WO2018050092A1 - 人工组织前体及制备其的方法 - Google Patents

人工组织前体及制备其的方法 Download PDF

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Publication number
WO2018050092A1
WO2018050092A1 PCT/CN2017/101738 CN2017101738W WO2018050092A1 WO 2018050092 A1 WO2018050092 A1 WO 2018050092A1 CN 2017101738 W CN2017101738 W CN 2017101738W WO 2018050092 A1 WO2018050092 A1 WO 2018050092A1
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Prior art keywords
tubular
component
microcapsules
solid support
sheet
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PCT/CN2017/101738
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English (en)
French (fr)
Inventor
康裕建
左潇
杜明春
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四川蓝光英诺生物科技股份有限公司
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Application filed by 四川蓝光英诺生物科技股份有限公司 filed Critical 四川蓝光英诺生物科技股份有限公司
Priority to EP17850294.4A priority Critical patent/EP3514228A4/en
Priority to US15/780,301 priority patent/US20190328935A1/en
Priority to CN201780003356.2A priority patent/CN108138134B/zh
Priority to JP2018528046A priority patent/JP6755952B2/ja
Priority to CN201910183820.1A priority patent/CN109735434B/zh
Priority to CN201910255721.XA priority patent/CN109913400B/zh
Priority to CN201910268261.4A priority patent/CN109913402B/zh
Publication of WO2018050092A1 publication Critical patent/WO2018050092A1/zh
Priority to US16/113,509 priority patent/US11439731B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/507—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials for artificial blood vessels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • 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
    • A61L27/3604—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 characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A61L27/3625—Vascular tissue, e.g. heart valves
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    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/08—Bioreactors or fermenters specially adapted for specific uses for producing artificial tissue or for ex-vivo cultivation of tissue
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/14—Scaffolds; Matrices
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    • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
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    • C12N5/0012—Cell encapsulation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/04—Hollow or tubular parts of organs, e.g. bladders, tracheae, bronchi or bile ducts
    • A61F2/06—Blood vessels
    • A61F2/062—Apparatus for the production of blood vessels made from natural tissue or with layers of living cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2210/00—Particular material properties of prostheses classified in groups A61F2/00 - A61F2/26 or A61F2/82 or A61F9/00 or A61F11/00 or subgroups thereof
    • A61F2210/0004—Particular material properties of prostheses classified in groups A61F2/00 - A61F2/26 or A61F2/82 or A61F9/00 or A61F11/00 or subgroups thereof bioabsorbable
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2230/00—Geometry of prostheses classified in groups A61F2/00 - A61F2/26 or A61F2/82 or A61F9/00 or A61F11/00 or subgroups thereof
    • A61F2230/0063—Three-dimensional shapes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/16—Particles; Beads; Granular material; Encapsulation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2513/00—3D culture
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N2533/30—Synthetic polymers
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N2533/50—Proteins
    • C12N2533/56—Fibrin; Thrombin
    • C—CHEMISTRY; METALLURGY
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C—CHEMISTRY; METALLURGY
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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
    • C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
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    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/069—Vascular Endothelial cells
    • C12N5/0691—Vascular smooth muscle cells; 3D culture thereof, e.g. models of blood vessels
    • C—CHEMISTRY; METALLURGY
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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
    • C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents

Definitions

  • the present invention relates to the field of tissue engineering and 3D printing.
  • the present invention relates to an artificial tissue precursor comprising a solid support and a plurality of microcapsules, wherein at least one microcapsule is attached to a solid support comprising cells and biological cells encapsulating the cells a capacitive material; a method for preparing the artificial tissue precursor; a kit and a kit for preparing an artificial tissue precursor; an artificial tissue obtained by culturing the artificial tissue precursor, such as an artificial lumen; a luminal implant or lumen model of a tissue precursor or artificial lumen; the use of an artificial tissue precursor for preparing an artificial tissue, a lumen implant or a lumen model; and an artificial tissue for preparing a lumen implant The use of an in-vivo or lumen model.
  • Vascular grafting and vascular patch repair can be used to replace, reconstruct, or repair stenotic, occluded, dilated, damaged, or deformed blood vessels.
  • Common vascular grafts or vascular patches are derived from the patient's own arteries or veins, but in cases where the patient's autologous vascular supply is insufficient (eg, the patient has vascular disease or has previously undergone vascular grafting), An artificial blood vessel (patch) or a heterologous blood vessel (patch) is required as an alternative.
