Particle composite, and method for preparing cell culture composition and cell therapeutic agent using same

Abstract

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

Description

Technical Field

[0001] Cross-references to related applications

[0002] This application claims priority and benefit to Korean Patent Application No. 10-2023-0152073, filed with the Korean Intellectual Property Office on November 6, 2023; Korean Patent Application No. 10-2024-0021286, filed with the Korean Intellectual Property Office on February 14, 2024; and Korean Patent Application No. 10-2024-0154399, filed with the Korean Intellectual Property Office on November 4, 2024, the disclosures of which are incorporated herein by reference in their entirety.

[0003] This disclosure relates to particulate complexes, cell culture compositions using the same, and methods for preparing cell therapeutic agents. Background Technology

[0004] With the expansion of applications in biopharmaceuticals and regenerative medicine, the demand for large-scale cell culture technologies that can efficiently produce cells, tissues, microorganisms, etc., is increasing.

[0005] Meanwhile, existing anti-cancer therapies use T cells (autologous cells) derived from each patient, which requires a long and expensive time to culture, and cancer patients are generally in a state of reduced immune cell function. Therefore, they have the drawback of not being able to guarantee their quality for therapeutic use.

[0006] To address this problem, attempts have been made to differentiate and use stem cells, such as allogeneic induced pluripotent stem cells (iPSCs). This differentiation requires stimulation with proteins, but there are limitations in large-scale production and automation when applying it to a planar (2D) environment.

[0007] Therefore, with the development of 3D transformation using particle methods, there is a need to develop techniques that can completely separate particles from cells after culture. Summary of the Invention

[0008] Technical issues

[0009] This disclosure relates to a technology for providing particulate complexes that can covalently immobilize proteins required for cell differentiation to ensure the stability of the differentiation platform, and can be completely isolated after cell culture to ensure the stability of cell therapeutics.

[0010] This disclosure also relates to cell culture compositions using particulate complexes.

[0011] Furthermore, this disclosure relates to a method for preparing a cell therapy agent using a particulate complex.

[0012] Technical solution

[0013] To achieve the above objectives, this article provides a particle complex comprising: spherical particles; and proteins fixed on the surface of the particles.

[0014] This article also provides cell culture compositions comprising a cell and particle complex.

[0015] A method for preparing a cell therapy agent is further provided, comprising the steps of: culturing a cell culture composition; and removing particulate complexes from the culture product.

[0016] The particulate complexes according to specific embodiments of the present disclosure, as well as cell culture compositions using the same and methods for preparing cell therapeutic agents, will be described in more detail.

[0017] Unless otherwise expressly stated herein, the technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0018] As used in this article, unless the context clearly indicates otherwise, nouns without quantifiers are intended to include plural forms as well.

[0019] It should be understood that the terms “comprising,” “including,” “having,” etc., used herein are used to indicate the presence of the stated features, regions, integers, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and / or groups thereof.

[0020] As used herein, the term “substituted” means that another functional group replaces a hydrogen atom in a compound for bonding, and there is no restriction on the position to be substituted, as long as the position is where a hydrogen atom is substituted, i.e., the position where the substituent can be substituted, and when two or more are substituted, the two or more substituents can be the same or different from each other.

[0021] As used herein, the term "substituted or unsubstituted" means unsubstituted or substituted with one or more substituents selected from: deuterium, halogen group, cyano, nitro, hydroxyl, carbonyl, ester group, imide group, amide group, primary amino group, carboxyl group, sulfonic acid group, sulfonamide group, phosphine oxide group, alkoxy group, aryloxy group, alkyl thio group, aryl thio group, alkyl sulfonyl group, aryl sulfonyl group, silyl group, boron group, alkyl group, cycloalkyl group, alkenyl group, aryl group, aralkyl group, arene group, alkylaryl group, alkoxysilylalkyl group, arylphosphine group, or a heterocyclic group containing at least one of N, O, and S atoms, or unsubstituted or substituted with two or more substituents linked together from the substituents exemplified above. For example, "substituents linked together from two or more substituents" can be biphenyl. That is, biphenyl can also be aryl and can be interpreted as substituents linked together from two phenyl groups.

[0022] As used in this article, symbols or The term "bond" refers to a bond connected to another substituent, while "direct bond" refers to a bond in which no other atoms are present in the portion represented by "L".

[0023] As used herein, alkyl groups are monovalent functional groups derived from alkanes and can be straight-chain or branched. The number of carbon atoms in straight-chain alkyl groups is not particularly limited, but is preferably 1 to 20. Furthermore, the number of carbon atoms in branched alkyl groups is 3 to 20. Specific examples of alkyl groups include, but are not limited to, 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, 5-methylhexyl, 2,6-dimethylheptane-4-yl, etc. Alkyl groups can be substituted or unsubstituted, and when substituted, the examples of substituents are the same as described above.

[0024] As used in this article, (meth)acrylates include both acrylates and methacrylates.

[0025] As used herein, the term "dispersed phase composition" means a composition that, when mixed with a continuous phase composition, can form a dispersed phase (or droplets).

[0026] As used herein, the term "continuous phase composition" means a composition that, when mixed with a dispersed phase composition, can form a continuous phase.

[0027] Unless otherwise specifically defined or interpreted herein, the temperature at which the preparation process (or each preparation step) is carried out, or the temperature at which the numerical characteristics of the prepared particles are calculated or measured, can be room temperature. Specifically, as used herein, "room temperature" means a temperature under conditions where the temperature has not been particularly increased or decreased, such as a temperature in the range of 15°C to 30°C.

[0028] The contents of this disclosure will be described in more detail below.

[0029] 1. Particulate complex

[0030] According to one embodiment of this disclosure, a particulate complex is provided, comprising: spherical particles; and proteins fixed on the surface of the particles.

[0031] Through experiments, the inventors discovered that, in the case of a particle complex in one embodiment, proteins can be fixed to the surface of spherical particles by chemical covalent bonds or physical bonds, such as adsorption, to ensure the structural stability of the particle complex. Furthermore, a cell filter can be used to completely separate the particle complex from the cell culture medium to ensure the stability of the cell therapy agent, thereby completing the present disclosure.

[0032] Specifically, the particulate complex of one embodiment may include particles. The particles may be spherical. 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 improve protein immobilization efficiency.

[0033] SEM can be used to visually determine whether particles have a spherical shape. Spherical particles can include both true spheres, which correspond to a theoretically perfect sphere, and particles that are not true spheres but have a shape close to that of a true sphere.

[0034] More specifically, the roundness of the spherical particles associated with this embodiment is typically 0.88 or greater, or 0.90 or greater, or 0.91 or greater, or 0.92 or greater, 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] If the roundness of spherical particles is within the above range, there is a tendency to inhibit the degradation of particle durability and protein fixation properties. Furthermore, roundness is defined by the following equation, and when roundness is 1, it becomes a theoretically true sphere. Conversely, the further the particle shape deviates from a true sphere, the smaller the value of roundness.

