culture conditions are not particularly limited, and various shaking culture conditions known in the art can be applied without limitation. However, as an example, the reaction can be carried out for 7 hours or more while shaking at room temperature at a speed of 250 rpm or more in a shaking incubator.

[0171] The above examples of centrifugation conditions are not particularly limited, and various centrifugation conditions known in the art can be applied without limitation. However, as an example, centrifugation can be performed at 10,000 xg for 10 minutes.

[0172] Examples of methods for measuring the mass of protein released from the above particle complex are not particularly limited, and various conventionally known quantitative analysis methods can be applied without limitation. However, an example is ELISA (Enzyme-Linked Immunosorbent Assay).

[0173] Meanwhile, the particle complex of the above embodiment may be composed of spherical particles; and a protein fixed to the particle surface. The spherical particles and the protein fixed to the particle surface are the same as described above. If the particle complex includes other components (e.g., magnetic particles) in addition to the spherical particles and the protein fixed to the particle surface, it is difficult to remove them from the particle complex, making it difficult to maintain quality, and there is a limitation that the process efficiency deteriorates when a separate removal process is performed for reuse.

[0174] However, if necessary, the particle complex of the above-described embodiment may further include magnetic particles. Specifically, when magnetic particles are further included within the particles of the particle complex, the particle complex and cells can be separated and purified more easily using magnetism after cell culture.

[0175] The above magnetic particles refer to particles that exhibit magnetic properties. All substances interact with a magnetic field, generating an attractive or repulsive force. In other words, when a magnetic field is applied to a substance, it becomes magnetized, and depending on the way the substance becomes magnetized, it is classified as a ferromagnetic substance, a paramagnetic substance, a diamagnetic substance, or a ferrimagnetic substance.

[0176] The above magnetic particles can be prepared by, but are not limited to, solution synthesis, co-precipitation, sol-gel method, high-energy grinding, hydrothermal synthesis, microemulsion synthesis, synthesis by pyrolysis, or sonochemical synthesis.

[0177] The type of the magnetic particles is not particularly limited, and metal nanoparticles may be used. For example, the magnetic particles may include one or more metals or oxides thereof selected from the group consisting of gold (Au), silver (Ag), cobalt (Co), copper (Cu), iron (Fe), chromium (Cr), nickel (Ni), palladium (Pd), platinum (Pt), and tin (Sn). Specifically, the magnetic particles may include iron (Fe) particles.

[0178]

[0179] 2. Cell culture composition

[0180] According to another embodiment of the invention, a cell culture composition comprising cells and a particle complex of the above embodiment may be provided. The content of the particle complex includes all of the content described above in the above embodiment.

[0181] The above cells may include stem cells.

[0182] The above cell culture composition may further include a medium solution. The medium solution may include various additives to sufficiently satisfy environmental conditions such as pH, temperature, and osmotic pressure, as well as nutrients similar to those of a living organism based on body fluids such as plasma or lymph. Various materials widely known in the art of cell culture may be used without limitation.

[0183]

[0184] 3. Method for manufacturing cell therapy products

[0185] According to another embodiment of the invention, a method for producing a cell therapeutic agent may be provided, comprising: culturing a cell culture composition of the other embodiment; and removing a particle complex from the culture result. The contents of the cell culture composition include all of the contents described above in the other embodiment.

[0186] In the step of culturing the cell culture composition of the above-described other embodiment, the cells included in the cell culture composition can be differentiated into immune cells. Specific culture conditions are not particularly limited, and any culture condition capable of differentiating stem cells into immune cells can be applied without limitation.

[0187] Meanwhile, in the step of removing particle complexes from the culture results, the safety of the cell therapeutic agent can be ensured by separating and removing the particle complexes from the cell culture results. The method for removing the particle complexes from the culture results is not particularly limited, and various conventionally known methods and conditions can be applied without limitation. However, for example, the particle complexes can be separated and removed using a cell strainer.

[0188] According to the present invention, a particle complex capable of securing the stability of a differentiation platform by fixing a protein necessary for cell differentiation and completely separating after cell culture to secure the stability of a cell therapeutic agent, and a cell culture composition using the same and a method for manufacturing a cell therapeutic agent can be provided.

[0189] Figure 1 shows an SEM image of the particles obtained in (1) of Example 1.

[0190] Figure 2 shows an SEM image of the particles obtained in (1) of Example 6.

