Preparation method and reaction device of microcarrier suitable for three-dimensional cell culture

Abstract

This article provides a method for preparing microcarrier particles, comprising passing a dispersed phase liquid through a porous plate at low temperature to form liquid microspheres in the continuous phase, and allowing the synthetic polymers and / or natural biomacromolecules within the liquid microspheres to undergo a low-temperature solidification reaction to form particles. Also provided are a method for preparing an emulsion, an apparatus for preparing microcarrier particles, and a process system for preparing the microcarrier particles, which can be used for large-scale preparation of emulsions and microcarrier particles.

Description

Technical Field

[0001] The present disclosure relates to a method and apparatus for preparing microcarriers, and in particular to a method and process system for preparing microcarriers on a large scale. Background Art

[0002] 3D TableTrix microslides (Beijing Huakan Biotechnology Co., Ltd.) are a novel cell expansion carrier material customized for stem cell production. This technology utilizes an innovative microcarrier tablet design. Each microslide is individually sterilized and ready for use, eliminating the tedious weighing and sterilization procedures required by traditional microcarriers. This technology offers advantages over traditional microcarrier-based cell culture.

[0003] Microcarriers can be prepared in the form of microslides. When the microslide comes into contact with water, it will 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 compared to the microcarriers before being made into microslides. The porosity of the microcarriers is >90%, the particle size can be controlled in the range of 50-500μm, the uniformity is ≤100μm, and the biochemical and physical properties can be customized to achieve precise and controllable 3D bionic culture. The raw materials are of pharmaceutical grade and meet clinical application standards. The 3DTableTrix microslide, combined with reagents such as 3DFloTrixDigest lysis buffer (Beijing Huakan Biotechnology Co., Ltd.), can lyse the microcarriers and achieve gentle and non-destructive recovery of cells without any harmful substances remaining.

[0004] Given the large market demand for the microslide and the limited production capacity of existing production methods, there is an urgent need to adopt new methods and equipment to expand the production scale. Summary of the Invention

[0005] In one aspect, the present invention provides a method for preparing an emulsion, comprising allowing a dispersed phase liquid to flow from one side of a porous plate through a plurality of micropores on the porous plate to the other side of the porous plate, while allowing a continuous phase liquid to flow parallel to the porous plate on the other side of the porous plate, and shearing the dispersed phase liquid passing through the porous plate to form liquid microspheres in the flowing continuous phase liquid.

[0006] In some embodiments, the diameter of the micropores is 0.1 μm to 500 μm.

[0007] In some embodiments, the diameter of the micropores is 30 μm to 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 adjusted by adjusting the flow rate of the dispersed phase liquid and / or the continuous phase liquid.

[0010] In one aspect, provided herein is a method of preparing microcarrier particles comprising:

[0011] 1) Prepare dispersed phase liquid and continuous phase liquid, wherein:

[0012] The dispersed phase liquid includes artificial synthetic polymers and / or natural biomacromolecules; and a curing agent;

[0013] The continuous phase liquid includes an organic solvent and a nonionic surfactant;

[0014] 2) allowing the dispersed phase liquid to flow from one side of the porous plate through a plurality of micropores provided on the porous plate to the other side of the porous plate, while allowing the continuous phase liquid to flow parallel to the porous plate on the other side of the porous plate, and shearing the dispersed phase liquid passing through the porous plate to form liquid microspheres in the flowing continuous phase liquid;

[0015] 3) allowing the artificial synthetic polymer and / or natural biomacromolecule in the liquid microsphere to react with the curing agent to form particles; and

[0016] 4) collecting and washing the particles;

[0017] The temperature of the continuous phase liquid in step 2) is not higher than 0°C; and step 3) is performed at a temperature not higher than 0°C for 2-72 hours.

[0018] In some embodiments, step 2) is carried out in a container including the porous plate, wherein the porous plate divides the interior of the container into a first part and a second part, the dispersed phase liquid enters the first part through a dispersed phase inlet provided on the container and communicating with the first part, and then flows through the porous plate into the second part; the continuous phase liquid enters the second part through a continuous phase inlet provided on the container and communicating with the second part; the mixed liquid containing the liquid microspheres after the dispersed phase liquid and the continuous phase liquid are mixed leaves the container through a container outlet provided on the container and communicating with the second part; the container outlet and the continuous phase inlet are provided on opposite sides of the container.

[0019] In some embodiments, the dispersed phase liquid enters the first portion of the container and flows through the porous plate by gas pressurization; the continuous phase liquid enters the second portion of the container and flows parallel to the porous plate by a gear pump.

[0020] In some embodiments, step 3) is performed in a tank equipped with a stirring device.

[0021] In some embodiments, step 4) is performed by vacuuming a tank provided with a filtering device.

[0022] In some embodiments, the diameter of the micropores is 0.1 μm-500 μm.

[0023] In some embodiments, the micropores have a diameter of 30 μm-50 μm.

[0024] In some embodiments, the flow rate of the continuous phase liquid is 5-20 times the flow rate of the dispersed phase liquid in the same time.

[0025] 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 acid alkyd copolymer, polydimethylsiloxane, polyanhydride, polyester, polyamide, polyamino acid, polyacetal, polycyanoacrylate, polyurethane, polypyrrole, polyester, polymethacrylate, polyethylene, polycarbonate and polyethylene oxide.

[0026] In some embodiments, the natural biomacromolecule 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.

[0027] In some embodiments, 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, carbon tetrachloride and tetrachloroethylene.

[0028] In some embodiments, the nonionic surfactant is at least one selected from sorbitan fatty acid esters, fatty acid glycerides, lauric acid esters, alkylphenol polyoxyethylene ethers, high-carbon fatty alcohol polyoxyethylene ethers, Span, PO-500, monooleate, and Tween.

[0029] 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.

[0030] In some embodiments, the dispersed phase liquid further comprises a buffer selected from at least one of carboxymethyl cellulose, sodium chloride, polyacrylamide, potassium chloride, polyvinyl pyrrolidone, sodium sulfate, calcium chloride, sodium chloride, sodium carbonate, and sodium bicarbonate.

[0031] In some embodiments, the particles are washed with a cleaning agent selected from at least one of acetone, anhydrous copper sulfate, calcium chloride, sodium sulfate, anhydrous ethanol, medical alcohol, hydrofluoroether, sodium alkylbenzene sulfonate, sodium fatty alcohol sulfate, sodium tripolyphosphate and deionized water.

[0032] In some embodiments, the ratio of the organic solvent to the non-ionic surfactant in the continuous phase liquid is 5:1 to 20:1 by weight.

