Microcarriers and uses thereof

Abstract

The present application relates to a kind of microcarrier and its application, the microcarrier has three-dimensional structure, the microcarrier has shell and inside is hollow or solid, the shell outer wall is uneven and has multiple concave points.This microcarrier improves the permeability of traditional microcarrier, is conducive to the material exchange between cell and external environment, improves cell survival rate, provides good mechanical protection for cell proliferation and constructs good three-dimensional structure for cell growth, realizes the friendly connection of cell, is conducive to the large-scale expansion of cell in limited space, and maintains the stability of cell physicochemical properties in long-term culture.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological materials, in particular to a microcarrier and application thereof. BACKGROUND

[0002] With the rapid development of life medicine field, two-dimensional cell culture cannot meet the demand of laboratory and enterprise for cell quantity. Compared with three-dimensional cell culture, the disadvantages of two-dimensional cell culture are more and more prominent, mainly in that: (1) two-dimensional cell culture can only use culture dish to culture cells, and the proliferation of adherent cells is limited to the size of dish bottom, and a large amount of manpower and material resources are consumed in long-term culture, and large-scale expansion in a short time cannot be realized, and resource loss is large; (2) two-dimensional cell culture cannot construct a friendly microenvironment for cell growth, and cells proliferate on the plane, and long-term culture will affect the structure of cells, and bring difficulties to subsequent research.

[0003] Therefore, the carrier structure capable of providing three-dimensional culture for cells still needs further research. SUMMARY

[0004] The present application aims to solve one of the above-mentioned problems of two-dimensional cell culture. The present application provides a microcarrier and a preparation method thereof, and the application thereof is described. The microcarrier provided by the present application improves the permeability of the traditional microcarrier, is beneficial to the material exchange between cells and the external environment, improves the cell survival rate, provides good mechanical protection for cell proliferation, and constructs a good three-dimensional structure for cell growth, realizes the friendly connection of cells, is beneficial to the large-scale expansion of cells in limited space, and maintains the stability of the physicochemical properties of cells in long-term culture; the microcarrier of the present application has more flexible structure characteristics, and can meet various application scenarios such as biology, medicine and clinical treatment.

[0005] Therefore, in the first aspect of the present application, the present application provides a microcarrier, wherein the microcarrier has a three-dimensional structure, the microcarrier has a shell layer and is hollow or solid inside, and the outer wall of the shell layer is uneven and has multiple concave points.

[0006] In some embodiments, the ratio of the concave area of the outer wall of the shell layer to the total surface area of the outer wall of the shell layer is 15% or more, for example 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more, or for example 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, or 90-95%, and specifically for example 15%, 20%, 50%, 60%, 80%, 85%, or 90%.

[0007] In some embodiments, the outer wall of the shell layer has a number of concave points in the range of 2-800, optionally in the range of 2-700, optionally in the range of 2-600, optionally in the range of 2-500, optionally in the range of 2-400, optionally in the range of 2-300, optionally in the range of 2-200, optionally in the range of 5-200, or optionally in the range of 5-100.

[0008] In some embodiments, the outer wall of the shell layer has a number of concave points in the range of 5-60, for example 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, or 55-60. In some specific embodiments, the outer wall of the shell layer has a number of concave points in the range of 10-60 (and specifically for example 10, 20, 25, 30, 40, 50, or 60).

[0009] In some embodiments, the shape of the concave points of the outer wall of the shell layer is selected from the group consisting of circular arc-shaped concave points (or arc-shaped concave points), thin slot-shaped concave points (or elongated concave points), spherical concave points, polygonal concave points, irregular concave points, or any combination thereof.

[0010] In some embodiments, the average radius of the circular arc-shaped concave points is in the range of 1-500 μm, optionally in the range of 1-400 μm, optionally in the range of 1-300 μm, optionally in the range of 10-300 μm, optionally in the range of 50-300 μm, optionally in the range of 80-300 μm, or optionally in the range of 80-200 μm.

[0011] In some embodiments, the average radius of the circular arc-shaped recesses is 1-100 μιη, for example, 1-10 μιη, 10-20 μιη, 20-30 μιη, 30-40 μιη, 40-50 μιη, 50-60 μιη, 60-70 μιη, 70-80 μιη, 80-90 μιη, or 90-100 μιη.

[0012] In some embodiments, the average width of the recessed surface of the fine groove-shaped recesses is 1-500 μιη, optionally 1-400 μιη, optionally 1-300 μιη, optionally 10-300 μιη, optionally 50-300 μιη, optionally 80-300 μιη, or optionally 80-200 μιη.

[0013] In some embodiments, the average width of the fine groove-shaped recesses is 1-100 μιη, for example, 1-10 μιη, 10-20 μιη, 20-30 μιη, 30-40 μιη, 40-50 μιη, 50-60 μιη, 60-70 μιη, 70-80 μιη, 80-90 μιη, or 90-100 μιη.

[0014] In some embodiments, the average width of the recessed surface of the polygonal-shaped recesses is 1-500 μιη, optionally 1-400 μιη, optionally 1-300 μιη, optionally 10-300 μιη, optionally 50-300 μιη, optionally 80-300 μιη, or optionally 80-200 μιη.

[0015] In some embodiments, the average width of the polygonal-shaped recesses is 1-100 μιη, for example, 1-10 μιη, 10-20 μιη, 20-30 μιη, 30-40 μιη, 40-50 μιη, 50-60 μιη, 60-70 μιη, 70-80 μιη, 80-90 μιη, or 90-100 μιη.

[0016] In some embodiments, the average width of the recessed surface of the irregular-shaped recesses is 1-500 μιη, optionally 1-400 μιη, optionally 1-300 μιη, optionally 10-300 μιη, optionally 50-300 μιη, optionally 80-300 μιη, or optionally 80-200 μιη.

[0017] In some embodiments, the irregularly shaped recesses have an average width of the recessed surface of 1-100 μm, for example, 1-10 μm, 10-20 μm, 20-30 μm, 30-40 μm, 40-50 μm, 50-60 μm, 60-70 μm, 70-80 μm, 80-90 μm, or 90-100 μm.

[0018] In some embodiments, the average radius of the spherical recesses is less than 500 μm, optionally less than 400 μm, optionally less than 300 μm, optionally less than 200 μm, optionally less than 100 μm, optionally less than 50 μm, optionally less than 20 μm, and optionally less than 10 μm.

[0019] In some embodiments, the average radius of the spherical recesses is less than 100 μm, for example, less than 90 μm, less than 80 μm, less than 70 μm, less than 60 μm, less than 50 μm, less than 40 μm, less than 30 μm, less than 20 μm, or less than 10 μm.

[0020] It is noted that the average width refers to the average length of the shortest side, for example, the average width of the slot-shaped recesses refers to the average length of the shortest side of the slot-shaped recesses.

[0021] In some embodiments, the average particle size of the microcarriers is 1-3000 μm, optionally 1-2000 μm, optionally 1-1000 μm, optionally 1-800 μm, optionally 1-600 μm, optionally 10-600 μm, optionally 50-500 μm, optionally 100-500 μm, and optionally 100-400 μm.

[0022] In some embodiments, the average particle size of the microcarriers is 10-500 μm, for example, 10-50 μm, 50-100 μm, 100-150 μm, 150-200 μm, 200-250 μm, 250-300 μm, 300-350 μm, 350-400 μm, 400-450 μm, or 450-500 μm.

[0023] In some embodiments, the average particle size of the microcarriers is 30-400 μm.

[0024] In some embodiments, the thickness of the shell layer is 1-100 μm, optionally 1-90 μm, optionally 1-80 μm, optionally 1-70 μm, optionally 1-60 μm, and optionally 1-50 μm.

[0025] In some embodiments, the shell layer has a thickness of 1-100 μm, such as 1-10 μm, 10-20 μm, 20-30 μm, 30-40 μm, 40-50 μm, 50-60 μm, 60-70 μm, 70-80 μm, 80-90 μm, or 90-100 μm.

[0026] In some embodiments, the microcarrier has a three-dimensional structure of a round sphere, a shuttle shape, an oval sphere, a rod shape, a flat sphere, or an irregular sphere. In some specific embodiments, the microcarrier has a three-dimensional structure of a round sphere.

[0027] In some embodiments, the microcarrier has a hollow structure inside, and the microcarrier has an open three-dimensional structure. In some embodiments, the shell layer of the microcarrier has one or more through holes penetrating the shell layer to communicate the inside and the outside of the shell layer.

[0028] In some embodiments, the hollow structure inside the microcarrier is obtained by dissolving a space-occupying material.

[0029] In some embodiments, the outer wall and / or the inner wall of the shell layer is surface-modified.

[0030] In some embodiments, the outer wall and / or the inner wall of the shell layer is RGD surface-modified.

[0031] In some embodiments, the microcarrier is loaded with cells.

[0032] In some embodiments, the cells are disposed on the inner wall of the shell layer of the microcarrier, or on the outer wall of the shell layer of the microcarrier, or on both the inner wall of the shell layer of the microcarrier and the outer wall of the shell layer of the microcarrier.