  • the normal blood vessels do not have a thrombus because of a layer of endothelial cells on the inner wall of the lumen. Therefore, in order for the artificial blood vessel to achieve the same function as the normal blood vessel, the most fundamental solution is to endothelialize the artificial blood vessel, that is, to form a complete endothelial cell layer on the inner wall surface of the artificial blood vessel.
  • Endothelial seeding of polytetrafluoroethylene femoral popliteal bypasses the failure of low-density seeding to improve patency.
  • the method for constructing artificial blood vessels using stem cells as seed cells comprises: manufacturing a blood vessel scaffold material, and inducing stem cells into vascular cells (including endothelial cells, smooth muscle cells and fibroblasts) in vitro, In the stent material, in vivo implantation; or directly plant stem cells into the vascular stent material.
  • vascular cells including endothelial cells, smooth muscle cells and fibroblasts
  • the specific process of the latter is as follows: first, a blood vessel stent is prepared, and the cultured seed cells are added to the surface of the blood vessel stent as a cell suspension, and the cells are adhered to the surface of the stent and then implanted in the body.
  • the cells need to go through the migration process to enter the inside of the stent. Therefore, the artificial blood vessels manufactured by the two methods tend to have a large amount of cell aggregation on the surface of the stent, and only a small amount of cells exist inside, or the cells are unevenly distributed, so that the manufactured artificial blood vessels are difficult to form a complete structure and function. If a variety of cells are planted in the scaffold, there will be a phenomenon of disordered cell distribution. Therefore, the artificial blood vessels manufactured by these two methods have disordered internal vascular cells, and it is difficult to form a complete endothelial cell layer and a structurally smooth muscle cell layer, and it is still not clinically applicable.
  • the inventors of the present application have developed a new method for preparing an artificial tissue precursor.
  • the artificial tissue precursor is an artificial lumen precursor that can form an artificial lumen (eg, an artificial blood vessel).
  • the present invention is directed to an artificial tissue precursor comprising a solid support and a plurality of microcapsules, wherein at least one microcapsule is attached to a solid support comprising cells and biological cells encapsulating the cells Capacitive material.
  • the artificial tissue precursor is a lumen (eg, a circulating lumen, a digestive lumen, a sinus lumen, a urinary lumen, or a genital lumen) precursor.
  • a lumen eg, a circulating lumen, a digestive lumen, a sinus lumen, a urinary lumen, or a genital lumen
  • the lumen is a lumen comprising epithelial cells (eg, blood vessels, esophagus, trachea, stomach, bile duct, intestinal tract (including the small and large intestines, eg, duodenum, jejunum, ileum, The cecum (including the appendix), the ascending colon, the right colon of the colon, the transverse colon, the left collateral, the descending colon, the sigmoid colon, the rectum, the fallopian tube, the vas deferens, the ureter, the bladder or the lymphatic vessels).
  • epithelial cells eg, blood vessels, esophagus, trachea, stomach, bile duct, intestinal tract (including the small and large intestines, eg, duodenum, jejunum, ileum, The cecum (including the appendix), the ascending colon, the right colon of the colon, the transverse colon, the left collateral, the descending colon, the sigmoid colon,
  • the artificial tissue precursor is tubular or flaky.
  • a plurality of said microcapsules constitute one or more biological constructs.
  • one or more biological constructs are attached to a solid support.
  • the invention relates to a method of preparing the artificial tissue precursor, the artificial tissue precursor being tubular; comprising the steps of:
  • tubular biological construct is attached to the inner wall of the tubular solid support.
  • the preparation of the tubular biological construct is carried out by a method comprising the steps of:
  • microcapsules having a first component attached to all or part of its surface; preferably, the first component is contained in the first reagent;
  • the temporary support is tubular or columnar (for example, a circular tubular shape in which the side wall is not open, a circular tubular shape in which the side wall is open, a cylindrical shape or a columnar shape disposed along a partial circumference), and the predetermined area is temporarily supported. a curved surface of the object; optionally, coating the substrate material on a predetermined area of the surface of the temporary support prior to coating the second reagent;
  • step (1) placing all or part of the microcapsules to which the first component is attached in the step (1) on a predetermined region coated with the second reagent, so that the first component on the surface of the microcapsule is pre- Contacting the second component on the region to produce a blocking effect, thereby assembling (bonding) the microcapsule into a first layer structure, the first layer structure being a tubular structure;
  • the method further comprises the steps of:
  • the invention relates to another method of preparing the artificial tissue precursor, the artificial tissue precursor being tubular, comprising the steps of:
  • tubular biological construct is attached to the inner wall of the tubular solid support.
