Preparation method and reactor for microcarriers applied to three-dimensional cell culture

Abstract

A method for preparing microcarrier particles is provided, which includes passing a dispersed phase liquid through a perforated plate at a low temperature to form liquid microspheres in a continuous phase, and curing synthetic polymers and / or natural biopolymers in the liquid microspheres at a low temperature to form particulate matter. Methods for preparing emulsions, and apparatus and process systems for preparing microcarrier particles are also provided.

Description

[Technical Field]

[0001] The present invention relates to methods and apparatus for preparing microcarriers, and in particular to methods and process systems that allow for the large scale preparation of microcarriers. [Background technology]

[0002] 3D TableTrix microcarrier tablets (Beijing CytoNiche Biotechnology Co., Ltd.) are a novel cell growth support material customized for stem cell production and preparation. This technological product employs an innovative microcarrier tablet design, with each microcarrier tablet having a defined weight and being independently sterilized for immediate use. This eliminates the need for the cumbersome weighing and sterilization procedures required for conventional microcarriers, making it superior to conventional microcarrier-based cell culture.

[0003] Microcarriers can be prepared in the form of microcarrier tablets. When exposed to water, the microcarrier tablets disperse into tens of thousands of elastic, three-dimensional porous microcarrier particles. The 3D structure and physical properties of the dispersed elastic microcarriers remain unchanged from those of the microcarriers prepared into microcarrier tablets. Microcarriers have a porosity of over 90%, particle diameters controllable between 50 and 500 μm, and uniformity of less than 100 μm. Their biochemical and physical properties can be customized, enabling precise and controllable 3D bionic culture. Their raw materials are medical-grade and comply with clinical standards. 3D TableTrix microcarrier tablets, combined with reagents such as 3DfloTrixDigest digestion and cleavage solution (Beijing CytoNiche Biotechnology Co., Ltd.), decompose and cleave the microcarriers, enabling gentle, non-destructive cell recovery without leaving any harmful residues. Summary of the Invention [Problem to be solved by the invention]

[0004] In view of the large market demand for these microcarrier tablets, the production volume is limited by conventional production methods, so it is necessary to adopt new methods and equipment to expand the production scale. [Means for solving the problem]

[0005] In one aspect, the present application provides a method for preparing an emulsion, comprising flowing a dispersed phase liquid from one side of a perforated plate through a plurality of microwells in the perforated plate to the other side of the perforated plate, and flowing a continuous phase liquid on the other side of the perforated plate parallel to the perforated plate, thereby forming liquid microspheres in the flowing continuous phase liquid by applying shear to the dispersed phase liquid passing through the perforated plate.

[0006] In some embodiments, the diameter of the microwells is between 0.1 μm and 500 μm.

[0007] In some embodiments, the diameter of the microwells is between 30 μm and 50 μm.

[0008] In some embodiments, the emulsion is a water-in-oil emulsion.

[0009] In some embodiments, the size of the liquid microspheres is controlled by adjusting the flow rate of the dispersed phase liquid and / or the continuous phase liquid.

[0010] In one aspect, the present application provides: Step 1) providing a dispersed phase liquid and a continuous phase liquid, the dispersed phase liquid comprises an artificial synthetic polymer and / or a natural biopolymer, and a curing agent; the continuous phase liquid comprises an organic solvent and a nonionic surfactant; Step 2) causing the dispersed phase liquid to flow from one side of a perforated plate to the other side of the perforated plate through a plurality of microwells disposed in the perforated plate, while causing the continuous phase liquid to flow parallel to the perforated plate on the other side of the perforated plate, thereby forming liquid microspheres in the flowing continuous phase liquid by applying shear to the dispersed phase liquid passing through the perforated plate; Step 3) forming particulate matter by reacting the artificial synthetic polymer and / or natural biopolymer in the liquid microsphere with a curing agent; and step 4) collecting and washing the particulate matter; The method for preparing microcarrier particles is provided, wherein the temperature of the continuous phase liquid in step 2) is 0°C or lower, and step 3) is carried out at 0°C or lower for 2 to 72 hours.

[0011] In some embodiments, step 2) is carried out in a vessel comprising a perforated plate, the perforated plate dividing the interior of the vessel into a first section and a second section, the dispersed phase liquid entering the first section through a dispersed phase inlet in the vessel communicating with the first section and flowing through the perforated plate into the second section, the continuous phase liquid entering the second section through a continuous phase inlet in the vessel communicating with the second section, the mixture containing the liquid microspheres after mixing of the dispersed phase liquid and the continuous phase liquid flows out of the vessel through a vessel outlet communicating with the second section of the vessel, the vessel outlet and the continuous phase inlet being on opposite sides of the vessel.

[0012] In some embodiments, the dispersed phase liquid enters the first portion of the vessel by gas pressurization and flows through the perforated plate, and the continuous phase liquid enters the second portion of the vessel by a gear pump and flows parallel to the perforated plate.

[0013] In some embodiments, step 3) is carried out in a tank equipped with a stirring device.

[0014] In some embodiments, step 4) is performed by vacuum suction in a tank in which the filtration device is placed.

[0015] In some embodiments, the diameter of the microwells is between 0.1 μm and 500 μm.

[0016] In some embodiments, the diameter of the microwells is between 30 μm and 50 μm.

[0017] In some embodiments, the flow rate of the continuous phase liquid is 5 to 20 times the flow rate of the dispersed phase liquid over the same period of time.

[0018] In some embodiments, the artificial synthetic polymer is selected from at least one of polyethylene glycol, polyethylene glycol derivatives, polyethylene glycol diacrylate, polypropylene, polystyrene, polyacrylamide, polylactic acid, polyhydroxy acid, polylactic alcohol acid copolymer, polydimethylsiloxane, polyanhydride, polyacid ester, polyamide, polyamino acid, polyacetal, polycyanoacrylate, polyurethane, polypyrrole, polyester, polymethacrylate, polyethylene, polycarbonate, and polyethylene oxide.

[0019] In some embodiments, the natural biopolymer is selected from at least one of collagen, proteoglycan, glycoprotein, gelatin, gelatin derivatives, chitin, alginate, alginate derivatives, agar, fibrinogen, Matrigel, hyaluronic acid, laminin, and fibronectin.

[0020] In some embodiments, the organic solvent is selected from at least one of hydrofluoroethers, carbon tetrachloride, petroleum ether, cyclohexane, liquid paraffin, edible oil, soybean oil, olive oil, chloroform, dichloromethane, carbon tetrachloride, and tetrachloroethylene.

[0021] In some embodiments, the nonionic surfactant is selected from at least one of sorbitan fatty acid esters, glycerin fatty acid esters, lauric acid esters, polyoxyethylene alkylphenyl ethers, polyoxyethylene high carbon fatty alcohol ethers, Span, PO-500, monooleic acid esters, and Tween.

[0022] In some embodiments, the curing agent is selected from at least one of divinylbenzene, diisocyanate, N-hydroxysuccinimide, N,N-methylenebisacrylamide, formaldehyde, glutaraldehyde, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, calcium ions, tetramethylethylenediamine, ammonium sulfate, genipin, and transglutaminase.