[0036] Roundness = circumference of the equivalent circle with the same area as the projected particle shape / actual circumference of the projected particle shape

[0037] Specifically, there are no particular limitations on the method for measuring roundness, but as an example, it can be measured using cross-sectional SEM images. Specifically, the particle area S [μm] obtained from the cross-sectional SEM image is used. 2 The roundness of the circumference L [μm] is calculated using the following equation.

[0038]

[0039] There are no particular limitations on the method for obtaining particle boundaries, and it can be done automatically or manually using commercially available analysis software. Preferably, it approximates a polygon, and in this case, more preferably, a shape with 15 or more angles. This is because if the shape is less than 15 angles, there is a possibility that the background portion might be considered inside the particle when approximating curved sections.

[0040] The particle cross-section images were obtained using reflectance electron microscopy (SEM) at an accelerating voltage of 5 kV. There are no particular limitations on the method for preparing the sample for SEM observation to obtain cross-sectional images of the particles; the particle cross-section is cut to prepare the sample, and then an SEM image of the particle cross-section is acquired. Furthermore, the imaging magnification is typically 10x or greater, or 50x or greater, or 100x or greater, or 1000x or less.

[0041] On the other hand, when using conventional, widely used culture plates instead of spherical particles, a limitation exists: because proteins are immobilized through simple adsorption rather than covalent bonds, it is difficult to control the amount and uniformity of proteins. Furthermore, since it is difficult to mass-produce and automate the production of proteins coated on plates, immobilizing proteins onto particles is essential for large-scale culture.

[0042] Furthermore, the D50 diameter of the particles can be greater than 20 μm or less than 180 μm, or greater than 20 μm and less than 180 μm, or 30 μm to 175 μm, or 50 μm to 160 μm, or 50 μm to 130 μm, or 50 μm to 80 μm, or 80 μm to 120 μm, or 120 μm to 160 μm. When the D50 diameter of the particles meets the above ranges, the cell differentiation and culture performance is excellent.

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

[0044] There are no particular limitations on the methods for measuring the D50 diameter of particles, and various well-known methods for measuring the D50 diameter of particles can be applied without restriction. However, as an example, it can be measured by analyzing SEM (scanning electron microscopy) images.

[0045] If the D50 diameter of the particles increases excessively beyond the range mentioned above, the surface area per unit volume decreases, which reduces the interaction between cells and particles and may therefore lead to problems with reduced cell differentiation efficiency.

[0046] On the other hand, if the D50 diameter of the particles is reduced excessively beyond the aforementioned range, it becomes difficult to completely separate the particle complex from the cell culture medium using a cell filter, which may lead to safety issues with the final cell therapy agent.

[0047] Particles may include single particles or groups of particles consisting of multiple single particles. A single particle means a single type of particle, and a group of particles means a plurality of particle matrices in which two or more types of particles are mixed.

[0048] In this context, a single particle may include proteins fixed to its surface. That is, it may include proteins fixed to the surface of a single particle, i.e., one type of particle. Furthermore, it may include proteins fixed to the surface of each single particle in a particle group containing two or more types of particles.

[0049] The particles can be suspension polymerized particles. Suspension polymerized particles refer to particles obtained by mixing a continuous phase composition and a dispersed phase composition and polymerizing them in a suspended state.

[0050] A dispersed phase composition means a composition which, when mixed with a continuous phase composition, can form a dispersed phase (or droplets), and a continuous phase composition means a composition which, when mixed with a dispersed phase composition, can form a continuous phase.

[0051] In one exemplary embodiment, the dispersed phase composition may contain polymerizable monomers.

[0052] In one embodiment of this application, the polymerizable monomer is a monomer having unsaturated bonds between carbon atoms, and may also contain epoxy, amide, carboxyl, alkoxy, sulfonic acid, mercapto, amino, or hydroxyl groups. These groups may be referred to as hydrophilic groups.

[0053] In one exemplary embodiment, the polymerizable monomer may be a (meth)acrylate-based monomer. In this case, the (meth)acrylate-based monomer having a hydrophilic group may contain one or more of the aforementioned hydrophilic groups.

[0054] Although there are no particular limitations, examples of (meth)acrylate-based monomers having an epoxy group as a hydrophilic group may include glycidyl (meth)acrylate, 4,5-epoxybutyl (meth)acrylate, 9,10-epoxystearyl (meth)acrylate, etc.

[0055] Although there are no particular limitations, examples of (meth)acrylate-based monomers having an amide group as a hydrophilic group may include (meth)acrylamide or N-hydroxymethyl (meth)acrylamide.

[0056] Although there are no particular limitations, examples of (meth)acrylate-based monomers having a carboxyl group as a hydrophilic group can include acrylic acid, methacrylic acid, maleic acid, itaconic acid, etc.

[0057] Although there are no particular restrictions, monomers based on (meth)acrylates that have an alkoxy group as a hydrophilic group can have a methoxy or ethoxy group. For example, methoxyethyl (meth)acrylate can be used.

[0058] Although there are no particular restrictions, monomers based on (meth)acrylates that have hydroxyl groups as hydrophilic groups can be, for example, hydroxyethyl (meth)acrylate, hydroxypropyl (meth)acrylate, or hydroxybutyl (meth)acrylate.

[0059] One or more of the monomers listed above can be used as (meth)acrylate monomers having hydrophilic groups. In one specific embodiment of this application, a monomer having unsaturated bonds and epoxy groups between carbon atoms can be used as a polymerizable monomer.

[0060] In one exemplary embodiment, the dispersed phase composition may further comprise a crosslinking agent. There are no particular limitations on the crosslinking agents that can be used, but for example, polyfunctional (meth)acrylates such as ethylene glycol dimethacrylate, trimethylolpropane trimethacrylate, pentaerythritol trimethacrylate, dipentaerythritol hexamethacrylate, dipentaerythritol hexamethacrylate, or trimethylpropane trimethacrylate can be used. Alternatively, one or more of the crosslinking agents listed above can be used in combination. Ethylene glycol dimethacrylate is preferred, taking into account the formation of a hydrophilic surface of the particles, etc.

[0061] In one exemplary embodiment, based on 100 parts by weight of polymerizable monomer, the dispersed phase composition may contain a crosslinking agent in an amount ranging from 50 parts by weight to 500 parts by weight. Specifically, the content of the crosslinking 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. Furthermore, its 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 content of the crosslinking agent meets the above requirements, it is advantageous to stably form an emulsion through the desired level of crosslinking and to ensure particle strength and morphological properties.

[0062] In one exemplary embodiment, the dispersed phase composition may further comprise an initiator. The type of initiator is not particularly limited, as long as it does not impede ensuring the particulate properties according to the preparation method of this application. For example, initiators such as organic peroxide initiators or azo initiators can be used. Specifically, compounds such as benzoyl peroxide, di-tert-amyl peroxide, tert-butyl peroxybenzoate, 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane, 2,5-dimethyl-2,5-di(tert-butylperoxy)hexyne-3, or dicumyl peroxide, and mixtures thereof, can be used, but are not limited thereto.

