Method for producing microparticles

Abstract

The present invention relates to a method for producing microparticles. The method for producing microparticles can produce microparticles with uniform size and narrow density range. Therefore, the method for producing microparticles can show a higher production yield than existing methods for producing microparticles. The microparticles have a density lower than that of water and a very narrow range, so that when used as microcarriers, they enable highly efficient cell culture without the problem of floating on the surface of the culture medium during cell culture, and can be easily separated and collected after the culture process.

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of priority based on Korean Patent Application No. 10-2022-0133483, filed on October 17, 2022, and all contents disclosed in the documents of said Korean patent application are incorporated herein by reference.

[0002] The present invention relates to a method for producing microparticles. [Background technology]

[0003] With the expansion of the fields of biopharmaceuticals and regenerative medicine, there is an increasing demand for mass cell culture technologies that can efficiently produce cells, tissues, microorganisms, etc. For example, there has been active development of cell culture technologies using microcarriers.

[0004] In microcarrier-based cell culture technology, adherent cells are cultured using microcarriers in a 3D bioreactor. Specifically, cells, culture medium, and microcarriers are placed in the bioreactor, and the culture medium is stirred to bring the cells into contact with the microcarriers, allowing the cells to adhere to the microcarrier surface and then cultured. To be suitable for large-scale cell culture, the microcarriers must have a high surface area / volume ratio, allowing cells to attach and grow.

[0005] On the other hand, currently commercially available microcarriers have a size of 100-300 μm and a density of approximately 1.1-1.3 g / cm 3 The density of the cells cultured after attachment to the carriers was approximately 1.2 g / cm. 3Due to the density of microcarriers and cells, it is advantageous to attach cells to microcarriers in the initial stage of culturing cells in a bioreactor, but it is difficult to apply centrifugation when separating and recovering cells after culturing. Therefore, in addition to centrifugation, another filtering method that can separate and recover cells based on the size of the microcarriers and cells must be used. However, filtering methods based on the size of the microcarriers and cells have problems such as clogging of the filter as the process is repeated, lengthening the process time, inducing physical damage and contamination of the cells, and losing cells during the filtering process. To solve these problems, a method was developed to filter cells with a density of 1.0 g / cm 3 Less than 1.3g / cm 3 One approach to fabricating microcarriers is to utilize the properties of larger materials (polymers or polymeric components that form the microcarrier backbone). However, even in this case, the range of achievable microcarrier densities is limited, resulting in insufficient adhesion between the microcarriers and cells, leading to poor culture efficiency. For example, when a culture medium containing microcarriers and cells is stirred, if the microcarrier density is too low, most of the microcarriers float to the surface of the culture medium despite stirring; if the microcarrier density is too high, most of the microcarriers sink to the bottom despite stirring, resulting in poor cell adhesion to the carriers and poor culture efficiency.

[0006] Therefore, it is necessary to develop a microcarrier-related technology that can be dispersed evenly in the culture medium, which is advantageous for cell attachment and culture, and that also allows for easy cell separation and recovery after culture. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention provides a method for producing microparticles. [Means for solving the problem]

[0008] Hereinafter, a method for producing microparticles according to a specific embodiment of the present invention and the microparticles produced therefrom will be described.

[0009] According to one embodiment of the invention, there is provided a method for producing microparticles, comprising the steps of: a) injecting a dispersed phase composition containing a polymerizable monomer into a continuous phase composition through a microchannel to generate droplets composed of the dispersed phase composition in the continuous phase composition; b) photopolymerizing the droplets; and c) thermally polymerizing the photopolymerized droplets.

[0010] As used herein, the term "microparticles" refers to particles having a size (diameter) at the micron level, a particle group containing such particles, or a particle group consisting of such particles. For example, a particle group containing individual particles having a particle diameter in the range of 10 to 500 μm is provided. The diameter can be measured by a method similar to that described below.

[0011] As used herein, "dispersed phase composition" means a composition that is capable of forming a dispersed phase (or droplets) after being mixed with a continuous phase composition.

[0012] As used herein, "continuous phase composition" means a composition that can form a continuous phase after being mixed with a dispersed phase composition.

[0013] Unless otherwise defined or explained in this specification, the temperature at which the manufacturing process (or each manufacturing step) is carried out or the temperature at which the numerical properties of the manufactured particles are calculated or measured is room temperature. Specifically, in this specification, "room temperature" refers to a temperature in a state where the temperature is not particularly increased or decreased, and may mean, for example, a temperature in the range of 15 to 30°C.

[0014] The inventors have confirmed through experiments that when droplets manufactured to a uniform size and shape by a specific method are continuously photopolymerized and thermally polymerized, microparticles with high uniformity in shape and size, lower density than typically cultured cells, a very narrow density range, and excellent surface flatness can be obtained in high yield.

[0015] The method for producing microparticles according to the embodiment will be described in detail below.