[0191] Figure 3 is a plan view of a microfluidic device.

[0192] The invention is described in more detail in the following examples. However, the following examples are merely illustrative of the present invention, and the scope of the present invention is not limited by the following examples.

[0193]

[0194] <Example: Preparation of particle composite>

[0195] Example 1

[0196] (1) Manufacturing of particles

[0197] After stirring the monomer GMA (glycidyl methacrylate) and the crosslinking agent EGDMA (ethylene glycol dimethacrylate), the thermal initiator V-65 was additionally added and stirred at room temperature for about 5 minutes to prepare a dispersed phase. At this time, the weight ratio of monomer: crosslinking agent: initiator was 1:3:0.05.

[0198] A continuous phase was prepared by dissolving PVA (polyvinyl alcohol; having a weight average molecular weight (Mw) of 85,000 to 124,000 and a hydrolysis rate of 87 to 89%) in distilled water at a concentration of 2 wt%.

[0199] 720 g of the above-described continuous phase was added to a reactor of a given capacity, and 72 g of the dispersed phase was added, followed by stirring at about 800 rpm at room temperature until a uniform dispersion was obtained. Thereafter, the reactor temperature was increased to about 80°C, about 800 rpm, and under nitrogen purging conditions, polymerization was performed for about 6 hours.

[0200] The polymerized particles were recovered and washed twice with distilled water and five times with ethanol. They were then dried in an oven at approximately 80°C to produce particles. The D50 diameter (D50 is the size of the 50th particle when a total of 100 particles are counted based on SEM (scanning electron microscopy) images) of the produced particles was measured to be 130 μm.

[0201] (2) Preparation of particle composite

[0202] The particles obtained in the above (1) were washed three times with PBS (Phosphate buffered saline) by centrifugation at 10,000 xg for 10 minutes. Thereafter, proteins for immune cell differentiation (GM-CSF, IL4, IL-1b, TNFa, PGE2, DLL1, DLL4, TGFb, etc. were each independently applied) were dissolved in PBS and mixed at a ratio of 0.05 mg to 10 mg per 1 g of particles.

[0203] The particles obtained in (1) above and the protein for immune cell differentiation were mixed with a PBS solution containing the same volume of 3 M ammonium sulfate as the PBS solution, so that the final solution became 1.5 M ammonium sulfate. Afterwards, the solution was reacted for more than 16 hours while shaking at a speed of 250 rpm or more at room temperature in a shaking incubator, thereby covalently bonding the epoxy group exposed on the surface of the particles obtained in (1) above with the amine group or thiol group of the protein.

[0204] Afterwards, the protein-covalently bound particles were separated from the mixed solution by centrifugation at 10,000 xg for 10 minutes, the supernatant was removed, and pH 12 Tris buffer was added and vortexed. After removing the buffer by centrifugation, pH 4 Acetate buffer was added and vortexed. Afterwards, the obtained protein-covalently bound particles were washed three more times with Tween 20 0.5 wt% PBS and used as the particle complex of Example 1. The particle complex was stored in PBS at 4°C.

[0205]

[0206] Example 2

[0207] (1) Manufacturing of particles

[0208] Particles were manufactured using the same method as (1) of Example 1, except that the D50 diameter of the particles was changed to 100 μm.

[0209] (2) Preparation of particle composite

[0210] A particle complex was manufactured in the same manner as in (2) of Example 1, except that the particles obtained in (1) of Example 2 were used instead of the particles obtained in (1) of Example 1.

[0211]

[0212] Example 3

[0213] (1) Manufacturing of particles

[0214] Particles were manufactured using the same method as (1) of Example 1, except that the D50 diameter of the particles was changed to 50 μm.

[0215] (2) Preparation of particle composite

[0216] A particle complex was manufactured in the same manner as in (2) of Example 1, except that the particles obtained in (1) of Example 3 were used instead of the particles obtained in (1) of Example 1.

[0217]

[0218] Example 4

[0219] (1) Manufacturing of particles

[0220] Particles were manufactured using the same method as (1) of Example 1, except that the D50 diameter of the particles was changed to 38 μm.

[0221] (2) Preparation of particle composite

[0222] A particle complex was manufactured in the same manner as in (2) of Example 1, except that the particles obtained in (1) of Example 4 were used instead of the particles obtained in (1) of Example 1.