[0063] There is no particular limitation on the initiator content. The initiator can be used in appropriate amounts, within a range that does not hinder the attainment of desired particle characteristics. For example, based on 100 parts by weight of the total polymerizable monomer and crosslinking agent, the dispersed phase composition may contain 0.1 parts by weight or more, specifically 0.2 parts by weight or more, 0.3 parts by weight or more, 0.4 parts by weight or more, 0.5 parts by weight or more, 0.6 parts by weight or more, 0.7 parts by weight or more, 0.8 parts by weight or more, 0.9 parts by weight or more, or 1.0 parts by weight or more of initiator. Furthermore, the upper limit for the initiator content 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 exemplary embodiment, the continuous phase composition may comprise water and a polymeric surfactant.

[0065] Although there are no particular restrictions, the water can be distilled or deionized.

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

[0067] In one specific embodiment of this application, the polymeric surfactant may have a weight-average molecular weight (Mw) within a predetermined range and / or a degree of hydration (degree of hydrolysis) within a predetermined range.

[0068] In one exemplary embodiment, the weight-average molecular weight of the polymeric surfactant can be 60,000 or greater. Specifically, the lower limit of the weight-average molecular weight can be, for example, 65,000 or greater, 70,000 or greater, 80,000 or greater, or 85,000 or greater. Furthermore, the upper limit of the weight-average molecular weight can 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 exemplary embodiment, the polymeric surfactant may have a degree of hydrolysis in the range of 80% to 99%. More specifically, the lower limit of the degree of hydrolysis may be, for example, 81% or greater, 82% or greater, 83% or greater, 84% or greater, 85% or greater, or 87% or greater, and its 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. Polyvinyl alcohol may be used as a surfactant having said degree of hydrolysis, for example. The degree of hydrolysis can be measured using 1H-NMR.

[0070] Examples of available polymer surfactants include polyvinyl alcohol (PVA) or polyvinylpyrrolidone.

[0071] In one exemplary embodiment, the concentration of the polymeric surfactant in the continuous phase composition can be 1.0% or greater. For example, when using a continuous phase composition that is a mixture of water and a polymeric surfactant, the content of the polymeric surfactant can be 1.0% by weight or greater based on the total weight (100% by weight) of the continuous phase composition. Specifically, the lower limit of the concentration of the polymeric surfactant in the continuous phase composition can be 1.5% by weight or greater or 2.0% by weight or greater. Furthermore, the upper limit of the concentration of the polymeric surfactant can be less than 4.0%, specifically, for example, 3.5% or less, 3.0% or less, or 2.5% or less. When using polymeric surfactants at concentrations meeting the above requirements, uniform and stable droplets can be formed when the continuous and dispersed phases are mixed, which is advantageous for obtaining the desired final particle shape in this application.

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

[0073] In one exemplary embodiment, the mixture comprising a dispersed phase and a continuous phase can satisfy a density in the range of 0.99 g / cm³ to 1.05 g / cm³.

[0074] Suspension polymerization can be carried out under conditions that do not impede ensuring the particle characteristics of the preparation method according to this 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 temperatures, stirring can be performed at a speed of 600 rpm to 1,000 rpm.

[0075] In one exemplary embodiment, the method may further include a washing step following the completion of the suspension polymerization reaction. Washing can remove impurities unrelated to the particles of the suspended polymer. The washing method is not particularly limited, and known washing methods can be used. For example, washing can be performed by adding the suspended polymer to a washing solution containing an alcohol (e.g., an alcohol containing ethanol) and / or water (e.g., distilled water) and stirring it. Although not particularly limited, such washing can be repeated, for example, two or more times.

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

[0077] In one exemplary embodiment, the method may further include steps of centrifuging, washing, and drying the suspension polymer after the suspension polymerization reaction is complete. The description of washing and drying is the same as above.

[0078] In addition, the particulate composite of one embodiment may include particles with a particle size deviation coefficient 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%.

[0079] There are no restrictions on the method for measuring the deviation coefficient of particles, but it can be obtained, for example, according to the following mathematical equation 2.

[0080] [Mathematical Equation 2]

[0081]

[0082] The standard deviation and average particle size in mathematical equation 2 can be measured by analyzing SEM (scanning electron microscopy) images.

[0083] As the particle size deviation coefficient is reduced to the above range, particles with uniform size can be prepared, thereby maximizing particle performance.

[0084] On the other hand, if the particle size deviation coefficient increases excessively, for example, greater than 20%, the particle size deviation is large and uneven, which leads to the production of large particles. This results in reduced reproducibility during the preparation of cell therapy agents and the need for a separate fractionation process, which reduces preparation efficiency.

[0085] Simultaneously, the particles can be microfluidic chip particles. That is, particles with a particle size deviation coefficient 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% can be microfluidic chip particles. On the other hand, when the particles are suspended polymer particles, the particle size deviation coefficient increases to greater than 20%.

[0086] Specifically, one embodiment of the particle complex may be a particle complex comprising spherical particles and proteins immobilized on the particle surface, wherein the particles have a particle size deviation coefficient of 20% or less. Furthermore, one embodiment of the particle complex may be a particle complex comprising spherical particles and proteins immobilized on the particle surface, wherein the particles have a particle size deviation coefficient of 20% or less, and the particles may be microfluidic chip particles.

[0087] More specifically, the microfluidic chip particles can be particles obtained by methods including: a) injecting a dispersed phase composition containing polymerizable monomers into a continuous phase composition via a microfluidic path to generate droplets composed of the dispersed phase composition within the continuous phase composition; and b) photopolymerizing the droplets.

[0088] In step a) of generating droplets, droplets with uniform size and shape can be prepared by a specific method.

[0089] In step a) of generating droplets, droplets with uniform size can be generated because the dispersed phase composition that forms droplets is injected into the continuous phase composition through a microfluidic path to generate droplets.

[0090] Specifically, in step a) of generating droplets, a microfluidic device including microflow paths can be used.

[0091] As an example, a microfluidic device may include: a first supply unit through 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 through 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 microfluidic paths connecting the sides of the first and second flow paths. The sides of a flow path refer to directions other than the flow direction of the fluid flowing within the flow path.

[0092] More specifically, refer to Figure 3 The microfluidic device may include: a first supply unit 10 for supplying a dispersed phase composition; a first flow path 11 through which the dispersed phase composition supplied from the first supply unit can flow; a second supply unit 20 for supplying a continuous phase composition; a second flow path 21 through which the continuous phase composition supplied from the second supply unit can flow; and a plurality of microfluidic paths 12 connecting the sides of the first flow path and the sides of the second flow path. Figure 1 A diagram shows a microfluidic device with the following structure: In this structure, a first flow path 11 through which the dispersed phase composition flows is disposed between two second flow paths 21 through which the continuous phase composition flows, and the two sides of the first flow path 11 are connected to the sides of the two second flow paths 21 by a plurality of microflow paths, which is an example of a plurality of microflow paths arranged as densely as possible. The structure of the microfluidic device is not limited to this. Figure 3 And can refer to Figure 3 You are free to modify this content to the extent that it achieves the purpose of this disclosure.

