WO2023138389A1 - 一种微载体及其应用 - Google Patents

一种微载体及其应用 Download PDF

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WO2023138389A1
WO2023138389A1 PCT/CN2023/070671 CN2023070671W WO2023138389A1 WO 2023138389 A1 WO2023138389 A1 WO 2023138389A1 CN 2023070671 W CN2023070671 W CN 2023070671W WO 2023138389 A1 WO2023138389 A1 WO 2023138389A1
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microcarrier
cells
optional
shell
derivatives
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PCT/CN2023/070671
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English (en)
French (fr)
Inventor
顾奇
赵喜源
吴骏
高婷婷
丁声龙
高子力
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中国科学院动物研究所
北京干细胞与再生医学研究院
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Publication of WO2023138389A1 publication Critical patent/WO2023138389A1/zh

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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0062—General methods for three-dimensional culture
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
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    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • C12N5/0075—General culture methods using substrates using microcarriers
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0655—Chondrocytes; Cartilage
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0668—Mesenchymal stem cells from other natural sources
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    • C12N2513/00—3D culture
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    • C12N2531/00—Microcarriers
    • C—CHEMISTRY; METALLURGY
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    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/70—Polysaccharides
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    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/70—Polysaccharides
    • C12N2533/76—Agarose, agar-agar
    • C—CHEMISTRY; METALLURGY
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    • C12N2533/00—Supports or coatings for cell culture, characterised by material
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    • C12N2533/80—Hyaluronan
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    • C12N2535/00—Supports or coatings for cell culture characterised by topography

Definitions

  • the invention relates to the field of biological materials, in particular to a microcarrier and its application.
  • two-dimensional cell culture cannot meet the needs of laboratories and enterprises for the number of cells.
  • the disadvantages of two-dimensional cell culture are becoming more and more prominent, mainly in the following aspects: (1) two-dimensional cell culture can only be cultured in petri dishes, and the proliferation of adherent cells is limited by the size of the bottom of the dish. Long-term culture requires a lot of manpower and material resources, and it is impossible to achieve large-scale expansion in a short period of time, and the resource consumption is large; (2) two-dimensional culture cells cannot build a friendly microenvironment for cell growth.
  • the present invention aims to solve one of the above outstanding problems of two-dimensional cell culture.
  • the invention provides a microcarrier and its preparation method and describes its application.
  • the microcarrier provided by the present invention improves the permeability of traditional microcarriers, facilitates material exchange between cells and the external environment, improves cell viability, provides good mechanical protection for cell proliferation and constructs a good three-dimensional structure for cell growth, realizes friendly connection of cells, facilitates large-scale expansion of cells in a limited space, and maintains stable physical and chemical properties of cells in long-term culture; the microcarrier of the present invention has more flexible structural characteristics, and can meet various application scenarios such as biology, medical treatment, and clinical treatment.
  • the present invention provides a kind of microcarrier, wherein, described microcarrier has three-dimensional structure, and described microcarrier has shell and interior is hollow or solid, and the outer wall of described shell is uneven and has multiple sunken points.
  • the ratio of the concave area of the outer wall of the shell to the total surface area of the outer wall of the shell is more than 15%, such as more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90% or more than 95%, 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%, specifically such as 15%, 20%, 50%, 60%, 80%, 85% or 90%.
  • the outer wall of the shell has 2-800 depressions, 2-700 optional depressions, 2-600 depressions, 2-500 depressions, 2-400 depressions, 2-300 depressions, 2-200 depressions.
  • the number is 5-200, and the number of optional recessed points is 5-100.
  • the outer wall of the shell has 5-60 depressions, such as 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55 or 55-60.
  • the outer wall of the shell has 10-60 depressions (specifically, for example, 10, 20, 25, 30, 40, 50 or 60).
  • the shape of the depressions on the outer wall of the shell is selected from arc-shaped depressions (or called arc-shaped depressions), fine groove-shaped depressions (or called elongated depressions), spherical depressions, polygonal depressions, irregular depressions or any combination thereof.
  • the arc-shaped depression has an average radius of 1-500 ⁇ m, an optional average radius of 1-400 ⁇ m, an optional average radius of 1-300 ⁇ m, an optional average radius of 10-300 ⁇ m, an optional average radius of 50-300 ⁇ m, an optional average radius of 80-300 ⁇ m, and an optional average radius of 80-200 ⁇ m.
  • the average radius of the arc-shaped depression is 1-100 ⁇ m, for example, 1-10 ⁇ m, 10-20 ⁇ m, 20-30 ⁇ m, 30-40 ⁇ m, 40-50 ⁇ m, 50-60 ⁇ m, 60-70 ⁇ m, 70-80 ⁇ m, 80-90 ⁇ m or 90-100 ⁇ m.
  • the average width of the concave surface of the fine groove type depression is 1-500 ⁇ m
  • the average width of the optional concave surface is 1-400 ⁇ m
  • the average width of the optional concave surface is 1-300 ⁇ m
  • the optional average width of the concave surface is 10-300 ⁇ m
  • the optional average width of the concave surface is 50-300 ⁇ m
  • the optional average width of the concave surface is 80-300 ⁇ m
  • the optional average width of the concave surface is 80-300 ⁇ m.
  • the average width is 80-200 ⁇ m.
  • the average width of the groove-shaped depressions is 1-100 ⁇ m, for example, 1-10 ⁇ m, 10-20 ⁇ m, 20-30 ⁇ m, 30-40 ⁇ m, 40-50 ⁇ m, 50-60 ⁇ m, 60-70 ⁇ m, 70-80 ⁇ m, 80-90 ⁇ m or 90-100 ⁇ m.
  • the average width of the concave surface of the polygonal depression is 1-500 ⁇ m
  • the optional average width of the concave surface is 1-400 ⁇ m
  • the optional average width of the concave surface is 1-300 ⁇ m
  • the optional average width of the concave surface is 10-300 ⁇ m
  • the optional average width of the concave surface is 50-300 ⁇ m
  • the optional average width of the concave surface is 80-300 ⁇ m
  • the optional depression The average width of the surface is 80 to 200 ⁇ m.
  • the average width of the concave faces of the polygonal depressions is 1-100 ⁇ m, for example, 1-10 ⁇ m, 10-20 ⁇ m, 20-30 ⁇ m, 30-40 ⁇ m, 40-50 ⁇ m, 50-60 ⁇ m, 60-70 ⁇ m, 70-80 ⁇ m, 80-90 ⁇ m or 90-100 ⁇ m.
  • the average width of the concave surface of the irregular depression is 1-500 ⁇ m
  • the average width of the optional concave surface is 1-400 ⁇ m
  • the average width of the optional concave surface is 1-300 ⁇ m
  • the average width of the optional concave surface is 10-300 ⁇ m
  • the average width of the optional concave surface is 50-300 ⁇ m
  • the average width of the optional concave surface is 80-300 ⁇ m
  • the average width of the optional concave surface The width is 80 to 200 ⁇ m.
  • the average width of the concave surface of the irregular depression is 1-100 ⁇ m, for example, 1-10 ⁇ m, 10-20 ⁇ m, 20-30 ⁇ m, 30-40 ⁇ m, 40-50 ⁇ m, 50-60 ⁇ m, 60-70 ⁇ m, 70-80 ⁇ m, 80-90 ⁇ m or 90-100 ⁇ m.
  • the spherical depressions have an average radius of less than 500 ⁇ m, an optional average radius of less than 400 ⁇ m, an optional average radius of less than 300 ⁇ m, an optional average radius of less than 200 ⁇ m, an optional average radius of less than 100 ⁇ m, an optional average radius of less than 50 ⁇ m, an optional average radius of less than 20 ⁇ m, and an optional average radius of less than 10 ⁇ m.
  • the average radius of the spherical depressions is less than 100 ⁇ m, such as less than 90 ⁇ m, less than 80 ⁇ m, less than 70 ⁇ m, less than 60 ⁇ m, less than 50 ⁇ m, less than 40 ⁇ m, less than 30 ⁇ m, less than 20 ⁇ m, or less than 10 ⁇ m.
  • the average width refers to the average length of the shortest side, for example, the average width of the narrow groove-shaped depressions refers to the average length of the shortest sides of the narrow groove-shaped depressions.
  • the average particle diameter of the microcarrier is 1-3000 ⁇ m
  • the optional average particle diameter is 1-2000 ⁇ m
  • the optional average particle diameter is 1-1000 ⁇ m
  • the optional average particle diameter is 1-800 ⁇ m
  • the optional average particle diameter is 1-600 ⁇ m
  • the optional average particle diameter is 10-600 ⁇ m
  • the optional average particle diameter is 50-500 ⁇ m
  • the optional average particle diameter is 100-5 00 ⁇ m
  • the optional average particle size is 100-400 ⁇ m.
  • the microcarriers have an average particle size of 10-500 ⁇ m, such as 10-50 ⁇ m, 50-100 ⁇ m, 100-150 ⁇ m, 150-200 ⁇ m, 200-250 ⁇ m, 250-300 ⁇ m, 300-350 ⁇ m, 350-400 ⁇ m, 400-450 ⁇ m or 450-500 ⁇ m.
  • the microcarriers have an average particle size of 30-400 ⁇ m.
  • the shell layer has a thickness of 1-100 ⁇ m, an optional thickness of 1-90 ⁇ m, an optional thickness of 1-80 ⁇ m, an optional thickness of 1-70 ⁇ m, an optional thickness of 1-60 ⁇ m, and an optional thickness of 1-50 ⁇ m.
  • the shell layer has a thickness of 1-100 ⁇ m, such as 1-10 ⁇ m, 10-20 ⁇ m, 20-30 ⁇ m, 30-40 ⁇ m, 40-50 ⁇ m, 50-60 ⁇ m, 60-70 ⁇ m, 70-80 ⁇ m, 80-90 ⁇ m or 90-100 ⁇ m.
  • the three-dimensional structure of the microcarrier is a sphere, a fusiform, an ellipsoid, a rod, an oblate spheroid or an irregular sphere. In some specific embodiments, the three-dimensional structure of the microcarrier is spherical.
  • the interior of the microcarrier is hollow, and the microcarrier has a non-closed three-dimensional structure.
  • the shell of the microcarrier has one or more through holes penetrating the shell to communicate the interior and exterior of the shell.
  • the hollow structure inside the microcarrier is obtained by stripping out space-occupying materials.
  • the outer and/or inner walls of the shell are surface-modified.
  • the outer and/or inner walls of the shell are surface-modified with RGD.
  • the microcarriers are loaded with cells.
  • the cells are disposed on the inner shell of the microcarrier, or on the outer shell of the microcarrier, or on both the inner shell of the microcarrier and the outer shell of the microcarrier.
  • the types of cells disposed on the inner shell wall and the outer shell wall can be the same or different.
  • the cells are arranged at the recessed point of the outer wall of the shell, or at the smooth surface of the outer wall of the shell (i.e. at a non-sag point), or at the same time at the recessed point of the outer wall of the shell and at the smooth surface.
  • the cells are arranged at the depressions on the outer shell of the microcarrier.
  • the cells are disposed on the inner wall of the shell of the microcarrier through the through holes.
  • the cells are disposed on the inner wall of the shell of the microcarrier by cell injection.
  • the cells are disposed on the outer shell of the microcarriers by co-culturing with the microcarriers.
  • the cells are selected from prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, cells of unicellular eukaryotic organisms, protozoan cells, plant cells, animal cells, algal cells, fungal cells, artificial cells, or any combination thereof.
  • the cells are selected from stem cells, somatic cells, germ cells, or any combination thereof.
  • the stem cells are selected from embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, mesenchymal stem cells, neural stem cells, muscle stem cells, hematopoietic stem cells, epithelial stem cells, mammary gland stem cells, intestinal stem cells, mesoderm stem cells, endothelial stem cells, or any combination thereof.