[0016] The manufacturing method of one embodiment includes the step of: a) injecting a dispersed phase composition containing a polymerizable monomer into a continuous phase composition through a microchannel to generate droplets composed of the dispersed phase composition in the continuous phase composition.

[0017] In the step a) of generating droplets, droplets of uniform size and shape can be produced by a specific method.

[0018] Oil-in-water (O / W) droplets, in which existing oil components are dispersed within water, have been formed by pouring a dispersed phase composition into a continuous phase composition and physically mixing it, such as by stirring. However, this method produces droplets of non-uniform size, resulting in microparticles with non-uniform size and physical properties.

[0019] In contrast, in the step a) of generating droplets, droplets of uniform size can be produced by injecting a droplet-forming dispersed phase composition into a continuous phase composition through a microchannel to generate droplets.

[0020] Specifically, in the step a) of generating droplets, a microfluidic device including a microchannel is used.

[0021] For example, the microfluidic device may include a first supply unit to which a dispersed phase composition is supplied, a first flow path through which the dispersed phase composition supplied from the first supply unit flows, a second supply unit to which a continuous phase composition is supplied, a second flow path through which the continuous phase composition supplied from the second supply unit flows, and a plurality of microfluidic channels connecting side surfaces of the first and second flow paths to each other. The side surfaces of the flow paths refer to a direction other than the flow direction of the fluid flowing through the flow paths.

[0022] More specifically, referring to FIG. 1, the microfluidic device may include a first supply unit 10 through which a dispersed phase composition is supplied, a first channel 11 through which the dispersed phase composition supplied from the first supply unit flows, a second supply unit 20 through which a continuous phase composition is supplied, a second channel 21 through which the continuous phase composition supplied from the second supply unit flows, and a plurality of microchannels 12 connecting the sides of the first and second channels. FIG. 1 illustrates a microfluidic device having a structure in which the first channel 11 through which the dispersed phase composition flows is disposed between two second channels 21 through which the continuous phase composition flows, and both sides of the first channel 11 are connected to the sides of the two second channels 21 via a plurality of microchannels 12, and this is an example in which the plurality of microchannels 12 are arranged as closely as possible. The structure of the microfluidic device is not limited to that shown in FIG. 1 and can be freely modified within the scope of the objectives of the present invention.

[0023] The first and second flow channels are spaced apart by a desired length of the microchannel. The height and length of the microchannel are not particularly limited and can be appropriately adjusted depending on the desired droplet size.

[0024] In the step a) of generating droplets, the dispersed phase composition may be supplied to a first supply unit of the microfluidic device, and the continuous phase composition may be supplied to a second supply unit of the microfluidic device. The dispersed phase composition and the continuous phase composition may be injected into the first and second supply units, respectively, via pumps, but are not limited thereto.

[0025] 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. At this time, the velocity of the dispersed phase composition is adjusted to 1 μl / min to 10 ml / min. The velocity of the continuous phase composition is adjusted to 0.1 μl / min to 100 ml / min.

[0026] The dispersed phase composition flowing through the first flow path flows through a plurality of microchannels to a second flow path where it meets the continuous phase composition flowing through the second flow path.

[0027] The dispersed phase composition flowing through the plurality of microchannels in the direction from the first channel to the second channel generates droplets at the boundary between the microchannel and the second channel, and the generated droplets flow through the second channel together with the continuous phase composition.

[0028] The microfluidic device may further include a discharge part through which droplets formed from the dispersed phase composition can be discharged. In this case, the microfluidic device may further include a third channel 31 connecting the second channel 21 and the discharge part 40, as shown in FIG.

[0029] The dispersed phase composition is a precursor composition for forming low-density microparticles and may be an oil phase that is insoluble in the continuous phase composition, which is a water phase.

[0030] Specifically, the dispersed phase composition can include a polymerizable monomer, a crosslinker, a low density oil, a photoinitiator, and a thermal initiator.

[0031] 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-based monomer having a vinyl group, or a mixture thereof. In one example, to form microparticles having a density lower than that of water, the polymerizable monomer may include a styrene-based monomer as a vinyl-based monomer.

[0032] The crosslinking agent forms a crosslinked structure with the polymerizable monomer or a prepolymer formed therefrom, allowing the microparticles to maintain their spherical shape. When the microparticles have a spherical shape, a cell culture microcarrier exhibiting excellent cell culture efficiency due to a large specific surface area can be provided. For example, an ethylenically unsaturated crosslinking agent having two or more unsaturated bonds can be used as the crosslinking agent. More specifically, considering the density of the microparticles and the cell culture process, it is preferable to use, for example, divinylbenzene as the crosslinking agent.

[0033] The dispersed phase composition may contain the crosslinker in an amount of 10 parts by weight or more, 50 parts by weight or more, 100 parts by weight or more, 200 parts by weight or more, 250 parts by weight or more, or 300 parts by weight or more, and 1000 parts by weight or less, 800 parts by weight or less, 700 parts by weight or less, 600 parts by weight or less, 500 parts by weight or less, or 450 parts by weight or less, per 100 parts by weight of the polymerizable monomer.