[0093] The first and second flow paths can be configured to be spaced apart from each other by the desired length of the micropaths. The height and length of the micropaths are not particularly limited and can be appropriately adjusted according to the desired droplet size.

[0094] In step a) of generating droplets, the dispersed phase composition can be supplied to a first supply unit of the microfluidic device, and the continuous phase composition can be supplied to a second supply unit. The dispersed phase composition and the continuous phase composition can be injected into the first and second supply units respectively using a pump, but are not limited thereto.

[0095] The dispersed phase composition supplied to the first supply unit flows along a first flow path, and the continuous phase composition supplied to the second supply unit flows along a second flow path. In this case, the flow rate of the dispersed phase composition can be adjusted from 1 μl / min to 100 ml / min. The flow rate of the continuous phase composition can be adjusted from 10 μl / min to 500 ml / min.

[0096] The dispersed phase composition flowing through the first flow path flows into the second flow path through a plurality of microflow paths and encounters the continuous phase composition flowing through the second flow path.

[0097] The dispersed phase composition flowing from the first flow path through multiple microflow paths to the second flow path generates droplets at the boundary between the microflow path and the second flow path, and the generated droplets flow together with the continuous phase composition through the second flow path.

[0098] The microfluidic device may also include a discharge unit through which droplets formed from the dispersed phase composition are discharged. In this case, the microfluidic device may also include, as in Figure 3 The third flow path 31 shown connects the second flow path 21 and the discharge unit 40.

[0099] The dispersion path composition is a precursor composition for forming particles and may be an oil phase insoluble in the continuous phase composition which is an aqueous phase.

[0100] Specifically, the dispersed phase composition may contain polymerizable monomers, crosslinking agents, and photoinitiators.

[0101] Polymerizable monomers can be monomers having one or more unsaturated bonds. As an example, a polymerizable monomer can be a (meth)acrylate monomer or a (meth)acrylamide monomer having a (meth)acrylyl group, or an ethylene-based monomer having a vinyl group, or a mixture thereof.

[0102] Specifically, as epoxy-containing monomers, glycidyl methacrylate, glycidyl acrylate, and glycidyl acrylamide can be used alone or in mixtures with epoxy-free monomers.

[0103] Monomers that do not contain epoxy groups include methyl methacrylate, methyl acrylate, ethyl acrylate, ethylhexyl acrylate, butyl acrylate, hydroxyethyl methacrylate, trimethylolpropane triacrylate, N-isopropylacrylamide, n-butyl acrylate, etc.

[0104] Crosslinking agents can form crosslinked structures with polymerizable monomers or prepolymers formed therefrom, thereby allowing particles to maintain a spherical shape. When particles have a spherical shape, they can exhibit excellent cell culture efficiency due to their large specific surface area. As an example, an olefinic unsaturated crosslinking agent with two or more unsaturated bonds can be used as the crosslinking agent. More specifically, as the crosslinking agent, polyfunctional (meth)acrylates, such as ethylene glycol di(meth)acrylate, ethylene glycol di(meth)acrylate, trimethylolpropane tri(meth)acrylate, pentaerythritol tri(meth)acrylate, or dipentaerythritol hexa(meth)acrylate, can be used. Alternatively, one or more components of the crosslinking agents listed above can be used together. Considering the formation of a hydrophilic surface of the particles, ethylene glycol di(meth)acrylate is preferred.

[0105] The crosslinking agent content per 1 part by weight of monomer can be from 0.1 parts by weight to 10 parts by weight, or from 0.5 parts by weight to 2 parts by weight. When the above contents are met, it is beneficial to stably form the emulsion through the desired level of crosslinking and to ensure particle strength and morphological properties.

[0106] In one exemplary embodiment, the dispersed phase 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.

[0107] There are no particular limitations on the type of photoinitiator, and various initiators known in the art to which this disclosure pertains can be used, as long as the initiator does not hinder the achievement of the particle characteristics described above.

[0108] As photoinitiators, for example, initiators such as ketone initiators, organic peroxide initiators, or azo initiators can be used. Specifically, compounds such as 2,2-dimethoxy-2-phenylacetophenone, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone, 1-hydroxycyclohexylphenyl ketone, benzoyl peroxide, di-tert-amyl peroxide, tert-butyl peroxybenzoate, 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane, 2,5-dimethyl-2,5-di(tert-butylperoxy)hexyne-3, or dicumyl peroxide, and mixtures thereof, can be used, but are not limited thereto.

[0109] Based on 100 parts by weight of polymerizable monomer, the photoinitiator may be included in amounts 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.

[0110] Within this range, microparticles exhibiting appropriate polymerization rates and desired properties can be prepared.

[0111] In addition to the components mentioned above, the dispersed phase composition may include various additives known in the art to which this disclosure pertains, without interfering with the purpose of this disclosure.

[0112] Meanwhile, the continuous phase composition can be an aqueous phase. Specifically, the continuous phase composition can be an aqueous solution containing a surfactant and water.

[0113] There are no particular restrictions on the water, but it can be distilled water or deionized water.

[0114] Surfactants can be ionic or nonionic. For example, ionic surfactants can be sodium dodecyl sulfate (SDS), and nonionic surfactants can be Tween 20, Tween 40, Tween 60, Tween 80, Triton X-100, polyvinyl alcohol (PVA), polyethylene glycol (PEG), etc.

[0115] Based on the overall continuous phase composition, the continuous phase composition may contain 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 of a surfactant. Within this range, it is advantageous to prepare microparticles with desired properties.

[0116] Simultaneously, the droplets prepared in step a) of droplet generation can be introduced into step b) of droplet photopolymerization. In this case, the droplets can be mixed with another continuous phase composition before being introduced into the photopolymerization process.

[0117] As an example, a droplet-containing suspension discharged through the outlet of a microfluidic device can be mixed with another continuous phase composition and then introduced into a photopolymerization process. This process allows for uniform control of the particle shape.

[0118] The additional continuous phase composition can be the same as or different from the continuous phase composition used in step a) of droplet generation. As an example, in step a) of droplet generation, an aqueous solution of sodium dodecyl sulfate can be used as the continuous phase composition, and the suspension containing the droplets can be mixed with an aqueous solution of polyvinyl alcohol. However, it is not limited to this; the droplets generated in step a) of droplet generation can be directly transferred to the photopolymerization process.