[0033] In some embodiments, when the cells are disposed on both the inner wall of the shell layer of the microcarrier and the outer wall of the shell layer of the microcarrier, the types of the cells disposed on the inner wall and the outer wall of the shell layer can be the same or different.

[0034] In some embodiments, when the outer wall of the shell layer of the microcarrier is provided with cells, the cells are disposed at the concave points of the outer wall of the shell layer, or at the smooth surface of the outer wall of the shell layer (i.e. non-concave points), or at both the concave points and the smooth surface of the outer wall of the shell layer.

[0035] In some embodiments, when the outer wall of the shell layer of the microcarrier is provided with cells, the cells are disposed at the concave points of the outer wall of the shell layer.

[0036] In some embodiments, the cells are disposed within the shell inner wall of the microcarrier via the through-hole.

[0037] In some embodiments, the cells are disposed within the shell inner wall of the microcarrier via cell injection.

[0038] In some embodiments, the cells are disposed outside the shell wall of the microcarrier via co-culturing with the microcarrier.

[0039] In some embodiments, the cells are selected from a prokaryotic cell, a eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a unicellular eukaryotic organism, a protozoan cell, a plant cell, an animal cell, an algal cell, a fungal cell, an artificial cell, or any combination thereof.

[0040] In some embodiments, the cells are selected from a stem cell, a somatic cell, a germ cell, or any combination thereof.

[0041] In some embodiments, the stem cell is selected from an embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell, a mesenchymal stem cell, a neural stem cell, a muscle stem cell, a hematopoietic stem cell, an epithelial stem cell, a mammary stem cell, an intestinal stem cell, a mesodermal stem cell, an endothelial stem cell, or any combination thereof.

[0042] In some embodiments, the somatic cell is selected from an epithelial cell, an endothelial cell, a mesothelial cell, a fibroblast cell, an osteoblast cell, a chondrocyte cell, a muscle cell, an osteocyte cell, a hepatocyte cell, a pancreatic cell, an exogenous cell, an endogenous cell, a cardiomyocyte cell, a skeletal cell, a cardiac myoblast cell, a skeletal myoblast cell, an oligodendrocyte cell, a glial cell, a hematopoietic cell, a neuron, or any combination thereof.

[0043] In some embodiments, the germ cell is selected from an oocyte, a sperm, an oogonium, a spermatogonium, or any combination thereof.

[0044] In some embodiments, the shell or the microcarrier is made of a biomaterial. That is, if the microcarrier is hollow inside, the shell is made of a biomaterial; if the microcarrier is solid inside, the microcarrier is made of a biomaterial.

[0045] In some embodiments, the biomaterial is selected from an artificially synthesized biomaterial, a natural biomaterial, or a combination thereof.

[0046] In some embodiments, the synthetic biomaterial is selected from polyethylene glycol, polyethylene glycol derivatives, polylactic acid, polylactic-glycolic acid copolymer, polyanhydrides, polyesters, polyamino acids, polyethylene oxide, polyesters, polymethyl methacrylate, polycarbonates, polyurethanes, polycaprolactone, polyhydroxyalkanoates, polysiloxanes, polyethylene, polyvinyl chloride, polytetrafluoroethylene, polystyrene, polypropylene, maleic anhydride grafted copolymers, polyacrylamide, polyacetals, polypyrrole, or any combination thereof.

[0047] In some embodiments, the natural biomaterial is selected from natural proteins, collagen and collagen derivatives, gelatin and gelatin derivatives, agar and agar derivatives, proteoglycans, alginate and alginate derivatives thereof, Matrigel, propolis, cellulose and cellulose derivatives, chitin and chitin derivatives, silk fibroin and derivatives thereof, laminin and derivatives thereof, fibronectin and derivatives thereof, sodium hyaluronate and hyaluronic acid derivatives, agarose and derivatives thereof, dextran and derivatives thereof, sucrose and sucrose derivatives, starch, chitosan and chitosan derivatives, or any combination thereof.

[0048] In some embodiments, the biomaterial is selected from sodium hyaluronate, gelatin.

[0049] In a second aspect of the present application, the present application provides a microcarrier aggregate, wherein the aggregate is formed by aggregation of the aforementioned microcarrier particles.

[0050] In some embodiments, the aggregate is a collection of microcarriers of a single trait, or a collection of microcarriers of various traits in any proportion.

[0051] In a third aspect of the present application, the present application provides a method for preparing the aforementioned microcarriers, comprising: Figure 5 (a) providing an organic phase solution;

[0052] (b) providing an aqueous phase solution;

[0053] (c) adding the aqueous phase solution to the organic phase solution to form a mixture; emulsifying the mixture to form a uniform water-in-oil system;

[0054] (d) filtering the water-in-oil system, and the filter cake is a carrier matrix to be processed;

[0055] (e) post-treating the carrier matrix to be processed to dissolve the occupying material, and conjugating a short peptide or protein comprising an RGD amino acid sequence to the post-treated carrier matrix to obtain the microcarriers;

[0056] (f) collecting the microcarriers.

[0057] Optionally, the conjugation further comprises the steps of washing, sieving and lyophilizing after the conjugation.

[0058] In some embodiments, the emulsification is performed at a temperature of 4-200 °C, such as 20-75 °C, or such as 20-25 °C.

[0059] In some embodiments, the emulsification is performed for a time of 4-72 h, preferably 5-30 h, such as 5-10 h, 10-15 h, 15-20 h, 20-25 h or 25-30 h, in particular such as 10 h.

[0060] In some embodiments, the emulsification is achieved by a stirring method, an ultrasonic method, a shaking method or a microfluidic method. In some embodiments, the stirring speed during emulsification is 100-5000 rpm / min, preferably 300-1000 rpm / min, such as 300-400 rpm / min, 400-500 rpm / min, 500-600 rpm / min, 600-700 rpm / min, 700-800 rpm / min, 800-900 rpm / min or 900-1000 rpm / min, in particular such as 600 rpm / min.

[0061] In some embodiments, the organic phase solution is obtained by mixing an organic solvent with a non-ionic surfactant. In some embodiments, the mixing is performed at a temperature of 4-200 °C, preferably 20-75 °C (e.g. 20-25 °C, 25-30 °C, 30-35 °C, 35-40 °C, 40-45 °C, 45-50 °C, 50-55 °C, 55-60 °C, 60-65 °C, or 65-70 °C, specifically 60 °C). In some embodiments, the mixing is performed for 10-120 min, preferably 10-60 min. In some embodiments, the mixing is performed with the aid of a stirring paddle. In some embodiments, the stirring paddle is operated at a speed of 100-2000 rpm / min, preferably 300-1000 rpm / min, e.g. 300-700 rpm / min. In some embodiments, the organic solvent is selected from the group consisting of liquid paraffin, petroleum ether, carbon tetrachloride, dimethyl sulfoxide, trichloromethane, dichloromethane, edible oil, silicone oil, soybean oil, mineral oil, or any combination thereof. In some embodiments, the organic solvent is liquid paraffin. In some embodiments, the non-ionic surfactant is selected from the group consisting of Tween, Span 80, fatty acid glyceride, sodium dodecyl benzene sulfonate, PO-500, hydrofluoroether, polyethylene glycol, block polyoxyethylene-polyoxypropylene ether (PO-EO copolymer), polyol ester, or any combination thereof. In some embodiments, the non-ionic surfactant is Span 80. In some embodiments, the volume ratio of the organic solvent to the non-ionic surfactant is 200-400: 1, e.g. 200: 1, 210: 1, 220: 1, 230: 1, 240: 1, 250: 1, 260: 1, 270: 1, 280: 1, 290: 1, 300: 1, 310: 1, 320: 1, 330: 1, 340: 1, 350: 1, 360: 1, 370: 1, 380: 1, 390: 1, or 400: 1, specifically 300: 1.

[0062] In some embodiments, the aqueous phase solution is obtained by dissolving biomaterials in water to obtain a solution, and suspending the space-occupying material and the solidifying agent in the solution to form a uniformly dispersed suspension, which is the aqueous phase solution. In some embodiments, the dissolving is performed at a temperature of 4-200 °C, preferably 10-80 °C. In some embodiments, the uniformly dispersed suspension is achieved by stirring, shaking, ultrasonication, or shaking, preferably at a speed of 100-2000 rpm / min, preferably 300-700 rpm / min.

[0063] In some embodiments, the mass fraction of the biomaterial in the aqueous solution is 3-10%, for example 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, preferably 5-10%, in particular 5% or 10%.

[0064] In some embodiments, the mass fraction of the space-occupying material in the aqueous solution is 0.01-1%, for example 0.01%, 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, or 1%, preferably 0.05-1%, in particular 0.05%, 0.1%, 0.5%, or 1%.

[0065] In some embodiments, the mass fraction of the solidifying agent in the aqueous solution is 0.5-7%, for example 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, or 7%, preferably 0.5-5%, in particular 0.5% or 5%.

[0066] In some embodiments, the biomaterial is selected from the group consisting of an artificially synthesized biomaterial, a natural biomaterial, or a combination thereof.