[0023] In some embodiments, the dispersed phase liquid further comprises a buffering agent, wherein the buffering agent is selected from at least one of carboxymethylcellulose, sodium chloride, polyacrylamide, potassium chloride, polyvinylpyrrolidone, sodium sulfate, calcium chloride, sodium chloride, sodium carbonate, and sodium bicarbonate.

[0024] Some embodiments include washing the particles with a detergent, wherein the detergent is selected from at least one of acetone, anhydrous copper sulfate, calcium chloride, sodium sulfate, absolute ethanol, medical grade alcohol, hydrofluoroether, sodium alkylbenzene sulfonate, sodium fatty alcohol sulfate, sodium tripolyphosphate, and deionized water.

[0025] In some embodiments, the ratio of organic solvent to nonionic surfactant in the continuous phase liquid is 5:1 to 20:1 by weight.

[0026] On the other hand, the present application 1) Container, 2) a perforated plate including a plurality of microwells disposed within the vessel, the perforated plate dividing the interior of the first vessel into a first portion and a second portion; 3) a dispersed phase inlet in communication with the first portion for introducing a dispersed phase liquid; 4) a continuous phase inlet in communication with the second portion for introducing a continuous phase liquid; and 5) a vessel outlet in communication with the second portion; wherein the vessel outlet and the continuous phase inlet are arranged on opposite sides of the vessel such that the continuous phase liquid introduced from the continuous phase inlet can flow through the second portion in a direction parallel to the perforated plate and then flow out from the vessel outlet.

[0027] In some embodiments, the diameter of the microwells is between 0.1 μm and 500 μm.

[0028] In some embodiments, the diameter of the microwells is between 30 μm and 50 μm.

[0029] In some embodiments, the container is rectangular shaped.

[0030] In some embodiments, the continuous phase inlet is two or more and the vessel outlet is two or more.

[0031] In some embodiments, the continuous phase inlet and the vessel outlet have the same horizontal height relative to the bottom of the vessel.

[0032] In some embodiments, the emulsion is a water-in-oil emulsion.

[0033] On the other hand, the present application 1) Container, a perforated plate including a plurality of microwells disposed within the vessel, the perforated plate dividing the interior of the first vessel into a first portion and a second portion; a dispersed phase inlet in communication with the first portion for introducing a dispersed phase liquid; a continuous phase inlet in communication with the second portion for introducing a continuous phase liquid; and a vessel outlet in communication with the second portion; wherein the vessel outlet and the continuous phase inlet are arranged on opposite sides of the vessel such that the continuous phase liquid introduced through the continuous phase inlet can flow through the second portion in a direction parallel to the perforated plate and then flow out of the vessel outlet; 2) a first tank for containing the dispersed phase liquid, in communication with the dispersed phase inlet, the first tank further in communication with a pressurizer or gas cylinder to allow the dispersed phase liquid to flow under pressure into the emulsion preparation apparatus; 3) a second tank in communication with the continuous phase inlet for containing the continuous phase liquid and equipped with a cooling device for cooling the continuous phase liquid; 4) a third tank for carrying out an emulsion reaction, the third tank being equipped with an agitator and a cooling device and communicating with the outlet of the vessel; and 5) a fourth tank in communication with the third tank equipped with a filtration device for collecting particles formed by the emulsion reaction; The present invention provides a process system for preparing microcarrier particles, comprising:

[0034] In some embodiments, the container is rectangular shaped.

[0035] In some embodiments, the continuous phase inlet is two or more and the vessel outlet is two or more.

[0036] In some embodiments, the continuous phase inlet and the vessel outlet have the same horizontal height relative to the bottom of the vessel.

[0037] In some embodiments, a gear pump is provided between the continuous phase inlet and the second tank to transport the continuous phase liquid from the second tank to the emulsion preparation apparatus.

[0038] In some embodiments, the process system further includes a fifth tank in communication with the fourth tank equipped with a filtration device for washing the particulate matter.

[0039] In some embodiments, the fourth tank and the fifth tank each include a vacuum device.

[0040] In some embodiments, the diameter of the microwells is between 0.1 μm and 500 μm.

[0041] In some embodiments, the diameter of the microwells is between 30 μm and 50 μm.

[0042] The methods and apparatus provided by the present invention are useful for the large-scale preparation of emulsions and microcarrier particles. [Brief explanation of the drawings]

[0043] [Figure 1] FIG. 1 is a schematic diagram showing the structure of an emulsion preparation apparatus (or microsphere forming machine). [Figure 2] FIG. 2 is a schematic diagram showing the structure of a perforated plate. [Figure 3] 1 shows various devices and connections included in a process system for preparing microcarrier particles. [Figure 4] 1 shows an electron microscope photograph of the microcarrier particles prepared in Example 1. [Figure 5] 1 shows an electron microscope photograph of the microcarrier particles prepared in Example 2. DETAILED DESCRIPTION OF THE INVENTION

[0044] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art.

[0045] An "emulsion," also known as an emulsion, is a dispersed system formed from two immiscible liquids. The liquid present in the form of suspended droplets is called the dispersed phase (or internal phase), and the liquid continuously distributed as a dispersion medium is called the continuous phase (or external phase). Many organic substances that are immiscible with water exist in nature, and many emulsions exist consisting of an aqueous phase (mainly composed of water or water-soluble components) and an organic phase (mainly composed of water-insoluble organic substances, also known as the oil phase). Typically, emulsions with an aqueous phase as the dispersed phase and an organic phase as the continuous phase are called "water-in-oil emulsions." In contrast, emulsions with an organic phase as the dispersed phase and an aqueous phase as the continuous phase are called "oil-in-water emulsions." Suspended droplets formed from the dispersed phase liquid in the continuous phase liquid are also referred to herein as "liquid microspheres."

[0046] In this application, the term "emulsion reaction" refers to a chemical reaction that occurs between components in a liquid microsphere. For example, in the process of preparing microcarrier particles described below, polymeric compounds such as artificial polymers are hardened in the liquid microsphere to obtain particulate matter that is insoluble in both the aqueous and organic phases.

[0047] In this application, the term "perforated plate" refers to a plate-like structure having multiple microwells formed therein. In the method and apparatus provided by the present invention, a "perforated plate" is used to restrict the flow of the dispersed phase liquid so that passage of the dispersed phase liquid from one side of the perforated plate to the other can only be achieved by passing through multiple microwells. Therefore, the perforated plate is not limited to a specific shape for its intended use; it may have an irregular shape as long as the intended purpose is achieved. However, for emulsion preparation, a plate-like shape is preferred because of its ease of processing and its advantageous effect on achieving uniform liquid microsphere sizes. The microwells typically have a diameter of 0.1 μm to 500 μm. When the perforated plate has a substantial thickness, the microwells are actually microtubes having a certain length. For simplicity, however, the term "microwell" is intended to include such microtubes. Similarly, in the present invention, the material of the microwell plate is not particularly limited as long as it achieves the above-mentioned objectives and is chemically inert to both the dispersed phase liquid and the continuous phase liquid. Materials such as plastics and ceramics can be used.