[0033] In another aspect, there is provided an emulsion preparation apparatus comprising:

[0034] 1) Container;

[0035] 2) a porous plate comprising a plurality of micropores disposed within the container, wherein the porous plate separates the interior of the first container into a first portion and a second portion;

[0036] 3) a dispersed phase inlet communicating with the first portion for inputting a dispersed phase liquid;

[0037] 4) a continuous phase inlet communicating with the second portion for inputting a continuous phase liquid; and

[0038] 5) a container outlet communicating with the second portion,

[0039] The container outlet and the continuous phase inlet are arranged on opposite sides of the container so that the continuous phase liquid input from the continuous phase inlet can flow through the second part in a direction parallel to the porous plate and then flow out from the container outlet.

[0040] In some embodiments, the diameter of the micropores is 0.1 μm - 500 μm.

[0041] In some embodiments, the diameter of the micropores is 30 μm to 50 μm.

[0042] In some embodiments, the container is in the shape of a cuboid.

[0043] In some embodiments, the number of the continuous phase inlets is two or more, and the number of the container outlets is two or more.

[0044] In some embodiments, the continuous phase inlet and the vessel outlet are at the same level relative to the bottom of the vessel.

[0045] In some embodiments, the emulsion is a water-in-oil emulsion.

[0046] In another aspect, the present invention provides a process system for preparing microcarrier particles, comprising:

[0047] 1) Emulsion preparation device, comprising:

[0048] container;

[0049] A porous plate comprising a plurality of micropores disposed in the container, wherein the porous plate divides the interior of the first container into a first part and a second part;

[0050] a dispersed phase inlet communicating with the first portion for inputting a dispersed phase liquid;

[0051] a continuous phase inlet communicating with the second portion for inputting a continuous phase liquid; and

[0052] a container outlet communicating with the second portion,

[0053] wherein the container outlet and the continuous phase inlet are arranged on opposite sides of the container so that the continuous phase liquid input from the continuous phase inlet can flow through the second portion in a direction parallel to the porous plate and then flow out from the container outlet;

[0054] 2) a first tank body in communication with the dispersed phase inlet, for containing the dispersed phase liquid, the first tank body also being in communication with a pressurizing device or a gas cylinder so as to allow the dispersed phase liquid to enter the emulsion preparation device under pressure;

[0055] 3) a second tank body connected to the continuous phase inlet and equipped with a cooling device, for containing the continuous phase liquid and cooling the continuous phase liquid;

[0056] 4) a third tank body connected to the outlet of the container, wherein the third tank body is equipped with an agitator and a cooling device for performing an emulsion reaction; and

[0057] 5) a fourth tank body connected to the third tank body, wherein the fourth tank body is equipped with a filtering device for collecting particulate matter formed by the emulsion reaction.

[0058] In some embodiments, the container is in the shape of a cuboid.

[0059] In some embodiments, the number of the continuous phase inlets is two or more, and the number of the container outlets is two or more.

[0060] In some embodiments, the continuous phase inlet and the vessel outlet are at the same level relative to the bottom of the vessel.

[0061] In some embodiments, a gear pump is provided between the continuous phase inlet and the second tank body for inputting the continuous phase liquid from the second tank body into the emulsion preparation device.

[0062] In some embodiments, the process elution further includes a fifth tank body connected to the fourth tank body, and the fifth tank body is equipped with a filtering device for washing the particulate matter.

[0063] In some embodiments, the fourth tank and the fifth tank are respectively equipped with a vacuum pumping device.

[0064] In some embodiments, the diameter of the micropores is 0.1 μm to 500 μm.

[0065] In some embodiments, the diameter of the micropores is 30 μm to 50 μm.

[0066] The methods and apparatus provided by the present disclosure can be used for large-scale preparation of emulsions and microcarrier particles. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 It is a schematic diagram of the structure of the emulsion preparation device (or microsphere forming machine).

[0068] Figure 2 Schematic diagram of the structure of the porous plate.

[0069] Figure 3 Shows the various equipment and connection relationships included in the process system for preparing microcarrier particles.

[0070] Figure 4 An electron microscope photograph of the microcarrier particles prepared in Example 1 is shown.

[0071] Figure 5 An electron microscope photograph of the microcarrier particles prepared in Example 2 is shown. DETAILED DESCRIPTION

[0072] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0073] "Emulsion", also known as emulsion, is a dispersed system formed by two immiscible liquids, in which the liquid in the form of suspended droplets is called the dispersed phase (or internal phase), and the continuously distributed liquid as the dispersion medium is called the continuous phase (or external phase). In nature, there are a large number of organic substances that are immiscible with water, and many emulsions are composed of an aqueous phase (the main component is water or water-soluble components) and an organic phase (the main component is a water-insoluble organic substance, also called an oil phase). Usually, an emulsion with an aqueous phase as the dispersed phase and an organic phase as the continuous phase is called an "oil-in-water emulsion". Correspondingly, an emulsion with an organic phase as the dispersed phase and an aqueous phase as the continuous phase is called an "oil-in-water emulsion". The suspended droplets formed by the dispersed phase liquid in the continuous phase liquid are also referred to as "liquid microspheres" in this article.

[0074] As used herein, "emulsion reaction" refers to a chemical reaction between components within a liquid microsphere. For example, in the microcarrier particle preparation process described below, a macromolecular compound, such as an artificial polymer, is solidified within the liquid microsphere to obtain particles that are insoluble in both aqueous and organic phases.

[0075] "Porous plate" herein refers to a plate-like object with a plurality of micropores formed therein. In the methods and apparatus provided herein, the "porous plate" is used to restrict the flow of the dispersed phase liquid, so that the dispersed phase liquid can only reach the other side of the porous plate by passing through the plurality of micropores. Therefore, in terms of application, the porous plate is not limited to a specific shape and can even adopt an irregular shape as long as the purpose is achieved. However, for preparing emulsions, a plate-like shape is preferred, as it is both easier to process and facilitates the formation of uniform liquid microspheres. The size of the micropores is generally 0.1μm to 500μm in diameter. When the porous plate has a considerable thickness, the micropores are actually microchannels of a certain length. However, for the sake of simplicity, the term "micropores" also includes such microchannels. Similarly, the present invention does not impose specific restrictions on the material of the microporous plate; as long as it can achieve the above-mentioned purpose and is chemically inert to both the dispersed phase liquid and the continuous phase liquid, materials such as plastic and ceramic can be used.