[0067] In some embodiments, the artificially synthesized biomaterial is selected from the group consisting of polyethylene glycol, polyethylene glycol derivative, polylactic acid, polylactic-glycolic acid copolymer, polyanhydride, polyacid ester, polyamino acid, polyethylene oxide, polyester, polymethyl methacrylate, polycarbonate, polyurethane, polycaprolactone, polyhydroxyalkanoate, polysiloxane, polyethylene, polyvinyl chloride, polytetrafluoroethylene, polystyrene, polypropylene, maleic anhydride graft copolymer, polyacrylamide, polyacetal, polypyrrole, or any combination thereof.

[0068] In some embodiments, the natural biomaterial is selected from the group consisting of a natural protein, collagen and collagen derivative, gelatin and gelatin derivative, agar and agar derivative, proteoglycan, alginate and alginate derivative thereof, matrigel, propolis, cellulose and cellulose derivative, chitin and chitin derivative, silk fibroin and derivative thereof, laminin and derivative thereof, fibronectin and derivative thereof, sodium hyaluronate and hyaluronic acid derivative, agarose and derivative thereof, dextran and derivative thereof, sucrose and sucrose derivative, starch, chitosan and chitosan derivative, or any combination thereof.

[0069] In some embodiments, the biomaterial is selected from the group consisting of sodium hyaluronate, gelatin.

[0070] In some embodiments, the space occupying material is selected from the group consisting of polyethylene glycol and polyethylene glycol derivatives, paraffin spheres, oxidized alginate and derivatives thereof, polycaprolactone, silicon dioxide, beeswax, propolis, agar, agarose, alginate and derivatives thereof, soy lecithin, egg lecithin, phospholipids, dextran, chitosan, starch, gelatin, sodium hyaluronate and hyaluronic acid derivatives or any combination thereof.

[0071] In some embodiments, the space occupying material is selected from the group consisting of agarose, dextran, sodium hyaluronate.

[0072] In some embodiments, the solidification agent is selected from the group consisting of N,N-methylenebisacrylamide, l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, N-hydroxysulfosuccinimide, diisocyanate, glutaraldehyde, Kyonini, ammonium sulfate, calcium ions, butanediol diglycidyl ether, transglutaminase, divinylbenzene, adipic acid dihydrazide or any combination thereof.

[0073] In some embodiments, the solidification agent is selected from the group consisting of l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, glutaraldehyde.

[0074] In some embodiments, the filtration is achieved by a filtration device having a mesh diameter of 30-1000 microns.

[0075] In some embodiments, the filtration device has a four-layer structure, the first layer has a mesh diameter of 500-800 microns, the second layer has a mesh diameter of 300-400 microns, the third layer has a mesh diameter of 200-300 microns, and the fourth layer has a mesh diameter of 100-200 microns.

[0076] In some embodiments, the step of dissolving the space occupying material is achieved by mixing the carrier matrix to be treated with an organic solvent to dissolve the space occupying material. In some embodiments, the mixing is carried out at a temperature of 15-150°C, preferably 25-100°C (such as 25-30°C, 30-35°C, 35-40°C, 40-45°C or 45-50°C, in particular 25°C) for 4-48h, preferably 6-24h (such as 6-8h, 8-10h, 10-12h, 12-14h, 14-16h, 16-18h or 18-20h, in particular 6h). In some embodiments, the organic solvent is selected from the group consisting of acetone, absolute ethanol, petroleum ether, trichloromethane, dimethyl sulfoxide, dichloromethane, petroleum ether, carbon tetrachloride, acetonitrile, toluene, methanol or any combination thereof. In some embodiments, the organic solvent is absolute ethanol.

[0077] In some embodiments, the conjugation of the short peptide or protein comprising RGD amino acid sequence with the post-processed carrier matrix is achieved by mixing the post-processed carrier matrix with the short peptide or protein comprising RGD amino acid sequence so as to achieve RGD surface modification of the post-processed carrier matrix. In some embodiments, the mixing is performed at a temperature of 15-25 °C, preferably 20 °C. In some embodiments, the mixing is performed for a time period of 4-10 hours, preferably 6 hours.

[0078] In some embodiments, the short peptide or protein comprising RGD amino acid sequence is selected from collagen or collagen derivative, gelatin or gelatin derivative, fibronectin, fibroin, laminin, matrigel or any combination thereof.

[0079] In some embodiments, the short peptide or protein comprising RGD amino acid sequence is selected from collagen, gelatin.

[0080] In some embodiments, the short peptide or protein comprising RGD amino acid sequence is provided in a solution. In some embodiments, the solvent of the solution is a solvent routinely used by a person skilled in the art for RGD modification, for example, in some embodiments, the solvent is water (e.g. deionized water). In addition, in some embodiments, the mass fraction of the short peptide or protein comprising RGD amino acid sequence in the solution is a mass fraction routinely used by a person skilled in the art for RGD modification, for example, in some embodiments, the mass fraction of the short peptide or protein comprising RGD amino acid sequence in the solution is 0.1%-20%, for example 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5% or 20%.

[0081] In a fourth aspect of the present application, the present application provides a kit comprising the aforementioned microcarriers or the aforementioned microcarrier aggregates or the aforementioned microcarriers prepared by the aforementioned method.

[0082] In a fifth aspect of the present application, the present application provides the use of the aforementioned microcarriers or the aforementioned microcarrier aggregates or the aforementioned microcarriers prepared by the aforementioned method in the preparation of a kit for cell three-dimensional culture and / or expansion, 3D bioprinting, biomimetic structure construction, cell therapy, drug loading or medical injection.

[0083] In some embodiments, the biomimetic construct is a three-dimensional construct, a tissue precursor, a tissue, an organ or an embryo (e.g. a mouse embryo).

[0084] In some embodiments, the biomimetic construct is constructed as an embryonic biomimetic simulation construct or a cartilage biomimetic construct.

[0085] In some embodiments, the biomimetic construct is a cartilage lacuna structure of articular cartilage.

[0086] In some embodiments, the cell therapy is a skin surface treatment, a clinical corneal treatment, a bone treatment or a cartilage repair and bone injury treatment.

[0087] In a sixth aspect of the present application, the present application provides the use of the aforementioned microcarriers or the aforementioned microcarrier aggregates or the aforementioned microcarriers prepared by the aforementioned method for in vitro three-dimensional cell culture and / or expansion, in vitro 3D bioprinting, in vitro biomimetic construct construction, in vitro drug loading; preferably, the use is for non-diagnostic or therapeutic purposes.

[0088] In some embodiments, the biomimetic construct is a three-dimensional construct, a tissue precursor, a tissue, an organ or an embryo (e.g. a mouse embryo).

[0089] In some embodiments, the biomimetic construct is constructed as an embryonic biomimetic simulation construct or a cartilage biomimetic construct.

[0090] In some embodiments, the biomimetic construct is a cartilage lacuna structure of articular cartilage.

[0091] In a seventh aspect of the present application, the present application provides a method for in vitro three-dimensional cell culture and / or expansion, comprising:

[0092] 1) inoculating cells on the aforementioned microcarriers or the aforementioned microcarrier aggregates or the aforementioned microcarriers prepared by the aforementioned method for culture (e.g. in a 37°C carbon dioxide incubator);

[0093] Optionally, after step 1), further comprising:

[0094] 2-A) subjecting the mixture obtained in step 1) to in situ freezing (e.g. -80°C freezing);

[0095] Or optionally, after step 1), further comprising:

[0096] 2-B) subjecting the cells loaded on the microcarriers obtained in step 1) to digestion and collection.

[0097] In some embodiments, the culture is a dynamic culture.

[0098] In some embodiments, the dynamic culture is a stirred dynamic culture or a suspension dynamic culture.

[0099] In some embodiments, the cells are as described in the first aspect.

[0100] Advantages of the invention

[0101] 1、The microcarrier of the present application can well make up for the shortcomings of two-dimensional cell culture, and has a large number of advantages: (1) The cells are no longer dependent on the plane, but grow in three-dimensional space, and a large number of cells can be harvested in a short period of time; (2) The microcarrier and the recessed structure provide a large amount of adhesion area for the cells, so that the cells have better connectivity and build a more friendly growth microenvironment for the cells, which further simulates the in-vivo cell survival environment and is beneficial to maintaining the physicochemical properties of the cells; (3) The cavity structure of the three-dimensional cell culture microcarrier has more flexible structural characteristics, which can provide a friendly survival environment for the cells while providing more unique structural support, compared with the traditional microcarrier, the existence of the cavity structure improves the permeability of the microcarrier, accelerates the exchange and transportation of nutrients, greatly improves the cell survival rate and expansion rate, and is beneficial to the rapid expansion of cells in the limited space of the laboratory; The more flexible structural characteristics of the cavity structure of the three-dimensional cell culture microcarrier can meet the application scenarios of biology, medicine, clinic and the like.

[0102] 2、The microcarrier of the present application has good biocompatibility, can realize good adhesion of cells, and realize growth and proliferation of cells.