[0048] The continuous phase is referred to herein as flowing "parallel" to the perforated plate within the emulsion preparation apparatus. Ideally, the dispersed phase liquid passes through the microwells perpendicular to the perforated plate, but the continuous phase liquid flows perpendicular to the flow of the dispersed phase liquid through the perforated plate (i.e., parallel to the perforated plate), thereby cutting the dispersed phase liquid flow into liquid microspheres. Of course, those skilled in the art will recognize that the continuous phase liquid does not need to flow strictly parallel to achieve the goal of cutting the dispersed phase liquid flow into liquid microspheres. Therefore, in most cases during emulsion formation, the continuous phase liquid inlet and outlet of the emulsion preparation apparatus may be located on opposite sides of the emulsion preparation apparatus, i.e., "oppositely located." In some embodiments, the continuous phase liquid inlet and outlet have the same height. In some embodiments, multiple continuous phase liquid inlets and multiple vessel outlets may be horizontally arranged on opposite sides of a generally rectangular vessel to facilitate parallel flow of the continuous phase liquid.

[0049] "Microcarrier particles" or "microcarriers" refer to particulate materials in the micrometer-scale size range suitable for cell attachment and growth. Microcarrier particle sizes are preferably 50-500 μm. Porosity is typically greater than 80%, e.g., 90% or 95%. While many cells can grow simply by adhering to the surface of a solid substrate, the microcarriers prepared in the present invention possess the characteristics of microcarriers, such as porosity, large surface area, and biocompatibility, allowing cells to grow within them, and can form three-dimensional culture patterns. These biomimetic three-dimensional culture patterns are becoming increasingly widely used.

[0050] "Large-scale" means that the amount of product prepared can meet the needs of laboratory research, up to the needs of industrial production. For example, emulsions of 1 mL to 1000 L (e.g., 5 mL to 100 L, 100 mL to 10 L, 500 mL to 1 L, etc.) or even larger volumes can be prepared at one time. For microcarrier particles, "large-scale" includes, for example, 1 mg to 1000 kg (e.g., 100 mg to 100 kg, 1 g to 10 kg, 200 g to 1 kg, etc.) or even larger amounts of microcarrier particles per preparation.

[0051] In some embodiments of the present invention, there is provided a method for preparing an emulsion for large-scale emulsion preparation. The method includes flowing a dispersed phase liquid from one side of a perforated plate to the other side of the perforated plate through a plurality of microwells in the perforated plate, while flowing a continuous phase liquid on the other side of the perforated plate parallel to the perforated plate, thereby forming liquid microspheres in the flowing continuous phase liquid by applying shear to the dispersed phase liquid passing through the perforated plate. Because a large number of microwells can be densely provided in the perforated plate, large amounts of emulsion can be continuously and rapidly prepared by alternately flowing the dispersed phase liquid and the continuous phase liquid in a substantially vertical direction near the perforated plate.

[0052] In some specific embodiments, the emulsion formation process is carried out in a vessel including a perforated plate. More specifically, the perforated plate disposed within the vessel divides the interior of the vessel into a first section and a second section, the dispersed phase liquid entering the first section through a dispersed phase inlet provided in the vessel and communicating with the first section, then flowing through the perforated plate to enter the second section, the continuous phase liquid entering the second section through a continuous phase inlet provided in the vessel and communicating with the second section, and the mixture containing the liquid microspheres after mixing of the dispersed phase liquid and the continuous phase liquid flows out of the vessel through a vessel outlet provided in the vessel and communicating with the second section. The vessel outlet and the continuous phase inlet are provided on opposite sides of the vessel so that the continuous phase passes through the second section substantially parallel to the perforated plate.

[0053] Typically, the amount of dispersed phase liquid used is less than or equal to the amount of continuous phase liquid used. For example, the ratio of dispersed phase liquid to mobile phase liquid in the prepared emulsion is 1:1 to 1:30, preferably 1:5 to 1:20. A ratio outside this range may affect the yield of the final product. The size of the liquid microspheres formed can be controlled by adjusting the size of the microwells. The flow rates of the dispersed phase and the continuous phase, and the relative flow rates between them, also affect the size of the liquid microspheres. To ensure that the dispersed phase liquid and the continuous phase liquid flow at substantially the same rate, the liquid flows can be controlled using a device such as a pressurized gas or a gear pump. For example, in one specific embodiment, the uniform flow of the dispersed phase liquid is promoted by pressurized gas, and the uniform flow of the continuous phase liquid is promoted by the rotation of a gear pump. In some embodiments of the present invention, the dispersed phase is an aqueous phase containing a reactant, and the continuous phase is an organic phase containing an organic substance.

[0054] The present invention also provides a method for preparing microcarrier particles using the emulsion formation process described above. The method is suitable for large-scale preparation of microcarrier particles. In the method, a dispersed phase liquid containing a compound to be hardened (e.g., an artificial synthetic polymer and / or a natural biopolymer) and a hardener is used. A continuous phase containing an organic solvent and a non-ionic surfactant is used. The liquid microspheres produced in the emulsion formation process are reacted under stirring to form particulate matter. The particulate matter is then recovered by filtration and washed with a detergent.

[0055] The present inventors have found that by carrying out the above emulsion formation process and reaction process at low temperatures (0°C or below, e.g., -10°C or below, e.g., -30°C), the resulting particulate material has better porosity and specific surface area, making it more suitable as a three-dimensional carrier for cell culture. Generally, the lower the temperature, the smaller the pore size of the microcarrier particles produced. For example, the pore size of microcarrier particles prepared by reaction at -30°C is approximately 5 μm to 20 μm.

[0056] To ensure that the dispersed phase liquid and the continuous phase liquid flow at substantially the same rate, the liquid flows can be controlled using a device such as a pressurized gas or a gear pump. For example, in one specific embodiment, the uniform flow of the dispersed phase liquid is promoted by pressurized gas, and the uniform flow of the continuous phase liquid is promoted by the rotation of a gear pump. Generally, the greater the pressure applied to the dispersed phase and the faster the flow rate, the larger or smaller the particle size of the prepared microcarrier particles. Furthermore, the slower the flow rate of the continuous phase, the larger or smaller the particle size of the prepared microcarrier particles. Therefore, the particle size of the final microcarrier particles can be adjusted by flexibly controlling the diameter of the microwells in the perforated plate and the flow rates of the dispersed phase liquid and the continuous phase liquid.

[0057] As an example, the artificial synthetic polymer includes at least one of polyethylene glycol, polyethylene glycol derivatives, polyethylene glycol diacrylate, polypropylene, polystyrene, polyacrylamide, polylactic acid, polyhydroxy acid, polylactic alcohol acid copolymer, polydimethylsiloxane, polyacid anhydride, polyacid ester, polyamide, polyamino acid, polyacetal, polycyanoacrylate, polyurethane, polypyrrole, polyester, polymethacrylate, polyethylene, polycarbonate, and polyethylene oxide.