[0119] In photopolymerization step b), the droplets generated in step a) are photopolymerized to prepare particles. In photopolymerization step b), the 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 exemplary embodiments and conditions widely used in conventional particle preparation fields can be applied without limitation.

[0120] Following photopolymerization step b), a washing step may also be included. Impurities unrelated to the particles can be removed by washing. There are no particular limitations on the washing method, and known washing methods can be used. For example, washing can be performed by adding the particles to an alcohol such as ethanol and stirring it. Although there are no particular limitations, this washing can be repeated, for example, three or more times.

[0121] After washing, a drying step may also be included. Drying can remove residual solvents, etc. There are no particular limitations on the drying method, and known drying methods can be used. For example, drying can be carried out in an oven or at room temperature. Furthermore, although there are no particular limitations, drying can be performed in a vacuum.

[0122] Depending on the particle preparation method, particles with uniform size and shape can be prepared. In conventional particle preparation methods, after particle formation, a process is performed to sort particles with the desired shape and characteristics from those with undesirable shapes or properties, resulting in low preparation yields. However, particle preparation methods can obtain particles with the desired shape and characteristics without undergoing this sorting process, thus exhibiting high preparation yields.

[0123] Additionally, the particle complex in one embodiment may include proteins immobilized on the surface of the particles.

[0124] The protein in question can be a protein used for immune cell differentiation. Proteins used for immune cell differentiation can play a role in providing the stimulation required for stem cells (such as allogeneic induced pluripotent stem cells (iPSCs)) to differentiate into immune cells.

[0125] Specific examples of proteins used for immune cell differentiation are not particularly limited, and examples include GM-CSF, IL4, IL-1β, TNFα, PGE2, DLL1, DLL4, TGFβ, or mixtures of two or more thereof. Furthermore, well-known proteins may be used without restriction.

[0126] Because the protein has the function of differentiating immune cells, when the particle complex of one embodiment is cultured with stem cells, the stem cells can differentiate into immune cells to prepare cell therapeutic agents for anti-cancer treatment, etc. By introducing such particles, the technical limitations of surface protein adsorption used in existing immune cell differentiation platforms can be effectively overcome, thereby enabling the economical production of large quantities of immune cells and possessing high market value as off-the-shelf drugs.

[0127] The protein content per 1 g of particles can range from 0.05 mg to 10 mg. If the protein content per 1 g of particles decreases excessively beyond this range, it becomes difficult to achieve adequate stem cell differentiation through protein. On the other hand, if the protein content per 1 g of particles increases excessively beyond this range, the protein may become higher than the density of ligands used for fixation on the cell surface, which may lead to technical problems such as the generation of proteins that cannot be fixed.

[0128] Proteins can be anchored to particle surfaces via covalent or physical bonds. In one embodiment, the particle complex can ensure structural stability by anchoring proteins to the particle surface via chemical bonds (e.g., covalent bonds) or physical bonds (e.g., adsorption). In particular, if proteins are anchored to the particle surface via chemical bonds (e.g., covalent bonds), large-scale production and automation can be stably achieved through a 3D process compared to conventional techniques, thereby improving process efficiency.

[0129] A covalent bond is a bond formed when atoms share electrons during chemical bonding, and proteins can be fixed to the surface of particles by covalent bonds when the particles are dispersed in water.

[0130] Specifically, covalent bonds may include bonding functional groups represented by the following chemical formula 1:

[0131] [Chemical Formula 1]

[0132]

[0133] In chemical formula 1, R1 is hydrogen or alkyl.

[0134] That is, the covalent bond can have a structure in which the particle and the protein are bonded via a bonding functional group represented by Formula 1. More specifically, the particle can be bonded to one end of the bonding functional group represented by Formula 1, and the protein can be bonded to the other end of the bonding functional group represented by Formula 1.

[0135] As an example, a covalent bond can include a bond represented by the following chemical formula 2.

[0136] [Chemical Formula 2]

[0137]

[0138] In chemical formula 2, R1 is hydrogen or alkyl, X is a particle, and Y is a protein.

[0139] The covalent bond represented by Formula 2 can be formed through the reaction between epoxy groups on the particle surface and amino groups contained in the protein. There are no particular limitations on the reaction conditions between the epoxy groups on the particle surface and the amino groups contained in the protein, and various well-known covalent bonding conditions can be applied without restriction. However, as an example, ammonium sulfate can be added to a solution in which the particles and protein are mixed in PBS (phosphate-buffered saline), and the reaction can be carried out for 16 hours or longer while shaking at 250 rpm or higher at room temperature.

[0140] Furthermore, covalent bonds may include bonding functional groups represented by the following chemical formula 3.

[0141] [Chemical Formula 3]

[0142]

[0143] That is, the covalent bond can have a structure in which the particle and the protein are bonded via a bonding functional group represented by Formula 3. More specifically, the particle can be bonded to one end of the bonding functional group represented by Formula 3, and the protein can be bonded to the other end of the bonding functional group represented by Formula 3.

[0144] As an example, a covalent bond can include a bond represented by the following chemical formula 4.

[0145] [Chemical Formula 4]

[0146]

[0147] In chemical formula 4, X represents particles and Y represents proteins.

[0148] The covalent bond represented by Formula 4 can be formed through the reaction between epoxy groups on the particle surface and thiol groups contained in the protein. There are no particular restrictions on the reaction conditions between the epoxy groups on the particle surface and the thiol groups contained in the protein, and various known covalent bonding conditions can be applied without limitation. However, as an example, ammonium sulfate can be added to a solution in which the particles and protein are mixed in PBS (phosphate-buffered saline), and the reaction can be carried out for 16 hours or longer while shaking at 250 rpm or higher at room temperature.

[0149] In addition, covalent bonds may include bonding functional groups represented by the following chemical formula 5.

[0150] [Chemical Formula 5]

[0151]

[0152] In chemical formula 5, R2 is hydrogen or alkyl.

[0153] That is, the covalent bond can have a structure in which the particle and the protein are bonded via a bonding functional group represented by Formula 5. More specifically, the particle can be bonded to one end of the bonding functional group represented by Formula 5, and the protein can be bonded to the other end of the bonding functional group represented by Formula 5.

[0154] As an example, a covalent bond can include a bond represented by the following chemical formula 6.

[0155] [Chemical Formula 6]

[0156]

[0157] In chemical formula 6, R2 represents hydrogen or an alkyl group, X represents particles, and Y represents proteins.

[0158] The covalent bond represented by Formula 6 can be formed through the reaction between carboxyl groups on the particle surface and amino groups contained in the protein. There are no particular restrictions on the reaction conditions between the carboxyl groups on the particle surface and the amino groups contained in the protein, and various well-known covalent bonding conditions can be applied without limitation. However, as an example, ammonium sulfate can be added to a solution in which the particles and protein are mixed in PBS (phosphate-buffered saline), and the reaction can be carried out for 16 hours or longer while shaking at 250 rpm or higher at room temperature.