[0103] 3、The microcarrier of the present application can realize the penetration of the culture medium in a short time, which is beneficial to the material exchange between the cells and the outside world, and after the cells grow on the surface of the microcarrier, new microcarriers are put in, and when the balls contact each other, the cells can realize cross-ball growth. The microcarrier is used for long-term culture of cells, and the cells still maintain a high survival rate and proliferation rate after being cultured for a long time (for example, 7 days).

[0104] 4、The microcarrier of the present application is used for culture and expansion of stem cells, and the stem cells have good adhesion, and long-term culture of the stem cells maintains a high survival rate and proliferation rate.

[0105] 5、The microcarrier of the present application is used for culture and expansion of rat chondrocytes, which can realize large-scale expansion and culture, can simulate in-vivo culture environment, and maintain the biological function of the chondrocytes.

[0106] 6、The microcarrier of the present application has a large recessed structure on the surface, which is used for inoculating rat chondrocytes on the microcarrier to promote the aggregation, growth and proliferation of the chondrocytes, thereby simulating the cartilage pit structure of the joint cartilage in-vivo, and the microcarrier can be modified by polypeptides and factors to promote the secretion function of the chondrocytes.

[0107] 7. The microcarrier of the present application is used for encapsulation and culture of stem cells. The stem cells encapsulated in the microcarrier have good biological activity. After the addition of inducing factors, the stem cells can be directionally differentiated in the microcarrier. After a long time of culture (for example, 7 days), the cells still have a high survival rate and proliferation.

[0108] 8. The microcarrier of the present application is used for simulation of mouse embryonic structure. Mouse embryonic stem cells are encapsulated in the microcarrier. The microcarrier has the potential to induce differentiation into three germ layers in vitro. The surface of the microcarrier is viscous to mouse trophoblast stem cells, so that the mouse trophoblast stem cells can interact with the mouse embryonic stem cells encapsulated in the microcarrier, and the microcarrier has the potential to implant mouse embryos.

[0109] 9. For those skilled in the art, conventional cell cryopreservation refers to cryopreservation of cells together with a cryopreservation solution. The cryopreservation solution contains 10% CPA (antifreeze, which is toxic to cells). The microcarrier of the present application can be directly cryopreserved (i.e., together with the cells) at -80°C after cell culture. The concentration of CPA in the cryopreservation solution can be reduced by 50% from 10% to 5%. After thawing, the survival rate of the cells can be more than 70%.

[0110] 10. Some microcarriers in the prior art cannot completely separate the cells by digestion after the cells proliferate and grow. Some microcarriers change the functionality of the cells after the cells proliferate and grow and are then digested. For example, stem cells no longer have stemness. The microcarrier of the present application can be used for large-scale expansion of cells. After the expansion, the cells can be directly digested and collected. The microcarrier is used for cell expansion. After 7 days of culture, the cells are expanded by 12 times. More importantly, the functionality of the cells (for example, the stemness of the cells, i.e., the differentiation ability) remains stable.

[0111] 11. The microcarrier of the present application can be used for large-scale dynamic culture of cells, including stirring dynamic culture and suspension dynamic culture. During the dynamic culture, the microcarrier can tolerate a large shear force, can protect the cells from shear damage, and has good permeability. After the cells adhere to the microcarrier, the cells can exchange substances in time. The cells expanded by the method have a good state, have a high survival rate and proliferation rate, and more importantly, the functionality of the cells remains stable. BRIEF DESCRIPTION OF DRAWINGS

[0112] Figure 1 SEM image of the microcarrier structure of the embodiment of the present application;

[0113] Figure 2 Microscopic morphology image of the microcarrier of the embodiment of the present application;

[0114] Figure 3 These are microscopic images of the microcarriers according to embodiments of the present invention.

[0115] Figure 4 Image A shows the microscopic morphology of a mouse cartilage section, and image B shows the cell adhesion of chondrocytes after one day of co-culture with microcarriers.

[0116] Figure 5 This is a schematic diagram of the microcarrier preparation process of the present invention;

[0117] Figure 6 These are microscopic images of the microcarriers according to embodiments of the present invention.

[0118] Figure 7 These are microscopic images of the microcarriers according to embodiments of the present invention.

[0119] Figure 8 These are microscopic images of the microcarriers according to embodiments of the present invention.

[0120] Figure 9 These are microscopic images of the microcarriers according to embodiments of the present invention.

[0121] Figure 10 These are microscopic images of the microcarriers according to embodiments of the present invention.

[0122] Figure 11 These are microscopic images of the microcarriers according to embodiments of the present invention.

[0123] Figure 12 These are microscopic images of the microcarriers according to embodiments of the present invention.

[0124] Figure 13 This is a diagram showing the cell viability and mortality staining results after cryopreservation of the microcarriers according to an embodiment of the present invention.

[0125] Figure 14 This is a diagram showing the results of dynamic cell culture using microcarriers according to an embodiment of the present invention;

[0126] Figure 15 This is a diagram showing the dynamic cell proliferation curve of the microcarrier in an embodiment of the present invention.

[0127] Figure 16 This is a characterization diagram of cell stemness after dynamic cell culture using microcarriers, according to an embodiment of the present invention.

[0128] Figure 17 These are microscopic images of the microcarriers according to embodiments of the present invention.

[0129] Figure 18 This is a microcarrier permeability characterization diagram from an embodiment of the present invention. Detailed Implementation

[0130] Embodiments of the present application will be described in detail below with reference to the attached drawings and examples, but those skilled in the art will understand that the following drawings and examples are only intended to illustrate the present application, and not to limit the scope of the present application. According to the following detailed description of the drawings and preferred embodiments, various objects and advantages of the present application will become apparent to those skilled in the art.

[0131] The present application will now be described with reference to the following examples, which are intended to illustrate the present application (but not to limit the present application). Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents used in the examples of the present application are commercially available.

[0132] It should be noted that in the following examples, the shape and structure of the microcarriers do not change before and after the microcarriers are hydrated and swollen.

[0133] Example 1: Preparation of microcarriers

[0134] The mixed organic phase liquid paraffin and span 80 were placed in a reaction device with a stirrer, the volume ratio of liquid paraffin to span 80 was 300:1, heated to above 60°C, and stirred to mix them evenly. The aqueous phase was formed by mixing HA (sodium hyaluronate) (5wt%), water-insoluble space-occupying material, i.e. agarose with uniform particle size (1wt%), and crosslinking agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (5wt%). After the organic phase was mixed evenly, it was transferred to room temperature, and the mixed aqueous phase was slowly added to the organic phase under the action of the stirring paddle. The mixture was emulsified at room temperature. After 10 hours of reaction at room temperature, the mixture was filtered, the stirring speed was controlled at 600 rpm / min, and after the microcarriers were formed, the organic phase was washed away. The microcarriers were filtered, and the filtered carrier material was transferred to a reagent bottle containing anhydrous ethanol, the organic solvent was completely immersed in the carrier, and the carrier was shaken in a shaking bed at 25°C for 6 hours, so that the space-occupying material inside the carrier was fully dissolved and completely dissolved. Then the material was filtered and washed with deionized water to further wash away the excess organic solvent. Finally, the material was placed in a solution of amino acids containing RGD sites (collagen solution) and stirred at 20°C for 6 hours. After that, the material was washed, sieved, and freeze-dried to obtain the microcarriers.

[0135] The obtained microcarriers were observed by scanning electron microscopy, and the diameter of the microcarriers was between 30-400 microns, and after freeze-drying, the microcarriers were spherical or granular. In addition, after the microcarriers were hydrated overnight, the microcarriers swelled, and further observation showed that they were regular spherical with concave surface, as shown in Figure 1 , the proportion of concave was about 85%, and the number of concave was about 40.

[0136] Example 2: Preparation of microcarriers

[0137] The mixed organic phase liquid paraffin and Span 80 were placed in a reaction device with a stirrer, the volume ratio of liquid paraffin to Span 80 was 300:1, heated to above 60°C, and stirred to mix evenly. The aqueous phase was formed by mixing HA (sodium hyaluronate) (5wt%), water-insoluble space-occupying material, i.e. agarose with uniform particle size (0.5wt%), and crosslinking agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (5wt%). After the organic phase was mixed evenly, it was transferred to room temperature, and the mixed aqueous phase was slowly added to the organic phase under the action of a stirring paddle. The mixture was emulsified at room temperature. After 10 hours of reaction at room temperature, the mixture was filtered, and the stirring speed was controlled at 600 rpm / min. After the microcarriers were formed into spheres, the organic phase was washed away. The microcarriers were filtered, and the filtered carrier material was transferred to a reagent bottle containing anhydrous ethanol. The organic solvent completely immersed the carrier, and the carrier was shaken in a shaking bed at 25°C for 6 hours, so that the space-occupying material inside the carrier was fully dissolved and completely eluted. Then the material was filtered and washed with deionized water to further wash away the excess organic solvent. Finally, the material was placed in an amino acid solution containing RGD sites (collagen solution) and stirred at 20°C for 6 hours. After washing, screening, and freeze-drying, the microcarriers were obtained.