[0058] By way of example, the natural biopolymer includes at least one of collagen, proteoglycan, glycoprotein, gelatin, gelatin derivatives, chitin, alginate, alginate derivatives, agar, fibrinogen, Matrigel, hyaluronic acid, laminin, and fibronectin.

[0059] By way of example, the curing agent includes at least one of divinylbenzene, diisocyanate, N-hydroxysuccinimide, N,N-methylenebisacrylamide, formaldehyde, glutaraldehyde, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, calcium ions, tetramethylethylenediamine, ammonium sulfate, genipin, and transglutaminase. As an example, the organic solvent includes at least one of hydrofluoroether, carbon tetrachloride, petroleum ether, cyclohexane, liquid paraffin, edible oil, soybean oil, olive oil, chloroform, dichloromethane, carbon tetrachloride, and tetrachloroethylene.

[0060] By way of example, the nonionic surfactant includes at least one of sorbitan fatty acid esters, glycerin fatty acid esters, lauric acid esters, polyoxyethylene alkylphenyl ethers, polyoxyethylene high carbon fatty alcohol ethers, Span, PO-500, monooleic acid esters, and Tween.

[0061] As an example, the cleaning agent used to clean the particulate matter includes at least one of acetone, anhydrous copper sulfate, calcium chloride, sodium sulfate, absolute ethanol, medical alcohol, hydrofluoroether, sodium alkylbenzene sulfonate, sodium aliphatic alcohol sulfate, sodium tripolyphosphate, and deionized water.

[0062] In some embodiments, the dispersed phase liquid further comprises a buffer, such as at least one of carboxymethylcellulose, sodium chloride, polyacrylamide, potassium chloride, polyvinylpyrrolidone, sodium sulfate, calcium chloride, sodium chloride, sodium carbonate, and sodium bicarbonate.

[0063] Usually, the concentration of the compound to be cured in the dispersed phase is 1 wt % to 20 wt %, but the amount of curing agent used may be adjusted depending on the properties of the compound to be cured and the properties of the curing agent itself.

[0064] It should be noted that the above ingredients are merely examples of some of the raw materials suitable for preparing microcarrier particles, and those skilled in the art can easily replace these materials with other similar materials depending on their physicochemical properties and conduct simple experiments to verify the feasibility of following the method provided by the present invention. It will be apparent that the method for preparing microcarrier particles using the substitutions according to the method provided by the present invention and the product thereof are also within the scope of the present invention.

[0065] On the other hand, the present invention further provides an emulsion preparation apparatus useful for large-scale preparation of emulsions, the emulsion preparation apparatus comprising: container, a perforated plate including a plurality of microwells disposed within the vessel, the perforated plate dividing the interior of the first vessel into a first portion and a second portion; a dispersed phase inlet in communication with the first portion for introducing a dispersed phase liquid; a continuous phase inlet in communication with the second portion for introducing a continuous phase liquid; and a vessel outlet in communication with the second portion; the vessel outlet and the continuous phase inlet are arranged on opposite sides of the vessel such that the continuous phase liquid introduced through the continuous phase inlet can flow through the second portion in a direction parallel to the perforated plate and then flow out of the vessel outlet.

[0066] FIG. 1 is a cross-sectional view showing the main components of an emulsion preparation apparatus of the present invention, and schematically illustrates a portion of the configuration of this emulsion preparation apparatus and the flow direction of each liquid. As shown in FIG. 1, a vessel 1 includes an internal space surrounded by a vessel wall 11. This internal space is divided into an upper first section 12 and a lower second section 13 by a perforated plate 14 provided within the vessel 1. The vessel 1 is provided with a dispersed phase inlet 15 communicating with the first section 12, and a continuous phase inlet 16 and vessel outlet 17 communicating with the second section 13. The continuous phase inlet 16 and vessel outlet 17 are provided on both sides of the vessel 1. In some embodiments, the continuous phase inlet 16 and vessel outlet 17 are at substantially the same height relative to the bottom 18 of the vessel 1. The perforated plate 14 is provided with a plurality of microwells 141 (see FIG. 2). Dispersed phase liquid is introduced under pressure into the first section 12 through the dispersed phase inlet 15 and continues under pressure through the microwells 141 of the perforated plate 14 into the lower second section 13. Simultaneously, continuous phase liquid enters the second section 13 through the continuous phase inlet 16, flows roughly parallel to the perforated plate 14, and exits through the vessel outlet 17. Below the perforated plate 14, the flowing continuous phase liquid shears the dispersed phase liquid passing through the microwells 141 in the perforated plate 14 into liquid microspheres, forming a multitude of liquid microspheres that are not dissolved in the continuous phase liquid and are suspended in the continuous phase liquid. These liquid microspheres exit the vessel outlet 17 along with the continuous phase flow. The liquid mixture exiting the vessel outlet 17 is collected to obtain the prepared emulsion.

[0067] The pore size of the microwells 141 in the perforated plate 14 can be adjusted to obtain a suitable size for the liquid microspheres. When the diameter of the microwells is 0.1 μm to 500 μm, uniform liquid microspheres can be prepared with a particle size error of 100 μm or less in the range of 1 μm to 1000 μm.

[0068] In some embodiments, the dispersed phase liquid is an aqueous phase liquid, the continuous phase liquid is an organic phase liquid, and the emulsion prepared is a water-in-oil emulsion.

[0069] In some embodiments, the present invention provides a process system for preparing microcarrier particles useful for large-scale preparation of emulsions, the process system including, in addition to the emulsion preparation apparatus described above, multiple tanks.

[0070] One of the tanks is connected to the dispersed phase inlet to contain the dispersed phase liquid. The tank may be equipped with a stirrer to prepare the dispersed phase liquid. The tank may be connected to a pressure device or a gas cylinder containing compressed gas, and the dispersed phase liquid can be introduced into the emulsion preparation device by pressurizing the tank. The gas used may be any gas that does not react with the dispersed phase liquid or the continuous phase liquid, and can be selected from, for example, air, nitrogen gas, carbon dioxide gas, oxygen gas, argon gas, etc. The gas pressure used may be, for example, 1 kPa to 100 kPa.

[0071] The other tank contains the continuous phase liquid and is connected to the continuous phase inlet for cooling the continuous phase liquid. This tank can also be used to prepare the continuous phase liquid, or the prepared continuous phase liquid can be supplied to the tank and then cooled. Methods for cooling the contents of the tank are well known in the art, and include, for example, providing a jacket on the tank and circulating a refrigerant liquid through the jacket using a refrigerator. The refrigerant liquid may include, for example, at least one of liquid nitrogen, ethanol, trichloroethane, isopropyl alcohol, dichloromethane, ethyl acetate, ethylene glycol, propylene glycol, isobutane, n-hexane, chloroform, tetrahydrofuran, bromohexane, and acetonitrile.

[0072] The other tank is connected to the outlet of the container and can receive the liquid mixture containing the liquid microspheres discharged from the emulsion preparation device. The tank is equipped with an agitator and a refrigerator, and reacts the reactants (e.g., biopolymers and corresponding hardeners) contained in the liquid microspheres under low-temperature conditions (e.g., −10° C.) to form a particulate material.