[0076] Mention is made herein that in the emulsion preparation device, the continuous phase is allowed to flow "parallel" to the porous plate. Under ideal conditions, the dispersed phase liquid passes through the micropores perpendicular to the porous plate, and the flow direction of the continuous phase liquid is perpendicular to the dispersed phase liquid flow passing through the micropores (i.e., parallel to the porous plate), thereby cutting the dispersed phase liquid flow into liquid microspheres. Of course, those skilled in the art will appreciate that, in most cases, even if it is not a strictly parallel flow, the purpose of the continuous phase liquid cutting the dispersed phase liquid flow into liquid microspheres can be achieved. Therefore, in the emulsion formation process, in most cases, the continuous phase liquid inlet and outlet on the emulsion preparation device are arranged on the opposite sides of the emulsion preparation equipment, i.e., "opposite side arrangement". In some embodiments, the continuous phase liquid inlet and outlet have the same horizontal height. In some embodiments, a plurality of continuous phase liquid inlets and a plurality of container outlets can be horizontally arranged on the opposite sides of a container that is roughly in the shape of a cube, to facilitate the parallel flow of the continuous phase liquid.

[0077] "Microcarrier particles" or "microcarriers" refer to particles in the micrometer range that are suitable for cell attachment and growth. Microcarrier particles are preferably between 50 and 500 μm in size. The porosity is typically greater than 80%, for example, 90% or 95%. Most cells can only proliferate when attached to a solid substrate. However, the porous, large surface area, and biocompatibility of microcarriers allow cells to grow within the microcarriers prepared herein, forming a three-dimensional culture model. This biomimetic three-dimensional culture model is gaining increasing application.

[0078] "Large-scale" refers to the production of products in quantities sufficient for laboratory studies up to industrial production needs. For example, 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 more, of emulsion can be produced at a time. For microcarrier particles, "large-scale" includes, for example, the production of 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 more, of microcarrier particles at a time.

[0079] In some aspects of the present disclosure, a method for preparing an emulsion is provided, which can be used for large-scale emulsion preparation. The method includes allowing a dispersed phase liquid to flow from one side of a porous plate through multiple micropores on the porous plate to the other side of the porous plate, while allowing a continuous phase liquid to flow parallel to the porous plate on the other side of the porous plate, and shearing the dispersed phase liquid passing through the porous plate to form liquid microspheres in the flowing continuous phase liquid. Because a large number of micropores can be densely arranged on the porous plate, a large amount of emulsion can be continuously and rapidly prepared by cross-flowing the dispersed phase liquid and the continuous phase liquid in substantially perpendicular directions near the porous plate.

[0080] In some specific embodiments, the emulsion formation process is performed in a container comprising a porous plate. More specifically, the porous plate disposed within the container divides the interior of the container into a first portion and a second portion. The dispersed phase liquid enters the first portion through a dispersed phase inlet disposed on the container and communicating with the first portion, and then flows through the porous plate into the second portion. The continuous phase liquid enters the second portion through a continuous phase inlet disposed on the container and communicating with the second portion. The mixed liquid containing the liquid microspheres, resulting from the mixing of the dispersed phase liquid and the continuous phase liquid, exits the container through a container outlet disposed on the container and communicating with the second portion. To allow the continuous phase to pass through the second portion substantially parallel to the porous plate, the container outlet and the continuous phase inlet are disposed on opposite sides of the container.

[0081] Typically, the amount of dispersed phase liquid used is not greater than the amount of continuous phase liquid, for example, in the prepared emulsion, the ratio of dispersed phase liquid to mobile phase liquid is 1:1 to 1:30, preferably 1:5 to 1:20. Exceeding this ratio 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 micropores. In addition, the flow rate of the dispersed phase and the flow rate of the continuous phase, as well as the relative flow rate between them, can also affect the size of the liquid microspheres. In order to make the dispersed phase liquid and the continuous phase liquid flow at a substantially uniform speed, the liquid flow can be controlled by using pressurized gas or a device such as a gear pump. For example, in a specific embodiment, the dispersed phase liquid is forced to flow at a uniform speed by gas pressurization, and the continuous phase liquid is driven to flow at a uniform speed by the rotation of the gear pump. In some embodiments of the present invention, the dispersed phase is an aqueous phase comprising reactants, and the continuous phase is an organic phase comprising organic matter.

[0082] The present disclosure also provides a method for preparing microcarrier particles using the above-mentioned emulsion formation process. This method is suitable for large-scale preparation of microcarrier particles. In this method, the dispersed phase liquid used includes a compound to be solidified (e.g., a synthetic polymer and / or a natural biomacromolecule) and a curing agent. The continuous phase used includes an organic solvent and a non-ionic surfactant. The liquid microspheres produced by the emulsion formation process react under stirring to form particulate matter. The particulate matter is then collected by filtration and washed with a detergent.

[0083] The present inventors have discovered that when the emulsion formation and reaction processes are carried out at low temperatures (below 0°C, for example, below -10°C, for example -30°C), the resulting particles have improved porosity and specific surface area, making them more suitable as three-dimensional carriers for cell culture. Generally, the lower the temperature, the smaller the pore size of the resulting microcarrier particles. For example, when the reaction is carried out at -30°C, the pore size of the resulting microcarrier particles is approximately 5 μm to 20 μm.

[0084] In order to make the dispersed phase liquid and the continuous phase liquid flow at a substantially uniform speed, the liquid flow can be controlled by using pressurized gas or a device such as a gear pump. For example, in one embodiment, the dispersed phase liquid is forced to flow at a uniform speed by gas pressurization, and the continuous phase liquid is pushed to flow at a uniform speed by the rotation of the gear pump. Generally, the greater the pressure on the dispersed phase and the faster the flow rate, the larger the particle size of the prepared microcarriers, and vice versa. In addition, the lower the continuous phase flow rate, the larger the particle size of the prepared microcarrier particles, and vice versa. Therefore, the particle size of the finally prepared microcarrier particles can be adjusted by flexibly controlling the diameter of the micropores on the porous plate and the flow rates of the dispersed phase liquid and the continuous phase liquid.

[0085] 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 acid alkyd copolymer, polydimethylsiloxane, polyanhydride, polyester, polyamide, polyamino acid, polyacetal, polycyanoacrylate, polyurethane, polypyrrole, polyester, polymethacrylate, polyethylene, polycarbonate and polyethylene oxide.

[0086] As an example, the natural biomacromolecule includes at least one of collagen, proteoglycan, glycoprotein, gelatin, gelatin derivatives, chitin, alginate, alginate derivatives, agar, fibrinogen, matrigel, hyaluronic acid, laminin, and fibronectin.

[0087] As an 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 ion, tetramethylethylenediamine, ammonium sulfate, genipin, and transglutaminase.

[0088] 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.

[0089] As an example, the nonionic surfactant includes at least one of sorbitan fatty acid ester, fatty acid glyceride, lauric acid ester, alkylphenol polyoxyethylene ether, high-carbon fatty alcohol polyoxyethylene ether, Span, PO-500, monooleate, and Teflon.