[0138] The obtained microcarriers were observed by scanning electron microscopy. The microcarriers had a diameter of 30-400 microns and were spherical or granular after freeze-drying. After the microcarriers were hydrated overnight, the microcarriers swelled. Further observation showed that they were regular spherical with concave surfaces, as shown in Figure 2 The proportion of concave surfaces was about 50%, and the number of concave surfaces was about 30.

[0139] Example 3: Preparation of microcarriers

[0140] The mixed organic phase liquid paraffin and Span 80 were placed in a reaction device with a stirrer, the volume ratio of liquid paraffin to Span 80 was 300:1, heated to above 60°C, and stirred to mix evenly. The aqueous phase was formed by mixing HA (sodium hyaluronate) (5wt%), water-insoluble space-occupying material, i.e. agarose with uniform particle size (0.1wt%), and crosslinking agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (5wt%). After the organic phase was mixed evenly, it was transferred to room temperature, and the mixed aqueous phase was slowly added to the organic phase under the action of a stirring paddle. The mixture was emulsified at room temperature. After 10 hours of reaction at room temperature, the mixture was filtered, and the stirring speed was controlled at 600 rpm / min. After the microcarriers were formed into spheres, the organic phase was washed away. The microcarriers were filtered, and the filtered carrier material was transferred to a reagent bottle containing anhydrous ethanol. The organic solvent completely immersed the carrier, and the carrier was shaken in a shaking bed at 25°C for 6 hours, so that the space-occupying material inside the carrier was fully dissolved and completely eluted. Then the material was filtered and washed with deionized water to further wash away the excess organic solvent. Finally, the material was placed in an amino acid solution containing RGD sites (collagen solution) and stirred at 20°C for 6 hours. After washing, screening, and freeze-drying, the microcarriers were obtained.

[0141] The obtained microcarriers were observed by scanning electron microscopy. The microcarriers had a diameter of 30-400 microns and were spherical or granular after freeze-drying. After the microcarriers were hydrated overnight, the microcarriers swelled. Further observation showed that they were regular spherical with concave depressions, as shown in Figure 3 The proportion of concave depressions was about 20%, and the number of concave depressions was about 10.

[0142] Example 4: Preparation of microcarriers

[0143] The mixed organic phase liquid paraffin and Span 80 were placed in a reaction device with a stirrer, the volume ratio of liquid paraffin to Span 80 was 300:1, heated to above 60°C, and stirred to mix evenly. The aqueous phase was composed of HA (sodium hyaluronate) (5wt%), water-insoluble space-occupying material, i.e. agarose with uniform particle size (0.5wt%), and crosslinking agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (5wt%). After the organic phase was mixed evenly, it was transferred to room temperature, and the mixed aqueous phase was slowly added to the organic phase under the action of a stirring paddle. The mixture was emulsified at room temperature. After 10 hours of reaction, the mixture was filtered, and the stirring speed was controlled at 600 rpm / min. After the microcarriers were formed into spheres, the organic phase was washed away. The microcarriers were filtered, and the filtered carrier material was transferred to a reagent bottle containing anhydrous ethanol. The organic solvent completely immersed the carriers, and the carriers were shaken in a shaking bed at 25°C for 6 hours, so that the space-occupying material inside the carriers was fully dissolved and completely eluted. Then the material was filtered and washed with deionized water to further wash away the excess organic solvent. Finally, the material was placed in an amino acid solution containing RGD sites (collagen solution) and stirred at 20°C for 6 hours. After washing, sieving, and freeze-drying, the microcarriers were obtained.

[0144] The obtained microcarriers were observed by scanning electron microscopy, and the diameter of the microcarriers was between 30-400 microns. After freeze-drying, the microcarriers were spherical or granular, as shown in Figure 6 After ultraviolet light sterilization, the microcarriers were hydrated in a basic medium. After hydration overnight, the microcarriers swelled. Further observation showed that the microcarriers were regular spheres with large-radius arc-shaped depressions on the surface, and the average radius of the arc-shaped depressions was about 30-40 μm.

[0145] Example 5: Preparation of microcarriers

[0146] The mixed organic phase liquid paraffin and Span 80 were put into a reaction device with a stirrer, the volume ratio of liquid paraffin to Span 80 was 300:1, heated to above 60℃, stirred to mix them evenly, and the water phase was formed by mixing gelatin (10wt%), water-insoluble space-occupying material (1wt%) with uniform particle size agarose, and crosslinking agent glutaraldehyde (0.5wt%); after the organic phase was mixed evenly, the mixed water phase was slowly added to the organic phase under the action of the stirring paddle at room temperature, and emulsified under stirring at room temperature. After 10 hours of reaction at room temperature, the microcarriers were filtered, and the stirring speed was controlled at 600 rpm / min. After the microcarriers were formed into balls, the organic phase was washed away. The filtered microcarriers were transferred to a reagent bottle containing anhydrous ethanol, the organic solvent completely immersed the carriers, and the carriers were shaken in a shaking bed at 25℃ for 6 hours, so that the space-occupying material in the carriers was fully dissolved and completely dissolved out. Then the material was filtered and washed with deionized water to further wash away the excess organic solvent. Finally, the material was put into a solution containing RGD site amino acids (collagen solution) and stirred at 20℃ for 6 hours. After that, the material was washed, sieved and freeze-dried to obtain the microcarriers.

[0147] The obtained microcarriers were observed by scanning electron microscope, and the diameter of the microcarriers was between 30-400 microns, and after freeze-drying, the microcarriers were spherical or granular, as shown in Figure 7 , the proportion of long and narrow type concave was about 90%, and the number of concave was about 50.

[0148] In addition, the inventors also prepared more microcarriers through the following examples. The specific embodiments are shown in Table A as follows.

[0149] Table A: Example 6-Example 10

[0150]

[0151]

[0152] Test Example 1: Bionic construction of cartilage lacuna structure

[0153] The inventors inoculated GFP green fluorescent labeled rat primary chondrocytes (from 4-week-old rat cartilage) on the microcarriers prepared in Example 4 at a density of 1×10 5 / mg, and placed the inoculated microcarriers in a 37℃ constant temperature incubator for culture. After 24 hours of culture, laser scanning confocal microscope was used for observation, and it was found that the chondrocytes adhered and proliferated on the microcarriers, and first adhered to the concave, as shown in Figure 4 After the human cartilage developed and matured, the chondrocytes aggregated and secreted matrix proteins to form cartilage lacuna structure, and the physiological diameter was also about 30μm, as shown in Figure 4 A.

[0154] Analysis Figure 4 As can be seen in Figure B, chondrocytes (green fluorescence) preferentially adhere to the depressions, and in most cases, multiple cells adhere to the depressions together (shown by the green fluorescent regions in B), while no cells adhere to the microcarriers without depressions. Figure 4

[0155] Therefore, the microcarriers of the present application can simulate the normal cartilage lacuna structure, and effectively inhibit the dedifferentiation caused by the in vitro proliferation of chondrocytes.

[0156] Test Example 2: In-situ cryopreservation of microcarriers and cells

[0157] The inventors inoculated human umbilical cord mesenchymal stem cells at a density of 1 x 10 5 The microcarriers were placed in a 37°C constant-temperature incubator for culture, and after 72 hours of culture (with medium replacement every other day), the cells had grown on the microcarriers. The culture medium was discarded, and a cryopreservation solution was added, which was prepared by mixing 90% FBS and 10% glycerol, and 95% FBS and 5% glycerol. After 1 day of cryopreservation, the cells were recovered, and after 24 hours of recovery, live & dead staining was performed and observed using a laser scanning confocal microscope, and the results are shown in Figure Figure 13

[0158] As shown in Figure Figure 13 , green indicates live cells and red indicates dead cells, and the microcarriers show no color. The 10% glycerol group is the control group, and the commonly used cryopreservation solution is prepared by mixing 90% FBS and 10% glycerol. In this experiment, the inventors reduced the glycerol concentration to 50% of the original, i.e., 5% glycerol and 95% FBS, and found that the cell viability was almost the same as the control group, and the number of live cells (green fluorescence) reached more than 70% of the total number.

[0159] Therefore, the microcarriers of the present application can be directly cryopreserved in-situ after cell culture, and can effectively inhibit the use of CPA (mainly DMSO and glycerol), and the cell viability after cryopreservation is high.

[0160] Test Example 3: Microcarriers can maintain the functionality of cultured cells

[0161] The inventors resuspended human umbilical cord mesenchymal stem cells in culture medium to obtain a cell suspension. 1.6 x 10 7 cells were inoculated into a bioreactor containing microcarriers and 80 ml of culture medium (the ratio of microcarriers to culture medium was 3 g / L), and the cell density was 2 x 10 5 ​​ml. The inoculated bioreactor was placed in a 37℃ carbon dioxide incubator for culture, and the reactor stirring program was 40 rpm / min, 3 min; 1 rpm / min, 1 h; and the circulation program was 24 times. After 24 h of inoculation, the program was changed to a constant speed of 40 rpm. The cell culture was treated with liquid exchange once a day and once every two days, and the liquid exchange amount was 60 ml / time. Among them, commercially available microcarriers cytodex 3 from the United States cytiva company were selected as a control group for comparison. The experimental group used the microcarriers prepared in Example 3. After 4 days, the cells in the experimental group were harvested, stained with live & dead staining solution, and observed under a laser scanning confocal microscope (as shown in Figure 14 , the proliferation results of the cells on the microcarriers were measured using a CCK8 detection kit (as shown in Figure 15 , and the cell function (stemness) expression was tested after dynamic culture and cell digestion (as shown in Figure 16 .