[0034] Within this range, the microparticles can stably maintain a spherical shape and have a particle density suitable for cell culture and centrifugation.

[0035] The low-density oil is included in the dispersed phase composition so that the microparticles have a density lower than that of water. As used herein, "low-density oil" refers to a hydrocarbon oil having a density lower than that of water at room temperature. For example, low-density oil refers to a hydrocarbon oil having a density of 0.700 to 0.997 g / cm at 21°C. 3 "Hydrocarbon oil" means a hydrocarbon oil having a density of

[0036] In the prior art, expanded polystyrene particles were produced using a blowing agent to reduce the density of microcarrier particles. However, the use of a blowing agent resulted in an excessively broad distribution range of particle diameters and densities, making it difficult to obtain microcarriers with diameters and densities suitable for cell culture applications in sufficient yield. However, in one embodiment of the manufacturing method, low-density microparticles can be produced without a foaming step by using a dispersed phase composition containing a low-density oil. In other words, the microparticles produced by this manufacturing method are non-expanded particles.

[0037] The low-density oil escapes from the droplets during the polymerization process, and some of it is trapped in the polymer, contributing to a reduction in the density of the microparticles. Furthermore, the microparticles obtained using the low-density oil do not sink to the bottom of the culture medium or float on the surface of the culture medium, but remain evenly dispersed in the culture medium. As a result, when the microparticles are used as microcarriers for cell culture, the degree of suspension of the microcarriers in the culture medium is improved, increasing the adhesion between the microcarriers and the cells and improving the culture efficiency.

[0038] The type of low-density oil that can be contained in the dispersed phase composition is not particularly limited, but can be selected in consideration of ease of carrying out the production method and ensuring the properties of the microparticles described below. For example, 3 or less than 0.790g / cm 3 The following low-density hydrocarbon oils can be used. At this time, the lower limit of the density of the low-density hydrocarbon oil is not particularly limited, but for example, it is 0.750 g / cm 3 or more than 0.760 g / cm 3 It may be more than that.

[0039] In one example, the low-density oil can contain one or more linear or branched saturated hydrocarbon compounds having from 8 to 50 carbon atoms. Specifically, the low-density oil can contain, for example, a normal alkane having from 8 to 16 carbon atoms, an isoalkane having from 8 to 16 carbon atoms, or a mixture thereof. Specific examples that can be used as the low-density oil include dodecane having from 12 carbon atoms, hexadecane having from 16 carbon atoms, and Isopar M (a mixture of an isoalkane having from 12 to 14 carbon atoms and an isoalkane having from 13 to 16 carbon atoms).

[0040] The dispersed phase composition may include the low-density oil in an amount of 5 parts by weight or more, 10 parts by weight or more, 20 parts by weight or more, 30 parts by weight or more, 40 parts by weight or more, or 50 parts by weight or more, and 100 parts by weight or less, 97 parts by weight or less, 95 parts by weight or less, 94 parts by weight or less, 93 parts by weight or less, or 92 parts by weight or less, per 100 parts by weight of the polymerizable monomer.

[0041] When used as a microcarrier for cell culture within this range, it is possible to provide microparticles of an appropriate density that do not sink to the bottom of the culture medium or float on the surface of the culture medium, but remain evenly dispersed in the culture medium, exhibiting high cell attachment rates and culture efficiency, and that can be easily separated and recovered by centrifugation after cell culture.

[0042] In one embodiment, the manufacturing method produces microparticles through successive photopolymerization and thermal polymerization steps, whereby the dispersed phase composition contains both a photoinitiator and a thermal initiator.

[0043] The types of the photoinitiator and thermal initiator are not particularly limited, and various initiators known in the art to which the present invention pertains can be used as long as they do not cause any problems in ensuring the particle characteristics of the microparticles described above.

[0044] The photoinitiator may be, for example, one or more selected from the group consisting of benzoin ether, dialkyl acetophenone, hydroxyl alkyl ketone, phenyl glyoxylate, benzyl dimethyl ketal, acyl phosphine, and α-aminoketone. A specific example of an acyl phosphine is the commercially available IRGACURE 819, i.e., bis(2,4,6-trimethylbenzoyl)-phenylphosphine oxide.

[0045] 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, and 30 parts by weight or less, 27 parts by weight or less, 25 parts by weight or less, 23 parts by weight or less, 20 parts by weight or less, or 18 parts by weight or less, relative to 100 parts by weight of the polymerizable monomer.

[0046] Within this range, an appropriate polymerization rate can be exhibited to produce microparticles with desired properties.