[0073] Another tank is used to separate the formed particulate matter from the mixed liquid, and may be equipped with a filtering device such as a vacuum filter to facilitate the removal of the liquid component and leaving the particulate matter behind, for example by vacuum suction using a vacuum pump.

[0074] If necessary, a tank for washing the particulate matter may be included. The tank may also be provided with a vacuum filter, and the particulate matter may be washed multiple times (e.g., 3 to 5 times) by filling the tank with a cleaning agent and drawing a vacuum.

[0075] In one specific embodiment, the process system for preparing microcarrier particles of the present invention is shown in Figure 3. A mixing tank is used to prepare the continuous phase liquid (organic phase). After preparation, the continuous phase liquid may be transferred, for example, by pressure, to a pre-cooling tank equipped with a refrigerator, where the temperature of the continuous phase liquid is lowered to 0°C or below (e.g., -10°C or below). An aqueous phase tank is used to prepare the dispersed phase liquid. The prepared dispersed phase liquid and continuous phase liquid are transported to an emulsion preparation device using pressure and a pump, respectively. Next, the emulsion containing liquid microspheres produced in the emulsion preparation device is transferred to a reaction tank for emulsion reaction. The particulate matter produced by the reaction is further separated from other components in the emulsion in an oil filtration tank by vacuum filtration. Finally, the mixture is washed in a water washing tank using a detergent to obtain microcarrier particulate matter.

[0076] The construction and mode of operation of the process system will now be described in more detail with reference to FIG.

[0077] This system mainly consists of six tanks: a mixing tank, pre-cooling tank, aqueous phase tank, reaction tank, oil filtration tank, and water washing tank, and is used in combination with other devices such as a vacuum pump, water cooler, and control panel. It also consists of piping connecting multiple tanks and external piping for transporting drinking water, purified water, water for injection, compressed air, high-temperature steam, etc. The entire system, including some valves and links, is made of 316L stainless steel, which ensures that the material is in contact with 316L stainless steel and prevents the introduction of other foreign matter.

[0078] The tanks are arranged from left to right according to the process steps, and the height positions of some tanks are determined according to important process control points and appear as work tables. Each piece of equipment is briefly described in Table 1.

[0079] [Table 1]

[0080] 1. Blending organic phase stock solution 1. First, check the cleanliness and clearance log of the mixing tank, pre-cooling tank, liquid sterilization filter, and gauges to ensure they are clean and usable within the clearance validity period. Weigh out the raw materials and additives, and then use a peristaltic pump to add multiple raw materials and additives to the mixing tank in a set ratio. The mixing tank is opened and agitated, with the rotation speed set to 10-150 rpm and the agitation time set to 0.5-4 hours. The tank is closed, and with only the compressed air vent open, stock solution 1 is poured into the liquid sterilization filter until the internal tank pressure reaches 0.1-1 MPa. Then, the stock solution is poured into the pre-cooling tank, and a refrigerant liquid is placed in the outer wall of the pre-cooling tank. The temperature of stock solution 1 is lowered to 0°C-196°C by continuous cooling using a water chiller. Meanwhile, the pre-cooling tank is kept stirring at 10-2000 rpm for 0.5-24 hours.

[0081] Organic phase stock solution 1 is a mixed organic phase and contains at least one organic solvent selected from the group consisting of hydrofluoroether, carbon tetrachloride, petroleum ether, cyclohexane, liquid paraffin, edible oil, soybean oil, olive oil, chloroform, dichloromethane, carbon tetrachloride, and tetrachloroethylene, and at least one nonionic surfactant selected from the group consisting of sorbitan fatty acid esters, fatty acid glycerin esters, lauric acid esters, polyoxyethylene alkylphenyl ethers, polyoxyethylene high-carbon fatty alcohol ethers, Span, PO-500, monooleate esters, and Tween.

[0082] The refrigerant liquid includes, but is not limited to, at least one of liquid nitrogen, ethanol, trichloroethane, isopropanol, dichloromethane, ethyl acetate, ethylene glycol, propylene glycol, isobutane, n-hexane, chloroform, tetrahydrofuran, bromohexane, and acetonitrile, and is cooled to 0 to -196°C by a low-temperature refrigerator.

[0083] 2. Blending of stock solution 2. First, check the cleanliness of the aqueous phase tank and instruments and the clearance log to ensure they are clean and usable within the clearance validity period. Weigh out the raw materials and add the main stock solution 2-1 to the water for injection so that it is blended into a stock solution 2-1 solution of a certain concentration and volume. Add the stock solution 2-1 solution to the aqueous phase tank using a peristaltic pump, then add stock solution 2-2. Set the rotation speed to 10 to 300 rpm and mix for 5 to 120 minutes.

[0084] The stock solution 2-1 contains an artificially synthesized biomaterial and / or a natural biomaterial, and a buffer solution.

[0085] The artificially synthesized biomaterial is at least one selected from the group consisting of polyethylene glycol, polyethylene glycol derivatives, polyethylene glycol diacrylate, polypropylene, polystyrene, polyacrylamide, polylactic acid, polyhydroxy acid, polylactic acid alcohol acid copolymer, polydimethylsiloxane, polyacid anhydride, polyacid ester, polyamide, polyamino acid, polyacetal, polycyanoacrylate, polyurethane, polypyrrole, polyester, polymethacrylate, polyethylene, polycarbonate, and polyethylene oxide.

[0086] The natural biomaterial is at least one selected from the group consisting of collagen, proteoglycan, glycoprotein, gelatin, gelatin derivatives, chitin, alginate, alginate derivatives, agar, fibrinogen, Matrigel, hyaluronic acid, laminin, and fibronectin.

[0087] The buffer comprises at least one of carboxymethylcellulose, sodium chloride, polyacrylamide, potassium chloride, polyvinylpyrrolidone, sodium sulfate, calcium chloride, sodium chloride, sodium carbonate, and sodium bicarbonate.

[0088] Stock solution 2-2 includes, but is not limited to, divinylbenzene, diisocyanate, N-hydroxysuccinimide, N,N-methylenebisacrylamide, formaldehyde, glutaraldehyde, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, calcium ions, tetramethylethylenediamine, ammonium sulfate, genipin, transglutaminase, etc.

[0089] 3. Place in the reaction tank and mix. First, check the cleanliness and clearance log of the reaction tank to ensure they meet the acceptable standards. A refrigerant liquid is poured into the middle layer of the outer wall of the reaction tank. The reaction tank is then continuously cooled with a water chiller to lower the tank temperature to 0 to -196°C. Once the required temperature is reached and both stock solutions 1 and 2 are prepared, stock solution 1 is passed through a microsphere extrusion machine (i.e., emulsion preparation device) at a flow rate of 50 to 5,000 ml / min and mixed with the rest of the mixture before being poured into the reaction tank. Stock solution 2 is then passed through a microsphere extrusion machine under gas pressure at 1 kPa to 100 kPa and introduced into the reaction tank. Gas compositions include, but are not limited to, air, nitrogen, carbon dioxide, oxygen, and argon. The reaction tank is opened and stirred, with the rotation speed set to 10 to 1,200 rpm for 2 to 72 hours.