[0162] Figure 14 Results: Green is live cells, and red is dead cells. The results show that the prepared microcarriers have excellent biocompatibility, allowing cells to adhere and proliferate, and have excellent shear resistance. In the case of stirring, the cells can be protected, and the damage of shear force to the cells is reduced. The cells proliferate on the microcarriers and maintain a high survival rate.

[0163] Figure 15 The results show that the cells proliferate on the microcarriers after 1, 2, 3, and 4 days of cell culture. The number of cells harvested on the fourth day is 12 times that of the first day.

[0164] Figure 16 The results show that after the cells are collected by trypsin digestion from the microcarriers, the cells are incubated with antibodies. The CD105 marker expression of the experimental group is 99.3%, which is higher than that of the control group 99.1%. This shows that the stemness of the stem cells is maintained very well after proliferation on the microcarriers, and is better than that of the commercially available microcarriers.

[0165] Figures 14-16 The results prove that the microcarriers of the application can well expand the cells, collect them by digestion, and maintain excellent functionality, which has potential for clinical use.

[0166] Test Example 4: Microcarrier Permeability Test

[0167] The microcarriers prepared in Example 6 were taken for permeation test characterization, and the main operation method was as follows: about 10 mg of the freeze-dried microcarriers were taken in a 15 ml centrifuge tube, PBS was added for hydration overnight, then the supernatant was discarded, and the microcarriers were washed with PBS for 2-3 times, and the supernatant was discarded. The microcarriers were placed in a fluorescently labeled (488) culture medium (the culture medium should immerse the microcarriers), and the laser scanning confocal microscope was used for observation at 10 min, 20 min and 40 min, respectively. The results are shown in Figure 18

[0168] Among them, Figure 18 a is the microcarrier permeation result at 10 min, wherein the green fluorescent part is the labeled culture medium, and the black part is the microcarrier without fluorescent labeling, Figure 18 b is the permeation result at 20 min, and it can be seen that the culture medium has gradually permeated into the cavity, Figure 18 c is the permeation result at 40 min, and it can be seen that the culture medium has completely permeated into the cavity of the microcarrier.

[0169] The results show that the microcarriers of the application have good permeability, and the complete permeation is completed in 40 min, which is beneficial to the culture of cells and the exchange of substances, and the microcarriers with the cavity structure of the application have the condition to realize the culture of cells in the cavity.

[0170] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application and not to limit the protection scope of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.​

Claims

1. A microcarrier, characterized in that, The microcarrier has a three-dimensional structure, and has a shell layer and is hollow or solid inside, and the outer wall of the shell layer is uneven and has multiple concave points, The preparation method of the microcarrier comprises: (a) providing an organic phase solution; (b) providing an aqueous phase solution; (c) adding the aqueous phase solution into the organic phase solution to form a mixed solution, and emulsifying the mixed solution to form a uniform water-in-oil system, wherein the emulsification is carried out at a temperature of 20-75°C; (d) filtering the water-in-oil system, and the filter cake is a carrier substrate to be treated; (e) post-treating the carrier substrate to be treated to dissolve the occupying material, and conjugating a short peptide or protein containing an RGD amino acid sequence with the post-treated carrier substrate to obtain the microcarrier.

2. The microcarriers of claim 1, wherein, The proportion of the concave area of the outer wall of the shell layer to the total surface area of the outer wall of the shell layer is more than 15%.

3. The microcarrier according to claim 1, wherein, The outer wall of the shell layer has 2-800 concave points.

4. The microcarrier according to claim 1, wherein, The outer wall of the shell layer has 2-700 concave points.

5. The microcarrier according to claim 1, wherein, The outer wall of the shell layer has 2-600 concave points.

6. The microcarrier according to claim 1, wherein, The outer wall of the shell layer has 2-500 concave points.

7. The microcarrier according to claim 1, wherein, The outer wall of the shell layer has 2-400 concave points.

8. The microcarrier according to claim 1, wherein, The outer wall of the shell layer has 2-300 concave points.

9. The microcarrier according to claim 1, wherein, The outer wall of the shell layer has 2-200 concave points.

10. The microcarrier according to claim 1, wherein, The outer wall of the shell layer has 5-200 concave points.

11. The microcarrier according to claim 1, wherein, The outer wall of the shell layer has 5-100 concave points.

12. The microcarrier according to claim 1, wherein, The outer wall of the shell layer has 5-60 concave points.

13. The microcarrier according to claim 1, wherein, The outer wall of the shell layer has 10-60 concave points.

14. The microcarrier according to claim 1, wherein, The shape of the concave points on the outer wall of the shell layer is selected from circular arc-shaped concave points, fine groove-shaped concave points, polygonal concave points, irregular concave points, spherical concave points or any combination thereof.

15. The microcarrier according to claim 14, wherein, The average radius of the circular arc-shaped concave points is 1-500 μm.

16. The microcarrier according to claim 14, wherein, The average radius of the circular arc-shaped concave points is 1-400 μm.

17. The microcarrier according to claim 14, wherein, The average radius of the circular arc-shaped concave points is 1-300 μm.

18. The microcarrier of claim 14, wherein, The average radius of the circular arc-shaped concave points is 10-300 μm.

19. The microcarrier of claim 14, wherein, The average radius of the circular arc-shaped concave points is 50-300 μm.

20. The microcarrier of claim 14, wherein, The average radius of the circular arc-shaped concave points is 80-300 μm.

21. The microcarrier of claim 14, wherein, The average radius of the circular arc-shaped concave points is 80-200 μm.

22. The microcarrier of claim 14, wherein, The average width of the concave surface of the fine groove-shaped concave points is 1-500 μm.

23. The microcarrier of claim 14, wherein, The average width of the concave surface of the fine groove-shaped concave points is 1-400 μm.

24. The microcarrier of claim 14, wherein, The average width of the concave surface of the fine groove-shaped concave points is 1-300 μm.

25. The microcarrier of claim 14, wherein, The average width of the concave surface of the fine groove-shaped concave points is 10-300 μm.

26. The microcarrier of claim 14, wherein, The average width of the concave surface of the fine groove-shaped concave points is 50-300 μm.

27. The microcarrier of claim 14, wherein, The average width of the concave surface of the fine groove-shaped concave points is 80-300 μm.

28. The microcarrier of claim 14, wherein, The average width of the concave surface of the fine groove-shaped concave points is 80-200 μm.

29. The microcarrier of claim 14, wherein, The average width of the concave surface of the polygonal concave points is 1-500 μm.

30. The microcarrier of claim 14, wherein, The average width of the concave surface of the polygonal concave points is 1-400 μm.

31. The microcarrier of claim 14, wherein, The average width of the concave surface of the polygonal concave points is 1-300 μm.

32. The microcarrier of claim 14, wherein, The average width of the concave surface of the polygonal concave is 10-300 μm.

33. The microcarrier of claim 14, wherein, The average width of the concave surface of the polygonal concave is 50-300 μm.

34. The microcarrier of claim 14, wherein, The average width of the concave surface of the polygonal concave is 80-300 μm.

35. The microcarrier of claim 14, wherein, The average width of the concave surface of the polygonal concave is 80-200 μm.

36. The microcarrier of claim 14, wherein, The average width of the concave surface of the irregular concave is 1-500 μm.

37. The microcarrier of claim 14, wherein, The average width of the concave surface of the irregular concave is 1-400 μm.

38. The microcarrier of claim 14, wherein, The average width of the concave surface of the irregular concave is 1-300 μm.

39. The microcarrier of claim 14, wherein, The average width of the concave surface of the irregular concave is 10-300 μm.

40. The microcarrier of claim 14, wherein, The average width of the concave surface of the irregular concave is 50-300 μm.

41. The microcarrier of claim 14, wherein, The average width of the concave surface of the irregular concave is 80-300 μm.

42. The microcarrier of claim 14, wherein, The average width of the concave surface of the irregular concave is 80-200 μm.