[0159] Furthermore, since the particulate complex of one embodiment has the function of differentiating immune cells, when the particulate complex of one embodiment is cultured with stem cells, the stem cells can differentiate into immune cells to prepare cell therapeutic agents for anti-cancer treatment, etc. By introducing such particles, the technical limitations of surface protein adsorption used in existing immune cell differentiation platforms can be effectively overcome, thereby enabling the economical preparation of large quantities of immune cells and possessing high market value as off-the-shelf drugs.

[0160] The protein release ratio of the particulate complex in one embodiment, according to the following mathematical equation 1, is 0.1% or less or 0.0001% to 0.1%.

[0161] [Mathematical Equation 1]

[0162]

[0163] In mathematical 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.

[0164] This is believed to be because, in one implementation, the particle complex covalently anchors the protein to the particle surface to ensure structural stability. Therefore, compared to conventional techniques, large-scale production and automation can be stably achieved through a 3D process, thereby improving process efficiency.

[0165] On the other hand, if the protein release rate according to mathematical equation 1 increases excessively by more than 0.1%, the protein may not be stably fixed by the particle complex, which could lead to a significant reduction in process efficiency when mass-producing and automating through 3D processes.

[0166] The solution containing the particle complex is a mixture of the particle complex and a solvent. There are no particular limitations on the examples of solvents, and various known solvents can be used without restriction. However, as an example, PBS (phosphate-buffered saline) can be used. The particle complex can be added such that the protein concentration in the solution containing the particle complex meets the requirement of 1 μg / mL.

[0167] There are no particular limitations on the examples of shaking culture conditions, and various well-known shaking culture conditions can be applied without restriction. However, as an example, the reaction can be carried out for 7 hours or longer while shaking at 250 rpm or higher in a shaking incubator at room temperature.

[0168] There are no particular limitations on the examples of centrifugation conditions, and various well-known centrifugation conditions can be applied without restriction. However, as an example, centrifugation at 10,000 xg for 10 minutes is possible.

[0169] There are no particular limitations on the examples of methods used to measure the quality of proteins released from particulate complexes, and various well-known quantitative analytical methods can be applied without limitation. However, ELISA (enzyme-linked immunosorbent assay) can be mentioned as an example.

[0170] Meanwhile, the particulate complex of one embodiment may include spherical particles and proteins fixed to the surface of the particles. The contents of the spherical particles and the proteins fixed to the particle surface are the same as described above. If the particulate complex includes other components (e.g., magnetic particles) in addition to the spherical particles and the proteins fixed to the particle surface, it is difficult to remove them from the particulate complex, making it difficult to maintain quality, and there is a limitation that a separate removal process is required for reuse, which reduces process efficiency.

[0171] However, if necessary, the particulate complex of one embodiment may also include magnetic particles. Specifically, if the magnetic particles are further included within the particles of the particulate complex, the magnetic properties can be used to more easily separate and purify the particulate complex from the cells during separation and recovery after cell culture.

[0172] Magnetic particles are particles that exhibit magnetic properties. All materials interact with magnetic fields to produce attractive or repulsive forces. That is, when a magnetic field is applied to a material, the material becomes magnetized, and materials are classified according to the way they are magnetized, such as ferromagnetic materials, paramagnetic materials, semimagnetic materials, and ferrimagnetic materials.

[0173] Magnetic particles can be prepared by solution synthesis, coprecipitation, sol-gel method, high-energy milling, hydrothermal synthesis, microemulsion synthesis, thermal decomposition synthesis or sonochemical synthesis, but are not limited to these methods.

[0174] There are no particular limitations on the type of magnetic particles, and metal nanoparticles can be used. For example, magnetic particles may include one or more metals selected from gold (Au), silver (Ag), cobalt (Co), copper (Cu), iron (Fe), chromium (Cr), nickel (Ni), palladium (Pd), platinum (Pt), and tin (Sn), or oxides thereof. Specifically, magnetic particles may include iron (Fe) particles.

[0175] 2. Cell culture composition

[0176] According to another embodiment of this disclosure, a cell culture composition comprising cells and a particle complex according to an embodiment can be provided. The content regarding the particle complex includes all the above-described contents of the embodiments above.

[0177] Cells can include stem cells.

[0178] Cell culture compositions may also include culture medium solutions. Culture medium solutions may be based on bodily fluids (such as plasma or lymph) and contain various additives to adequately meet environmental conditions similar to those in vivo, such as pH, temperature, osmotic pressure, and nutrients, and may use a wide variety of materials widely known in the field of cell culture without limitation.

[0179] 3. Methods for preparing cell therapy agents

[0180] According to another embodiment of this disclosure, a method for preparing a cell therapy agent can be provided, the method comprising the steps of: culturing the cell culture composition of the other embodiment described above; and removing the particulate complex from the culture result. Content related to the cell culture composition includes all of the above-described contents in other embodiments.

[0181] In the step of culturing cell culture compositions according to other embodiments, the cells contained in the cell culture composition can differentiate into immune cells. Specific culture conditions are not particularly limited, and culture conditions that enable stem cells to differentiate into immune cells can be used without restriction.

[0182] Meanwhile, in the step of removing particulate complexes from the culture product, the safety of the cell therapy agent can be ensured by separating and removing the particulate complexes from the product obtained from the cell culture step. There are no particular limitations on the method for removing particulate complexes from the culture product, and various methods and conditions known in the art can be applied without restriction. However, as an example, the particulate complexes can be separated and removed using a cell filter.

[0183] Beneficial effects

[0184] According to this disclosure, a particulate complex, a cell culture composition using the same, and a method for preparing a cell therapeutic agent are available. The particulate complex not only immobilizes proteins required for cell differentiation to ensure the stability of the differentiation platform, but can also be completely isolated after cell culture to ensure the stability of the cell therapeutic agent. Attached Figure Description

[0185] Figure 1 SEM images of the particles obtained in Example 1 (1) are shown.

[0186] Figure 2 SEM images of the particles obtained in Example 6 (1) are shown.

[0187] Figure 3 This is a plan view of a microfluidic device. Detailed Implementation

[0188] The present disclosure will be described in more detail below with reference to embodiments. However, the following embodiments are provided for illustrative purposes only and are not intended to limit the scope of the present disclosure.

[0189] <Example: Preparation of particulate complex>

[0190] Example 1

[0191] (1) Preparation of particles

[0192] GMA (glycidyl methacrylate) as a monomer and EGDMA (ethylene glycol dimethacrylate) as a crosslinking agent were stirred, and V-65 as a thermal initiator was further added. The mixture was stirred at room temperature for about 5 minutes to prepare a dispersed phase. In this case, the weight ratio of monomer:crosslinking agent:initiator was 1:3:0.05.

[0193] PVA (polyvinyl alcohol; weight average molecular weight (Mw): 85,000 to 124,000, with a hydrolysis rate of 87% to 89%) was dissolved in distilled water at a concentration of 2% by weight to prepare a continuous phase.