[0223]

[0224] Example 5

[0225] (1) Manufacturing of particles

[0226] Glycidyl methacrylate (GMA) as a monomer and ethylene glycol dimethacrylate (EGDMA) as a crosslinking agent were stirred at a weight ratio of GMA:EGDMA = 1:1, and then irgacure 651 as a photoinitiator was additionally added at 1 wt% based on the weight of the stirred solution, and stirred at room temperature for about 5 minutes to prepare a dispersed composition.

[0227] A continuous phase composition was prepared by dissolving SDS (sodium dodecyl sulfate) in deionized water at a concentration of 0.5 wt%.

[0228] The previously prepared dispersed phase composition (injection rate of 100 μl / min) and continuous phase composition (injection rate of 300 μl / min) were respectively injected through a pump into the first supply section (10) and the second supply section (20) of a microfluidic device (40 μm step emulsification chip) as shown in FIG. 3 below. The dispersed phase composition injected into the first supply section (10) flowed along the first flow path (11) and was supplied to the second flow path (21) through the micro flow path (12) to form droplets within the continuous phase composition. The suspension including the droplets was discharged through the discharge section (40).

[0229] Afterwards, the suspension containing the above droplets was collected in a PVA (polyvinyl alcohol; molecular weight 85,000 to 125,000) aqueous solution, and the droplets were dispersed in a final 3 wt% PVA. After shaking to evenly disperse the droplets, photopolymerization was performed using a UV spot curing device.

[0230] 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 produce particles.

[0231] (2) Preparation of particle composite

[0232] A particle complex was manufactured in the same manner as in (2) of Example 1, except that the particles obtained in (1) of Example 5 were used instead of the particles obtained in (1) of Example 1.

[0233]

[0234] Example 6

[0235] (1) Manufacturing of particles

[0236] Particles were manufactured in the same manner as in (1) of Example 5, except that the following four conditions were changed.

[0237] 1) GMA: EGDMA = weight ratio of 1:2

[0238] 2) Dispersed phase injection rate: 10 ㎕ / min

[0239] 3) Continuous injection rate: 15 μl / min

[0240] 4) Microfluidic device: 100 ㎛ flow focusing chip

[0241] (2) Preparation of particle composite

[0242] A particle complex was manufactured in the same manner as in (2) of Example 1, except that the particles obtained in (1) of Example 6 were used instead of the particles obtained in (1) of Example 1.

[0243]

[0244] Example 7

[0245] (1) Manufacturing of particles

[0246] Particles were manufactured in the same manner as in (1) of Example 5, except that the following four conditions were changed.

[0247] 1) GMA: EGDMA = weight ratio of 1:2

[0248] 2) Dispersed phase injection rate: 15 μl / min

[0249] 3) Continuous injection rate: 225 μl / min

[0250] 4) Microfluidic device: 190 ㎛ flow focusing chip

[0251] (2) Preparation of particle composite

[0252] A particle complex was manufactured in the same manner as in (2) of Example 1, except that the particles obtained in (1) of Example 7 were used instead of the particles obtained in (1) of Example 1.

[0253]

[0254] <Comparative Example>

[0255] Comparative Example 1

[0256] Using a flat-bottom tissue culture plate (a polystyrene culture plate that does not have a particle-shaped support and in which proteins are fixed directly to the bottom), proteins for immune cell differentiation (GM-CSF, IL4, IL-1b, TNFa, PGE2, DLL1, DLL4, TGFb, etc.) were diluted to the target concentration in PBS at 4°C and then added to a 96-well plate at 50 uL / well. After storing flat at 4°C for more than 16 hours to induce adsorption, the plates were washed twice with PBS to remove any residual proteins, thereby preparing protein complexes.

[0257]

[0258] <Reference Example: Manufacturing of Particle Composites>

[0259] Reference Example 1

[0260] Commercial particles (D50 diameter 180 ㎛) were prepared in PBS at a concentration of 0.1 mg / mL, and proteins for immune cell differentiation (GM-CSF, IL4, IL-1b, TNFa, PGE2, DLL1, DLL4, TGFb, etc.) were added at 0.05 mg to 5 mg per 1 g of particles. Afterwards, the particles were reacted for more than 16 hours while shaking at room temperature at a speed of 250 rpm or higher, and washed three times with PBS to remove any residual proteins, thereby preparing particle complexes.