43. The microcarrier of claim 14, wherein, The average radius of the spherical concave is less than 500 μm.

44. The microcarrier of claim 14, wherein, The average radius of the spherical concave is less than 400 μm.

45. The microcarrier of claim 14, wherein, The average radius of the spherical concave is less than 300 μm.

46. The microcarrier of claim 14, wherein, The average radius of the spherical concave is less than 200 μm.

47. The microcarrier of claim 14, wherein, The average radius of the spherical concave is less than 100 μm.

48. The microcarrier of claim 14, wherein, The average radius of the spherical concave is less than 50 μm.

49. The microcarrier of claim 14, wherein, The average radius of the spherical concave is less than 20 μm.

50. The microcarrier of claim 14, wherein, The average radius of the spherical concave is less than 10 μm.

51. The microcarrier of claim 1, wherein, The average particle size of the microcarrier is 1-3000 μm.

52. The microcarrier of claim 1, wherein, The average particle size of the microcarrier is 1-2000 μm.

53. The microcarrier of claim 1, wherein, The average particle size of the microcarrier is 1-1000 μm.

54. The microcarrier of claim 1, wherein, The average particle size of the microcarrier is 1-800 μm.

55. The microcarrier of claim 1, wherein, The average particle size of the microcarrier is 1-600 μm.

56. The microcarrier of claim 1, wherein, The average particle size of the microcarrier is 10-600 μm.

57. The microcarrier of claim 1, wherein, The average particle size of the microcarrier is 50-500 μm.

58. The microcarrier of claim 1, wherein, The average particle size of the microcarrier is 100-500 μm.

59. The microcarrier of claim 1, wherein, The average particle size of the microcarrier is 100-400 μm.

60. The microcarrier of claim 1, wherein, The average particle size of the microcarrier is 30-400 μm.

61. The microcarrier of claim 1, wherein, The thickness of the shell layer is 1-100 μm.

62. The microcarrier of claim 1, wherein, The thickness of the shell layer is 1-90 μm.

63. The microcarrier of claim 1, wherein, The thickness of the shell layer is 1-80 μm.

64. The microcarrier of claim 1, wherein, The thickness of the shell layer is 1-70 μm.

65. The microcarrier of claim 1, wherein, The thickness of the shell layer is 1-60 μm.

66. The microcarrier of claim 1, wherein, The thickness of the shell layer is 1-50 μm.

67. The microcarrier of claim 1, wherein, The microcarrier has a three-dimensional structure of a sphere, a shuttle, an oval sphere, a rod, a flat sphere or an irregular sphere.

68. The microcarrier of claim 1, wherein, When the microcarrier is hollow, the outer wall and the inner wall of the shell layer are surface-modified; when the microcarrier is solid, the outer wall of the shell layer is surface-modified.

69. The microcarrier of claim 1, wherein, When the microcarrier is hollow, the outer wall and the inner wall of the shell layer are RGD surface-modified; when the microcarrier is solid, the outer wall of the shell layer is RGD surface-modified.

70. The microcarrier of claim 1, wherein, The microcarrier is loaded with cells.

71. The microcarrier of claim 70, wherein, When the microcarrier is hollow, the cells are arranged on the inner wall of the shell layer of the microcarrier.

72. The microcarrier of claim 70, wherein, The cells are arranged on the outer wall of the shell layer of the microcarrier.

73. The microcarrier of claim 70, wherein, When the microcarrier is hollow, the cells are arranged on both the inner wall of the shell layer of the microcarrier and the outer wall of the shell layer of the microcarrier.

74. The microcarrier of claim 73, wherein, The cells are disposed on the inner wall of the shell of the microcarrier and the outer wall of the shell of the microcarrier at the same time.

75. The microcarrier of claim 72 or 73, wherein, The cells are disposed on the outer wall of the shell of the microcarrier at the concave points of the outer wall of the shell of the microcarrier, or at the smooth surface of the outer wall of the shell of the microcarrier, or at both the concave points and the smooth surface of the outer wall of the shell of the microcarrier.

76. The microcarrier of claim 71 or 73, wherein, The cells are disposed on the inner wall of the shell of the microcarrier by cell injection.

77. The microcarrier of claim 72, wherein, The cells are disposed on the outer wall of the shell of the microcarrier by co-culturing with the microcarrier.

78. The microcarrier of claim 70, wherein, The cells are selected from prokaryotic cells, eukaryotic cells, or any combination thereof.

79. The microcarrier of claim 70, wherein, The cells are selected from bacterial cells, archaeal cells.

80. The microcarrier of claim 70, wherein, The cells are selected from plant cells, animal cells, or any combination thereof.

81. The microcarrier of claim 70, wherein, The cells are algal cells.

82. The microcarrier of claim 70, wherein, The cells are fungal cells.

83. The microcarrier of claim 70, wherein, The cells are cells of unicellular eukaryotic organisms.

84. The microcarrier of claim 70, wherein, The cells are protozoan cells.

85. The microcarrier of claim 70, wherein, The cells are artificial cells.

86. The microcarrier of claim 70, wherein, The cells are selected from stem cells, somatic cells, or any combination thereof.

87. The microcarrier of claim 86, wherein, The stem cells are mouse embryonic stem cells.

88. The microcarrier of claim 86, wherein, The stem cells are induced pluripotent stem cells.

89. The microcarrier of claim 86, wherein, The stem cells are selected from mesenchymal stem cells, neural stem cells, muscle stem cells, hematopoietic stem cells, epithelial stem cells, or any combination thereof.

90. The microcarrier of claim 86, wherein, The stem cells are endothelial stem cells.

91. The microcarrier of claim 86, wherein, The stem cells are selected from mammary stem cells, intestinal stem cells, or any combination thereof.

92. The microcarrier of claim 86, wherein, The stem cells are mesodermal stem cells.

93. The microcarrier of claim 86, wherein, The somatic cells are epithelial cells.

94. The microcarrier of claim 86, wherein, The somatic cells are endothelial cells.

95. The microcarrier of claim 86, wherein, The somatic cells are mesothelial cells.

96. The microcarrier of claim 86, wherein, The somatic cells are selected from fibroblasts, osteoblasts, chondrocytes, myocytes, hepatocytes, pancreatic cells, or any combination thereof.

97. The microcarrier of claim 86, wherein, The somatic cells are bone cells.

98. The microcarrier of claim 86, wherein, The somatic cells are selected from cardiomyocytes, skeletal cells, or any combination thereof.

99. The microcarrier of claim 86, wherein, The somatic cells are selected from cardiac myoblasts, skeletal myoblasts, or any combination thereof.

100. The microcarrier of claim 86, wherein, The somatic cells are oligodendrocytes.

101. The microcarrier of claim 86, wherein, The somatic cells are glial cells.

102. The microcarrier of claim 86, wherein, The somatic cells are hematopoietic cells.

103. The microcarrier of claim 86, wherein, The somatic cells are neurons.

104. The cell of claim 86, wherein, The somatic cells are selected from exogenous cells, endogenous cells, or any combination thereof.

105. The microcarrier of claim 1, wherein, The shell or the microcarrier is made of biomaterials.

106. The microcarrier of claim 105, wherein, The biomaterials are selected from artificially synthesized biomaterials, natural biomaterials, or a combination thereof.

107. The microcarrier of claim 106, wherein, The artificially synthesized biomaterials are selected from polyethylene glycol, polyethylene glycol derivatives, polylactic acid, polylactic-glycolic acid copolymer, polyanhydride, polyacid ester, polyamino acid, polyethylene oxide, polyester, polymethyl methacrylate, polycarbonate, polyurethane, polycaprolactone, polyhydroxyalkanoate, polysiloxane, polyethylene, polyvinyl chloride, polytetrafluoroethylene, polystyrene, polypropylene, maleic anhydride graft copolymer, polyacrylamide, polyacetal, polypyrrole, or any combination thereof.

108. The microcarrier of claim 106, wherein, The natural biomaterial is selected from natural proteins, collagen and collagen derivatives, gelatin and gelatin derivatives, agar and agar derivatives, proteoglycans, alginate and alginate derivatives thereof, Matrigel, propolis, cellulose and cellulose derivatives, chitin and chitin derivatives, silk fibroin and derivatives thereof, laminin and derivatives thereof, fibronectin and derivatives thereof, sodium hyaluronate and hyaluronic acid derivatives, agarose and derivatives thereof, dextran and derivatives thereof, sucrose and sucrose derivatives, starch, chitosan and chitosan derivatives, or any combination thereof.

109. A microcarrier aggregate, characterized in that, The aggregate is formed by aggregation of the microcarrier particles of any one of claims 1-108.

110. The microcarrier aggregate of claim 109, wherein, The aggregate is a collection of microcarriers of a single property, or a collection of microcarriers of various properties in any ratio.

111. A method for preparing the microcarriers of any one of claims 1-108, comprising: (a) providing an organic phase solution; (b) providing an aqueous phase solution; (c) adding the aqueous phase solution to the organic phase solution to form a mixture, and emulsifying the mixture to form a uniform water-in-oil system, the emulsifying being performed at a temperature of 20-75°C; (d) filtering the water-in-oil system, and the filter cake being a carrier matrix to be processed; (e) post-processing the carrier matrix to be processed to dissolve the occupying material, and conjugating a short peptide or protein comprising an RGD amino acid sequence to the post-processed carrier matrix to obtain the microcarriers; wherein the aqueous phase solution is obtained by the following steps: dissolving the biomaterial in water to obtain a solution; suspending the occupying material and the solidifying agent in the solution to form a uniformly dispersed suspension, the suspension being the aqueous phase solution; the uniformly dispersed suspension is achieved by stirring, shaking, ultrasonic or shaking, at a speed of 100-2000 rpm / min; the mass fraction of the occupying material in the aqueous phase solution is 0.01-1%.