[0047] The thermal initiator may be one or more selected from the group consisting of persulfate initiators, azo initiators, hydrogen peroxide, and ascorbic acid. Specific examples of persulfate initiators include sodium persulfate (NaSO), potassium persulfate (KSO), and ammonium persulfate ((NHSO). Examples of azo initiators include 2,2-azobis(2-amidinopropane) dihydrochloride and 2,2-azobis(N,N-dimethylene)isobutyramidine dihydrochloride. dihydrochloride), 2-(carbamoylazo)isobutylonitrile, 2,2-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride, 4,4-azobis-(4-cyanovaleric acid), and 2,2-azobis(2,4-dimethylvaleronitrile; trade name: V-65).

[0048] The thermal initiator may be included in an amount of 0.1 parts by weight or more, 0.5 parts by weight or more, 1 part by weight or more, 1.5 parts by weight or more, 2 parts by weight or more, 2.5 parts by weight or more, or 3 parts by weight or more, and 30 parts by weight or less, 25 parts by weight or less, 20 parts by weight or less, 15 parts by weight or less, 10 parts by weight or less, 8 parts by weight or less, or 6 parts by weight or less, relative to 100 parts by weight of the polymerizable monomer.

[0049] Within this range, an appropriate polymerization rate can be exhibited to produce microparticles with desired properties.

[0050] In addition to the components described above, the dispersed phase composition may contain various additives known in the art to which the present invention pertains, as long as they do not impair the object of the present invention.

[0051] On the other hand, the continuous phase composition may be an aqueous phase. Specifically, the continuous phase composition may be an aqueous solution containing a surfactant and water.

[0052] The water is not particularly limited, but may be distilled water or deionized water.

[0053] The surfactant may be an ionic surfactant or a non-ionic surfactant, for example, sodium dodecyl sulfate (SDS) as an ionic surfactant, or Tween 20, Tween 40, Tween 60, Tween 80, Triton X-100, polyvinyl alcohol (PVA), polyethylene glycol (PEG), etc.

[0054] The continuous phase composition may contain the surfactant in an amount of at least 0.01 wt %, at least 0.05 wt %, at least 0.1 wt %, at least 0.2 wt %, or at least 0.3 wt %, and at most 5 wt %, at most 4 wt %, at most 3 wt %, at most 2 wt %, or at most 1 wt %, based on the total continuous phase composition. Such a range is advantageous for producing microparticles with desired properties.

[0055] Meanwhile, the droplets produced in the step a) of forming droplets are introduced into the step b) of photopolymerizing the droplets, where the droplets are mixed with an additional continuous phase composition before being introduced into the photopolymerization process.

[0056] For example, the suspension containing droplets discharged from the outlet of the microfluidic device is mixed with an additional continuous phase composition and then introduced into the photopolymerization process, which allows for uniform control of the shape of the microparticles.

[0057] The additional continuous phase composition may have the same composition as or a different composition from the continuous phase composition used in the droplet-forming step a) . For example, in the droplet-forming step a) , an aqueous solution of sodium dodecyl sulfate is used as the continuous phase composition, and the suspension containing the droplets is mixed with an aqueous polyvinyl alcohol solution. However, the droplets formed in the droplet-forming step a) can be immediately transferred to the photopolymerization step.

[0058] In the step b), photopolymerization is performed to photopolymerize the droplets produced in the step a) to produce preliminary microparticles. In the step b), photopolymerization can be induced by irradiating the droplets with UV light using an ultraviolet lamp. Referring to Comparative Example 3 described below, it can be seen that in the step b), even with sufficient UV irradiation, complete polymerization and crosslinking of the droplets are not achieved, and solidified particles cannot be obtained. However, complete polymerization and crosslinking are achieved in the subsequent step c), thermal polymerization.

[0059] The photopolymerized droplets that have undergone the photopolymerization step (b) are introduced into the thermal polymerization step (c) to achieve complete polymerization and crosslinking. For example, the thermal polymerization is performed at a temperature of 40°C or higher, 45°C or higher, 50°C or higher, 55°C or higher, or 60°C or higher, and 100°C or lower, 90°C or lower, 80°C or lower, 75°C or lower, or 70°C or lower. The thermal polymerization time can be controlled to 1 hour or higher, 2 hours or higher, 2.5 hours or higher, 3 hours or higher, 3.5 hours or higher, or 4 hours or higher, and 48 hours or lower, 36 hours or lower, 24 hours or lower, 12 hours or lower, 10 hours or lower, or 8 hours or lower. When the thermal polymerization temperature and time are controlled within the above-mentioned ranges, microparticles of uniform shape and size can be obtained without particle agglomerations.

[0060] In the method for producing microparticles according to the embodiment, the droplet production process, the photopolymerization process, and the thermal polymerization process are performed continuously, which makes the production process easy and provides excellent productivity.

[0061] When the microparticles are used as microcarriers, the manufacturing method of the embodiment may further include forming a primer layer and / or a cell attachment-inducing layer on the surface of the microparticles.

[0062] The primer layer allows the introduction of functional polymers onto the surface of microparticles lacking functional groups, and acts as a so-called adhesive layer, i.e., a cell adhesion-inducing layer or a layer that allows cells to be stably maintained on the particles.