  • the preparation of the tubular biological construct is carried out by a method comprising the steps of:
  • microcapsules having a first component attached to all or part of its surface; preferably, the first component is contained in the first reagent;
  • the temporary support has at least one plane, the annular pattern being located in a plane of the temporary support;
  • step (1) placing all or part of the microcapsules to which the first component is attached in the step (1) on a predetermined circular pattern drawn by the second reagent, so that the first component on the surface of the microcapsule is Contacting the second component on the annular pattern to produce a blocking effect, thereby assembling (bonding) the microcapsule into a first layer structure, the first layer structure being a ring structure;
  • the invention relates to another method of preparing the artificial tissue precursor, the artificial tissue precursor being in the form of a sheet comprising the following steps:
  • the preparation of the flaky biological construct is carried out by a method comprising the steps of:
  • microcapsules having a first component attached to all or part of its surface; preferably, the first component is contained in the first reagent;
  • step (1) placing all or part of the microcapsules to which the first component is attached in the step (1) on a predetermined region coated with the second reagent, so that the first component on the surface of the microcapsule is pre- Contacting the second component on the region to produce a blocking effect, thereby assembling (bonding) the microcapsule into a first layer structure, the first layer structure being a planar sheet-like structure;
  • the method further comprises the steps of:
  • the method further comprises bending the planar sheet-like biological construct to obtain a curved sheet-like biological construct.
  • the invention relates to another method of preparing the artificial tissue precursor, the artificial tissue precursor being in the form of a sheet comprising the following steps:
  • a material e.g., a biocompatible material for preparing a solid support is provided, and a sheet-like solid support is prepared on the sheet-like biological construct.
  • the sheet-like solid support is prepared by 3D printing or spraying.
  • the invention relates to another method of preparing the artificial tissue precursor, the artificial tissue precursor being tubular, comprising the steps of:
  • tubular biological construct is attached to the inner wall of the tubular solid support.
  • the invention relates to another method of preparing the artificial tissue precursor, the artificial tissue precursor being tubular, comprising the steps of:
  • a sheet-like biological construct is prepared by the method of preparing a sheet-like biological construct as defined above; thereafter, the obtained sheet-like biological construct is bent, and/or the edge of the sheet-like biological construct is adhered Combine to obtain a tubular biological construct;
  • a material e.g., a biocompatible material for preparing a solid support is provided, and a tubular solid support is prepared on the outer wall of the tubular biological construct.
  • the tubular solid support is prepared by 3D printing or spraying.
  • the invention relates to another method of making the artificial tissue precursor comprising the steps of:
  • microcapsules having a first component attached to all or part of its surface; preferably, the first component is contained in the first reagent;
  • step (1) placing all or part of the microcapsules to which the first component is attached in the step (1) on a predetermined region coated with the second reagent, so that the first component on the surface of the microcapsule is pre- Contacting the second component on the region to produce a blocking effect, thereby assembling (bonding) the microcapsule into a first layer structure on the surface of the solid support;
  • the method further comprises the steps of:
  • the solid support is tubular or flaky.
  • the solid support is a tubular solid support, the predetermined region being located on the inner wall of the solid support.
  • the first component and/or the second component is a biocompatible material, is a bio-derived material, and/or is biodegradable. material.
  • the blocking effect produced by contacting the first component with the second component can be used to bond the two microcapsules together to form a biological construct; and the resulting organism
  • the tensile modulus of the construct is not less than 10 Pa, for example, not less than 20 Pa, not less than 30 Pa, not less than 40 Pa, not less than 50 Pa, not less than 60 Pa, not less than 70 Pa, not less than 80 Pa, not low At 90 Pa, not less than 100 Pa, not less than 200 Pa, not less than 300 Pa, not less than 400 Pa, not less than 500 Pa, not less than 600 Pa, not less than 700 Pa, not less than 800 Pa, not less than 900 Pa or not low At 1000Pa.