  • ES embryonic stem
  • iPS induced pluripotent stem
  • mesenchymal stem cells mesenchymal stem cells
  • neural stem cells neural stem cells
  • muscle stem cells hematopoietic stem cells
  • epithelial stem cells epithelial stem cells
  • mammary gland stem cells intestinal stem cells
  • mesoderm stem cells mesoderm stem cells
  • endothelial stem cells or any combination thereof.
  • the somatic cells are selected from epithelial cells, endothelial cells, mesothelial cells, fibroblasts, osteoblasts, chondrocytes, myocytes, bone cells, hepatocytes, pancreatic cells, exogenous cells, endogenous cells, cardiomyocytes, skeletal cells, cardiac myoblasts, skeletal myoblasts, oligodendrocytes, glial cells, hematopoietic cells, neurons, or any combination thereof.
  • the germ cells are selected from oocytes, sperm, oogonia, spermatogonia, or any combination thereof.
  • the shell or the microcarriers are made of biological material. That is, if the inside of the microcarrier is hollow, the shell layer is made of biological material; if the inside of the microcarrier is solid, the microcarrier is made of biological material.
  • the biomaterial is selected from synthetic biomaterials, natural biomaterials, or combinations thereof.
  • the synthetic biomaterial is selected from polyethylene glycol, polyethylene glycol derivatives, polylactic acid, polylactic acid alcohol copolymer, polyanhydride, polyester, polyamino acid, polyethylene oxide, polyester, polymethylmethacrylate, polycarbonate, polyurethane, polycaprolactone, polyhydroxyalkanoate, polysiloxane, polyethylene, polyvinyl chloride, polytetrafluoroethylene, polystyrene, polypropylene, maleic anhydride graft copolymer, polyacrylamide, polyacetal, polypyrrole or any combination thereof.
  • the natural biological material is selected from natural protein, collagen and collagen derivatives, gelatin and gelatin derivatives, agar and agar derivatives, proteoglycan, alginate and alginate derivatives, matrigel, propolis, cellulose and cellulose derivatives, chitin and chitin derivatives, silk protein 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 Glycan derivatives or any combination thereof.
  • the biological material is selected from sodium hyaluronate, gelatin.
  • the present invention provides a microcarrier aggregate, wherein the aggregate is formed by aggregating the aforementioned microcarrier particles.
  • the aggregate is an aggregate of microcarriers with a single trait, or an aggregate of microcarriers with various traits in any proportion.
  • the present invention provides a method for preparing the aforementioned microcarrier, as shown in Figure 5, which includes:
  • the steps of washing, sieving and freeze-drying are further included after the conjugation.
  • the emulsification is performed at a temperature of 4-200°C, such as 20-75°C, or such as 20-25°C.
  • the emulsification time is 4-72h, preferably 5-30h, such as 5-10h, 10-15h, 15-20h, 20-25h or 25-30h, specifically 10h.
  • the emulsification is achieved by agitation, sonication, shaking or microfluidic methods.
  • the rotation speed during emulsification is 100-5000rpm/min, preferably 300-1000rpm/min, such as 300-400rpm/min, 400-500rpm/min, 500-600rpm/min, 600-700rpm/min, 700-800rpm/min, 800-900rpm/min or 900-1000rpm pm/min, specifically 600rpm/min.
  • the organic phase solution is obtained by mixing an organic solvent and a nonionic surfactant.
  • the mixing is performed at a temperature of 4-200°C, preferably 20-75°C (eg, 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).
  • the mixing time is 10-120 min, preferably 10-60 min.
  • the mixing is performed under the action of a paddle.
  • the rotation speed of the stirring blade is 100-2000 rpm/min, preferably 300-1000 rpm/min, such as 300-700 rpm/min.
  • 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. In some embodiments, the organic solvent is liquid paraffin.
  • the nonionic surfactant is selected from Tween, Span 80, fatty acid glycerides, sodium dodecylbenzenesulfonate, PO-500, hydrofluoroether, polyethylene glycol, block polyoxyethylene-polyoxypropylene ether (PO-EO copolymer), polyol esters or any combination thereof.
  • the nonionic surfactant is Span 80.
  • the volume ratio of the organic solvent to the nonionic surfactant is 200-400:1, such as 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, 3 50:1, 360:1, 370:1, 380:1, 390:1 or 400:1, specifically 300:1.
  • the aqueous phase solution is obtained through the following steps: dissolving the biological material in water to obtain a solution; suspending the space-occupying material and the curing agent in the solution to form a uniformly dispersed suspension, which is the aqueous phase solution.
  • the dissolution is performed at a temperature of 4-200°C, preferably 10-80°C.
  • the homogeneously dispersed suspension is achieved by stirring, vibrating, sonicating or shaking, preferably, the rotating speed is 100-2000 rpm/min, preferably 300-700 rpm/min.
  • the mass fraction of the biological material is 3-10%, such as 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%, preferably 5%-10%, specifically 5% or 10%.
  • the mass fraction of the site-occupied material is 0.01-1%, such as 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%, specifically 0.05%, 0.1%, 0.5% or 1%.
  • the mass fraction of the curing agent is 0.5-7%, such as 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%, specifically 0.5% or 5%.
  • the biomaterial is selected from synthetic biomaterials, natural biomaterials, or combinations thereof.
  • the natural biological material is selected from natural proteins, collagen and collagen derivatives, gelatin and gelatin derivatives, agar and agar derivatives, proteoglycans, alginates and alginate derivatives, matrigel, propolis, cellulose and cellulose derivatives, chitin and chitin derivatives, silk protein 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.
  • the biological material is selected from sodium hyaluronate, gelatin.
  • the space-occupied material is selected from polyethylene glycol and polyethylene glycol derivatives, paraffin spheres, oxidized alginate and its derivatives, polycaprolactone, silicon dioxide, beeswax, propolis, agar, agarose, alginate and its derivatives, soybean lecithin, egg yolk lecithin, phospholipids, dextran, chitosan, starch, gelatin, sodium hyaluronate and hyaluronic acid derivatives or any combination thereof.
  • the space-occupying material is selected from agarose, dextran, sodium hyaluronate.
  • the curing agent is selected from N,N-methylenebisacrylamide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, N-hydroxysulfosuccinimide, diisocyanate, glutaraldehyde, Jingpinil, ammonium sulfate, calcium ion, butanediol diglycidyl ether, transglutaminase, divinylbenzene, adipate dihydrazide or any combination thereof.
  • the curing agent is selected from 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, glutaraldehyde.
  • the filtering is achieved by a filter device with a mesh diameter of 30-1000 microns.
  • the filter device has a four-layer structure, the mesh diameter of the first layer is 500-800 microns, the mesh diameter of the second layer is 300-400 microns, the mesh diameter of the third layer is 200-300 microns, and the mesh diameter of the fourth layer is 100-200 microns.
  • the dissolution of the space-occupying material is achieved by mixing the carrier matrix to be treated with an organic solvent to dissolve the space-occupying material.
  • the mixing is carried out at a temperature of 15°C-150°C, preferably 25-100°C (such as 25-30°C, 30-35°C, 35-40°C, 40-45°C or 45-50°C, specifically 25°C) for 4-48h, preferably 6-24h (such as 6-8h, 8-10h, 10-12h, 12-14h, 14-16h, 1 6-18h or 18-20h, specifically as 6h).
  • the organic solvent is selected from acetone, absolute ethanol, petroleum ether, chloroform, dimethyl sulfoxide, dichloromethane, petroleum ether, carbon tetrachloride, acetonitrile, toluene, methanol or any combination thereof.
  • the organic solvent is absolute ethanol.
  • the conjugation of the post-treated carrier matrix with the short peptide or protein comprising the RGD amino acid sequence is achieved by the following steps: mixing the post-treated carrier matrix with the short peptide or protein comprising the RGD amino acid sequence, so as to achieve the RGD surface modification of the post-treated carrier matrix.
  • the mixing is performed at a temperature of 15-25°C, preferably 20°C.
  • the mixing time is 4-10 hours, preferably 6 hours.
  • the short peptide or protein comprising the amino acid sequence of RGD is selected from collagen or collagen derivatives, gelatin or gelatin derivatives, fibronectin, silk fibroin, laminin, matrigel or any combination thereof.
  • the short peptide or protein comprising the amino acid sequence of RGD is selected from collagen and gelatin.
  • the short peptide or protein comprising the RGD amino acid sequence is provided in the form of a solution.
  • the solvent of the solution is a solvent commonly used by those skilled in the art when modifying RGD, for example, in some embodiments, the solvent is water (such as deionized water).
  • the mass fraction of the short peptide or protein comprising the RGD amino acid sequence is the mass fraction commonly used by those skilled in the art when modifying RGD.
  • the mass fraction of the short peptide or protein comprising the RGD amino acid sequence in the solution is 0.1%-20%, such as 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%.
  • the present invention provides a kit comprising the aforementioned microcarriers or the aforementioned microcarrier aggregates or the microcarriers prepared by the aforementioned methods.
  • the present invention provides the use of the aforementioned microcarriers or the aforementioned microcarrier aggregates or the microcarriers prepared by the aforementioned method in the preparation of a kit for three-dimensional cell culture and/or expansion, 3D bioprinting, biomimetic structure construction, cell therapy, drug loading or medical injection.
  • the biomimetic structure is a three-dimensional construct, tissue precursor, tissue, organ, or embryo (such as a mouse embryo).
  • the biomimetic structure is an embryonic biomimetic construct or a cartilage biomimetic construct.
  • the biomimetic structure is a cartilage lacuna structure of articular cartilage.
  • the cell therapy is skin surface therapy, clinical corneal therapy, bone therapy, or cartilage repair and bone damage therapy.
  • the present invention provides the use of the aforementioned microcarriers or the aforementioned microcarrier aggregates or the microcarriers prepared by the aforementioned methods, which are used for in vitro cell three-dimensional culture and/or expansion, in vitro 3D bioprinting, in vitro biomimetic structure construction, and in vitro drug loading; preferably, the uses are for non-diagnostic or therapeutic purposes.
  • the biomimetic structure is a three-dimensional construct, tissue precursor, tissue, organ, or embryo (such as a mouse embryo).
  • the biomimetic structure is an embryonic biomimetic construct or a cartilage biomimetic construct.
  • the biomimetic structure is a cartilage lacuna structure of articular cartilage.
  • the present invention provides a method for three-dimensional cell culture and/or expansion in vitro, comprising:
  • step 1) further include:
  • step 1) further include:
  • the culture is a dynamic culture.
  • the dynamic culture is agitated dynamic culture or suspension dynamic culture.
  • the cell is as described in the first aspect.
  • the microcarriers of the present invention can well make up for the shortcomings of two-dimensional cell culture, and show a large number of advantages: (1) cells get rid of the limitation of plane culture space, no longer rely on the plane, but grow on three-dimensional space, and can harvest a large number of cells in a short period of time; (2) microcarriers and concave structures provide cells with a large amount of attachment area, so that the cells can be better connected and build a more friendly growth microenvironment for cells.
  • the more flexible structural features can provide a friendly living environment for cells and provide more unique structural support.
  • the presence of a cavity structure improves the permeability of microcarriers, speeds up the exchange and delivery of nutrients, and greatly improves cell survival. Rate and expansion rate is conducive to rapid expansion of cells in a limited space at the laboratory level; the more flexible structural characteristics of three-dimensional cell culture microcarriers with cavity structures can meet various application scenarios such as biology, medical treatment, and clinical practice.
  • the microcarrier of the present invention has good biocompatibility, can realize good adhesion of cells, and realize growth and proliferation of cells.
  • the microcarrier of the present invention can realize the penetration of the culture medium in a short time, which is beneficial to the exchange of substances between the cells and the outside world. After the cells are overgrown on the surface of the microcarrier, new microcarriers are put into it. When the ball contacts the ball, the cell can grow across the ball.
  • the microcarrier is used to carry out long-term culture of the cells, and the cells still maintain a high activity rate and proliferation after a long period of culture (for example, 7 days).
  • the stem cells When the microcarrier of the present invention is used to culture and expand stem cells, the stem cells have good adhesion, and the long-term culture of stem cells maintains a relatively high activity rate and proliferation rate.