[0063] Compounds that can form a primer layer include, but are not limited to, catechol derivatives capable of inducing aqueous phase adhesion. For example, one or more compounds selected from the group consisting of L-dihydroxyphenylalanine (L-DOPA), dopamine, norepinephrine, epinephrine, epigallocatechin, and their derivatives can be used to form the primer layer. A primer layer formed using such compounds can impart hydrophilicity to the particle surface, thereby further enhancing the dispersibility of the particles in the aqueous dispersion, which is the continuous phase composition.

[0064] In one example, the ratio of the radius of the microparticle to the thickness of the primer layer may be 1:0.00001 to 1:0.01, or 1:0.0001 to 1:0.001. If the ratio of the radius of the microparticle to the thickness of the primer layer is too low, the primer layer will be too thin compared to the microparticles, and the effect of modifying the surface of the microparticles to be hydrophilic will be minimal. If the ratio is too high, the primer layer will be too thick compared to the microparticles, and the adhesion efficiency between cells and the microparticles during cell culture may be reduced.

[0065] The cell attachment-inducing layer is composed of cell adhesive substances, which provide sites for the binding of cellular transmembrane proteins, thereby enabling stable attachment, spreading, and culture of adherent cells. The cell attachment-inducing layer-forming compound may be, but is not limited to, one or more selected from the group consisting of gelatin, collagen, fibronectin, chitosan, polydopamine, tannic acid, polyphenols, poly-L-lysine, vitronectin, RGD-containing peptides, lignin, cationic dextran, and derivatives thereof. Furthermore, the cell attachment-inducing layer formed using these compounds can modify the surface of microparticles to be hydrophilic, thereby improving the water dispersibility of the microparticles.

[0066] The method for producing microparticles according to one embodiment may further include a washing step after the thermal polymerization step (c). Impurities unrelated to the microparticles can be removed by washing. The washing method is not particularly limited, and known washing methods can be used. For example, the washing is performed by adding the microparticles to alcohol such as ethanol and stirring the mixture. Although not particularly limited, this washing may be repeated, for example, three or more times. For example, the washing is performed after forming a primer layer and / or a cell attachment-inducing layer on the surface of the microparticles.

[0067] The method for producing microparticles according to one embodiment may further include a drying step after the washing. Residual solvents can be removed by drying. The drying method is not particularly limited, and known drying methods can be used. For example, the drying can be performed using an oven or at room temperature. Also, although not particularly limited, the drying can be performed in a vacuum.

[0068] The manufacturing method of the present embodiment can produce microparticles having uniform size and shape and a narrow density range. Conventional methods for manufacturing microparticles have a low production yield due to the need to separate particles having desired shapes and properties from particles having undesired shapes or properties after particle formation. However, the manufacturing method of the present embodiment can produce microparticles having desired shapes and properties without the need for such separation, thereby achieving a high production yield.

[0069] The microparticles produced by the method of the embodiment have a size on the micrometer level. For example, the diameter of the microparticles may be 10 μm or more, 15 μm or more, 20 μm or more, 22 μm or more, 25 μm or more, 27 μm or more, 30 μm or more, or 35 μm or more, and 500 μm or less, 400 μm or less, 300 μm or less, 200 μm or less, 100 μm or less, 80 μm or less, 60 μm or less, or 50 μm or less. The diameter is determined by measuring the two-dimensional (2D) planar area of ​​the particle using an optical microscope and applying the equation for planar area (S=πr 2 ) to calculate the diameter. In this case, the diameter may be the arithmetic average of the diameter values ​​of at least 100 particles. When the microparticles have a diameter within the above range, they have a surface area suitable for cell culture and can exhibit good cell culture efficiency.

[0070] The microparticles may have a very uniform size and shape, resulting in a very small coefficient of variation in their diameter. For example, the coefficient of variation in the diameter of the microparticles may be 10% or less, 8% or less, 6% or less, or 5% or less. In this case, the coefficient of variation in the diameter may be a value calculated for at least 100 particles. The uniform particle size can provide an optimal surface for cell growth, contributing to improved culture efficiency. The lower limit of the coefficient of variation in the diameter of the microparticles is not particularly limited, but may be, for example, 0% or more.

[0071] Density is approximately 1.2 g / cm 3 Typical commercial microcarriers used for high-level cell culture have a density of approximately 1.1–1.3 g / cm. 3 Although microcarriers with such a density are advantageous for initial cell attachment, there is a problem in that cell separation and recovery by centrifugation after culture is difficult.

[0072] The microparticles produced by the production method of the embodiment have a density lower than that of water. Therefore, when the microparticles are used as microcarriers, the difference in sedimentation velocity due to gravity between the microparticles and the cultured cells is greater than when conventional microcarriers are used, making it easier to separate the microparticles from the cells.

[0073] On the other hand, if the microcarrier density is too low, the cells will have difficulty attaching to the microcarriers in the early stages of cell culture, and the microcarriers will float in the culture medium during the cell culture process, reducing the culture efficiency.