  • the first component and the second component are selected from the group consisting of:
  • Alginate such as sodium alginate
  • oxidized alginate such as oxidized sodium alginate
  • substances containing Ca 2+ , Mg 2+ , Ba 2+ , Sr 2+ , or Fe 3+ eg, a solution containing a Ca 2+ , Mg 2+ , Ba 2+ , Sr 2+ , or Fe 3+ or a semi-solid (eg, a gel)
  • a semi-solid eg, a gel
  • a molecule containing a maleimide group for example, a polyethylene glycol (MAL-PEG) containing a maleimide group
  • a molecule containing a free thiol group for example, a polyethylene glycol containing a free thiol group (PEG-SH)
  • An anion-containing substance for example, an anion-containing solution or a semi-solid (e.g., a gel)
  • an ⁇ -cyanoacrylate e.g., ⁇ -cyanoacrylate, ⁇ -cyanoacrylate, ⁇ -cyanide
  • Fibrinogen and ⁇ -cyanoacrylate for example, ⁇ -cyanoacrylate, ⁇ -cyanoacrylate, ⁇ -cyanoacrylate, iso- ⁇ -cyanoacrylate, ⁇ - N-octyl cyanoacrylate;
  • serum albumin for example, bovine serum albumin
  • glutaraldehyde glutaraldehyde
  • the invention relates to a biological construct obtained by the method of preparing a biological construct as defined in any one of the above.
  • the present invention relates to a kit for preparing an artificial tissue precursor, the kit comprising microcapsules, and first and second reagents separated from each other, wherein the microcapsules comprise cells and packages a biocompatible material of a cell, the first reagent comprising a first component, the second reagent comprising a second component, and capable of producing a blocking effect when the first component is contacted with the second component To achieve adhesion.
  • the blocking effect produced by contacting the first component with the second component can be used to bond the two microcapsules together to form a biological construct; and the resulting organism
  • the tensile modulus of the construct is not less than 10 Pa (for example, not less than 100 Pa).
  • the first component and/or the second component are biocompatible materials; are bio-derived materials; and/or are biodegradable materials.
  • the first component and the second component are selected from the group consisting of:
  • Alginate such as sodium alginate
  • oxidized alginate such as oxidized sodium alginate
  • substances containing Ca 2+ , Mg 2+ , Ba 2+ , Sr 2+ , or Fe 3+ eg, a solution containing a Ca 2+ , Mg 2+ , Ba 2+ , Sr 2+ , or Fe 3+ or a semi-solid (eg, a gel)
  • a semi-solid eg, a gel
  • a molecule containing a maleimide group for example, a polyethylene glycol (MAL-PEG) containing a maleimide group
  • a molecule containing a free thiol group for example, a polyethylene glycol containing a free thiol group (PEG-SH)
  • An anion-containing substance for example, an anion-containing solution or a semi-solid (e.g., a gel)
  • an ⁇ -cyanoacrylate e.g., ⁇ -cyanoacrylate, ⁇ -cyanoacrylate, ⁇ -cyanide
  • Fibrinogen and ⁇ -cyanoacrylate for example, ⁇ -cyanoacrylate, ⁇ -cyanoacrylate, ⁇ -cyanoacrylate, iso- ⁇ -cyanoacrylate, ⁇ - N-octyl cyanoacrylate;
  • serum albumin for example, bovine serum albumin
  • glutaraldehyde glutaraldehyde
  • the present application is directed to a kit for preparing an artificial tissue precursor comprising one or more kits of the invention.
  • the present application is directed to an artificial tissue obtained by culturing an artificial tissue precursor of the present invention (eg, in vitro culture or in vivo culture).
  • the artificial tissue is an artificial lumen.
  • the lumen is a lumen comprising epithelial cells (eg, blood vessels, esophagus, gas) Tube, stomach, bile duct, intestine (including small intestine and large intestine, such as duodenum, jejunum, ileum, cecum (including appendix), ascending colon, right colon, transverse colon, left colon, descending colon, sigmoid colon, rectum) , fallopian tubes, vas deferens, ureters, bladder or lymphatic vessels).
  • epithelial cells eg, blood vessels, esophagus, gas
  • Tube e.g, stomach, bile duct, intestine (including small intestine and large intestine, such as duodenum, jejunum, ileum, cecum (including appendix), ascending colon, right colon, transverse colon, left colon, descending colon, sigmoid colon, rectum) , fallopian tubes, vas deferens, ureters, bladder
  • the artificial lumen is a tubular artificial lumen or a sheet-like artificial lumen.
  • the artificial lumen is an artificial blood vessel or a blood vessel patch.
  • the present application is directed to a luminal implant comprising an artificial tissue precursor of the present invention (eg, a tubular artificial tissue precursor or a flaky artificial tissue precursor) or an artificial lumen.