[0090] As an example, the cleaning agent used to wash the particles includes at least one of acetone, anhydrous copper sulfate, calcium chloride, sodium sulfate, anhydrous ethanol, medical alcohol, hydrofluoroether, sodium alkylbenzene sulfonate, sodium fatty alcohol sulfate, sodium tripolyphosphate, and deionized water.

[0091] In some embodiments, the dispersed phase liquid further comprises a buffer. As an example, the buffer comprises at least one of carboxymethyl cellulose, sodium chloride, polyacrylamide, potassium chloride, polyvinyl pyrrolidone, sodium sulfate, calcium chloride, sodium chloride, sodium carbonate, and sodium bicarbonate.

[0092] Typically, the concentration of the compound to be cured in the dispersed phase is between 1% and 20% (wt), and the amount of the curing agent can be adjusted according to the properties of the compound to be cured and the properties of the curing agent itself.

[0093] It should be noted that the above components are merely examples of some of the raw materials suitable for preparing microcarrier particles. Those skilled in the art can readily substitute one or more of these materials with other similar substances based on their physical and chemical properties, and conduct simple experiments according to the methods provided herein to verify their feasibility. Obviously, methods for preparing microcarrier particles according to the methods provided herein using these substitutions and the products thereof are also intended to fall within the scope of the present invention.

[0094] In some aspects, the present disclosure also provides an emulsion preparation device, which can be used to prepare emulsions on a large scale.

[0095] The emulsion preparation device comprises:

[0096] container;

[0097] A porous plate comprising a plurality of micropores disposed in the container, wherein the porous plate divides the interior of the first container into a first part and a second part;

[0098] a dispersed phase inlet communicating with the first portion for inputting a dispersed phase liquid;

[0099] a continuous phase inlet communicating with the second portion for inputting a continuous phase liquid; and

[0100] a container outlet communicating with the second portion,

[0101] The container outlet and the continuous phase inlet are arranged on opposite sides of the container so that the continuous phase liquid input from the continuous phase inlet can flow through the second part in a direction parallel to the porous plate and then flow out from the container outlet.

[0102] Figure 1 This is a cross-sectional view of the main structure of the emulsion preparation device of the present invention, schematically showing some structures of the emulsion preparation device and various liquid flow directions. Figure 1 As shown, the container 1 includes an internal space surrounded by a container wall 11, and the internal space is divided into a first portion 12 on the upper side and a second portion 13 on the lower side by a porous plate 14 arranged in the container 1. The container 1 is provided with a dispersed phase inlet 15 communicating with the first portion 12, a continuous phase inlet 16 communicating with the second portion 13, and a container outlet 17. The continuous phase inlet 16 and the container outlet 17 are respectively arranged on both sides of the container 1. In some embodiments, the continuous phase inlet 16 and the container outlet 17 are substantially at the same height relative to the bottom 18 of the container 1. A plurality of micropores 141 (see Figure 2). The dispersed phase liquid can be input into the first part 12 from the dispersed phase inlet 15 by applying pressure. Under pressure, the dispersed phase liquid will continue to flow through the micropores 141 on the porous plate 14 and enter the second part 13 on the lower side. At the same time, the continuous phase liquid enters the second part 13 through the continuous phase inlet 16, flows basically parallel to the porous plate 14, and flows out from the container outlet 17. On the lower side of the porous plate 14, the flowing continuous phase liquid shears the dispersed phase liquid passing through the micropores 141 on the porous plate 14 into liquid microspheres. The liquid microspheres are insoluble in the continuous phase liquid and form numerous liquid microspheres suspended in the continuous phase liquid. These liquid microspheres leave the container outlet 17 along with the flow of the continuous phase. The prepared emulsion is obtained by collecting the liquid mixture flowing out of the container outlet 17.

[0103] In order to obtain liquid microspheres of suitable size, the pore diameter of the micropores 141 on the porous plate 14 can be adjusted. When the micropore diameter is 0.1 μm-500 μm, uniform liquid microspheres can be prepared within the range of 1 μm-1000 μm and the particle size error is within 100 μm.

[0104] In some embodiments, the dispersed phase liquid is an aqueous phase liquid, the continuous phase liquid is an organic phase liquid, and the prepared emulsion is a water-in-oil emulsion.

[0105] In some aspects, the present disclosure provides a process system for preparing microcarrier particles, which can be used to prepare microcarrier particles on a large scale. In addition to the above-mentioned emulsion preparation device, the process system also includes multiple tanks.

[0106] One of the tanks is connected to the dispersed phase inlet and is used to accommodate the dispersed phase liquid. The tank can be equipped with an agitator for preparing the dispersed phase liquid. The tank can also be connected to a pressurizing device or a gas cylinder containing compressed gas, and the dispersed phase liquid is allowed to enter the emulsion preparation device by applying pressure to the tank. The gas used should not react with the dispersed phase liquid and the continuous phase liquid, and can be, for example, selected from air, nitrogen, carbon dioxide, oxygen, and argon. The gas pressure used can be, for example, 1 kPa to 100 kPa.

[0107] Another tank is connected to the continuous phase inlet and is used to contain the continuous phase liquid and cool the continuous phase liquid. The tank can also be used to prepare the continuous phase liquid, or to cool the continuous phase liquid after the prepared continuous phase liquid is input into the tank. Methods for cooling the contents of the tank are well known in the art, for example, by providing a jacket to the tank and circulating a refrigerant in the jacket using a refrigerator. The refrigerant can, for example, include 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.

[0108] Another tank communicates with the container outlet and receives the mixed liquid containing the liquid microspheres from the emulsion preparation device. This tank is equipped with a stirrer and a refrigerator, which allow the reactants (e.g., biomacromolecules and corresponding curing agents) contained in the liquid microspheres to react under low-temperature conditions (e.g., -10°C) to form particles.

[0109] Another tank is used to separate the formed particles from the mixed liquid. The tank can be provided with a filter device, such as a vacuum filter, to facilitate the use of a vacuum pump to remove the liquid components by vacuuming, leaving the particles.

[0110] Optionally, a tank for washing the particles may be included. The tank may also be provided with a vacuum filter, and the particles may be washed repeatedly (e.g., 3 to 5 times) by adding detergent to the tank and then evacuating the tank.

[0111] In a specific embodiment, the process system for preparing microcarrier particles of the present invention is as follows Figure 3 As shown. The mixing tank is used for the preparation of the continuous phase liquid (organic phase). After the preparation is completed, the continuous phase liquid can be transferred to a pre-cooling tank equipped with a refrigerator, for example, by pressure, where the continuous phase liquid is reduced to below 0°C (for example, below -10°C). The aqueous phase tank is used for the preparation of the dispersed phase liquid. The prepared dispersed phase liquid and continuous phase liquid are respectively transported to the emulsion preparation device by pressure and pump. The emulsion including liquid microspheres generated in the emulsion preparation device is then transferred to the reaction tank for emulsion reaction. The particulate matter generated by the reaction is then separated from the other components of the emulsion by vacuum filtration in the oil filter tank. Finally, the microcarrier particles are washed with a detergent in a water washing tank to obtain microcarrier particles.