[0074] The microparticles produced by the production method of one embodiment have a density of 0.985 g / cm at 15 to 25°C. 3 More than 0.998g / cm 3 The density of the microparticles can be confirmed by preparing a solution of a predetermined density (e.g., deionized water or an aqueous ethanol solution), mixing the particles to be confirmed, and checking whether or not they float. Specifically, when the density is less than 0.985 g / cm at 15 to 25°C, 3 More than 0.998g / cm 3 Less than this means that the density is 0.985 g / cm at 15 to 25°C. 3 In the ethanol solution, there are no floating particles and the density is 0.998 g / cm at 15-25°C. 3 This can mean that there are no settled particles in deionized water.

[0075] The microparticles may have a narrower density distribution. For example, the microparticles may have a density of 0.985 g / cm at 15-25°C. 3 or more than 0.990g / cm 3 or more, 0.998 g / cm 3 Less than 0.995g / cm 3 or less, or 0.990 g / cm 3 As a more specific example, the microparticles may have a density of 0.985 g / cm at 15 to 25°C. 3 More than 0.990g / cm3 or less, or 0.990 g / cm 3 More than 0.995g / cm 3 It can have a narrow density distribution of:

[0076] The microparticles produced by the production method of the embodiment may be spherical. Spherical means that the particles have a shape that is nearly spherical, which can be confirmed with the naked eye. For example, spherical means that the sphericity value calculated by the following Equation 1 is about 0.80 or more. When particles have a spherical shape, a large surface area can be secured, and cell adhesion performance can be improved when used as a microcarrier.

[0077]

number

[0078] (In the above formula 1, V p is the volume of the particle, and A p is the surface area of ​​the particle.)

[0079] The microparticles produced by the method of the present invention can be used as microcarriers for cell culture. The type of cells is not particularly limited. For example, the cells may be adherent animal cells, such as fibroblasts, chondrocytes, mesenchymal stem cells, CHO, HEK293, Vero cells, BHK21, or MDCK cells.

[0080] The cells are cultured attached to the microparticles in a culture medium. The culture medium may contain nutrients based on bodily fluids such as plasma and lymph, similar to living body conditions, as well as various additives to fully satisfy environmental conditions such as pH, temperature, and osmotic pressure. Various substances widely known in the field of cell culture-related technologies can be used as such additives without limitation.

[0081] The microparticles and cells can have a density lower than that of the culture medium. Specifically, the microparticles have a density of 0.985 g / cm at 15 to 25°C. 3 More than 0.998g / cm 3 The cells may have a density of less than 1.10 to 1.25 g / cm 3 (approx. 1.2g / cm 3 ) range. Thus, the microparticles injected into the culture medium are suspended in the culture medium, and as the number of cells adhering to the surfaces of the suspended microparticles increases, the density of the microparticles with attached cells also gradually increases, allowing them to sink to the bottom of the container containing the culture medium. After cell culture, the cells can be separated from the microparticle-cell conjugates by centrifugation, thereby obtaining the cultured cells. [Effects of the Invention]

[0082] A method for producing microparticles according to one embodiment of the present invention can produce microparticles with a uniform size and a narrow density range. Therefore, the method can achieve a higher production yield than existing methods for producing microparticles. The microparticles have a density lower than that of water and a very narrow range. When used as microcarriers, the microparticles enable highly efficient cell culture without the problem of floating on the surface of the culture medium during cell culture, and can be easily separated and recovered after the culture process. [Brief explanation of the drawings]

[0083] [Figure 1] 1 is a schematic diagram of an apparatus capable of implementing a method for producing microparticles according to one embodiment. [Figure 2] 1 is an SEM image of the microparticles prepared in Example 1. [Figure 3] 1 is an SEM image of the microparticles prepared in Example 2. [Figure 4] 1 is a SEM image of microparticles prepared in Comparative Example 1. [Figure 5] 1 is a SEM image of microparticles prepared in Comparative Example 3. [Figure 6] 1 shows photographs taken to observe the degree of particle suspension after mixing the microparticles prepared in Example 1 with deionized water having a density of 0.998 g / cm3 and aqueous ethanol solutions having densities of 0.995 g / cm3, 0.990 g / cm3, and 0.985 g / cm3. The numbers on the photographs indicate density in g / cm3. [Figure 7] 1 shows photographs taken to observe the degree of particle suspension after mixing the microparticles prepared in Example 2 with deionized water having a density of 0.998 g / cm3 and aqueous ethanol solutions having densities of 0.995 g / cm3, 0.990 g / cm3, and 0.985 g / cm3. The numbers on the photographs indicate density in g / cm3. DETAILED DESCRIPTION OF THE INVENTION

[0084] The functions and effects of the present invention will be described in more detail below through specific examples of the present invention, but these are presented as examples of the present invention and do not limit the scope of the invention in any way.