  • microcarrier of the present invention for the culture and expansion of rat chondrocytes can realize large-scale expansion culture, can simulate the culture environment in vivo, and maintain the biological functions of chondrocytes.
  • the surface of the microcarrier of the present invention has a large concave structure. Using this structural feature, after the inoculation of rat chondrocytes on the microcarrier, the aggregation, growth and proliferation of chondrocytes can be promoted to simulate the cartilage lacuna structure of the articular cartilage in the body. At the same time, the microcarrier can be modified with polypeptides and factors to promote the secretory function of chondrocytes.
  • microcarrier of the present invention Utilize the microcarrier of the present invention to carry out the encapsulation and cultivation of stem cells. After the stem cells are encapsulated in the microcarriers, they have good biological activity. After adding inducing factors, they can carry out directional differentiation in the microcarriers. After cultivating for a long time (for example, 7 days), the cells still maintain a relatively high rate of activity and proliferation.
  • the microcarrier of the present invention is used to simulate the bionic structure of mouse embryos, and the mouse embryonic stem cells are encapsulated inside the microcarrier, which has the potential to induce differentiation into three germ layers in vitro; the surface viscosity of the microcarrier mouse trophoblast stem cells can realize the interaction with the internal mouse embryonic stem cells, and has the potential of mouse embryo implantation.
  • conventional cell cryopreservation refers to the cryopreservation of cells together with the cryopreservation solution, wherein the cryopreservation solution contains 10% CPA (antifreezing agent, which is toxic to cells).
  • CPA antifreezing agent
  • the microcarrier of the present invention can be directly (ie together with the cells) frozen at -80°C in situ after cell culture, which can reduce antifreeze (CPA) by 50%, and the cell viability after recovery can reach more than 70%.
  • the in situ cryopreservation means that the cells can be frozen together with the microcarriers and the cryopreservation solution after the cells proliferate on the microcarriers of the present invention, wherein the concentration of CPA in the cryopreservation solution can be reduced from the original 10% to 5%.
  • microcarriers cannot completely separate the cells through digestion after cell proliferation and overgrowth, and some microcarriers have changed cell functionality after cell proliferation and digestion, for example, stem cells no longer have stemness and so on.
  • the microcarrier of the present invention can carry out large-scale expansion of cells. After the amplification is completed, the cells can be directly digested and collected, and the microcarrier is used for cell amplification. After 7 days of culture, the cell amplification can reach 12 times. More importantly, the functionality of the cells (such as the stemness of the cells, that is, the ability to differentiate) remains stable.
  • the microcarriers of the present invention can be used for large-scale dynamic cell culture, including agitated dynamic culture and suspension dynamic culture.
  • the microcarriers can withstand large shear force, can provide protection to cells from shear damage, and the microcarriers have good permeability. After the cells adhere, material exchange can be carried out in time. Using this method to amplify cells, the cells are in good condition, have a high activity rate and proliferation rate, and more importantly, the functionality of the cells remains stable.
  • Fig. 1 is the microcarrier structure SEM figure of the embodiment of the present invention.
  • Fig. 2 is the microcarrier microtopography figure of the embodiment of the present invention.
  • Fig. 3 is the micro-topography figure of the microcarrier of the embodiment of the present invention.
  • A is the microtopography figure of the mouse cartilage section
  • B is the cell adhesion figure after co-culture of chondrocytes and microcarriers for 1 day;
  • Fig. 5 is the schematic flow chart of microcarrier preparation of the present invention.
  • Fig. 6 is the microscopic appearance figure of the microcarrier of the embodiment of the present invention.
  • Fig. 7 is the microscopic topography figure of the microcarrier of the embodiment of the present invention.
  • Fig. 8 is the micro-topography figure of the microcarrier of the embodiment of the present invention.
  • Fig. 9 is the microscopic appearance figure of the microcarrier of the embodiment of the present invention.
  • Fig. 10 is the microscopic appearance figure of the microcarrier of the embodiment of the present invention.
  • Fig. 11 is the micro-topography figure of the microcarrier of the embodiment of the present invention.
  • Fig. 12 is the micro-topography figure of the microcarrier of the embodiment of the present invention.
  • Fig. 13 is the cell death staining result figure after the microcarrier cryopreservation of the embodiment of the present invention.
  • Fig. 14 is the living and dead result figure of the microcarrier dynamic culture cell of the embodiment of the present invention.
  • Fig. 15 is the microcarrier dynamic culture cell proliferation curve diagram of the embodiment of the present invention.
  • Fig. 16 is a characterization diagram of cell stemness after the microcarrier dynamically cultures cells according to an embodiment of the present invention.
  • Fig. 17 is the microscopic appearance figure of the microcarrier of the embodiment of the present invention.
  • Fig. 18 is a microcarrier permeability characterization diagram of an embodiment of the present invention.
  • the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.
  • the reagents used in the examples of the present invention are commercially available.
  • the shape and structure of the microcarriers will not change before and after the hydration swelling of the microcarriers.
  • Embodiment 1 the preparation of microcarrier
  • the diameter of the micro-carrier is between 30-400 microns, and it is spherical or granular after freeze-drying.
  • the microcarrier swelled, and further observation showed that it had a regular spherical surface with depressions. As shown in Figure 1, the proportion of depressions was about 85%, and the number of depressions was about 40.
  • Embodiment 2 the preparation of microcarrier
  • the mixed organic phase liquid paraffin and Span 80 into a reaction device with a stirrer, wherein the volume ratio of liquid paraffin and Span 80 is 300:1, heat to above 60°C, stir to make it evenly mixed, and the water phase consists of HA (sodium hyaluronate) (5wt%) and water-insoluble material, that is, agarose with uniform particle size (0.5wt%) and cross-linking agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (5wt%) ) mixed to form; after the organic phase is mixed evenly, turn it to room temperature, slowly add the mixed water phase into the organic phase under the action of the stirring blade, and stir at room temperature to emulsify it.
  • HA sodium hyaluronate
  • water-insoluble material that is, agarose with uniform particle size (0.5wt%) and cross-linking agent 1-(3-dimethylaminopropyl)-3-ethylcarbodi
  • the diameter of the micro-carrier is between 30-400 microns, and it is spherical or granular after freeze-drying. After the microcarrier was hydrated overnight, the microcarrier swelled, and further observation showed that it had a regular spherical surface with depressions. As shown in Figure 2, the proportion of depressions was about 50%, and the number of depressions was about 30.
  • Embodiment 3 the preparation of microcarrier
  • the mixed organic phase liquid paraffin and Span 80 into a reaction device with a stirrer, wherein the volume ratio of liquid paraffin and Span 80 is 300:1, heat to above 60°C, stir to make it evenly mixed, and the water phase consists of HA (sodium hyaluronate) (5wt%) and water-insoluble material, namely agarose (0.1wt%) with a uniform particle size, and a cross-linking agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (5wt%) %) mixed to form; after the organic phase is evenly mixed, turn it to room temperature, slowly add the mixed water phase into the organic phase under the action of the stirring blade, and stir at room temperature to emulsify it.
  • HA sodium hyaluronate
  • water-insoluble material namely agarose (0.1wt%) with a uniform particle size
  • the diameter of the micro-carrier is between 30-400 microns, and it is spherical or granular after freeze-drying. After the microcarrier was hydrated overnight, the microcarrier swelled, and further observation showed that it had a regular spherical surface with depressions. As shown in Figure 3, the proportion of depressions was about 20%, and the number of depressions was about 10.
  • Embodiment 4 the preparation of microcarrier
  • the mixed organic phase liquid paraffin and Span 80 into a reaction device with a stirrer, wherein the volume ratio of liquid paraffin and Span 80 is 300:1, heat to above 60°C, stir to make it evenly mixed, and the water phase consists of HA (sodium hyaluronate) (5wt%) and water-insoluble material, namely agarose (0.5wt%) with uniform particle size, and cross-linking agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (5wt%) %) mixed to form a composition; after the organic phase is uniformly mixed, turn it to room temperature, slowly add the mixed water phase to the organic phase under the action of the stirring blade, and stir at room temperature to emulsify it.
  • HA sodium hyaluronate
  • water-insoluble material namely agarose (0.5wt%) with uniform particle size
  • the obtained microcarriers can be observed by scanning electron microscope.
  • the diameter of the microcarriers is between 30-400 microns, and they are spherical or granular after freeze-drying, as shown in FIG. 6 .
  • Embodiment 5 the preparation of microcarrier
  • the diameter of the microcarriers is between 30-400 microns, and they are spherical or granular after freeze-drying, as shown in Figure 7.
  • the proportion of elongated depressions is about 90%, and the number of depressions is about 50.
  • Test example 1 Bionic construction of cartilage lacuna structure
  • the inventors inoculated GFP green fluorescence-labeled primary rat chondrocytes (from 4-week-old rat cartilage) onto the microcarriers prepared in Example 4 at a density of 1 ⁇ 10 5 cells/mg, and cultured the inoculated microcarriers in a constant temperature incubator at 37°C. After culturing for 24 hours, they were observed using a laser scanning confocal microscope. It was found that chondrocytes adhered and proliferated on the microcarriers, and first adhered to the depression, as shown in Figure 4B. After normal human cartilage matures, chondrocytes gather and secrete matrix proteins to form a cartilage lacuna structure, and its physiological diameter is also about 30 ⁇ m, as shown in Figure 4A.
  • the microcarrier of the present invention can simulate the normal cartilage lacuna structure, and can effectively inhibit the dedifferentiation performance caused by the proliferation of chondrocytes in vitro.
  • Test Example 2 In situ cryopreservation of microcarriers and cells
  • the inventor inoculated human umbilical cord mesenchymal stem cells onto the microcarriers prepared in Example 4 at a density of 1 ⁇ 10 5 cells/mg, and placed the inoculated microcarriers in a 37°C constant temperature incubator for cultivation. After culturing for 72 hours (in which the medium was changed every other day), the cells had already overgrown the microcarriers. The culture medium was discarded, and the cryopreservation solution was added, and the configuration of the cryopreservation solution was 90% FBS, 10% glycerol, and 95% FBS, 5% glycerol. After being frozen for 1 day, it was revived, revived and cultured for 24 hours, and then stained with live&dead staining solution and observed with a laser scanning confocal microscope. The results are shown in Figure 13.
  • green means living cells
  • red means dead cells
  • microcarriers have no color display.
  • the 10% glycerol group is the control group
  • the commonly used cryopreservation solution ratio is 10% glycerol 90% FBS.
  • the inventors reduced the glycerol concentration to the original 50%, that is, using 5% glycerol 95% FBS. It was found that the cell viability was almost the same as that of the control group, and the number of living cells (green fluorescence) reached more than 70% of the total number.
  • the microcarrier of the present invention can be directly frozen in situ after cell culture, can effectively inhibit the use of CPA (mainly DMSO and glycerol), and has a higher cell viability after freezing.
  • CPA mainly DMSO and glycerol
  • Test Example 3 Microcarriers can maintain the functionality of cultured cells
  • the inventor resuspended the human umbilical cord mesenchymal stem cells in the culture medium to obtain a cell suspension.
  • 1.6 ⁇ 10 7 cells were inoculated into a bioreactor equipped with microcarriers and 80 ml medium (the ratio of microcarriers to medium was 3 g/L), and the cell density was 2 ⁇ 10 5 cells/ml.
  • the inoculated bioreactor was placed in a 37° C. carbon dioxide incubator for cultivation, and the stirring program of the reactor was 40 rpm/min, 3 min; 1 rpm/min, 1 h; the cycle program was 24 times. 24 hours after inoculation, the program was changed to a constant speed of 40rpm.
  • the medium was changed once respectively, and the volume of the medium changed was 60ml/time.
  • the commercially available microcarrier cytodex3 from Cytiva Company of the United States was selected as the control group for comparison.
  • the experimental group selected the microcarriers prepared in Example 3 above.