[0090] The structure of the microsphere molding machine can be divided into three parts, as shown in Figure 1.

[0091] The upper layer is the raw liquid 2 chamber, and the liquid flows vertically from top to bottom. Raw liquid 2 is driven by an inert gas. Due to the stable air pressure, raw liquid 2 passes through the perforated plate vertically from top to bottom at the same speed.

[0092] The intermediate layer is a perforated plate whose surface is made up of numerous cut microwells with diameters ranging from 0.1 μm to 500 μm, and the cut plate may be in the form of a single-layer sheet, a multi-layer sheet, a hollow pipe, a hollow block, etc. The size of the microwells affects the diameter of the emulsion droplets formed and the particle size of the final material, with the particle size of the final microcarrier particles being approximately 5 to 20 times the diameter of the microwells.

[0093] The lower layer is the flow layer for raw liquid 1, which flows horizontally. Raw liquid 1 is driven by a gear pump, and the flow rate per unit time is controlled to be 5 to 20 times that of raw liquid 2, which passes vertically through the cutting plate. The flow rate affects the stability of the final emulsion. If the flow rate is outside this range, significant demulsification may occur.

[0094] A stable air pressure is applied to the raw material 2 from top to bottom through an external vent located at the top of the aqueous phase tank. The gas pressure can be set between 1 kPa and 100 kPa, with a stable range of ±1 kPa. Gas compositions include, but are not limited to, air, nitrogen gas, carbon dioxide gas, oxygen gas, and argon gas. Under the action of the stable pressure, raw material 2 passes steadily from top to bottom through the perforated plate, forming initial liquids of circular droplets. At the same time, raw material 1 flows at the same speed below the perforated plate via a gear pump. After passing through the perforated plate, raw material 2 is sheared by raw material 1 to form stable microsphere droplets. Since raw material 1 and raw material 2 are mutually incompatible, stable microsphere droplets consisting of raw material 2 are suspended in raw material 1. The particle size of the liquid 2 microspheres is affected by the diameter of the microwell mold in the porous cutting plate. The diameter of the microwell mold ranges from 0.1 μm to 500 μm, and uniform liquid microspheres with a particle size error range of 1 μm to 1000 μm can be prepared.

[0095] 4. Cleaning. First, check that the oil filter tank and water rinse tank are clean, and check the instrument status and clearance log to ensure they are clean and within their validity period. The reacted material is poured into the oil filter tank using compressed air at 0.1MP to 1MP, and excess liquid in the material is removed using the continuous action of a vacuum pump. After the above process is completed, the detergent is first added, the oil filter tank is opened, and the stirring is set to 10-70 rpm to thoroughly mix the detergent and material. The stirring is turned off for 2-5 minutes, and the mixture is left to stand. Then the stirring is resumed and set to 20-50 rpm to thoroughly mix the effective material and detergent. The effective material and detergent are poured into the water rinse tank using compressed air at 0.1MP to 1MP. The detergent in the water rinse tank is removed using the continuous action of a vacuum pump. A certain volume and temperature of water for injection is added to the water rinse tank, and washing is repeated five times, with each stirring (washing) time being 10 minutes to 24 hours.

[0096] Detergents include, but are not limited to, acetone, anhydrous copper sulfate, calcium chloride, sodium sulfate, absolute ethanol, medical alcohol, hydrofluoroether, sodium alkylbenzene sulfonate, sodium fatty alcohol sulfate, sodium tripolyphosphate, deionized water, and the like.

[0097] 5. Freeze-drying and screening. First, check the cleanliness and clearance log of the freeze-drying cartridge, instrument, and freeze-dryer to ensure they are clean and can be used within their expiration date. The washed material is mixed with water for injection in a certain ratio based on its wet weight, frozen in a freezing device or freezing solution at -10 to -196°C, and then transferred to a freeze-dryer for 12 to 96 hours. The resulting three-dimensional porous microcarrier has microwells with a pore size of 20 to 200 μm and a porosity of 85 to 95%.

[0098] 6. Tablet preparation and packaging. First, the tablet press's cleanliness and clearance log are checked to ensure that the material to be tableted is clean and within its expiration date. The machine then debugs the various parameters to ensure that individual tablets of uniform quality and shape are extruded. During the tableting process, various tablet characteristics, such as water absorption, dispersibility, and stability, are observed, recorded, and tested in real time. Each tablet is then placed in a designated amount into the corresponding packaging bottle. Once packaged, the tablets are submitted for inspection and other follow-up work. [Example]

[0099] The processing steps of the microcarrier particles of the present invention will now be further illustrated by specific examples.

[0100] Example 1 Oil phase formulation 1.1 First, check the cleanliness and clearance log of the mixing tank, pre-cooling tank, liquid sterilization filter, and instrument, and ensure that they are clean and within their validity period.

[0101] 1.2 100 L of petroleum ether and 10 L of PO-500 reagent were added to the mixing tank using a peristaltic pump. The mixing tank was opened and stirred at a rotation speed of 60 rpm for 1 hour.

[0102] 1.3 Close the mixing tank, leave only the compressed air inlet open, and pour the oil phase into the pre-cooling tank until the internal pressure of the tank reaches 0.1 MPa. Use the water cooler to continuously cool the oil phase down to -10°C, while maintaining the pre-cooling tank at 40 rpm.

[0103] Water Phase Formulation 2.1 First, check the cleanliness of the aqueous phase tank and instruments and the clearance log to ensure they are clean and within their validity period.

[0104] 2.2 100 g of gelatin was weighed out and added to 5 L of deionized water. The gelatin solution was stirred at 60 rpm using a stirrer until it was fully dissolved, and then added to the aqueous phase tank using a peristaltic pump. The rotation speed was set to 150 rpm and the mixture was stirred for 90 minutes to prepare the solution.

[0105] mixture 3.1 First, check the cleanliness of the reaction tank and the clearance log, and ensure that both are usable up to the pass standard.

[0106] 3.2 The reaction tank temperature was continuously cooled to below -10°C using a water chiller. After the reaction tank was brought to the required temperature and the oil phase temperature was reduced to -10°C, 5 mL of 75% formaldehyde solution was added to the aqueous phase. The rotation speed was set to 80 rpm and the mixture was stirred for 10 minutes. The oil and aqueous phases were then mixed using a microsphere extrusion machine and then added to the reaction tank. The oil phase was driven by a gear pump (pumping speed 3000 mL / min), and the aqueous phase was driven by a constant pressure inert gas (air) at 5 KPa. The microsphere extrusion machine used a perforated plate with microwells of 30 μm diameter.

[0107] The microsphere molding machine consisted of a 40cm x 40cm x 30cm rectangular parallelepiped. A 40cm x 40cm, 1cm thick plastic plate was placed horizontally within the machine, 1cm above the horizontally positioned oil phase inlet and outlet. The plastic plate was microfabricated with numerous 30µm diameter circular holes, resulting in a 300x300 microwell array with 50µm spacing. The dispersed phase (aqueous phase) entered the microsphere molding machine perpendicular to the microwell plate, penetrating vertically through the microwells. Below the microwell plate, the oil phase solution rapidly entered the microsphere molding machine through the horizontal inlet. The aqueous solution passing through the microwells was continuously and rapidly sheared to form microspheres. The formed microspheres were dispersed in the oil phase and discharged through the horizontal outlet.