[0085] Example 1: Microparticle production A mixture was prepared by mixing 400 parts by weight of divinylbenzene and 68 parts by weight of Isopar M (low-density oil) with 100 parts by weight of styrene, and 17 parts by weight of IRGACURE 819 (photoinitiator) and 5.7 parts by weight of V-65 (thermal initiator) with 100 parts by weight of styrene to prepare a dispersed phase composition.

[0086] A continuous phase composition was prepared by dissolving sodium dodecyl sulfate (SDS) in deionized water to a concentration of 0.5 wt %.

[0087] The prepared dispersed phase composition and continuous phase composition were respectively pumped into first supply unit 10 and second supply unit 20 of the microfluidic device shown in Figure 1. The dispersed phase composition injected into first supply unit 10 flowed along first channel 11 and was supplied to second channel 21 through microchannel 12, forming droplets in the continuous phase composition. The suspension containing the droplets was discharged through discharge unit 40.

[0088] The suspension containing the droplets was then fed into a mixing zone via a T-connector together with a 4 wt% aqueous polyvinyl alcohol (PVA) solution supplied by another pump, where they were mixed. A static mixer was used in the mixing zone.

[0089] The mixture from the mixing zone was transferred to the photopolymerization zone, where the droplets were irradiated with UV light using a UV lamp to induce a photopolymerization reaction, and the droplets were photopolymerized for about 10 minutes.

[0090] The photopolymerized droplets were then transferred to a thermal polymerization zone, where they were thermally polymerized at 65°C for 4 hours.

[0091] After thermal polymerization, the produced particles were washed three times with deionized water and then washed three times with ethanol to obtain microparticles.

[0092] Example 2: Microparticle production Microparticles were produced in the same manner as in Example 1, except that 400 parts by weight of divinylbenzene, 89 parts by weight of Isopar M, 17.7 parts by weight of IRGACURE 819 (photoinitiator), and 5.9 parts by weight of V-65 (thermal initiator) were used per 100 parts by weight of styrene.

[0093] Comparative Example 1: Production of Microparticles A mixture was prepared by mixing 33 parts by weight of divinylbenzene and 34 parts by weight of Isopar M with 100 parts by weight of styrene, and a dispersed phase composition was prepared by adding 3.5 parts by weight of benzoyl peroxide and 0.5 parts by weight of tert-butyl peroxybenzoate with respect to 100 parts by weight of the styrene to the mixture.

[0094] A continuous phase composition was prepared by dissolving 2.5 g of polyvinyl alcohol (PVA) having a weight average molecular weight in the range of 85,000 to 125,000 and a hydrolysis rate of 87 to 89% in 250 g of distilled water.

[0095] 50 g of a 1 wt% aqueous polyvinyl alcohol (PVA) solution was mixed with the dispersed phase composition and stirred in an oil bath until a uniform dispersion was obtained. Specifically, the oil bath was gradually heated while stirring at 800 rpm at room temperature, and suspension polymerization was carried out at a temperature of 85 to 88°C and a speed of 600 to 800 rpm. The polymerization was carried out under a nitrogen purge.

[0096] After 6 hours of reaction, the produced particles were collected through a 100 μm sieve, washed five times with ethanol, and then dried at room temperature.

[0097] Comparative Example 2: Method for producing microparticles Microparticles were produced in the same manner as in Example 1, except that the photopolymerization step was not performed.

[0098] Comparative Example 3: Method for producing microparticles Microparticles were produced in the same manner as in Example 1, except that the thermal polymerization step was not performed.

[0099] Test example: Evaluation of the shape and physical properties of microparticles (1) Particle shape evaluation The shape of the microparticles was observed using a scanning electron microscope (SEM).

[0100] As a result of examining the SEM images, Examples 1 and 2 provided spherical particles with very uniform sizes and uniform surfaces (see Figures 2 and 3), whereas Comparative Example 1 provided particles with non-uniform particle sizes and non-uniform surfaces (see Figure 4), Comparative Example 2 caused coalescence due to a sudden increase in polymerization temperature, and Comparative Example 3 did not form solidified particles (see Figure 5).

[0101] (2) Diameter and its coefficient of variation evaluation The diameters of 100 particles were measured using an optical microscope, and the arithmetic mean value of the measured diameters was calculated. Specifically, the diameter of each particle was calculated by measuring the two-dimensional (2D) plane area of ​​the particle using an optical microscope, and then calculating the arithmetic mean value of the plane area (S = πr 2 ) was calculated by back-calculating. The coefficient of variation was also calculated from the diameters of 100 particles.

[0102] In Example 1, the particle diameter was 42.3±1.9 μm and the diameter variation coefficient was approximately 4.4%, while in Comparative Example 1, the particle diameter was 130±33 μm and the diameter variation coefficient was approximately 25.4%.