  • the cells in the experimental group were harvested, stained with live&dead staining solution and observed with a laser scanning confocal microscope (as shown in Figure 14), digested and counted, and measured the proliferation of the cells on the microcarrier with a CCK8 detection kit (as shown in Figure 15) and the results of dynamic culture and the expression of cell functionality (stemness) after the cells were digested (as shown in Figure 16).
  • Figure 14 Results Green is live cells, red is dead cells.
  • the results show that the prepared microcarrier has excellent biocompatibility, enables cells to adhere and proliferate, and has anti-shear performance. In the case of stirring, the cells can be protected, the damage to the cells by the shear force can be reduced, and the cells can proliferate on the microcarrier and maintain a high activity rate.
  • the main operation method is: take about 10 mg of the lyophilized microcarrier in a 15ml centrifuge tube, add PBS to hydrate overnight, then discard the supernatant, wash with PBS 2-3 times, and discard the supernatant. Place the microcarriers in the fluorescently labeled (488) medium (the medium needs to be submerged into the microcarriers), and observe them with a laser scanning confocal microscope at 10 minutes, 20 minutes, and 40 minutes respectively. The results are shown in Figure 18.
  • Fig. 18a is the microcarrier infiltration result figure when 10min, and wherein green fluorescent part is the substratum of mark, and black is the microcarrier of unlabeled fluorescence
  • Fig. 18b is the infiltration result when infiltration time is 20min, and it can be seen that the culture medium has penetrated into the cavity gradually
  • Fig. 18c is the infiltration result when infiltration time is 40min, it can be seen that the culture medium has completely penetrated into the microcarrier cavity.
  • microcarrier of the present invention has good permeability, complete penetration in 40 minutes, and is beneficial to cell culture and material exchange, and the microcarrier with cavity structure of the present invention has the conditions for cultivating cells inside the cavity.

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Abstract

本发明涉及一种微载体及其应用，该微载体具有三维立体结构，所述微载体具有壳层且内部为空心或实心，所述壳层外壁不平整且具有多处凹陷点。该微载体提高了传统微载体的渗透性，有利于细胞与外界环境进行物质交换，提高了细胞活率，为细胞增殖提供了良好的机械保护以及构建良好的细胞生长的三维结构，实现了细胞的友好连接，有利于细胞在有限空间中的大规模扩增，以及在长期培养中维持细胞理化性质的稳定。

Description

一种微载体及其应用 技术领域
本发明涉及生物材料领域，具体涉及一种微载体及其应用。
背景技术
随着生命医学领域的快速发展，二维细胞培养无法满足实验室及企业对于细胞数量的需求。相比于三维细胞培养，二维细胞培养的劣势越来越凸显，主要表现在：(1)二维细胞培养只能用培养皿进行细胞的培养，贴壁生长的细胞增殖受限于皿底的大小，长期培养需要耗费大量的人力物力，且无法实现短时间的大规模扩增，资源损耗较大；(2)二维培养细胞无法为细胞的生长构建一个友好的微环境，细胞在平面上增殖，长期培养会影响细胞的结构，为后续研究带来困难。
因此，能够为细胞提供三维培养的载体结构还需进一步研究。
发明内容
本发明旨在解决上述二维细胞培养的突出问题之一。本发明提供了一种微载体及其制备方法并对其应用进行了说明。本发明提供的微载体提高了传统微载体的渗透性，有利于细胞与外界环境进行物质交换，提高了细胞活率，为细胞增殖提供了良好的机械保护以及构建良好的细胞生长的三维结构，实现了细胞的友好连接，有利于细胞在有限空间中的大规模扩增，以及在长期培养中维持细胞理化性质的稳定；本发明的微载体具有更加灵活的结构特点，可以满足生物，医疗，临床治疗等多种应用场景。
为此，在本发明的第一方面，本发明提供了一种微载体，其中，所述微载体具有三维立体结构，所述微载体具有壳层且内部为空心或实心，所述壳层外壁不平整且具有多处凹陷点。
在一些实施方案中，所述壳层外壁的凹陷面积占壳层外壁总表面积的比例为15％以上，例如20％以上、25％以上、30％以上、35％以上、40％以上、45％以上、50％以上、55％以上、60％以上、65％以上、70％以上、75％以上、80％以上、85％以上、90％以上或95％以上，或者例如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％或90％-95％，具体例如15％、20％、50％、60％、80％、85％或90％。
在一些实施方案中，所述壳层外壁具有的凹陷点的数量为2～800个，可选的凹陷点的数量为2～700个，可选的凹陷点的数量为2～600个，可选的凹陷点的数量为2～500个，可选的凹陷点的数量为2～400个，可选的凹陷点的数量为2～300个，可选的凹陷点的数量为2～200个，可选的凹陷点的数量为5～200个，可选的凹陷点的数量为5～100个。
在一些实施方案中，所述壳层外壁具有的凹陷点的数量为5～60个，例如5-10、10-15、15-20、20-25、25-30、30-35、35-40、40-45、45-50、50-55或55-60个。在一些具体实施方案中，所述壳层外壁具有的凹陷点的数量为10～60个(具体例如为10个、20个、25个、30个、40个、50个或60个)。
在一些实施方案中，所述壳层外壁具有的凹陷点的形状选自圆弧状凹陷(或称为弧形凹陷)、细槽型凹陷(或称为狭长型凹陷)、球形凹陷、多边形凹陷、不规则凹陷或其任意组合。
在一些实施方案中，所述圆弧状凹陷的平均半径为1～500μm，可选的平均半径为1～400μm，可选的平均半径为1～300μm，可选的平均半径为10～300μm，可选的平均半径为50～300μm，可选的平均半径为80～300μm，可选的平均半径为80～200μm。
在一些实施方案中，所述圆弧状凹陷的平均半径为1-100μm，例如，1-10μm、10-20μm、20-30μm、30-40μm、40-50μm、50-60μm、60-70μm、70-80μm、80-90μm或90-100μm。
在一些实施方案中，所述细槽型凹陷的凹陷面的平均宽度为1～500μm，可选的凹陷面的平均宽度为1～400μm，可选的凹陷面的平均宽度为1～300μm，可选的凹陷面的平均宽度为10～300μm，可选的凹陷面的平均宽度为50～300μm，可选的凹陷面的平均宽度为80～300μm，可选的凹陷面的平均宽度为80～200μm。
在一些实施方案中，所述细槽型凹陷的平均宽度为1-100μm，例如，1-10μm、10-20μm、20-30μm、30-40μm、40-50μm、50-60μm、60-70μm、70-80μm、80-90μm或90-100μm。
在一些实施方案中，所述多边形凹陷的凹陷面的平均宽度为1～500μm，可选的凹陷面的平均宽度为1～400μm，可选的凹陷面的平均宽度为1～300μm，可选的凹陷面的平均宽度为10～300μm，可选的凹陷面的平均宽度为50～300μm，可选的凹陷面的平均宽 度为80～300μm，可选的凹陷面的平均宽度为80～200μm。
在一些实施方案中，所述多边形凹陷的凹陷面的平均宽度为1-100μm，例如，1-10μm、10-20μm、20-30μm、30-40μm、40-50μm、50-60μm、60-70μm、70-80μm、80-90μm或90-100μm。
在一些实施方案中，所述不规则凹陷的凹陷面的平均宽度为1～500μm，可选的凹陷面的平均宽度为1～400μm，可选的凹陷面的平均宽度为1～300μm，可选的凹陷面的平均宽度为10～300μm，可选的凹陷面的平均宽度为50～300μm，可选的凹陷面的平均宽度为80～300μm，可选的凹陷面的平均宽度为80～200μm。
在一些实施方案中，所述不规则凹陷的凹陷面的平均宽度为1-100μm，例如，1-10μm、10-20μm、20-30μm、30-40μm、40-50μm、50-60μm、60-70μm、70-80μm、80-90μm或90-100μm。
在一些实施方案中，所述球形凹陷的平均半径小于500μm，可选的平均半径小于400μm，可选的平均半径小于300μm，可选的平均半径小于200μm，可选的平均半径小于100μm，可选的平均半径小于50μm，可选的平均半径小于20μm，可选的平均半径小于10μm。
在一些实施方案中，所述球形凹陷的平均半径小于100μm，例如小于90μm、小于80μm、小于70μm、小于60μm、小于50μm、小于40μm、小于30μm、小于20μm或小于10μm。
需要说明的是，所述平均宽度指的是最短边的平均长度，例如，所述细槽型凹陷的平均宽度，指的是所述细槽型凹陷的最短边的平均长度。