[0112] Now combined Figure 3 The structure and working mode of the above process system are further described in detail.

[0113] The system consists of six tanks: a mixing tank, a pre-cooling tank, an aqueous phase tank, a reaction tank, an oil filter tank, and a water wash tank. These tanks are complemented by other equipment such as a vacuum pump, a chiller, and a control panel. Several inter-tank connecting pipes are also included, along with external pipelines for drinking water, purified water, water for injection, compressed air, and high-temperature steam. With the exception of some valves and connections, the entire system is constructed of 316L stainless steel, ensuring that all material contact surfaces are made of 316L stainless steel to prevent the introduction of foreign matter.

[0114] The tanks are arranged left and right according to the process steps, and the height positions of some tanks are determined according to important process control points, which are reflected in the form of workbenches. Table 1 briefly describes each piece of equipment.

[0115] Table 1 A brief description of each tank and other related equipment

[0116]

[0117]

[0118] 1. Proportion of organic phase stock solution 1. First check the clean status of the mixing tank, pre-cooling tank, liquid sterilizing filter, measuring instruments and clearance records. They can only be used if they are clean and within the clearance validity period. Measure the raw and auxiliary materials, and use a peristaltic pump to add a variety of raw and auxiliary materials in a certain proportion to the mixing tank in sequence. Open the mixing tank and stir, set the speed to 10-150rpm, and stir for 0.5-4h. Seal the tank body and leave only the compressed air port open until the internal pressure of the tank reaches 0.1MP-1MP. Pump the stock solution 1 into the liquid sterilizing filter, and then pass it into the pre-cooling tank. The outer wall interlayer of the pre-cooling tank is equipped with refrigerant. Through the continuous cooling effect of the chiller, the temperature of the stock solution 1 is reduced to 0℃ to -196℃ for standby use. During this period, the stirring of the pre-cooling tank is maintained at 10-2000rpm; the stirring time is 0.5-24h.

[0119] The organic phase stock solution 1 is a mixed organic phase containing an organic solvent and a non-ionic surfactant, wherein the organic solvent is at least one selected from hydrofluoroether, carbon tetrachloride, petroleum ether, cyclohexane, liquid paraffin, edible oil, soybean oil, olive oil, chloroform, dichloromethane, carbon tetrachloride and tetrachloroethylene, and the non-ionic surfactant is at least one selected from sorbitan fatty acid ester, fatty acid glyceride, lauric acid ester, alkylphenol polyoxyethylene ether, high carbon fatty alcohol polyoxyethylene ether, span, PO-500, monooleate, and soil temperature.

[0120] The refrigerant includes but is not limited to 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, and is cooled to 0 to -196 degrees Celsius using a low-temperature refrigerator.

[0121] 2. Prepare Solution 2. First, check the cleanliness of the aqueous phase tank, measuring instruments, and clearance records. Use only if they are clean and within the clearance period. Weigh the raw materials and add the main solution 2-1 to the water for injection to a solution of the specified concentration and volume. Use a peristaltic pump to add the solution 2-1 to the aqueous phase tank, then add solution 2-2. Set the speed to 10-300 rpm and stir for 5-120 minutes.

[0122] The stock solution 2-1 includes artificially synthesized biological materials and / or natural biological materials; and a buffer solution.

[0123] The artificially synthesized biomaterial is at least one selected from polyethylene glycol, polyethylene glycol derivatives, polyethylene glycol diacrylate, polypropylene, polystyrene, polyacrylamide, polylactic acid, polyhydroxy acid, polylactic acid alkyd copolymer, polydimethylsiloxane, polyanhydride, polyester, polyamide, polyamino acid, polyacetal, polycyanoacrylate, polyurethane, polypyrrole, polyester, polymethacrylate, polyethylene, polycarbonate and polyethylene oxide.

[0124] The natural biomaterial is at least one selected from collagen, proteoglycan, glycoprotein, gelatin, gelatin derivatives, chitin, alginate, alginate derivatives, agar, fibrinogen, matrigel, hyaluronic acid, laminin and fibronectin.

[0125] The buffer includes at least one of carboxymethyl cellulose, sodium chloride, polyacrylamide, potassium chloride, polyvinyl pyrrolidone, sodium sulfate, calcium chloride, sodium chloride, sodium carbonate, and sodium bicarbonate.

[0126] The 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 ion, tetramethylethylenediamine, ammonium sulfate, genipin, transglutaminase, etc.

[0127] 3. Enter the reaction tank for mixing. First, check the cleanliness of the reaction tank and the clearance record. It can be used only if they are qualified. The refrigerant flows in the interlayer of the outer wall of the reaction tank. First, the reaction tank is continuously acted on by a chiller to reduce the temperature of the tank body to 0-minus 196°C. After the temperature meets the requirements and the stock solution 1 and stock solution 2 are prepared, the stock solution 1 is mixed at a flow rate of 50-5000ml / min through a microsphere forming machine (i.e., an emulsion preparation device) and injected into the reaction tank. The stock solution 2 is pressurized by gas and enters the reaction tank through the microsphere forming machine. The gas pressure is 1Kpa-100Kpa. The gas composition includes but is not limited to air, nitrogen, carbon dioxide, oxygen and argon. Turn on the reaction tank for stirring and set the speed to 10-1200rpm, stirring for 2-72h.

[0128] The structure of microsphere forming machine can be found in Figure 1 , can be divided into 3 parts:

[0129] The upper layer is the stock solution 2 chamber, where the liquid flows vertically, from top to bottom. Stock solution 2 is driven by an inert gas. The stable gas pressure forces stock solution 2 to flow vertically from top to bottom through the porous plate at a constant speed.

[0130] The middle layer is a porous plate, whose surface is composed of numerous cut micropores with diameters ranging from 0.1μm to 500μm. The cut plate can be in the form of single-layer sheets, multi-layer sheets, hollow tubes, hollow blocks, etc. The size of the micropores affects the diameter of the formed emulsion droplets and the final material particle size. The final microcarrier particle size is approximately 5-20 times the micropore diameter.

[0131] The lower layer is where stock solution 1 flows horizontally. Driven by a gear pump, stock solution 1's flow rate is controlled to be 5-20 times that of stock solution 2, which flows vertically through the cutting plate, per unit time. The flow rate affects the stability of the resulting emulsion. Deviating from this range can result in severe demulsification.