  • an artificial tissue precursor of the present invention eg, a tubular artificial tissue precursor or a flaky artificial tissue precursor
  • the luminal implant comprises one or more artificial tissue precursors of the invention (eg, tubular artificial tissue precursors or flaky artificial tissue precursors), or one or more Inventive artificial lumen (eg tubular artificial lumen or flaky artificial lumen).
  • tissue precursors of the invention eg, tubular artificial tissue precursors or flaky artificial tissue precursors
  • Inventive artificial lumen eg tubular artificial lumen or flaky artificial lumen
  • the lumen implant is a linear tubular structure or a branched tubular structure.
  • the lumen implant is an X-shaped tubular, Y-shaped tubular or T-shaped tubular.
  • the lumen is a lumen comprising epithelial cells, such as a blood vessel.
  • the luminal implant is a vascular implant comprising an artificial blood vessel or a blood vessel patch of the present invention.
  • the present application is directed to a lumen (e.g., blood vessel) model comprising an artificial lumen (e.g., an artificial blood vessel) of the present invention.
  • a lumen e.g., blood vessel
  • an artificial lumen e.g., an artificial blood vessel
  • the lumen model comprises one or more artificial lumens (e.g., artificial blood vessels) of the present invention.
  • the present application relates to the use of the artificial tissue precursor of the present invention for the preparation of an artificial tissue, a lumen implant or a lumen model.
  • the present application relates to the use of the artificial tissue of the present invention for preparing a luminal implant or lumen model.
  • FIG. 1 schematically depicts an exemplary structure of a microcapsule of the present invention comprising: a cell and a biocompatible material encasing the cell; in a preferred embodiment, the cell may be uniformly dispersed in the microcapsule or may be concentrated in Together, it is located inside the microcapsules.
  • FIG. 2A-E schematically depict an exemplary structure of a biobrick of the present invention comprising: a cell, a core layer encasing the cell, and a shell layer encapsulating the core layer.
  • Figure 2A schematically depicts a structure of a bio-brick of the present invention comprising a core layer and a shell layer, wherein the core layer is coated with cells and the shell layer is located outside the core layer and encapsulated Nuclear layer.
  • FIG. 2B schematically depicts a structure of a biobrick of the present invention comprising, from the inside to the outside, a core layer encasing cells, a first shell encapsulating the core layer, and a second shell surrounding the first shell layer.
  • Floor schematically depicts a structure of a biobrick of the present invention comprising, from the inside to the outside, a core layer encasing cells, a first shell encapsulating the core layer, and a second shell surrounding the first shell layer.
  • Floor schematically depicts a structure of a biobrick of the present invention comprising, from the inside to the outside, a core layer encasing cells, a first shell encapsulating the core layer, and a second shell surrounding the first shell layer.
  • 2C schematically depicts a structure of a biobrick of the present invention comprising, from the inside to the outside, a first core layer encasing cells, a second core layer enclosing cells outside the first core layer, and encapsulating said a first shell layer of the first core layer and the second core layer.
  • 2D schematically illustrates a structure of the bio-brick of the present invention, which comprises, in order from the inside to the outside, a first nuclear layer enclosing the cells, a second nuclear layer enclosing the cells outside the first nuclear layer, and the encapsulation. a first shell layer of a core layer and a second core layer, and a second shell layer surrounding the first shell layer.
  • 2E schematically depicts a structure of a biobrick of the present invention comprising, from the inside to the outside, a first core layer encapsulating cells, a first shell layer encapsulating the first core layer, and a second core encapsulating cells a layer, and a second shell encapsulating the second core layer.
  • 3A-E exemplarily illustrate the structure of a tubular artificial tissue precursor comprising a plurality of tubular biological constructs of the present invention.
  • 3A is a side view of a tubular artificial tissue precursor comprising a tubular solid support and a tubular biological construct with a plurality of sidewalls that are not open, the tubular biostructures of the plurality of sidewalls that are not open.
  • the body is disposed inside the tubular solid support and arranged along the axial direction of the tubular solid support, and the outer wall of the tubular biological construct in which the plurality of side walls are not open is attached to the inner wall of the tubular solid support .
  • 3B is a top plan view of a tubular artificial tissue precursor comprising a tubular solid support and a plurality of tubular biological constructs that are not open to the side walls, the plurality of laterally open tubular biological constructs Inside the tubular solid support, an outer wall of the tubular biological construct that is disposed coaxially with the tubular solid support and only the outermost sidewall is not open is attached to the inner wall of the tubular solid support.