在一些实施方案中，所述微载体的平均粒径为1～3000μm，可选的平均粒径为1～2000μm，可选的平均粒径为1～1000μm，可选的平均粒径为1～800μm，可选的平均粒径为1～600μm，可选的平均粒径为10～600μm，可选的平均粒径为50～500μm，可选的平均粒径为100～500μm，可选的平均粒径为100～400μm。
在一些实施方案中，所述微载体的平均粒径为10-500μm，例如10-50μm、50-100μm、100-150μm、150-200μm、200-250μm、250-300μm、300-350μm、350-400μm、400-450μm或450-500μm。
在一些实施方案中，所述微载体的平均粒径为30-400μm。
在一些实施方案中，所述壳层的厚度为1-100μm，可选的厚度为1-90μm，可选的厚度为1-80μm，可选的厚度为1-70μm，可选的厚度为1-60μm，可选的厚度为1-50μm。
在一些实施方案中，所述壳层的厚度为1-100μm，例如1-10μm、10-20μm、20-30μm、30-40μm、40-50μm、50-60μm、60-70μm、70-80μm、80-90μm或90-100μm。
在一些实施方案中，所述微载体具有的三维立体结构为圆球、梭形、椭圆球、棒状、扁球体或不规则球体。在一些具体实施方案中，所述微载体具有的三维立体结构为圆球状。
在一些实施方案中，所述微载体的内部为空心，所述微载体具有非封闭式三维立体结构。在一些实施方案中，所述微载体的壳层具有一个或多个通孔，所述通孔穿透所述壳层以便联通壳层的内部和外部。
在一些实施方案中，所述微载体内部的空心结构通过溶出占位材料来得到。
在一些实施方案中，所述壳层的外壁和/或内壁经过表面修饰。
在一些实施方案中，所述壳层的外壁和/或内壁经过RGD表面修饰。
在一些实施方案中，所述微载体负载有细胞。
在一些实施方案中，所述细胞设置在所述微载体的壳层内壁，或者设置在所述微载体的壳层外壁，或者同时设置在所述微载体的壳层内壁和所述微载体的壳层外壁。
在一些实施方案中，所述细胞同时设置在所述微载体的壳层内壁和所述微载体的壳层外壁时，设置于壳层内壁和壳层外壁的细胞种类可以相同或不同。
在一些实施方案中，所述微载体的壳层外壁设置有细胞时，所述细胞设置在所述壳层外壁的凹陷点处，或者设置在所述壳层外壁的光滑表面处(即非凹陷点处)，或者同时设置在所述壳层外壁的凹陷点处和光滑表面处。
在一些实施方案中，所述微载体的壳层外壁设置有细胞时，所述细胞设置在所述壳层外壁的凹陷点处。
在一些实施方案中，所述细胞通过所述通孔设置在所述微载体的壳层内壁。
在一些实施方案中，所述细胞通过细胞注射设置在所述微载体的壳层内壁。
在一些实施方案中，所述细胞通过与所述微载体进行共培养设置在所述微载体的壳层外壁。
在一些实施方案中，所述细胞选自原核细胞、真核细胞、细菌细胞、古细菌细胞、单 细胞真核生物体的细胞、原生动物细胞、植物细胞、动物细胞、藻类细胞、真菌细胞、人造细胞或其任意组合。
在一些实施方案中，所述细胞选自干细胞、体细胞、生殖细胞或其任意组合。
在一些实施方案中，所述干细胞选自胚胎干(ES)细胞、诱导多能干(iPS)细胞、间充质干细胞、神经干细胞、肌肉干细胞、造血干细胞、上皮干细胞、乳腺干细胞、肠干细胞、中胚层干细胞、内皮干细胞或其任意组合。
在一些实施方案中，所述体细胞选自上皮细胞、内皮细胞、间皮细胞、成纤维细胞、成骨细胞、软骨细胞、肌细胞、骨细胞、肝细胞、胰腺细胞、外源细胞、内源细胞、心肌细胞、骨骼细胞、心脏成肌细胞、骨骼成肌细胞、少突胶质细胞、神经胶质细胞、造血细胞、神经元或其任意组合。
在一些实施方案中，所述生殖细胞选自卵母细胞、精子、卵原细胞、精原细胞或其任意组合。
在一些实施方案中，所述壳层或所述微载体由生物材料制成。即，若所述微载体内部为空心，则所述壳层由生物材料制成；若所述微载体内部为实心，则所述微载体由生物材料制成。
在一些实施方案中，所述生物材料选自人工合成的生物材料、天然生物材料或其组合。
在一些实施方案中，所述人工合成的生物材料选自聚乙二醇，聚乙二醇衍生物，聚乳酸，聚乳酸醇共聚物，聚酸酐，聚酸酯，聚氨基酸，聚氧化乙烯，聚酯，聚甲基丙烯酸甲酯，聚碳酸酯，聚氨基甲酸酯，聚己内酯，聚羟基脂肪酸酯，聚硅氧烷，聚乙烯，聚氯乙烯，聚四氟乙烯，聚苯乙烯，聚丙烯，马来酸酐接枝共聚物，聚丙烯酰胺，聚缩醛，聚吡咯或其任意组合。
在一些实施方案中，其中，所述天然生物材料选自天然蛋白质，胶原及胶原衍生物，明胶及明胶衍生物，琼脂及琼脂衍生物，蛋白多糖，海藻酸盐及其海藻酸盐衍生物，基质胶，蜂胶，纤维素及纤维素衍生物，甲壳素及甲壳素衍生物，蚕丝蛋白及其衍生物，层连接蛋白及其衍生物，纤维连接蛋白及其衍生物，透明质酸钠及透明质酸衍生物，琼脂糖及其衍生物，葡聚糖及其衍生物，蔗糖及蔗糖衍生物，淀粉，壳聚糖及壳聚糖衍生物或其任意组合。
在一些实施方案中，所述生物材料选自透明质酸钠、明胶。
在本发明的第二方面，本发明提供了一种微载体聚集体，其中，所述聚集体由前述的微载体颗粒聚集而成。
在一些实施方案中，所述聚集体是单一性状微载体的集合体，或各种性状微载体任意比例的集合体。
在本发明的第三方面，本发明提供了一种制备前述的微载体的方法，如图5，其包括：
(a)提供有机相溶液；
(b)提供水相溶液；
(c)将所述水相溶液加入所述有机相溶液，形成混合液；使所述混合液乳化，混合均匀，形成均一的油包水体系；
(d)将所述油包水体系进行过滤，滤饼为待处理的载体基质；
(e)将所述待处理的载体基质进行后处理，以便溶出占位材料，并用包含RGD氨基酸序列的短肽或蛋白与后处理后的载体基质进行缀合，获得所述微载体；
任选地，所述缀合后进一步包括清洗、筛分和冻干的步骤。
在一些实施方案中，所述乳化是在温度为4-200℃，如20-75℃，或者如20-25℃下进行的。
在一些实施方案中，所述乳化的时间是4-72h，优选5-30h，例如5-10h、10-15h、15-20h、20-25h或25-30h，具体如10h。
在一些实施方案中，所述乳化是通过搅拌法，超声法，震荡法或微流道法实现的。在一些实施方案中，乳化时的转速为100-5000rpm/min，优选为300-1000rpm/min，例如300-400rpm/min、400-500rpm/min、500-600rpm/min、600-700rpm/min、700-800rpm/min、800-900rpm/min或900-1000rpm/min，具体如600rpm/min。
在一些实施方案中，所述有机相溶液是通过将有机溶剂与非离子表面活性剂进行混合得到的。在一些实施方案中，所述混合是在温度为4-200℃，优选20-75℃(例如20-25℃、25-30℃、30-35℃、35-40℃、40-45℃、45-50℃、50-55℃、55-60℃、60-65℃或65-70℃，具体如60℃)下进行的。在一些实施方案中，所述混合的时间为10-120min，优选10-60min。在一些实施方案中，所述混合是在搅拌桨的作用下进行的。在一些实施方案中，所述搅拌桨的转速为100-2000rpm/min，优选为300-1000rpm/min，例如300-700 rpm/min。在一些实施方案中，所述有机溶剂选自液体石蜡，石油醚，四氯化碳，二甲基亚砜，三氯甲烷，二氯甲烷，食用油，硅油，大豆油，矿物油或其任意组合。在一些实施方案中，所述有机溶剂为液体石蜡。在一些实施方案中，所述非离子表面活性剂选自吐温，司班80，脂肪酸甘油酯，十二烷基苯磺酸钠，PO-500,氢氟醚，聚乙二醇，嵌段聚氧乙烯-聚氧丙烯醚(PO-EO共聚物)，多元醇酯类或其任意组合。在一些实施方案中，所述非离子表面活性剂为司班80。在一些实施方案中，所述有机溶剂与所述非离子表面活性剂的体积比为200-400:1，例如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或400:1，具体如300:1。
在一些实施方案中，所述水相溶液是通过如下步骤得到的：将生物材料溶解在水中，获得溶解液；占位材料和固化剂悬浮在所述溶解液中，形成分散均匀的悬浊液，所述悬浊液即为所述水相溶液。在一些实施方案中，所述溶解是在温度为4-200℃，优选10-80℃下进行的。在一些实施方案中，所述分散均匀的悬浊液是通过搅拌、震荡、超声或摇晃实现的，优选地，转速为100-2000rpm/min，优选为300-700rpm/min。
在一些实施方案中，所述水相溶液中，所述生物材料的质量分数为3-10％，例如为3％、4％、5％、6％、7％、8％、9％或10％，优选为5％-10％，具体如5％或10％。
在一些实施方案中，所述水相溶液中，所述占位材料的质量分数为0.01-1％，例如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％或1％，优选为0.05％-1％，具体如0.05％、0.1％、0.5％或1％。
在一些实施方案中，所述水相溶液中，所述固化剂的质量分数为0.5-7％，例如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％或7％，优选为0.5％-5％，具体如0.5％或5％。
在一些实施方案中，所述生物材料选自人工合成的生物材料、天然生物材料或其组合。
在一些实施方案中，所述人工合成的生物材料选自聚乙二醇，聚乙二醇衍生物，聚乳酸，聚乳酸醇共聚物，聚酸酐，聚酸酯，聚氨基酸，聚氧化乙烯，聚酯，聚甲基丙烯酸甲 酯，聚碳酸酯，聚氨基甲酸酯，聚己内酯，聚羟基脂肪酸酯，聚硅氧烷，聚乙烯，聚氯乙烯，聚四氟乙烯，聚苯乙烯，聚丙烯，马来酸酐接枝共聚物，聚丙烯酰胺，聚缩醛，聚吡咯或其任意组合。
在一些实施方案中，所述天然生物材料选自天然蛋白质，胶原及胶原衍生物，明胶及明胶衍生物，琼脂及琼脂衍生物，蛋白多糖，海藻酸盐及其海藻酸盐衍生物，基质胶，蜂胶，纤维素及纤维素衍生物，甲壳素及甲壳素衍生物，蚕丝蛋白及其衍生物，层连接蛋白及其衍生物，纤维连接蛋白及其衍生物，透明质酸钠及透明质酸衍生物，琼脂糖及其衍生物，葡聚糖及其衍生物，蔗糖及蔗糖衍生物，淀粉，壳聚糖及壳聚糖衍生物或其任意组合。
在一些实施方案中，所述生物材料选自透明质酸钠、明胶。
在一些实施方案中，所述占位材料选自聚乙二醇及聚乙二醇衍生物，石蜡球，氧化海藻酸盐及其衍生物，聚己内酯，二氧化硅，蜂蜡，蜂胶，琼脂，琼脂糖，海藻酸盐及其衍生物，大豆卵磷脂，蛋黄卵磷脂，磷脂，葡聚糖，壳聚糖，淀粉，明胶，透明质酸钠及透明质酸衍生物或其任意组合。
在一些实施方案中，所述占位材料选自琼脂糖、葡聚糖，透明质酸钠。
在一些实施方案中，所述固化剂选自N,N-亚甲基双丙烯酰胺，1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐，N-羟基琥珀酰亚胺，N-羟基硫代琥珀酰亚胺，二异氰酸酯，戊二醛，京平尼，硫酸铵，钙离子，丁二醇二缩水甘油醚，转谷酰胺酶，二乙烯基苯，己二酸二酰肼或其任意组合。
在一些实施方案中，所述固化剂选自1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐、戊二醛。
在一些实施方案中，所述过滤是通过过滤装置实现的，所述过滤装置的网孔直径为30-1000微米。
在一些实施方案中，所述过滤装置具有四层结构，第一层的网孔直径为500-800微米，第二层的网孔直径为300-400微米，第三层的网孔直径为200-300微米，第四层的网孔直径为100-200微米。