[0132] The stock solution 2 is delivered from top to bottom through the external gas line at the top of the water phase tank, with a stable gas pressure. The gas pressure can be set in the range of 1KPa-100KPa, and the stable range is ±1KPa. The gas composition costs include but are not limited to air, nitrogen, carbon dioxide, oxygen and argon. Under the action of stable pressure, the stock solution 2 passes through the porous plate stably from top to bottom, forming an initial liquid of circular droplets. At the same time, the stock solution 1 flows at a uniform speed through the lower part of the porous plate through the gear pump. The stock solution 1 shears the stock solution 2 passing through the porous plate into stable microsphere droplets. Since the stock solutions 1 and 2 are not miscible, stable microsphere droplets composed of the stock solution 2 are formed and suspended in the stock solution 1. The particle size of the stock solution 2 microspheres is affected by the diameter of the microporous mold on the porous cutting plate. The diameter of the microporous mold ranges from 0.1μm to 500μm, and can be prepared into uniform liquid microspheres in the range of 1μm to 1000μm with a particle size error range of less than 100μm.

[0133] 4. Cleaning. First, check the cleanliness of the oil filter tank and water washing tank, the status of the measuring instruments and the clearance records. They can only be used if they are clean and within the validity period. The reacted material is pumped into the oil filter tank through 0.1MP-1MP compressed air, and the excess liquid in the material is removed by continuous action of the vacuum pump. After the above process is completed, first add the cleaning agent, turn on the oil filter tank and stir, set to 10-70rpm to fully mix the material and the cleaning agent. Turn off the stirring for 2-5 minutes and let it stand, then turn on the stirring and set it to 20-50rpm to fully mix the effective material and the cleaning agent. Use 0.1MP-1MP compressed air to pump the effective material and the cleaning agent into the water washing tank. Remove the cleaning agent in the water washing tank through the continuous action of the vacuum pump. Add a certain volume and temperature of injection water to the water washing tank and wash it repeatedly five times. The stirring (cleaning) time for each time is 10 minutes to 24 hours.

[0134] Cleaning agents include but are not limited to acetone, anhydrous copper sulfate, calcium chloride, sodium sulfate, anhydrous ethanol, medical alcohol, hydrofluoroether, sodium alkylbenzene sulfonate, sodium fatty alcohol sulfate, sodium tripolyphosphate, deionized water, etc.

[0135] 5. Freeze-drying and screening. First, check the freeze-drying box, measuring tools, freeze-drying machine cleanliness, and clearance records. Use only if it is clean and within the expiration date. Mix the cleaned material with water for injection in a certain proportion based on wet weight. Freeze in a freezer or freezing liquid at -10 to -196 degrees Celsius. After freezing, transfer to a freeze-dryer and freeze-dry for 12 to 96 hours. The resulting three-dimensional porous microcarriers have pores with a diameter of 20 to 200 microns and a porosity of 85 to 95%.

[0136] 6. Tablet production and packaging. First, the tableting machine is inspected for cleanliness and clearance records. It can only be used if it is clean and within its expiration date. The materials to be tableted are added to the tableting machine. By adjusting the equipment parameters, individual tablets of uniform quality and shape are produced. During the tableting process, various properties of the tablets, such as water absorption, dispersibility, and stability, are observed and tested in real time. The finished tablets are promptly packaged into appropriate bottles according to the specified quantity. Once packaging is complete, the application for inspection and other subsequent procedures can be submitted.

[0137] The process of preparing microcarrier particles of the present invention is further described below through specific examples.

[0138] Example 1

[0139] Oil ratio

[0140] 1.1 First check the cleanliness of the mixing tank, pre-cooling tank, liquid sterilizing filter, measuring tools and the clearance records. They can only be used if they are clean and within the clearance validity period;

[0141] 1.2 Add 100L of petroleum ether and 10L of PO-500 reagent to the mixing tank using a peristaltic pump. Stir the mixing tank at 60 rpm for 1 hour.

[0142] 1.3 Close the mixing tank and leave only the compressed air port open until the internal pressure of the tank reaches 0.1MP, then pump the oil phase into the pre-cooling tank. Use the chiller to continuously cool the oil phase to -10℃ for standby use. During this period, the stirring in the pre-cooling tank can be maintained at 40rpm.

[0143] Water ratio

[0144] 2.1 First, check the cleanliness of the water phase tank and measuring instruments and the clearance records. They can only be used if they are clean and within the clearance validity period.

[0145] 2.2 Weigh 100g of gelatin and add it to 5L of deionized water. Stir the gelatin solution with a stirrer set at 60rpm until it is fully dissolved. Use a peristaltic pump to add it to the aqueous phase tank, set the speed to 150rpm and stir for 90 minutes.

[0146] mix

[0147] 3.1 First check the cleanliness of the reactor and the clearance records. It can be used only if they are qualified.

[0148] 3.2 The reaction tank is continuously cooled by a water chiller to reduce the tank temperature to below -10°C. When the reaction tank temperature reaches the required level and the oil phase temperature reaches -10°C, 5 mL of 75% formaldehyde solution is added to the aqueous phase and stirred at 80 rpm for 10 minutes. The oil phase and aqueous phase are then mixed through a microsphere forming machine and then enter the reaction tank. The oil phase is driven by a gear pump (pump speed is 3000 mL / min), and the aqueous phase is driven by a fixed pressure inert gas (air is selected as the gas), and the pressure is set to 5 kPa. The micropore diameter of the porous plate selected for the microsphere forming machine is 30 μm.

[0149] The microsphere forming machine is composed of a rectangular parallelepiped with dimensions of 40CM*40CM*30CM. Inside the forming machine, a 40cm*40cm plastic plate is horizontally set 1cm above the horizontal oil phase inlet and the forming machine outlet. The thickness of the plastic plate is 1cm. The plastic plate is made of a large number of transparent circular holes with a diameter of 30μm using micromachining technology. The micropore array is 300*300 and the micropore spacing is 50μm. The dispersed phase (aqueous phase) flows into the microsphere forming machine perpendicular to the microporous plate and vertically penetrates the micropores on the microporous plate. The oil phase solution below the microporous plate quickly flows into the microsphere forming machine from the horizontal inlet and continuously and rapidly shears the aqueous phase solution passing through the micropores to form microspheres. The formed microspheres are dispersed in the oil phase and flow out from the horizontal outlet.

[0150] 3.3 After the material enters the reaction tank, start stirring immediately and set the speed to 30 rpm for stirring and reaction for 48 hours.