[0108] 3.3 After the materials were placed in the reaction tank, stirring was started immediately, and the reaction was carried out for 48 hours with the rotation speed set at 30 rpm.

[0109] Cleaning 4.1 First, check that the oil filter tank and water flushing tank are clean, and check the instrument status and clearance log to ensure they are clean and within their validity period.

[0110] 4.2 The reacted material was poured into the oil filtration tank using 0.15MP compressed air, and the excess liquid in the material was removed by continuous vacuum pump action. After that, 50L of acetone, 10kg of calcium chloride, 100L of medical alcohol, and 100L of absolute ethanol were added as cleaning agents, and the mixture was washed for 20 minutes at a stirring speed of 100 rpm. After each wash, the excess liquid was removed by vacuum filtration. Using 0.10MP compressed air, the active material and alcohol were poured into the water washing tank.

[0111] 4.3 20 L of deionized water was placed in the washing tank, stirred thoroughly at 100 rpm for 50 minutes, and the water for injection was removed using a vacuum pump. The washing was repeated 3 to 5 times.

[0112] Freeze drying 5.1 First, check the cleanliness and clearance log of the freeze-drying cartridges, instruments, and freeze-drying machines to ensure they are clean and within their expiration dates.

[0113] 5.2 The washed material was mixed with 500 mL of water for injection in a lyophilization cartridge in a ratio of 50 g wet weight, and then frozen at -20°C for 48 hours.

[0114] 5.3 The frozen material was placed in a freeze-dryer and freeze-dried according to the freeze-dryer's operating instructions. The freeze-drying process was continued for at least 72 hours. The total weight of the freeze-dried material was measured and transferred to a temporary storage box for the material, which was then sent to the next process.

[0115] Sieving 6.1 First, check the cleanliness and clearance log of the mesh and sieve machine to ensure they are clean and within their validity period.

[0116] 6.2 The freeze-dried material was sieved to a size range of 50-500 microns. The sieved material was collected to obtain porous microcarrier particles with a certain particle size range.

[0117] As a result, the obtained microcarriers had an average particle size of 200 μm, an average pore size of 25 μm, and a porosity of 80%. The yield was approximately 50 g, and the yield was 50%. Figure 4 shows an electron microscope photograph of the microcarriers placed in an aqueous solution.

[0118] Example 2 Oil phase formulation 1.1 The device was tested as in Example 1.

[0119] 1.2 According to the dosage, 80 L of chloroform, 20 L of petroleum ether, and 10 L of Tween 20 reagent were taken and added to the mixing tank in sequence using a peristaltic pump. The mixing tank was opened and stirred, and the rotation speed was set to 60 rpm and stirred for 1 hour.

[0120] 1.3 Close the mixing tank, leave only the compressed air inlet open, and pour the oil phase into the pre-cooling tank until the internal pressure of the tank reaches 0.1 MPa. Use the water cooler to continuously cool the oil phase down to -30°C, while maintaining the pre-cooling tank at 40 rpm.

[0121] Water Phase Formulation 2.1 The device was tested as in Example 1.

[0122] 2.2 50 g of gelatin and 10 g of sodium alginate were weighed out and added to 1.5 L of deionized water. The gelatin solution was stirred at 60 rpm using a stirrer until fully dissolved, and then added to the aqueous phase tank using a peristaltic pump. 100 g of sodium chloride was added, heated to 60°C, and stirred at 150 rpm for 120 minutes to prepare the solution.

[0123] mixture 3.1 The device was tested as in Example 1.

[0124] The temperature of the 3.2 reaction tank was continuously cooled to below -30°C using a water chiller. The lower the temperature, the smaller the pore size of the prepared carrier material; the pore size of microcarriers prepared at -30°C was approximately 5 μm to 20 μm. After cooling to the specified temperature, 5 mL of 75% formaldehyde solution was added to the aqueous phase. The rotation speed was set to 80 rpm and stirred for 5 minutes. After that, the oil and aqueous phases were mixed at a constant flow rate using a microsphere molding machine and poured into the reaction tank. The oil phase was driven by a gear pump (pump speed: 1000 mL / min). The lower the pump speed, the larger or smaller the particle size of the prepared particulate material. The average particle size of the material prepared under these conditions was approximately 400 μm. The pressure used to pressurize the aqueous phase with nitrogen gas was 10 kPa. The higher the pressure, the larger or smaller the particle size of the microspheres. Under these conditions, the average particle size was 400 μm. The microsphere molding machine utilizes a perforated plate with microwell pores of 50 μm diameter. The microsphere molding machine used was the same as that used in Example 1, except that the microwell hole diameter of the perforated plate was set to 50 μm.

[0125] 3.3 After the materials were placed in the reaction tank, stirring was started immediately, and the rotation speed was set to 30 rpm for 48 hours. During this time, the reaction status of the materials in the tank and each parameter were regularly observed and recorded.

[0126] Cleaning 4.1 The device was tested as in Example 1.

[0127] 4.2 After the reaction was completed, the material was poured into the oil filtration tank using 0.15MP nitrogen gas, and the excess liquid in the material was removed using a vacuum pump. After that, 50L of acetone, 20kg of anhydrous magnesium sulfate, 100L of medical alcohol, and 100L of absolute ethanol were added as cleaning agents, and the mixture was washed for 20 minutes at a stirring speed of 100 rpm. After each wash, the excess liquid was removed by suction filtration using a vacuum pump. The effective material was then poured into the water washing tank using 0.10MP compressed air.

[0128] 4.3 20 L of deionized water was placed in the washing tank, stirred thoroughly at 100 rpm for 50 minutes, and the water for injection was removed using a vacuum pump. The washing was repeated 3 to 5 times.

[0129] Freeze drying 5.1 The device was tested as in Example 1.

[0130] 5.2 The washed material was mixed with 300 mL of water for injection in a lyophilization cartridge in a ratio of 50 g wet weight, and then frozen at -40°C for 96 hours.

[0131] 5.3 The frozen material was placed in a freeze-dryer and freeze-dried according to the freeze-dryer's operating instructions. Freeze-drying was continued for more than 72 hours. The total weight of the freeze-dried material was measured and transferred to a temporary storage box for the material, and then transferred to the next process.

[0132] Sieving 6.1 First, check the cleanliness and clearance log of the mesh and sieve machine to ensure they are clean and within their validity period.

[0133] 6.2 The freeze-dried material was sieved to a size range of 50-500 microns. The sieved material was collected to obtain porous microcarriers with a certain particle size range.

[0134] As a result, the obtained microcarriers had an average particle size of 400 μm, an average pore size of 15 μm, and a porosity of 90%. The yield was approximately 20 g, and the yield was approximately 30%. An electron microscope photograph of the microcarriers placed in an aqueous solution is shown in Figure 5.