[0194] 720 g of the obtained continuous phase was added to a reactor with a predetermined capacity, and 72 g of the dispersed phase was added thereto. The mixture was stirred at approximately 800 rpm at room temperature until a homogeneous dispersion was obtained. The reactor temperature was then increased, and polymerization was carried out at approximately 80°C, approximately 800 rpm, and under nitrogen purging conditions for approximately 6 hours.

[0195] The polymerized particles were recovered and washed twice with distilled water and five times with ethanol. The resulting material was then dried in an oven at approximately 80°C to prepare particles. The D50 diameter of the prepared particles was measured to be 130 μm (where D50 refers to the size of the 50th particle when analyzing a SEM (scanning electron microscope) image and counting the total number of particles as 100).

[0196] (2) Preparation of particulate complex

[0197] The particles obtained in (1) were washed three times with PBS (phosphate-buffered saline) by centrifugation at 10,000 xg for 10 minutes. Then, the proteins used for immune cell differentiation (GM-CSF, IL4, IL-1b, TNFa, PGE2, DLL1, DLL4, TGFb, etc., each applied independently) were dissolved in PBS and mixed at a ratio of 0.05 mg to 10 mg per 1 g of particles.

[0198] A PBS solution containing the particles obtained in (1) and the protein for immune cell differentiation was added in the same volume as 3 M ammonium sulfate, so that the final solution was 1.5 M ammonium sulfate. The resulting solution was then reacted in a shaking incubator at room temperature at 250 rpm or higher for 16 hours or longer, so that the epoxy groups exposed on the surface of the particles obtained in (1) were covalently bonded to the amino or thiol groups of the protein.

[0199] Then, the particles with covalently bonded proteins were separated from the mixture by centrifugation at 10,000 x g for 10 minutes. The supernatant was removed, and pH 12 Tris buffer was added and vortexed. The buffer solution was removed by centrifugation, and pH 4 acetate buffer was added and vortexed. The particles were then washed three times with PBS containing 0.5% by weight Tween 20, and the resulting particles with covalently bonded proteins were used as the particle complex of Example 1. The particle complex was placed in PBS and stored at 4°C.

[0200] Example 2

[0201] (1) Preparation of particles

[0202] The particles were prepared in the same manner as in (1) of Example 1, except that the D50 diameter of the particles was changed to 100 μm.

[0203] (2) Preparation of particulate complex

[0204] The particulate complex was prepared 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.

[0205] Example 3

[0206] (1) Preparation of particles

[0207] The particles were prepared in the same manner as in (1) of Example 1, except that the D50 diameter of the particles was changed to 50 μm.

[0208] (2) Preparation of particulate complex

[0209] The particulate complex was prepared 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.

[0210] Example 4

[0211] (1) Preparation of particles

[0212] The particles were prepared in the same manner as in (1) of Example 1, except that the D50 diameter of the particles was changed to 38 μm.

[0213] (2) Preparation of particulate complex

[0214] The particulate complex was prepared 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.

[0215] Example 5

[0216] (1) Preparation of particles

[0217] 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. Then, based on the weight of the stirred solution, Irgacure 651 as a photoinitiator was added at 1% by weight, and the mixture was stirred at room temperature for about 5 minutes to prepare a dispersed phase composition.

[0218] SDS (sodium dodecyl sulfate) was dissolved in deionized water at a concentration of 0.5% by weight to prepare a continuous phase composition.

[0219] The previously prepared dispersed phase composition (injection rate of 100 μl / min) and continuous phase composition (injection rate of 300 μl / min) were injected separately into the following... Figure 3The microfluidic device (40 μm step emulsification chip) shown in the diagram is divided into a first supply unit 10 and a second supply unit 20. The dispersed phase composition injected into the first supply unit 10 flows along a first flow path 11 and is supplied to the second flow path 21 via a microfluidic path 12, thereby forming droplets in the continuous phase composition. The suspension containing the droplets is discharged through a discharge unit 40.

[0220] The suspension containing the droplets was then collected in an aqueous solution of PVA (polyvinyl alcohol; molecular weight: 85,000 to 125,000), allowing the droplets to disperse in a final 3% by weight of PVA. After agitation to ensure uniform dispersion of the droplets, photopolymerization was performed using a UV spot curing device.

[0221] The polymerized particles were recovered and washed twice with distilled water and five times with ethanol. The resulting product was then dried in an oven at approximately 80°C to prepare granules.

[0222] (2) Preparation of particulate complex

[0223] The particulate complex was prepared 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.

[0224] Example 6

[0225] (1) Preparation of particles

[0226] The particles were prepared in the same manner as in Example 5 (1), except that the following four conditions were changed.

[0227] 1) Weight ratio of GMA:EGDMA = 1:2

[0228] 2) Dispersed phase injection rate: 10 μl / min

[0229] 3) Continuous phase injection rate: 15 μl / min

[0230] 4) Microfluidic device: 100 μm flow focusing chip

[0231] (2) Preparation of particulate complex

[0232] The particulate complex was prepared 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.

[0233] Example 7

[0234] (1) Preparation of particles

[0235] The particles were prepared in the same manner as in Example 5 (1), except that the following four conditions were changed.

[0236] 1) Weight ratio of GMA:EGDMA = 1:2

[0237] 2) Dispersed phase injection rate: 15 μl / min

[0238] 3) Continuous phase injection rate: 225 μl / min

[0239] 4) Microfluidic device: 190 μm flow focusing chip

[0240] (2) Preparation of particulate complex

[0241] The particulate complex was prepared 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.

[0242] <Comparative Example>

[0243] Comparative Example 1

[0244] Using flat-bottomed tissue culture plates (polystyrene culture plates, which are forms where proteins are directly fixed to the bottom without any particulate support), proteins for immune cell differentiation (GM-CSF, IL4, IL-1β, TNFα, PGE2, DLL1, DLL4, TGFβ, etc.) were diluted to target concentrations in PBS at 4°C and then added to 96-well plates at 50 μL / well. They were stored horizontally at 4°C for 16 hours or longer to induce adsorption, and then washed twice with PBS to remove residual proteins, thus preparing protein complexes.

[0245] <Reference Example: Preparation of Particulate Complexes>

[0246] Reference example 1

[0247] Commercially available particles (D50 diameter 180 μm) were prepared in PBS at a concentration of 0.1 mg / mL, and proteins for immune cell differentiation (GM-CSF, IL4, IL-1β, TNFα, PGE2, DLL1, DLL4, TGFβ, etc.) were added at a concentration of 0.05 mg to 5 mg per 1 g of particles. The reaction was then carried out at room temperature with shaking at 250 rpm or higher for 16 hours or longer. The reaction product was then washed three times with PBS to remove residual proteins, thus preparing the particle complex.