112. The method of claim 111, wherein, The conjugation further comprises the steps of washing, sieving and lyophilizing.

113. The method of claim 111, wherein, The emulsifying time is 4-72 h.

114. The method of claim 111, wherein, The emulsifying time is 5-30 h.

115. The method of claim 111, wherein, The emulsifying is achieved by stirring, ultrasonic, shaking or micro-channel method.

116. The method of claim 115, wherein, The emulsifying speed is 100-5000 rpm / min.

117. The method of claim 115, wherein, The emulsifying speed is 300-1000 rpm / min.

118. The method of claim 111, wherein, The organic phase solution is obtained by mixing an organic solvent and a non-ionic surfactant.

119. The method of claim 118, wherein, The mixing is performed at a temperature of 4-200°C.

120. The method of claim 118, wherein, The mixing is performed at a temperature of 20-75°C.

121. The method of claim 118, wherein, The mixing time is 10-120 min.

122. The method of claim 118, wherein, The mixing time is 10-60 min.

123. The method of claim 118, wherein, The mixing is performed under the action of a stirring paddle.

124. The method of claim 123, wherein the stirring paddle is rotated at a speed of 100-2000 rpm / min.

125. The method of claim 123, wherein, The stirring paddle is rotated at a speed of 300-1000 rpm / min.

126. The method of claim 118, wherein, The organic solvent is selected from liquid paraffin, petroleum ether, carbon tetrachloride, dimethyl sulfoxide, chloroform, dichloromethane, edible oil, silicone oil, soybean oil, mineral oil or any combination thereof.

127. The method of claim 118, wherein, The organic solvent is liquid paraffin.

128. The method of claim 118, wherein, The non-ionic surfactant is selected from Tween, Span 80, fatty acid glyceride, sodium dodecyl benzene sulfonate, PO-500, hydrofluoroether, polyethylene glycol, block polyoxyethylene-polyoxypropylene ether (PO-EO copolymer), polyol ester or any combination thereof.

129. The method of claim 118, wherein, The non-ionic surfactant is Span 80.

130. The method of claim 118, wherein, The volume ratio of the organic solvent to the non-ionic surfactant is 200-400:

1.

131. The method of claim 111, wherein, The dissolving is performed at a temperature of 4-200℃.

132. The method of claim 111, wherein, The dissolving is performed at a temperature of 10-80℃.

133. The method of claim 111, wherein, The uniformly dispersed suspension is achieved by stirring, shaking, ultrasonic or shaking, at a rotation speed of 300-700 rpm / min.

134. The method of claim 111, wherein, The mass fraction of the biomaterial in the aqueous solution is 3-10%.

135. The method of claim 111, wherein, The mass fraction of the solidifying agent in the aqueous solution is 0.5-7%.

136. The method of claim 111, wherein, The biomaterial is selected from artificially synthesized biomaterial, natural biomaterial or a combination thereof.

137. The method of claim 111, wherein, The biomaterial is selected from sodium hyaluronate, gelatin.

138. The method of claim 111, wherein, The space-occupying material is selected from polyethylene glycol and polyethylene glycol derivatives, paraffin balls, oxidized alginate and derivatives thereof, polycaprolactone, silicon dioxide, beeswax, propolis, agar, agarose, alginate and derivatives thereof, soybean lecithin, egg yolk lecithin, phospholipids, dextran, chitosan, starch, gelatin, sodium hyaluronate and hyaluronic acid derivatives or any combination thereof.

139. The method of claim 111, wherein, The space-occupying material is agarose, dextran, sodium hyaluronate.

140. The method of claim 111, wherein, The solidifying agent is selected from N,N-methylene bisacrylamide, 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride, N-hydroxysuccinimide, N-hydroxysulfosuccinimide, diisocyanate, glutaraldehyde, Kyonpin, ammonium sulfate, calcium ions, butanediol diglycidyl ether, transglutaminase, divinylbenzene, adipic acid dihydrazide or any combination thereof.

141. The method of claim 111, wherein, The solidifying agent is selected from 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride, glutaraldehyde.

142. The method of claim 111, wherein, The filtration is achieved by a filtering device, and the filtering device has a mesh diameter of 30-1000 microns.

143. The method of claim 142, wherein, The filtering device has a four-layer structure, the first layer has a mesh diameter of 500-800 microns, the second layer has a mesh diameter of 300-400 microns, the third layer has a mesh diameter of 200-300 microns, and the fourth layer has a mesh diameter of 100-200 microns.

144. The method of claim 111, wherein, The step of dissolving the space-occupying material is achieved by: Mixing the carrier substrate to be treated with an organic solvent to dissolve the space-occupying material.

145. The method of claim 144, wherein, The mixing is performed at a temperature of 15℃-150℃ for 4-48h.

146. The method of claim 144, wherein, The mixing is performed at a temperature of 15℃-150℃ for 6-24h.

147. The method of claim 144, wherein, The mixing is performed at a temperature of 25-100℃ for 4-48h.

148. The method of claim 144, wherein, The mixing is performed at a temperature of 25-100℃ for 6-24h.

149. The method of claim 144, wherein, The organic solvent is selected from the group consisting of acetone, absolute ethanol, petroleum ether, chloroform, dimethyl sulfoxide, dichloromethane, petroleum ether, carbon tetrachloride, acetonitrile, toluene, methanol or any combination thereof.

150. The method of claim 144, wherein, The organic solvent is absolute ethanol.

151. The method of claim 111, wherein, The conjugation of the short peptide or protein comprising the RGD amino acid sequence with the post-processed carrier matrix is achieved by the following steps: The post-processed carrier matrix is mixed with the short peptide or protein comprising the RGD amino acid sequence so as to achieve the RGD surface modification of the post-processed carrier matrix.

152. The method of claim 151, wherein, The mixing is performed at a temperature of 15-25 °C.

153. The method of claim 151, wherein, The mixing time is 4-10 hours.

154. The method of claim 111, wherein, The short peptide or protein comprising the RGD amino acid sequence is selected from the group consisting of collagen or collagen derivatives, gelatin or gelatin derivatives, fibronectin, fibroin, laminin, matrigel or any combination thereof.

155. The method of claim 111, wherein, The short peptide or protein comprising the RGD amino acid sequence is selected from the group consisting of collagen, gelatin.

156. A kit comprising the microcarriers of any one of claims 1-108, the microcarrier aggregates of any one of claims 109-110, or the microcarriers prepared by the method of any one of claims 111-155.

157. Use of the microcarriers of any one of claims 1-108, the microcarrier aggregates of any one of claims 109-110, or the microcarriers prepared by the method of any one of claims 111-155 for the manufacture of a kit for three-dimensional cell culture and / or expansion, 3D bioprinting, biomimetic construct building, cell therapy, drug loading or medical injection.

158. The use of claim 157, wherein, The biomimetic construct is a three-dimensional construct, a tissue precursor, a tissue, an organ or a mouse embryo.

159. The use of claim 157, wherein, The biomimetic construct is built as a mouse embryo biomimetic simulation construct or a cartilage biomimetic construct.

160. The use of claim 157, wherein, The biomimetic construct is a cartilage lacuna structure of articular cartilage.

161. The use of claim 157, wherein, The cell therapy is a skin surface treatment, a clinical corneal treatment, a bone treatment or a cartilage repair and bone injury treatment.

162. Use of the microcarriers of any one of claims 1-108, the microcarrier aggregates of any one of claims 109-110, or the microcarriers prepared by the method of any one of claims 111-155 for in vitro three-dimensional cell culture and / or expansion, in vitro 3D bioprinting, in vitro biomimetic construct building or in vitro drug loading; the use being for non-diagnostic or therapeutic purposes.

163. The use of claim 162, wherein, The biomimetic construct is a three-dimensional construct, a tissue precursor, a tissue, an organ or a mouse embryo.

164. The use of claim 162, wherein, The biomimetic construct is built as a mouse embryo biomimetic simulation construct or a cartilage biomimetic construct.

165. The use of claim 162, wherein, The biomimetic construct is a cartilage lacuna structure of articular cartilage.

166. A method for in vitro three-dimensional cell culture and / or expansion, comprising: 1) seeding cells on the microcarriers of any one of claims 1-108, the microcarrier aggregates of any one of claims 109-110, or the microcarriers prepared by the method of any one of claims 111-155 for culture.

167. The method of claim 166, wherein, Further comprising, after step 1): 2-A) subjecting the mixture obtained in step 1) to in situ cryopreservation.

168. The method of claim 166, wherein, After step 1) further comprises: 2-B) subjecting the cells obtained in step 1) to digestion, while being attached to the microcarriers.

169. The method of claim 166, wherein, The culture is a dynamic culture.

170. The method of claim 169, wherein, The dynamic culture is a stirred dynamic culture or a suspension dynamic culture.

171. The method of claim 166, wherein, The cells are as defined in any of claims 78-104.

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