[0151] Cleaning

[0152] 4.1 First check the cleanliness of the oil filter tank and water washing tank, the status of the measuring tools and the clearance records. They can only be used if they are clean and within the validity period;

[0153] 4.2 The reaction-completed material is pumped into the oil filter tank through 0.15MP compressed air, and the excess liquid in the material is continuously removed by the vacuum pump. Then, cleaning agents are added to the material, namely 50L of acetone, 10kg of calcium chloride, 100L of medical alcohol, and 100L of anhydrous ethanol. The stirring speed is 100rpm, and each is 20 minutes. After each cleaning, the excess liquid is removed by vacuum filtration. Use 0.10MP compressed air to pump the effective material and alcohol into the water washing tank;

[0154] 4.3 Add 20L of deionized water to the water washing tank, set the stirring setting to 100rpm and stir thoroughly for 50 minutes. Remove the injection water with the vacuum pump. Repeat the washing 3-5 times.

[0155] Freeze-dried

[0156] 5.1 First, check the cleanliness of the freeze-drying box, measuring tools, freeze-drying machine and the clearance records. They can only be used if they are clean and within the validity period;

[0157] 5.2 Add the cleaned material to 500 mL of water for injection in a freeze-drying container at a ratio of 50 g wet weight and mix well. Freeze at -20°C for 48 hours.

[0158] 5.3 Place the frozen material into the freeze dryer and perform freeze drying according to the freeze dryer operating procedures for at least 72 hours. Weigh the total weight of the freeze-dried material and transfer it to a temporary storage box for the next step.

[0159] Screening

[0160] 6.1 First check the cleanliness of the screen and the screening machine and the clearance records. They can only be used if they are clean and within the validity period;

[0161] 6.2 The freeze-dried material is sieved in the range of 50-500 microns. The sieved material is collected to obtain porous microcarrier particles with a certain particle size range.

[0162] Results: The average particle size of the obtained microcarriers was 200 μm, the average pore size was 25 μm, and the porosity was 80%. The yield was about 50 g, with a yield of 50%. The electron microscope photos of the microcarriers placed in the aqueous solution were shown in Figure 4 middle.

[0163] Example 2

[0164] Oil ratio

[0165] 1.1 Inspection device, same as in Example 1;

[0166] 1.2 Add 80 L of chloroform, 20 L of petroleum ether, and 10 L of Tween 20 reagent to the mixing tank using a peristaltic pump. Stir the mixing tank at 60 rpm for 1 hour.

[0167] 1.3 Close the mixing tank and leave only the compressed air port open until the internal pressure of the tank reaches 0.1MP, then pump the oil phase into the pre-cooling tank. Use the chiller to continuously cool the oil phase to -30℃ for standby use. During this period, the stirring in the pre-cooling tank can be maintained at 40rpm.

[0168] Water ratio

[0169] 2.1 Inspection device, same as in Example 1;

[0170] 2.2 Weigh 50g of gelatin and 10g of sodium alginate into 1.5L of deionized water and stir the gelatin solution at 60rpm until fully dissolved. Use a peristaltic pump to add the solution to the aqueous phase tank, then add 100g of sodium chloride, heat to 60℃, set the speed to 150rpm and stir for 120 minutes.

[0171] mix

[0172] 3.1 Inspection device, same as in Example 1;

[0173] 3.2 The reaction tank was continuously cooled by a water chiller to below -30°C. The lower the temperature, the smaller the pore size of the prepared carrier material. The pore size of the microcarriers prepared at -30°C was approximately 5μm-20μm. After the temperature was lowered to the preset level, 5ml of 75% formaldehyde solution was added to the aqueous phase and stirred at 80 rpm for 5 minutes. The oil and aqueous phases were then mixed at a constant flow rate through a microsphere forming machine and pumped into the reaction tank. The oil phase was driven by a gear pump (1000ml / min). The lower the pump speed, the larger the particle size of the prepared granules, while the lower the pump speed, the smaller the particle size. The average particle size of the prepared materials under these conditions was approximately 400μm. The aqueous phase was pressurized using nitrogen at a pressure of 10kPa. The higher the pressure, the larger the microsphere size, while the lower the pressure, the smaller the microsphere size. The average particle size under these conditions was 400μm. The microsphere forming machine used a multi-well plate with a pore size of 50μm.

[0174] The microsphere forming machine used is the same as that in Example 1, except that the pore size of the porous plate is 50 μm.

[0175] 3.3 After the material enters the reaction tank, start stirring immediately and set the speed to 30rpm for stirring reaction for 48 hours. During this period, regularly observe and record the reaction status and various parameters of the tank material.

[0176] Cleaning

[0177] 4.1 Inspection device, same as in Example 1;

[0178] 4.2 The reaction-completed material is pumped into the oil filter tank through 0.15MP nitrogen, and the excess liquid in the material is continuously removed by the vacuum pump. Then, cleaning agents are added to the material, namely 50L of acetone, 20kg of anhydrous magnesium sulfate, 100L of medical alcohol, and 100L of anhydrous ethanol. The stirring speed is 300rpm, and each is 20 minutes. After each cleaning, the excess liquid is removed by vacuum filtration. Use 0.10MP compressed air to pump the effective material into the water washing tank;

[0179] 4.3 Add 20L of deionized water to the water washing tank, set the stirring setting to 100rpm and stir thoroughly for 50 minutes. Remove the injection water using the vacuum pump. Repeat the washing 3-5 times.

[0180] Freeze-dried

[0181] 5.1 Inspection device, same as in Example 1;

[0182] 5.2 Add the cleaned material to a freeze-drying box at a ratio of 50 g wet weight to 300 mL of water for injection and mix well. Freeze at -40°C for 96 hours.

[0183] 5.3 Place the frozen material into the freeze dryer and perform freeze drying according to the freeze dryer operating procedures for at least 72 hours. Weigh the total weight of the freeze-dried material and transfer it to a temporary storage box for the next step.

[0184] Screening

[0185] 6.1 First check the cleanliness of the screen and the screening machine and the clearance records. They can only be used if they are clean and within the validity period;

[0186] 6.2 The freeze-dried material is sieved in the range of 50-500 microns. The sieved material is collected to obtain porous microcarriers with a certain particle size range.

[0187] Results: The average particle size of the obtained microcarriers was 400 μm, the average pore size was 15 μm, and the porosity was 90%. The yield was about 20 g, and the yield was about 30%. The electron microscope photos of the microcarriers placed in the aqueous solution were shown in Figure 5 middle.