在一些实施方案中，所述溶出占位材料是通过以下步骤实现的：将所述待处理的载体基质与有机溶剂进行混合，以便溶出占位材料。在一些实施方案中，所述混合是在温度为15℃-150℃，优选25-100℃(如25-30℃、30-35℃、35-40℃、40-45℃或45-50℃，具体如 25℃)下进行4-48h，优选6-24h(如6-8h、8-10h、10-12h、12-14h、14-16h、16-18h或18-20h，具体如6h)。在一些实施方案中，所述有机溶剂选自丙酮，无水乙醇，石油醚，三氯甲烷，二甲基亚砜，二氯甲烷，石油醚，四氯化碳，乙腈，甲苯，甲醇或其任意组合。在一些实施方案中，所述有机溶剂为无水乙醇。
在一些实施方案中，所述用包含RGD氨基酸序列的短肽或蛋白与后处理后的载体基质进行缀合是通过以下步骤实现的：将所述后处理后的载体基质与所述包含RGD氨基酸序列的短肽或蛋白进行混合，以便实现所述后处理后的载体基质的RGD表面修饰。在一些实施方案中，所述混合是在温度为15-25℃，优选20℃的条件下进行的。在一些实施方案中，所述混合的时间为4-10小时，优选6小时。
在一些实施方案中，所述包含RGD氨基酸序列的短肽或蛋白选自胶原或胶原衍生物，明胶或明胶衍生物，纤维连接蛋白，丝素蛋白，层连接蛋白，基质胶或其任意组合。
在一些实施方案中，所述包含RGD氨基酸序列的短肽或蛋白选自胶原、明胶。
在一些实施方案中，所述包含RGD氨基酸序列的短肽或蛋白是以溶液的形式提供的。其中，所述溶液的溶剂是本领域技术人员在RGD修饰时常规使用的溶剂，例如，在一些实施方案中，溶剂为水(如去离子水)。另外，所述溶液中，所述包含RGD氨基酸序列的短肽或蛋白的质量分数是本领域技术人员在RGD修饰时常规使用的质量分数，例如，在一些实施方案中，所述溶液中，所述包含RGD氨基酸序列的短肽或蛋白的质量分数为0.1％-20％，例如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％或20％。
在本发明的第四方面，本发明提供了一种试剂盒，其包含前述的微载体或者前述的微载体聚集体或者前述的方法制备得到的微载体。
在本发明的第五方面，本发明提供了前述的微载体或者前述的微载体聚集体或者前述的方法制备得到的微载体在制备试剂盒中的用途，所述试剂盒用于细胞三维培养和/或扩增、3D生物打印、仿生结构体构建、细胞治疗、载药或医疗注射。
在一些实施方案中，所述仿生结构体为三维构建体、组织前体、组织、器官或胚胎(如小鼠胚胎)。
在一些实施方案中，所述仿生结构体构建为胚胎仿生模拟构建或软骨仿生构建。
在一些实施方案中，所述仿生结构体为关节软骨的软骨陷窝结构。
在一些实施方案中，所述细胞治疗为皮肤表面治疗、临床角膜治疗、骨治疗或软骨修复与骨损伤治疗。
在本发明的第六方面，本发明提供了前述的微载体或者前述的微载体聚集体或者前述的方法制备得到的微载体的用途，其用于体外细胞三维培养和/或扩增、体外3D生物打印、体外仿生结构体构建、体外载药；优选地，所述用途用于非诊断或治疗目的。
在一些实施方案中，所述仿生结构体为三维构建体、组织前体、组织、器官或胚胎(如小鼠胚胎)。
在一些实施方案中，所述仿生结构体构建为胚胎仿生模拟构建或软骨仿生构建。
在一些实施方案中，所述仿生结构体为关节软骨的软骨陷窝结构。
在本发明的第七方面，本发明提供了一种体外细胞三维培养和/或扩增的方法，其包括：
1)将细胞接种于前述的微载体或者前述的微载体聚集体或者前述的方法制备得到的微载体上进行培养(例如在37℃二氧化碳恒温培养箱中培养)；
任选地，步骤1)后，进一步包括：
2-A)将步骤1)获得的混合物进行原位冻存(如-80℃冻存)；
或者任选地，步骤1)后，进一步包括：
2-B)将步骤1)获得的负载于微载体上的细胞进行消化、收集。
在一些实施方案中，所述培养为动态培养。
在一些实施方案中，所述动态培养为搅拌式动态培养或悬浮式动态培养。
在一些实施方案中，所述细胞如第一方面所述。
发明的有益效果
1、本发明的微载体可以很好的弥补二维细胞培养出现的缺点，表现出大量的优势：(1)细胞摆脱平面培养空间的限制，不再依赖于平面内，而是在三维空间上生长，短期内可收获大量细胞；(2)微载体及凹陷结构为细胞提供大量的贴附面积，使细胞连结性更好为细胞构建更加友好的生长微环境，更进一步仿生模拟体内细胞生存环境，有利于维持 细胞的理化性质；(3)空腔结构细胞三维培养微载体有更加灵活的结构特点，能够为细胞提供友好生存环境的同时提供更加独特的结构支撑，与传统微载体相比有空腔结构的存在提高了微载体的渗透性，加快了营养物质的交换与输送，大幅提高细胞存活率及扩增速率，有利于在实验室级别有限的空间内实现细胞的快速扩增；空腔结构细胞三维培养微载体更加灵活的结构特点可满足生物，医疗，临床等多种应用场景。
2、本发明的微载体生物相容性好，能够实现细胞的良好黏附，实现细胞的生长和增殖。
3、本发明的微载体能够在短时间内实现培养基的渗透，有利于细胞与外界进行物质交换，细胞在微载体表面长满后，投入新的微载体，当球与球接触后，细胞可以实现跨球生长。利用该微载体对细胞进行长期培养，培养较长时间(例如7天)后细胞仍保持较高活率和增殖。
4、利用本发明的微载体进行干细胞的培养和扩增，干细胞粘附性良好，长期培养干细胞保持较高活率和增殖率。
5、利用本发明的微载体用于大鼠软骨细胞进行培养和扩增能够实现大规模扩增培养，可以模拟体内培养环境，维持软骨细胞的生物功能。
6、本发明的微载体表面有大的凹陷结构，利用该结构特点，在微载体上接种大鼠软骨细胞后能够促进软骨细胞的聚集生长与增殖进而模拟体内关节软骨的软骨陷窝结构，同时可以对微载体进行多肽和因子修饰促进软骨细胞的分泌功能。
7、利用本发明的微载体进行干细胞的封装和培养，将干细胞封装在微载体后具有良好的生物活性，加入诱导因子后可在微载体内进行定向分化，培养较长时间(例如7天)后细胞仍保持较高活率和增殖。
8、本发明的微载体用于小鼠胚胎仿生结构模拟，将小鼠胚胎干细胞封装在微载体内部，具有体外诱导分化成三胚层的潜能；微载体表面粘度小鼠滋养层干细胞，可以实现与内部小鼠胚胎干细胞的相互作用，具备小鼠胚胎植入的潜能。
9、对于本领域技术人员来说，常规的细胞冻存都是指细胞和冻存液一起冻存,其中冻存液中包含10％CPA(防冻剂，对细胞有一定毒性)。而本发明的微载体在进行细胞培养后可直接(即连同细胞一起)进行原位-80℃冻存，可将防冻剂(CPA)减少50％，复苏后细胞活率可达70％以上。需要说明的是，这里的原位冻存是指细胞在本发明的微载 体上增殖长满以后可以连同微载体一起与冻存液冻存，其中冻存液中的CPA浓度可从原来的10％减少至5％。
10、现有技术中有些微载体在细胞增殖长满后，无法通过消化将细胞完全分离下来，还有些微载体在细胞增殖长满且消化后，细胞的功能性发生了改变，例如，干细胞不再具有干性等等。而本发明的微载体可以进行细胞大规模扩增，扩增完成后可直接对细胞进行消化收集，使用该微载体进行细胞扩增，培养7天后细胞扩增可达12倍，更重要的是，细胞的功能性(例如细胞的干性即分化能力)保持稳定。
11、本发明的微载体可以进行细胞大规模动态培养，包含搅拌式动态培养及悬浮式动态培养，在动态培养过程中，该微载体可耐受较大剪切力，可以对细胞提供保护使细胞免受剪切损害，且微载体渗透性良好，细胞粘附后可以及时进行物质交换，利用该方法扩增细胞，细胞状态良好，具备较高活率及增殖率，更重要的是，细胞的功能性保持稳定。
附图说明
图1为本发明实施例的微载体结构SEM图；
图2为本发明实施例的微载体显微形貌图；
图3为本发明实施例的微载体显微形貌图；
图4中A为鼠软骨切片显微形貌图，B为软骨细胞与微载体共培养1天后细胞粘附图；
图5为本发明微载体制备流程示意图；
图6为本发明实施例的微载体显微形貌图；
图7为本发明实施例的微载体显微形貌图；
图8为本发明实施例的微载体显微形貌图；
图9为本发明实施例的微载体显微形貌图；
图10为本发明实施例的微载体显微形貌图；
图11为本发明实施例的微载体显微形貌图；
图12为本发明实施例的微载体显微形貌图；
图13为本发明实施例的微载体冻存后细胞活死染色结果图；
图14为本发明实施例的微载体动态培养细胞活死结果图；
图15为本发明实施例的微载体动态培养细胞增殖曲线图；
图16为本发明实施例的微载体动态培养细胞后细胞干性表征图；
图17为本发明实施例的微载体显微形貌图；
图18为本发明实施例的微载体渗透性表征图。
具体实施方式
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。除非特别说明，本发明采用的试剂、方法和设备为本领域常规试剂、方法和设备。除非特别说明，本发明实施例所用试剂均为市购。
需要说明的是，以下实施例中，微载体水化溶胀之前和之后，微载体的形状构造不会发生变化。
实施例1：微载体的制备
将混合好的有机相液体石蜡与司班80放入带有搅拌器的反应装置中，其中液体石蜡与司班80体积比为300:1，加热至60℃以上，搅拌使其混合均匀，水相由HA(透明质酸钠)(5wt％)与不溶于水的占位材料即粒径均匀的琼脂糖(1wt％)以及交联剂1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(5wt％)混合形成；待有机相混合均匀后，转至室温条件下，在搅拌浆的作用下向有机相中缓慢滴加混合好的水相，常温搅拌使其乳化。室温下反应10小时后过滤，其中搅拌速度控制在600rpm/min,待微载体成球后洗去有机相。过滤微载体，并将过滤后的载体材料转移到装有无水乙醇的试剂瓶中，其中有机溶剂要完全浸没载体，25℃摇床摇6小时，使得载体内部占位材料充分溶解并完全溶出。之后过滤材料并对载体材料进行去离子水冲洗进一步洗去多余的有机溶剂，最后将材料投入到含有RGD位点的氨基酸溶液(胶原溶液)中，20℃下搅拌6小时，之后清洗、筛分、冻干，获得微载体。
将获得的微载体利用扫描电镜进行观察可得，该微载体直径在30-400微米之间，冻干后呈圆球状或颗粒状。另外，将该微载体水化一夜后微载体溶胀，进一步观察可得，其呈规则球状表面带有凹陷，如图1所示，凹陷比例占比约85％，凹陷个数约40个。
实施例2：微载体的制备
将混合好的有机相液体石蜡与司班80放入带有搅拌器的反应装置中，其中液体石蜡与司班80体积比为300:1，加热至60℃以上，搅拌使其混合均匀，水相由HA(透明质酸钠)(5wt％)与不溶于水的占位材料即粒径均匀的琼脂糖(0.5wt％)以及交联剂1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(5wt％)混合形成；待有机相混合均匀后，转至室温条件下，在搅拌浆的作用下向有机相中缓慢滴加混合好的水相，常温搅拌使其乳化。室温下反应10小时后过滤，其中搅拌速度控制在600rpm/min,待微载体成球后洗去有机相。过滤微载体，并将过滤后的载体材料转移到装有无水乙醇的试剂瓶中，其中有机溶剂要完全浸没载体，25℃摇床摇6小时，使得载体内部占位材料充分溶解并完全溶出。之后过滤材料并对载体材料进行去离子水冲洗进一步洗去多余的有机溶剂，最后将材料投入到含有RGD位点的氨基酸溶液(胶原溶液)中，20℃下搅拌6小时，之后清洗、筛分、冻干，获得微载体。
将获得的微载体利用扫描电镜进行观察可得，该微载体直径在30-400微米之间，冻干后呈圆球状或颗粒状。将该微载体水化一夜后微载体溶胀，进一步观察可得，其呈规则球状表面带有凹陷，如图2所示，凹陷比例占比约50％，凹陷个数约30个。
实施例3：微载体的制备