Claims

1. Step 1) Providing a dispersed phase liquid and a continuous phase liquid, the dispersed phase liquid comprises an artificial synthetic polymer and / or a natural biopolymer, and a curing agent; the continuous phase liquid comprises an organic solvent and a non-ionic surfactant; Step 2) causing the dispersed phase liquid to flow from one side of a perforated plate to the other side of the perforated plate through a plurality of microwells arranged on the perforated plate, and causing the continuous phase liquid to flow on the other side of the perforated plate in a direction parallel to the perforated plate, thereby forming liquid microspheres in the flowing continuous phase liquid by applying shear to the dispersed phase liquid passing through the perforated plate; Step 3) forming particulate matter by reacting the artificial synthetic polymer and / or natural biopolymer in the liquid microsphere with a curing agent; and step 4) collecting and washing said particulate matter; A method for preparing microcarrier particles, wherein the temperature of the continuous phase liquid is pre-cooled to 0°C or below before mixing with the dispersed phase liquid in step 2), and step 3) is carried out at 0°C or below for 2 to 72 hours.

2. 2. The method of claim 1, wherein step 2) is carried out in a vessel including a perforated plate, the perforated plate dividing the interior of the vessel into a first portion and a second portion, the dispersed phase liquid entering the first portion through a dispersed phase inlet provided in the vessel and communicating with the first portion, and then flowing through the perforated plate into the second portion, the continuous phase liquid entering the second portion through a continuous phase inlet provided in the vessel and communicating with the second portion, and the mixture containing the liquid microspheres after mixing of the dispersed phase liquid and the continuous phase liquid flows out of the vessel through a vessel outlet provided in the vessel and communicating with the second portion, the vessel outlet and the continuous phase inlet being provided on opposite sides of the vessel.

3. 3. The method of claim 2, wherein the dispersed phase liquid enters the first portion of the vessel by gas pressurization and flows through the perforated plate, and the continuous phase liquid enters the second portion of the vessel by a gear pump and flows parallel to the perforated plate.

4. 10. The method of claim 1, wherein step 3) is carried out in a tank equipped with an agitator.

5. 10. The method of claim 1, wherein step 4) is performed by vacuum suction in a tank in which the filtration device is located.

6. 2. The method of claim 1, wherein the diameter of the microwells is between 0.1 μm and 500 μm.

7. 2. The method of claim 1, wherein the flow rate of the continuous phase liquid is 5 to 20 times the flow rate of the dispersed phase liquid at the same time.

8. The artificial synthetic polymer is selected from at least one of polyethylene glycol, polyethylene glycol derivatives, polyethylene glycol diacrylate, polypropylene, polystyrene, polyacrylamide, polylactic acid, polyhydroxy acid, polylactic alcohol acid copolymer, polydimethylsiloxane, polyanhydride, polyacid ester, polyamide, polyamino acid, polyacetal, polycyanoacrylate, polyurethane, polypyrrole, polyester, polymethacrylate, polyethylene, polycarbonate, and polyethylene oxide; and / or the natural biopolymer is selected from at least one of collagen, proteoglycan, glycoprotein, gelatin, gelatin derivatives, chitin, alginate, alginate derivatives, agar, fibrinogen, matrigel, hyaluronic acid, laminin and fibronectin.

9. the organic solvent is selected from at least one of hydrofluoroether, carbon tetrachloride, petroleum ether, cyclohexane, liquid paraffin, edible oil, soybean oil, olive oil, chloroform, dichloromethane, and tetrachloroethylene; and / or the nonionic surfactant is selected from at least one of sorbitan fatty acid esters, glycerin fatty acid esters, lauric acid esters, polyoxyethylene alkylphenyl ethers, polyoxyethylene high carbon fatty alcohol ethers, Span, PO-500, monooleic acid esters, and Tween.

10. 10. The method of claim 1, wherein the curing agent is selected from at least one of divinylbenzene, diisocyanate, N-hydroxysuccinimide, N,N-methylenebisacrylamide, formaldehyde, glutaraldehyde, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, calcium ions, tetramethylethylenediamine, ammonium sulfate, genipin, and transglutaminase.

11. 10. The method of claim 1, wherein the dispersed phase liquid further comprises a buffering agent, the buffering agent being selected from at least one of carboxymethylcellulose, sodium chloride, polyacrylamide, potassium chloride, polyvinylpyrrolidone, sodium sulfate, calcium chloride, sodium carbonate, and sodium bicarbonate.

12. 10. The method of claim 1, comprising cleaning the particulate matter with a cleaning agent, the cleaning agent being selected from at least one of acetone, anhydrous copper sulfate, calcium chloride, sodium sulfate, absolute ethanol, medical grade alcohol, hydrofluoroether, sodium alkylbenzene sulfonate, sodium fatty alcohol sulfate, sodium tripolyphosphate, and deionized water.

13. 2. The method of claim 1, wherein the ratio of organic solvent to nonionic surfactant in the continuous phase liquid is from 5:1 to 20:1 by weight.

14. 1) Container, a perforated plate including a plurality of microwells disposed within the vessel, the perforated plate dividing the vessel into a first portion and a second portion; a dispersed phase inlet in communication with said first portion for introducing a dispersed phase liquid; a continuous phase inlet in communication with the second portion for introducing a continuous phase liquid; and a container outlet in communication with said second portion; wherein the vessel outlet and the continuous phase inlet are arranged on opposite sides of the vessel such that the continuous phase liquid introduced through the continuous phase inlet can flow through the second portion in a direction parallel to the perforated plate and then flow out of the vessel outlet; 2) a first tank in communication with the dispersed phase inlet for containing the dispersed phase liquid, the first tank further in communication with a pressurizer or gas cylinder for allowing the dispersed phase liquid to flow under pressure into the emulsion preparation apparatus; 3) a second tank communicating with the continuous phase inlet for containing the continuous phase liquid and equipped with a cooling device for cooling the continuous phase liquid, the inlet of the second tank communicating with a mixing tank, the mixing tank being movable and used to prepare the continuous phase liquid; 4) a third tank for carrying out an emulsion reaction, the third tank being equipped with an agitator and a cooling device and communicating with the vessel outlet; and 5) a fourth tank in communication with the third tank equipped with a filtration device for collecting particulate matter formed by the emulsion reaction; 1. A process system for preparing microcarrier particles, comprising:

15. the continuous phase inlet is two or more and the vessel outlet is two or more; and / or 15. The process system of claim 14, wherein the continuous phase inlet and the vessel outlet have the same horizontal elevation relative to the bottom of the vessel.

16. 15. The process system of claim 14, wherein the microwells have a diameter of 0.1 μm to 500 μm.

17. The processing system of claim 14, wherein the microwells have a diameter of 30 μm to 50 μm.

18. 15. The process system of claim 14, wherein a gear pump is provided between the continuous phase inlet and the second tank to transport the continuous phase liquid from the second tank to the emulsion preparation device.

19. 15. The process system of claim 14, further comprising a fifth tank in communication with the fourth tank equipped with a filtration device for washing the particulate matter.

20. 20. The process system of claim 19, wherein the fourth tank and the fifth tank each include a vacuum device.

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