[0248] Reference example 2

[0249] 5 mg of commercially available magnetic beads (D50 diameter 5 μm) were dispersed in 1 mL of PBS, and the supernatant was removed using a magnet. Proteins for immune cell differentiation (GM-CSF, IL4, IL-1β, TNFα, PGE2, DLL1, DLL4, TGFβ, etc.) were added from 1 μg to 100 μg to prepare a 1 mg / mL solution. The total solution and 3 M ammonium sulfate solution were added in equal volumes, and the mixture was slowly tilted and rotated at 37°C for 16 hours or longer. The supernatant was removed using a magnet, and the mixture was then washed four times with PBS to prepare the particle complex.

[0250] <Experimental Example 1>

[0251] The physical properties of the complexes obtained in the Examples, Comparative Examples and Reference Examples were measured using the following methods, and the results are shown in Table 1.

[0252] 1. Protein stability

[0253] The complexes obtained in the examples, comparative examples, and reference examples were dissolved in PBS at a protein concentration of 1 μg / mL in 1 mL EP tubes and reacted for 7 hours or longer at room temperature with shaking at 250 rpm or higher. The supernatant was then collected by centrifugation at 10,000 xg for 10 minutes. The supernatant was then used for ELISA (enzyme-linked immunosorbent assay) to measure the mass of protein released from the particulate complexes, and the protein release ratio was calculated according to the following mathematical equation 1. Protein stability was assessed according to the following criteria.

[0254] [Mathematical Equation 1]

[0255]

[0256] In mathematical equation 1,

[0257] W1 represents the mass of protein released from the particle complex after shaking culture and centrifugation of the solution containing the particle complex, and

[0258] W2 represents the mass of the protein fixed to the particle complex before shaking culture and centrifugation of the solution containing the particle complex.

[0259] [Evaluation Criteria]

[0260] Above: According to mathematical equation 1, the protein release rate is 0.1% or less.

[0261] In the middle: According to mathematical equation 1, the protein release rate is greater than 0.1% and less than 10%.

[0262] Below: According to mathematical equation 1, the protein release rate is greater than 10%.

[0263] 2. Particle separation performance

[0264] The cell-containing culture medium was placed into a 10 mL vial, and the complex obtained in the examples, comparative examples, and reference examples was injected. The mixture was then shaken for 30 minutes. After separating the particulate complex from the cell culture medium using a cell filter, the presence of any remaining particulate complex in the cell culture medium was analyzed using a cell culture microscope and a particle size analyzer (Beckman Coulter counter).

[0265] The particle separation performance of the particle complex was evaluated according to the following criteria.

[0266] Above: The residual amount of particulate complex is less than 0.1% of the feed amount.

[0267] In the middle: the residual amount of particulate complex is 0.1% or more and less than 5% of the feed amount.

[0268] Below: The residual amount of particulate complex is 5% or more of the feed amount.

[0269] [Table 1]

[0270] Measurement results of Experiment Example 1

[0271]

[0272] As shown in Table 1, it was confirmed that in the particulate complex of the examples, the proteins were stably immobilized under cell culture conditions and completely separated after cell culture, compared with the comparative examples.

[0273] <Experimental Example 2>

[0274] The physical properties of the complexes obtained in Examples 1 and 5 through 7 were measured using the following methods, and the results are shown in Table 2.

[0275] 1. Particle size and deviation coefficient

[0276] (1) Particle size

[0277] The particles obtained in Examples 1, 5 to 7 were observed by analyzing SEM (scanning electron microscope) images, and the particle size was measured.

[0278] The D50 diameter measurement is the size of the 50th particle when the total number of particles is converted to 100.

[0279] (2) Coefficient of variation (CV)

[0280] For the 100 particle samples obtained in Examples 1, 5 to 7, SEM (scanning electron microscopy) images were analyzed, and the deviation coefficient was obtained according to the following mathematical equation 2.

[0281] [Mathematical Equation 2]

[0282]

[0283] [Table 2]

[0284] Measurement results of Experiment Example 2

[0285]

Claims

1. A particulate complex, comprising: Spherical particles; and Proteins fixed on the surface of the particles.

2. The particulate complex according to claim 1, The D50 diameter of the particles is greater than 20 μm.

3. The particulate complex according to claim 1, The D50 diameter of the particles is less than 180 μm.

4. The particulate complex according to claim 1, The D50 diameter of the particles is 30 μm to 175 μm.

5. The particulate complex according to claim 1, The D50 diameter of the particles is 50 μm to 160 μm.

6. The particulate complex according to claim 1, The protein is fixed to the surface of the particle by covalent bonds or physical bonds.

7. The particulate complex according to claim 6, The covalent bonds mentioned therein include bonding functional groups represented by the following chemical formula 1: [Chemical Formula 1] ， In chemical formula 1, R1 is hydrogen or alkyl.

8. The particulate complex according to claim 7, The covalent bonds mentioned therein include bonds represented by the following chemical formula 2: [Chemical Formula 2] ， In chemical formula 2, R1 is hydrogen or an alkyl group. X represents particles, and Y represents proteins.

9. The particulate complex according to claim 6, The covalent bonds mentioned therein include bonding functional groups represented by the following chemical formula 3: [Chemical Formula 3] 。 10. The particulate composite according to claim 9, The covalent bonds mentioned therein include bonds represented by the following chemical formula 4: [Chemical Formula 4] ， In chemical formula 4, X represents particles, and Y represents proteins.

11. The particulate complex according to claim 6, The covalent bonds mentioned therein include bonding functional groups represented by the following chemical formula 5: [Chemical Formula 5] ， In chemical formula 5, R2 is hydrogen or alkyl.

12. The particulate composite according to claim 11, The covalent bonds mentioned therein include bonds represented by the following chemical formula 6: [Chemical Formula 6] ， In chemical formula 6, R2 is either hydrogen or an alkyl group. X represents particles, and Y represents proteins.

13. The particulate complex according to claim 1, The protein content per 1 g of the particles is from 0.05 mg to 10 mg.

14. The particulate complex according to claim 1, The protein mentioned above is a protein used for the differentiation of immune cells.

15. The particulate complex according to claim 1, The particles mentioned above are suspended polymer particles.

16. The particulate complex according to claim 1, The particles described therein have a particle size deviation coefficient of 20% or less.

17. The particulate complex according to claim 16, The particles mentioned above are microfluidic chip particles.

18. The particulate complex according to claim 1, The protein release ratio of the particle complex according to the following mathematical equation 1 is 0.1% or less. [Mathematical Equation 1] ， In mathematical equation 1, W1 is the mass of protein released from the particle complex after shaking culture and centrifugation of a solution containing the particle complex, and W2 is the mass of the protein fixed to the particle complex before shaking culture and centrifugation of the solution containing the particle complex.

19. A cell culture composition comprising cells and the particle complex according to claim 1.

20. A method for preparing a cell therapy agent, the method comprising the following steps: Cultivating the cell culture composition according to claim 19; and Remove the particulate complex from the culture product.

Images