将混合好的有机相液体石蜡与司班80放入带有搅拌器的反应装置中，其中液体石蜡与司班80体积比为300:1，加热至60℃以上，搅拌使其混合均匀，水相由HA(透明质酸钠)(5wt％)与不溶于水的占位材料即粒径均匀的琼脂糖(0.1wt％)，以及交联剂1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(5wt％)混合形成；待有机相混合均匀后，转至室温条件下，在搅拌浆的作用下向有机相中缓慢滴加混合好的水相，常温搅拌使其乳化。室温下反应10小时后过滤，其中搅拌速度控制在600rpm/min,待微载体成球后洗去有机相。过滤微载体，并将过滤后的载体材料转移到装有无水乙醇的试剂瓶中，其中有机溶剂 要完全浸没载体，25℃摇床摇6小时，使得载体内部占位材料充分溶解并完全溶出。之后过滤材料并对载体材料进行去离子水冲洗进一步洗去多余的有机溶剂，最后将材料投入到含有RGD位点的氨基酸溶液(胶原溶液)中，20℃下搅拌6小时，之后清洗、筛分、冻干，获得微载体。
将获得的微载体利用扫描电镜进行观察可得，该微载体直径在30-400微米之间，冻干后呈圆球状或颗粒状。将该微载体水化一夜后微载体溶胀，进一步观察可得，其呈规则球状表面带有凹陷，如图3所示，凹陷比例占比约20％，凹陷个数约10个。
实施例4：微载体的制备
将混合好的有机相液体石蜡与司班80放入带有搅拌器的反应装置中，其中液体石蜡与司班80体积比为300:1，加热至60℃以上，搅拌使其混合均匀，水相由HA(透明质酸钠)(5wt％)与不溶于水的占位材料即粒径均匀的琼脂糖(0.5wt％)，以及交联剂1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(5wt％)混合形成构成；待有机相混合均匀后，转至室温条件下，在搅拌浆的作用下向有机相中缓慢滴加混合好的水相，常温搅拌使其乳化。室温下反应10小时后过滤，其中搅拌速度控制在600rpm/min,待微载体成球后洗去有机相。过滤微载体，并将过滤后的载体材料转移到装有无水乙醇的试剂瓶中，其中有机溶剂要完全浸没载体，25℃摇床摇6小时，使得载体内部占位材料充分溶解并完全溶出。之后过滤材料并对载体材料进行去离子水冲洗进一步洗去多余的有机溶剂，最后将材料投入到含有RGD位点的氨基酸溶液(胶原溶液)中，20℃下搅拌6小时，之后清洗、筛分、冻干，获得微载体。
将获得的微载体利用扫描电镜进行观察可得，该微载体直径在30-400微米之间，冻干后呈圆球状或颗粒状，如图6所示。将该微载体紫外光照灭菌后，由基础培养基进行水化，水化一夜后微载体溶胀，进一步观察可得，其呈规则球状且表面带有曲率较大的圆弧状凹陷，所述圆弧状凹陷的平均半径约为30-40μm。
实施例5：微载体的制备
将混合好的有机相液体石蜡与司班80放入带有搅拌器的反应装置中，其中液体石蜡与司班80体积比为300:1，加热至60℃以上，搅拌使其混合均匀，水相由明胶(10wt％) 与不溶于水的占位材料即粒径均匀的琼脂糖(1wt％)以及交联剂戊二醛(0.5wt％)混合形成；待有机相混合均匀后，转至室温条件下，在搅拌浆的作用下向有机相中缓慢滴加混合好的水相，常温搅拌使其乳化。室温下反应10小时后过滤，其中搅拌速度控制在600rpm/min,待微载体成球后洗去有机相。过滤微载体，并将过滤后的载体材料转移到装有无水乙醇的试剂瓶中，其中有机溶剂要完全浸没载体，25℃摇床摇6小时，使得载体内部占位材料充分溶解并完全溶出。之后过滤材料并对载体材料进行去离子水冲洗进一步洗去多余的有机溶剂，最后将材料投入到含有RGD位点的氨基酸溶液(胶原溶液)中，20℃下搅拌6小时，之后清洗、筛分、冻干，获得微载体。
将获得的微载体利用扫描电镜进行观察可得，该微载体直径在30-400微米之间，冻干后呈圆球状或颗粒状，如图7所示，具有狭长型凹陷比例占比约90％，凹陷个数约50个。
另外，发明人还通过以下实施例制备获得了更多的微载体。具体如下表A所示。
表A：实施例6-实施例10
Figure PCTCN2023070671-appb-000001
Figure PCTCN2023070671-appb-000002
测试例1：软骨陷窝结构仿生构建
发明人将GFP绿色荧光标记的大鼠原代软骨细胞(来自4周大鼠软骨)以1×10 5个/mg的密度接种到实施例4制备得到的微载体上，将接种后的微载体放入37℃恒温培养箱中培养，培养24小时后使用激光扫描共聚焦显微镜观察，发现软骨细胞在微载体上粘附并增殖，且首先粘附在凹陷处，如图4B所示。人体正常软骨发育成熟后，软骨细胞聚集并分泌基质蛋白形成软骨陷窝结构，其生理直径也在30μm左右，如图4A所示。
分析图4B可知，软骨细胞(绿色荧光)在凹陷处优先粘附，且更多情况下多个细胞一起粘附在凹陷处(图4B中绿色荧光区域性显示)，而对于微载体没有凹陷的地方没有细胞粘附。
因此，本发明的微载体可以模拟正常软骨陷窝结构，能够有效地抑制软骨细胞在体外增值所导致的去分化表现。
测试例2：微载体与细胞的原位冻存
发明人将人脐间充质干细胞以1×10 5个/mg的密度接种到实施例4制备得到的微载体上，将接种后的微载体放入37℃恒温培养箱中培养，培养72小时后(其中隔天换液)，此时细胞已经在微载体上长满。弃培养基，加入冻存液，冻存液的配置为90％FBS、10％甘油，以及95％FBS、5％甘油。冻存1d后复苏，复苏培养24h,后使用live&dead染色液染色并使用激光扫描共聚焦显微镜观察，结果如图13所示。
如图13所示，绿色表示活细胞红色表示死细胞，微载体无颜色显示，其中10％甘油组为对照组，常用冻存液配比为10％甘油90％FBS，在本实验中发明人将甘油浓度降低至原来的50％，即采用5％甘油95％FBS，发现细胞活率与对照组相比几乎无差别，活细胞数量(绿色荧光)达到总数量的70％以上。
因此，本发明的微载体可以进行细胞培养后直接原位冻存，能够有效地抑制CPA(主要是DMSO与甘油)的使用，冻存后细胞活率较高。
测试例3：微载体能够保持培养细胞的功能性
发明人将人脐间充质干细胞通过培养基重悬，得到细胞悬液。将1.6×10 7个细胞接种至装有微载体和80ml培养基(微载体和培养基的比例为3g/L)的生物反应器，细胞密度为2×10 5个/ml。将接种后的生物反应器置于37℃二氧化碳培养箱进行培养，反应器搅拌程序为40rpm/min，3min；1rpm/min，1h；循环程序24次。接种24h后程序改为恒速40rpm。细胞培养第二天、第三天分别进行一次换液处理，换液量为60ml/次。其中选用市售微载体cytodex3,来自美国cytiva公司作为对照组进行对比。实验组选用上述实施例3所制备的微载体。4d后对实验组细胞进行收获，使用live&dead染色液染色并使用激光扫描共聚焦显微镜观察细胞活死情况(如图14所示)，消化计数并用CCK8检测试剂盒测量细胞在微载体上的增殖结果(如图15所示)以及动态培养并对细胞进行消化后细胞功能性(干性)表达进行测试结果(如图16所示)。
图14结果：绿色为活细胞，红色为死细胞。结果表明所制备的微载体具有优异的生物相容性，使细胞能够粘附并且出现了增殖，以及抗剪切性能。在搅拌的情况下能够保护细胞，减少剪切力对细胞的损害，细胞在微载体上出现增殖并保持高活率。
图15结果表明，细胞培养1,2,3,4天后细胞在微载体上出现了增殖，其中第四天收获的细胞与第一天收获的细胞数量相比，细胞增殖可达12倍。
图16结果表明，使用胰酶将细胞从微载体上消化收集后对细胞进行抗体孵育，其中实验组CD105marker表达量为99.3％高于对照组99.1％，说明干细胞在微载体上增殖后其功能性(干性)保持非常好，且优于市售微载体。
图14-16结果证明了本发明的微载体能够很好的进行细胞扩增，消化收集且其功能性保持优异，具有临床使用的潜力。
测试例4：微载体渗透性测试
取实施例6制备的微载体进行渗透测试表征，主要操作方法为：取冻干后的微载体约10mg于15ml离心管中，加入PBS水化过夜，之后弃上层清液，PBS洗2-3遍，弃上清。将微载体放置在荧光标记(488)的培养基中(培养基需浸没微载体)，分别在10min、20min、40min,使用激光扫描共聚焦显微镜观察。结果如图18所示。
其中，图18a为10min时微载体渗透结果图，其中绿色荧光部分为标记的培养基，黑色为未标记荧光的微载体，图18b为渗透时间为20min时渗透结果，可见培养基已逐渐渗透至空腔中，图18c为渗透时间为40min时渗透结果，可见培养基已完全渗透至微载体空腔中。
结果表明，本发明的微载体具有良好的渗透性，40min完成完全渗透，有利于细胞的培养及物质交换，且本发明具有空腔结构的微载体具备实现空腔内部培养细胞的条件。
最后应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。

Claims (27)

  1. 一种微载体，其特征在于，所述微载体具有三维立体结构，所述微载体具有壳层且内部为空心或实心，所述壳层外壁不平整且具有多处凹陷点。
  2. 根据权利要求1所述的微载体，其中，所述壳层外壁的凹陷面积占壳层外壁总表面积的比例为15％以上。
  3. 根据权利要求1-2任一项所述的微载体，其中，所述壳层外壁具有的凹陷点的数量为2～800个，可选的凹陷点的数量为2～700个，可选的凹陷点的数量为2～600个，可选的凹陷点的数量为2～500个，可选的凹陷点的数量为2～400个，可选的凹陷点的数量为2～300个，可选的凹陷点的数量为2～200个，可选的凹陷点的数量为5～200个，可选的凹陷点的数量为5～100个，可选的凹陷点的数量为5～60个，可选的凹陷点的数量为10～60个。
  4. 根据权利要求1-3任一项所述的微载体，其中，所述壳层外壁具有的凹陷点的形状选自圆弧状凹陷、细槽型凹陷、多边形凹陷、不规则凹陷、球形凹陷或其任意组合；
    优选地，所述圆弧状凹陷的平均半径为1～500μm，可选的平均半径为1～400μm，可选的平均半径为1～300μm，可选的平均半径为10～300μm，可选的平均半径为50～300μm，可选的平均半径为80～300μm，可选的平均半径为80～200μm；
    优选地，所述细槽型凹陷的凹陷面的平均宽度为1～500μm，可选的凹陷面的平均宽度为1～400μm，可选的凹陷面的平均宽度为1～300μm，可选的凹陷面的平均宽度为10～300μm，可选的凹陷面的平均宽度为50～300μm，可选的凹陷面的平均宽度为80～300μm，可选的凹陷面的平均宽度为80～200μm；
    优选地，所述多边形凹陷的凹陷面的平均宽度为1～500μm，可选的凹陷面的平均宽度为1～400μm，可选的凹陷面的平均宽度为1～300μm，可选的凹陷面的平均宽度为10～300μm，可选的凹陷面的平均宽度为50～300μm，可选的凹陷面的平均宽度为80～300μm，可选的凹陷面的平均宽度为80～200μm；
    优选地，所述不规则凹陷的凹陷面的平均宽度为1～500μm，可选的凹陷面的平均宽度为1～400μm，可选的凹陷面的平均宽度为1～300μm，可选的凹陷面的平均宽度为10～300μm，可选的凹陷面的平均宽度为50～300μm，可选的凹陷面的平均宽度为80～300μm，可选的凹陷面的平均宽度为80～200μm；
    优选地，所述球形凹陷的平均半径小于500μm，可选的平均半径小于400μm，可选的平均半径小于300μm，可选的平均半径小于200μm，可选的平均半径小于100μm，可选的平均半径小于50μm，可选的平均半径小于20μm，可选的平均半径小于10μm。
  5. 根据权利要求1-4任一项所述的微载体，其中，所述微载体的平均粒径为1～3000μm，可选的平均粒径为1～2000μm，可选的平均粒径为1～1000μm，可选的平均粒径为1～800μm，可选的平均粒径为1～600μm，可选的平均粒径为10～600μm，可选的平均粒径为50～500μm，可选的平均粒径为100～500μm，可选的平均粒径为100～400μm；
    优选地，所述微载体的平均粒径为30-400μm。
  6. 根据权利要求1-5任一项所述的微载体，其中，所述壳层的厚度为1-100μm，可选的厚度为1-90μm，可选的厚度为1-80μm，可选的厚度为1-70μm，可选的厚度为1-60μm，可选的厚度为1-50μm。