[0261]

[0262] Reference Example 2

[0263] 5 mg of commercially available magnetic beads (D50 diameter 5 ㎛) were dispersed in 1 mL of PBS, and the supernatant was removed using a magnet. 1 μg to 100 μg of immune cell differentiation proteins (GM-CSF, IL4, IL-1b, TNFa, PGE2, DLL1, DLL4, TGFb, etc.) were added to reach 1 mg / mL. An equal volume of 3 M ammonium sulfate solution as the total solution was added, and slow tilt rotation was performed at 37 °C for more than 16 hours. After removing the supernatant using a magnet, the beads were washed four times with PBS to prepare particle complexes.

[0264]

[0265] <Experimental Example 1>

[0266] For the composites obtained in the above examples, comparative examples and reference examples, the physical properties were measured using the following methods, and the results are shown in Table 1.

[0267]

[0268] 1. Protein stability

[0269] The complexes obtained in the above examples, comparative examples, and reference examples were dissolved in PBS at 1 ug / mL protein level in a 1 mL EP tube, and the mixture was allowed to react for more than 7 hours while shaking at a speed of 250 rpm or higher at room temperature in a shaking incubator. Afterwards, the supernatant was recovered by centrifugation at 10,000 xg for 10 minutes. Then, ELISA (Enzyme-Linked Immunosorbent Assay) was performed with the supernatant to measure the mass of protein released from the particle complex, and the free protein ratio was calculated according to the following mathematical formula 1, and the protein stability was evaluated under the following criteria.

[0270] [Mathematical Formula 1]

[0271] Free protein ratio (%) = (W1 / W2) * 100

[0272] In the above mathematical formula 1,

[0273] W1 is the mass of protein released from the particle complex after shaking and centrifugation of the solution containing the particle complex,

[0274] W2 is the mass of protein immobilized on the particle complex before shaking and centrifuging the solution containing the particle complex.

[0275] [Evaluation Criteria]

[0276] Top: The percentage of free protein according to Equation 1 is 0.1% or less.

[0277] Medium: The percentage of free protein according to Equation 1 is greater than 0.1% and less than 10%.

[0278] A: The percentage of free protein according to Equation 1 exceeds 10%.

[0279]

[0280] 2. Particle separation performance

[0281] A 10 mL vial was filled with a culture medium containing cells, and the complexes obtained in the above examples, comparative examples, and reference examples were injected and mixed for 30 minutes using a rocking shaker. After separating the particle complexes from the cell culture medium using a cell strainer, whether the particle complexes remained in the cell culture medium was analyzed using a cell culture microscope and a particle size analyzer (Beckman Coulter counter).

[0282] And, the particle separation performance of the particle complex was evaluated under the criteria below.

[0283] Top: Particle complex residue less than 0.1% of the input amount

[0284] Medium: Particle complex residue is 0.1% or more and less than 5% of the input amount.

[0285] A: The particle complex residue is 5% or more of the input amount.

[0286]

[0287] Experimental Example 1 Measurement ResultsExample 1Example 2Example 3Example 4Example 5Example 6Example 7Comparative Example 1Reference Example 1Reference Example 2Particle ShapeSphericalSphericalSphericalSphericalSpherical-(Not a particle)SphericalSphericalParticle Diameter (D50) 130 ㎛100 ㎛50 ㎛38 ㎛50 ㎛100 ㎛130 ㎛-180 ㎛5 ㎛Protein StabilityUpper Upper Upper Upper Upper Upper LoadMedium Upper Upper Particle Separation PerformanceUpper Upper Upper Middle Upper Upper Upper-(Not a particle)Upper Lower

[0288] As shown in Table 1 above, it was confirmed that the particle complex of the example stably immobilized the protein under cell culture conditions compared to the comparative example, and was completely separated after cell culture.

[0289]

[0290] <Experimental Example 2>

[0291] For the complexes obtained in Examples 1 and 5-7 above, the physical properties were measured using the following method, and the results are shown in Table 2.

[0292]

[0293] 1. Particle diameter and coefficient of variation

[0294] (1) Particle diameter

[0295] For the particles obtained in Examples 1, 5 to 7 above, the particles were observed by analyzing the SEM (scanning electron microscopy) images and the particle diameters were measured.

[0296] D50 diameter is the size of the 50th particle when the total number of particles is converted to 100.

[0297] (2) Coefficient of variation (CV)

[0298] The SEM (scanning electron microscopy) images of 100 particle samples obtained in Examples 1, 5 to 7 were analyzed and obtained using the following mathematical formula 2.