[0103] (3) Density evaluation At room temperature (approximately 21°C) and atmospheric pressure (1 atm), the density is 0.998 g / cm 3 of deionized water, density is 0.995g / cm 3 , 0.990g / cm 3 and 0.985 g / cm 3 The microparticles prepared in Examples 1 and 2 were added to each of the aqueous ethanol solutions. After the addition, the lids of the vials containing the solutions were closed and vigorously shaken, and after about 5 minutes, it was confirmed whether the particles were floating.

[0104] In the case of Example 1, as shown in FIG. 6, the density is 0.998 g / cm 3In deionized water, all particles are suspended without sinking particles and the density is 0.995 g / cm 3 In a solution with a density of 0.990 g / cm, a considerable number of particles are suspended and some are dispersed. 3 In a solution of 0.985g / cm3, most particles sink and some particles are dispersed, and the density is 0.985g / cm3. 3 In the solution of 0.990 to 0.995 g / cm, all particles sank. 3 It is confirmed that the density distribution is very narrow.

[0105] In the case of Example 2, as shown in FIG. 7, the density is 0.998 g / cm 3 of deionized water and density is 0.995g / cm 3 In a solution with a density of 0.990 g / cm, all particles are suspended without any sunk particles. 3 In a solution of 0.985g / cm3, most particles are suspended and some are dispersed, and the density is 0.985g / cm3. 3 In the solution of 0.985 to 0.990 g / cm, all particles sank. 3 It is confirmed that the density distribution is very narrow.

[0106] In contrast, the density of the microparticles of Comparative Example 1 was 0.990 g / cm 3 In a solution of 0.950 g / cm3, it floats and has a density of 0.950 g / cm3. 3 It sinks in a solution of 0.950-0.990 g / cm 3 It is confirmed that the density distribution is very wide.

[0107] Through the above test examples, it was confirmed that the microparticles produced by the method for producing microparticles according to one embodiment of the present invention have a uniform surface, very uniform size and shape, and a very narrow density range. In particular, the high uniformity of size and shape and the very narrow density range allow the production of the desired low-density particles with a high yield.

[0108] In the case of Comparative Example 1, which was produced by a suspension polymerization process, particles with a very low density were included, and there was a problem that they floated on the surface of the culture solution during the cell culture process. In contrast, the particles produced in Example 1 had a density of 0.990-0.995 g / cm 3 In the case of the particles produced in Example 2, the density is 0.985 to 0.990 g / cm 3 It has been confirmed that the present invention can provide microcarriers having a density of 100 μm, which enables highly efficient cell culture without the problem of floating on the surface of the culture medium during the cell culture process, and which can be easily separated and recovered after the culture process. [Explanation of symbols]

[0109] 10: 1st supply section 11: First flow path 12: Fine channel 20:Second supply section 21: Second flow path 31: Third flow path 40: Discharge section

Claims

1. a) injecting a dispersed phase composition containing a polymerizable monomer into a continuous phase composition through a microchannel to generate droplets of the dispersed phase composition in the continuous phase composition; b) photopolymerizing the droplets; c) thermally polymerizing the photopolymerized droplets; The step a) of generating droplets is a method for producing microparticles using a microfluidic device including a first supply unit to which a dispersed phase composition is supplied, a first flow path through which the dispersed phase composition supplied from the first supply unit flows, a second supply unit to which a continuous phase composition is supplied, a second flow path through which the continuous phase composition supplied from the second supply unit flows, and a plurality of microflow paths connecting the sides of the first and second flow paths to each other.

2. The method of claim 1 , wherein the dispersed phase composition comprises a polymerizable monomer, a cross-linking agent, a low density oil, a photoinitiator, and a thermal initiator.

3. The method for producing microparticles according to claim 2, wherein the crosslinking agent is contained in an amount of 10 to 1000 parts by weight based on 100 parts by weight of the polymerizable monomer.

4. The method for producing microparticles according to claim 2, wherein the low-density oil is contained in an amount of 5 to 100 parts by weight based on 100 parts by weight of the polymerizable monomer.

5. The method for producing microparticles according to claim 2, wherein the photoinitiator is contained in an amount of 0.1 to 30 parts by weight based on 100 parts by weight of the polymerizable monomer.

6. The method for producing microparticles according to claim 2, wherein the thermal initiator is contained in an amount of 0.1 to 30 parts by weight based on 100 parts by weight of the polymerizable monomer.

7. The method of claim 1 , wherein the continuous phase composition comprises a surfactant and water.

8. The method for producing microparticles according to claim 1 , further comprising the step of mixing the droplets with an additional continuous phase composition after the step a) of forming the droplets.

9. The method for producing microparticles according to claim 1 , which are used as microcarriers for cell culture.

10. The method for producing microparticles according to claim 1, wherein the microparticles have a diameter of 10 to 500 μm.

11. The method for producing microparticles according to claim 1, wherein the produced microparticles have a diameter coefficient of variation of 10% or less.

12. Density 0.985 g / cm at 15-25°C ３ 0.998g / cm or more ３ The method of claim 1 for producing microparticles of less than 10 ...

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