Claims

1. A method for preparing microcarrier particles, comprising: 1) Prepare dispersed phase liquid and continuous phase liquid, where: The dispersed phase liquid includes artificial synthetic polymers and / or natural biomacromolecules; and a curing agent; The continuous phase liquid includes an organic solvent and a nonionic surfactant; 2) allowing the dispersed phase liquid to flow from one side of a porous plate through a plurality of micropores provided on the porous plate to the other side of the porous plate, while allowing the continuous phase liquid to flow parallel to the porous plate on the other side of the porous plate, and shearing the dispersed phase liquid passing through the porous plate to form liquid microspheres in the flowing continuous phase liquid; 3) allowing the artificial synthetic polymer and / or natural biomacromolecule in the liquid microsphere to react with the curing agent to form particles; and 4) collecting and washing the particles; In step 2), the temperature of the continuous phase liquid is pre-cooled to no more than 0°C before being mixed with the dispersed phase liquid; and step 3) is performed at no more than 0°C for 2-72 hours.

2. The method of claim 1 , wherein step 2) is performed in a container comprising the porous plate, the porous plate dividing the interior of the container into a first part and a second part, the dispersed phase liquid enters the first part through a dispersed phase inlet provided on the container and communicating with the first part, and then flows through the porous plate into the second part; the continuous phase liquid enters the second part through a continuous phase inlet provided on the container and communicating with the second part; the mixed liquid containing the liquid microspheres after the dispersed phase liquid and the continuous phase liquid are mixed leaves the container through a container outlet provided on the container and communicating with the second part; the container outlet and the continuous phase inlet are provided on opposite sides of the container.

3. A method as claimed in claim 2, wherein the dispersed phase liquid enters the first part of the container and flows through the porous plate by gas pressurization; and the continuous phase liquid enters the second part of the container and flows parallel to the porous plate by a gear pump.

4. The method according to any one of claims 1 to 3, wherein step 3) is carried out in a tank provided with a stirring device.

5. The method according to any one of claims 1 to 3, wherein step 4) is performed by vacuuming in a tank provided with a filtering device.

6. The method according to any one of claims 1 to 3, wherein the diameter of the micropores is 0.1 μm - 500 μm.

7. The method according to any one of claims 1 to 3, wherein the diameter of the micropores is 30 μm - 50 μm.

8. The method according to any one of claims 1 to 3, wherein the flow rate of the continuous phase liquid is 5 to 20 times the flow rate of the dispersed phase liquid in the same time.

9. The method of any one of claims 1 to 3, wherein the artificially synthesized polymer is selected from at least one of polyethylene glycol, polyethylene glycol derivatives, polyethylene glycol diacrylate, polypropylene, polystyrene, polyacrylamide, polylactic acid, polyhydroxy acid, polylactic acid-alcohol copolymer, polydimethylsiloxane, polyanhydrides, polyesters, polyamides, polyamino acids, polyacetals, polycyanoacrylates, polyurethanes, polypyrroles, polyesters, polymethacrylates, polyethylene, polycarbonates, and polyethylene oxide.

10. The method of any one of claims 1 to 3, wherein the natural biomacromolecule 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.

11. The method according to any one of claims 1 to 3, wherein the organic solvent is at least one selected from the group consisting of hydrofluoroether, carbon tetrachloride, petroleum ether, cyclohexane, liquid paraffin, edible oil, soybean oil, olive oil, chloroform, dichloromethane and tetrachloroethylene.

12. The method according to any one of claims 1 to 3, wherein the nonionic surfactant is at least one selected from the group consisting of sorbitan fatty acid esters, fatty acid glycerides, lauric acid esters, alkylphenol polyoxyethylene ethers, higher carbon fatty alcohol polyoxyethylene ethers, Span, PO-500, monooleate, and Thuwen.

13. The method according to any one of claims 1 to 3, 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 ion, tetramethylethylenediamine, ammonium sulfate, genipin and transglutaminase.

14. The method of any one of claims 1 to 3, wherein the dispersed phase liquid further comprises a buffer selected from at least one of carboxymethyl cellulose, sodium chloride, polyacrylamide, potassium chloride, polyvinyl pyrrolidone, sodium sulfate, calcium chloride, sodium carbonate, and sodium bicarbonate.

15. The method according to any one of claims 1 to 3, further comprising washing the particulate matter with a cleaning agent selected from at least one of acetone, anhydrous copper sulfate, calcium chloride, sodium sulfate, anhydrous ethanol, medical alcohol, hydrofluoroether, sodium alkylbenzene sulfonate, sodium fatty alcohol sulfate, sodium tripolyphosphate, and deionized water.

16. The method according to any one of claims 1 to 3, wherein The ratio of the organic solvent to the nonionic surfactant in the continuous phase liquid is 5:1 to 20:1 by weight.

17. A process system for preparing microcarrier particles, comprising: 1) Emulsion preparation device, including: container; A porous plate comprising a plurality of micropores disposed in the container, wherein the porous plate divides the interior of the container into a first portion and a second portion; a dispersed phase inlet communicating with the first portion for inputting a dispersed phase liquid; a continuous phase inlet communicating with the second portion for inputting a continuous phase liquid; and a container outlet communicating with the second portion, wherein the container outlet and the continuous phase inlet are arranged on opposite sides of the container so that the continuous phase liquid input from the continuous phase inlet can flow through the second portion in a direction parallel to the porous plate and then flow out from the container outlet; 2) a first tank body in communication with the dispersed phase inlet, for containing the dispersed phase liquid, the first tank body also being in communication with a pressurizing device or a gas cylinder so as to allow the dispersed phase liquid to enter the emulsion preparation device under pressure; 3) a second tank body connected to the continuous phase inlet and equipped with a cooling device, for containing the continuous phase liquid and cooling the continuous phase liquid, wherein the inlet of the second tank body is connected to a movable mixing tank for preparing the continuous phase liquid; 4) a third tank body connected to the outlet of the container, wherein the third tank body is equipped with an agitator and a cooling device for performing an emulsion reaction; and 5) a fourth tank body connected to the third tank body, wherein the fourth tank body is equipped with a filtering device for collecting particulate matter formed by the emulsion reaction.

18. The process system according to claim 17, wherein the container is in the shape of a rectangular parallelepiped.

19. The process system according to claim 17 or 18, wherein the number of the continuous phase inlets is two or more, and the number of the container outlets is two or more.

20. The process system of claim 17 or 18, wherein the continuous phase inlet and the vessel outlet are at the same level relative to the vessel bottom.

21. The process system according to claim 17 or 18, wherein the diameter of the micropores is 0.1 μm to 500 μm.

22. The process system according to claim 17 or 18, wherein the diameter of the micropores is 30 μm to 50 μm.

23. The process system according to claim 17 or 18, wherein a gear pump is provided between the continuous phase inlet and the second tank body for inputting the continuous phase liquid from the second tank body into the emulsion preparation device.

24. The process system according to claim 17 or 18, further comprising a fifth tank body communicating with the fourth tank body, wherein the fifth tank body is equipped with a filtering device for washing the particulate matter.

25. The process system according to claim 24, wherein the fourth tank body and the fifth tank body are respectively equipped with a vacuum pumping device.

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