[0299] [Mathematical Formula 2]

[0300] Coefficient of variation (%) = (Standard deviation of particle diameter / Average particle diameter) X 100.

[0301]

[0302] Experimental Example 2 Measurement ResultsExample 1Example 5Example 6Example 7GMA:EGDMA1:31:11:21:2Polymerization ConditionsSuspension polymerizationPhotopolymerization1) Dispersed phase injection rate: 100 ㎕ / min2) Continuous phase injection rate: 300 ㎕ / min3) Microfluidic device: 40 ㎛ step emulsification chipPhotopolymerization1) Dispersed phase injection rate: 10 ㎕ / min2) Continuous phase injection rate: 15 ㎕ / min3) Microfluidic device: 100 ㎛ flow focusing chipPhotopolymerization1) Dispersed phase injection rate: 15 ㎕ / min2) Continuous phase injection rate: 225 ㎕ / min3) Microfluidic device: 190 ㎛ flow focusing chipParticle diameter135.9 ㎛ ± 28 ㎛53.1 ㎛ ± 4.1 ㎛99.0 ㎛ ± 2.2 ㎛131.0 ㎛ ± 2.8 ㎛Particle diameter (D50) 130 ㎛50 ㎛100 ㎛130 ㎛Coefficient of variation 21%7.7%2.2%2.1%

Claims

1. Spherical particles; and A particle complex comprising a protein fixed to the surface of the particle.

2. In paragraph 1, The above particles are a particle complex having a D50 diameter exceeding 20 ㎛.

3. In paragraph 1, The above particles are a particle complex having a D50 diameter of less than 180 μm.

4. In paragraph 1, The above particles are a particle complex having a D50 diameter of 30 μm to 175 μm.

5. In paragraph 1, The above particles are a particle complex having a D50 diameter of 50 μm to 160 μm.

6. In paragraph 1, A particle complex wherein the protein is fixed to the particle surface by covalent bonding or physical bonding.

7. In paragraph 6, The above covalent bond is a particle complex including a bonding functional group represented by the following chemical formula 1: [Chemical Formula 1] In the above chemical formula 1, R1 is hydrogen or alkyl.

8. In paragraph 7, The particle complex, wherein the covalent bond comprises a bond represented by the following chemical formula 2 or a bond represented by the following chemical formula 3: [Chemical Formula 2] In the above chemical formula 2, R1 is hydrogen or alkyl, X is a particle and Y is a protein.

9. In paragraph 6, The above covalent bond is a particle complex including a bonding functional group represented by the following chemical formula 3: [Chemical Formula 3] .

10. In paragraph 9, The above covalent bond is a particle complex including a bond represented by the following chemical formula 4: [Chemical Formula 4] In the above chemical formula 4, X is a particle and Y is a protein.

11. In paragraph 6, The above covalent bond is a particle complex including a bonding functional group represented by the following chemical formula 5: [Chemical Formula 5] In the above chemical formula 5, R2 is hydrogen or alkyl.

12. In paragraph 11, The above covalent bond is a particle complex including a bond represented by the following chemical formula 6: [Chemical Formula 6] In the above chemical formula 6, R2 is hydrogen or alkyl, X is a particle and Y is a protein.

13. In paragraph 1, A particle complex having a protein content ratio of 0.05 mg to 10 mg per 1 g of the particles.

14. In paragraph 1, The above protein is a particle complex that is a protein for immune cell differentiation.

15. In paragraph 1, The above particles are suspension polymer particles, a particle composite.

16. In paragraph 1, The above particles are a particle complex having a coefficient of variation of particle diameter of 20% or less.

17. In paragraph 16, The above particles are microfluidic chip particles, particle complex.

18. In paragraph 1, The above particle complex is A particle complex having a glass protein ratio of 0.1% or less according to the following mathematical formula 1: [Mathematical Formula 1] Free protein ratio (%) = (W1 / W2) * 100 In the above mathematical formula 1, W1 is the mass of protein released from the particle complex after shaking and centrifugation of the solution containing the particle complex, W2 is the mass of protein immobilized on the particle complex before shaking and centrifuging the solution containing the particle complex.

19. A cell culture composition comprising a cell and a particle complex of the first clause.

20. A step of culturing the cell culture composition of Article 19; and A method for producing a cell therapeutic agent, comprising: a step of removing a particle complex from the culture result.

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