  7. 根据权利要求1-6任一项所述的微载体，其中，所述微载体具有的三维立体结构为圆球、梭形、椭圆球、棒状、扁球体或不规则球体。
  8. 根据权利要求1-7任一项所述的微载体，其中，所述微载体的内部为空心，所述微载体具有非封闭式三维立体结构；
    优选地，所述微载体的壳层具有一个或多个通孔，所述通孔穿透所述壳层以便联通壳层的内部和外部。
  9. 根据权利要求1-8任一项所述的微载体，其中，所述微载体内部的空心结构通过溶出占位材料得到；
    或者，所述壳层的外壁和/或内壁经过表面修饰；
    优选地，所述壳层的外壁和/或内壁经过RGD表面修饰。
  10. 根据权利要求1-9任一项所述的微载体，其中，所述微载体负载有细胞；
    优选地，所述细胞设置在所述微载体的壳层内壁，或者设置在所述微载体的壳层外壁，或者同时设置在所述微载体的壳层内壁和所述微载体的壳层外壁；
    优选地，所述细胞同时设置在所述微载体的壳层内壁和所述微载体的壳层外壁时，设置于壳层内壁和壳层外壁的细胞种类可以相同或不同；
    优选地，所述微载体的壳层外壁设置有细胞时，所述细胞设置在所述壳层外壁的凹陷点处，或者设置在所述壳层外壁的光滑表面处(即非凹陷点处)，或者同时设置在所述壳层外壁的凹陷点处和光滑表面处；
    优选地，所述细胞通过所述通孔设置在所述微载体的壳层内壁；
    优选地，所述细胞通过细胞注射设置在所述微载体的壳层内壁；
    优选地，所述细胞通过与所述微载体进行共培养设置在所述微载体的壳层外壁。
  11. 根据权利要求10所述的微载体，其中，所述细胞选自原核细胞、真核细胞、细菌细胞、古细菌细胞、单细胞真核生物体的细胞、原生动物细胞、植物细胞、动物细胞、藻类细胞、真菌细胞、人造细胞或其任意组合；
    或者，所述细胞选自干细胞、体细胞、生殖细胞或其任意组合；
    优选地，所述干细胞选自胚胎干(ES)细胞、诱导多能干(iPS)细胞、间充质干细胞、神经干细胞、肌肉干细胞、造血干细胞、上皮干细胞、乳腺干细胞、肠干细胞、中胚层干细胞、内皮干细胞或其任意组合；
    优选地，所述体细胞选自上皮细胞、内皮细胞、间皮细胞、成纤维细胞、成骨细胞、软骨细胞、肌细胞、骨细胞、肝细胞、胰腺细胞、外源细胞、内源细胞、心肌细胞、骨骼细胞、心脏成肌细胞、骨骼成肌细胞、少突胶质细胞、神经胶质细胞、造血细胞、神经元或其任意组合；
    优选地，所述生殖细胞选自卵母细胞、精子、卵原细胞、精原细胞或其任意组合。
  12. 根据权利要求1-11任一项所述的微载体，其中，所述壳层或所述微载体由生物材料制成；
    优选地，所述生物材料选自人工合成的生物材料、天然生物材料或其组合。
  13. 根据权利要求12所述的微载体，其中，所述人工合成的生物材料选自聚乙二醇，聚乙二醇衍生物，聚乳酸，聚乳酸醇共聚物，聚酸酐，聚酸酯，聚氨基酸，聚氧化乙烯，聚酯，聚甲基丙烯酸甲酯，聚碳酸酯，聚氨基甲酸酯，聚己内酯，聚羟基脂肪酸酯，聚硅氧烷，聚乙烯，聚氯乙烯，聚四氟乙烯，聚苯乙烯，聚丙烯，马来酸酐接枝共聚物，聚丙烯酰胺，聚缩醛，聚吡咯或其任意组合。
  14. 根据权利要求12所述的微载体，其中，所述天然生物材料选自天然蛋白质，胶原及胶原衍生物，明胶及明胶衍生物，琼脂及琼脂衍生物，蛋白多糖，海藻酸盐及其海藻酸盐衍生物，基质胶，蜂胶，纤维素及纤维素衍生物，甲壳素及甲壳素衍生物，蚕丝蛋白及其衍生物，层连接蛋白及其衍生物，纤维连接蛋白及其衍生物，透明质酸钠及透明质酸衍生物，琼脂糖及其衍生物，葡聚糖及其衍生物，蔗糖及蔗糖衍生物，淀粉，壳聚糖及壳聚糖衍生物或其任意组合。
  15. 一种微载体聚集体，其特征在于，所述聚集体由权利要求1-14任一项的微载体颗粒聚集而成。
  16. 根据权利要求15所述的微载体聚集体，其中，所述聚集体是单一性状微载体的集合体，或各种性状微载体任意比例的集合体。
  17. 一种制备权利要求1-14任一项所述的微载体的方法，其包括：
    (a)提供有机相溶液；
    (b)提供水相溶液；
    (c)将所述水相溶液加入所述有机相溶液，形成混合液；使所述混合液乳化，混合均匀，形成均一的油包水体系；
    (d)将所述油包水体系进行过滤，滤饼为待处理的载体基质；
    (e)将所述待处理的载体基质进行后处理，以便溶出占位材料，并用包含RGD氨基酸序列的短肽或蛋白与后处理后的载体基质进行缀合，获得所述微载体；
    任选地，所述缀合后进一步包括清洗、筛分和冻干的步骤。
  18. 根据权利要求17所述的方法，其中，所述乳化是在温度为4-200℃，优选20-75℃下进行的；
    优选地，所述乳化的时间是4-72h，优选5-30h；
    优选地，所述乳化是通过搅拌法，超声法，震荡法或微流道法实现的，优选地，转速为100-5000rpm/min，优选为300-1000rpm/min。
  19. 根据权利要求17所述的方法，其中，所述有机相溶液是通过将有机溶剂与非离子表面活性剂进行混合得到的；
    优选地，所述混合是在温度为4-200℃，优选20-75℃下进行的；
    优选地，所述混合的时间为10-120min，优选10-60min；
    优选地，所述混合是在搅拌桨的作用下进行的；
    优选地，所述搅拌桨的转速为100-2000rpm/min，优选为300-1000rpm/min；
    优选地，所述有机溶剂选自液体石蜡，石油醚，四氯化碳，二甲基亚砜，三氯甲烷，二氯甲烷，食用油，硅油，大豆油，矿物油或其任意组合；
    更优选地，所述有机溶剂为液体石蜡；
    优选地，所述非离子表面活性剂选自吐温，司班80，脂肪酸甘油酯，十二烷基苯磺酸钠，PO-500,氢氟醚，聚乙二醇，嵌段聚氧乙烯-聚氧丙烯醚(PO-EO共聚物)，多元醇酯类或其任意组合；
    更优选地，所述非离子表面活性剂为司班80；
    优选地，所述有机溶剂与所述非离子表面活性剂的体积比为200-400:1。
  20. 根据权利要求17所述的方法，其中，所述水相溶液是通过如下步骤得到的：
    将生物材料溶解在水中，获得溶解液；
    将占位材料和固化剂悬浮在所述溶解液中，形成分散均匀的悬浊液，所述悬浊液即为所述水相溶液；
    优选地，所述溶解是在温度为4-200℃，优选10-80℃下进行的；
    优选地，所述分散均匀的悬浊液是通过搅拌、震荡、超声或摇晃实现的，优选地，转速为100-2000rpm/min，优选为300-700rpm/min；
    优选地，所述水相溶液中，所述生物材料的质量分数为3-10％；
    优选地，所述水相溶液中，所述占位材料的质量分数为0.01-1％；
    优选地，所述水相溶液中，所述固化剂的质量分数为0.5-7％；
    优选地，所述生物材料选自人工合成的生物材料、天然生物材料或其组合；
    更优选地，所述生物材料选自透明质酸钠、明胶；
    优选地，所述占位材料选自聚乙二醇及聚乙二醇衍生物，石蜡球，氧化海藻酸盐及其衍生物，聚己内酯，二氧化硅，蜂蜡，蜂胶，琼脂，琼脂糖，海藻酸盐及其衍生物，大豆卵磷脂，蛋黄卵磷脂，磷脂，葡聚糖，壳聚糖，淀粉，明胶，透明质酸钠及透明质酸衍生物或其任意组合；
    更优选地，所述占位材料为琼脂糖、葡聚糖、透明质酸钠；
    优选地，所述固化剂选自N,N-亚甲基双丙烯酰胺，1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐，N-羟基琥珀酰亚胺，N-羟基硫代琥珀酰亚胺，二异氰酸酯，戊二醛，京平尼，硫酸铵，钙离子，丁二醇二缩水甘油醚，转谷酰胺酶，二乙烯基苯，己二酸二酰肼或其任意组合；
    更优选地，所述固化剂选自1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐、戊二醛。
  21. 根据权利要求17所述的方法，其中，所述过滤是通过过滤装置实现的，所述过滤装置的网孔直径为30-1000微米；
    优选地，所述过滤装置具有四层结构，第一层的网孔直径为500-800微米，第二层的网孔直径为300-400微米，第三层的网孔直径为200-300微米，第四层的网孔直径为100-200微米。
  22. 根据权利要求17所述的方法，其中，所述溶出占位材料是通过以下步骤实现的：
    将所述待处理的载体基质与有机溶剂进行混合，以便溶出占位材料；
    优选地，所述混合是在温度为15℃-150℃，优选25-100℃下进行4-48h，优选6-24h；
    优选地，所述有机溶剂选自丙酮，无水乙醇，石油醚，三氯甲烷，二甲基亚砜，二氯甲烷，石油醚，四氯化碳，乙腈，甲苯，甲醇或其任意组合；
    更优选地，所述有机溶剂为无水乙醇。
  23. 根据权利要求17所述的方法，其中，所述用包含RGD氨基酸序列的短肽或蛋白与后处理后的载体基质进行缀合是通过以下步骤实现的：
    将所述后处理后的载体基质与所述包含RGD氨基酸序列的短肽或蛋白进行混合，以便实现所述后处理后的载体基质的RGD表面修饰；
    优选地，所述混合是在温度为15-25℃的条件下进行的；
    优选地，所述混合的时间为4-10小时；
    优选地，所述包含RGD氨基酸序列的短肽或蛋白选自胶原或胶原衍生物，明胶或明胶衍生物，纤维连接蛋白，丝素蛋白，层连接蛋白，基质胶或其任意组合；
    更优选地，所述包含RGD氨基酸序列的短肽或蛋白选自胶原、明胶。
  24. 一种试剂盒，其包含权利要求1-14任一项所述的微载体、权利要求15-16任一项所述的微载体聚集体或者权利要求17-23任一项所述的方法制备得到的微载体。
  25. 权利要求1-14任一项所述的微载体、权利要求15-16任一项所述的微载体聚集体或者权利要求17-23任一项所述的方法制备得到的微载体在制备试剂盒中的用途，所述试剂盒用于细胞三维培养和/或扩增、3D生物打印、仿生结构体构建、细胞治疗、载药或医疗注射；
    优选地，所述仿生结构体为三维构建体、组织前体、组织、器官或胚胎(如小鼠胚胎)；
    优选地，所述仿生结构体构建为胚胎仿生模拟构建或软骨仿生构建；
    优选地，所述仿生结构体为关节软骨的软骨陷窝结构；
    优选地，所述细胞治疗为皮肤表面治疗、临床角膜治疗、骨治疗或软骨修复与骨损伤治疗。
  26. 权利要求1-14任一项所述的微载体、权利要求15-16任一项所述的微载体聚集体或者权利要求17-23任一项所述的方法制备得到的微载体的用途，其用于体外细胞三维培养和/或扩增、体外3D生物打印、体外仿生结构体构建、体外载药；优选地，所述用途用于非诊断或治疗目的；
    优选地，所述仿生结构体为三维构建体、组织前体、组织、器官或胚胎(如小鼠胚胎)；
    优选地，所述仿生结构体构建为胚胎仿生模拟构建或软骨仿生构建；
    优选地，所述仿生结构体为关节软骨的软骨陷窝结构。
  27. 一种体外细胞三维培养和/或扩增的方法，其包括：
    1)将细胞接种于权利要求1-14任一项所述的微载体、权利要求15-16任一项所述的微载体聚集体或者权利要求17-23任一项所述的方法制备得到的微载体上进行培养；
    任选地，步骤1)后，进一步包括：
    2-A)将步骤1)获得的混合物进行原位冻存；
    或者任选地，步骤1)后，进一步包括：
    2-B)将步骤1)获得的负载于微载体上的细胞进行消化、收集；
    优选地，所述培养为动态培养；
    优选地，所述动态培养为搅拌式动态培养或悬浮式动态培养；
    优选地，所述细胞如权利要求11所限定。
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