  • 3C is a side view of a tubular artificial tissue precursor comprising a tubular solid support and a plurality of sidewall open tubular biological constructs, the plurality of laterally open tubular biological constructs
  • the inside of the tubular solid support is aligned along the axial direction of the tubular solid support, and the outer walls of the tubular living structures of the plurality of side walls are each attached to the inner wall of the tubular solid support.
  • 3D is a top plan view of a tubular artificial tissue precursor comprising a tubular solid support and a plurality of sidewall open tubular biological constructs, the plurality of laterally open tubular biological constructs in the The inside of the tubular solid support is disposed coaxially with the tubular solid support and arranged in the radial direction, and the outer wall of the tubular biological construct located at only the outermost side wall is fitted to the inner wall of the tubular support.
  • 3E is a top plan view of a tubular artificial tissue precursor comprising a tubular solid support and a plurality of sidewall open tubular biological constructs, the plurality of laterally open tubular biological constructs in the The inside of the tubular solid support is disposed coaxially with the tubular solid support, and the outer walls of the plurality of side wall open tubular biological constructs are bonded to the inner wall of the tubular solid support.
  • 4A-E exemplarily illustrate a column as a temporary support in a method of preparing a tubular biological construct of the present invention, a predetermined area on the unfolded side and side of the column.
  • Figure 4A exemplifies a cylindrical shape as a temporary support in the method of preparing a tubular biological construct of the present invention.
  • FIG. 4B exemplarily depicts a predetermined area on the unfolded side and side of the cylinder of FIG. 4A, wherein the predetermined area is the entire side of the cylinder.
  • FIG. 4C exemplarily depicts a predetermined area on the unfolded side and side of the cylinder of FIG. 4A, wherein the predetermined area is a rectangle on the side of the unfolded cylinder, and the predetermined area The side of the cylinder is penetrated in the axial direction of the cylinder.
  • FIG. 4D exemplarily depicts a predetermined area on the unfolded side and side of the cylinder of FIG. 4A, wherein the predetermined area is a rectangle on the side of the unfolded cylinder, and the predetermined area The side of the cylinder is penetrated in the circumferential direction of the cylinder.
  • FIG. 4E exemplarily depicts a predetermined area on the unfolded side and side of the cylinder of FIG. 4A, wherein The predetermined area is a rectangle on the side of the unfolded cylinder and does not penetrate the side of the cylinder in the circumferential direction and the axial direction of the cylinder.
  • Fig. 5A exemplarily describes a circular pattern and a fan ring pattern as a preset region in the method 2 of preparing an artificial tissue precursor of the present invention.
  • Figure 5B exemplarily shows the shape of a luminal implant or lumen model of the present invention.
  • Figure 6 is a photomicrograph of primary cultured fourth generation adipose-derived mesenchymal stem cells in Example 1. It can be seen from the figure that the cell morphology is uniform and the growth state is good.
  • Fig. 7 is a view showing the form of a biobrick containing rhesus adipose-derived mesenchymal stem cells in Example 1.
  • Example 8 is a photograph of a bio-brick containing rhesus adipose-derived mesenchymal stem cells photographed by a laser confocal microscope in Example 1, wherein green fluorescence is a shell of microcapsules, and red fluorescence is adipose-derived mesenchymal stem cells.
  • Fig. 9 is a photograph of an artificial blood vessel precursor obtained in Example 2.
  • FIG. 10A-C are artificial blood vessels obtained by implanting the artificial blood vessel precursor prepared in Example 2 into a rhesus monkey, 5 days after surgery.
  • Fig. 10A shows the overall shape of the artificial blood vessel.
  • Fig. 10B shows the structure obtained by removing the tubular support, and
  • Fig. 10C shows the form in which the tissue is longitudinally sectioned. As can be seen from the figure, the surface of the tubular support has formed endothelial tissue.
  • Fig. 11 is a result of HE staining of artificial blood vessel tissue in Example 2
  • Fig. 11A is a normal blood vessel
  • Fig. 11B is an artificial blood vessel.
  • the artificial blood vessel has a similar cell arrangement to the normal blood vessel, the endothelial cell layer (referred to by the thin arrow) and the smooth muscle cell layer (referred to by the thick arrow).
  • Fig. 12 is a graph showing the results of ⁇ -SMA staining of artificial blood vessel tissue in Example 2, in which ⁇ -SMA positive cells were smooth muscle cells.
  • Fig. 12A is a normal blood vessel
  • Fig. 12B is an artificial blood vessel.
  • adipose-derived mesenchymal stem cells constituting an artificial blood vessel differentiate into smooth muscle cells, and have a cell morphology, arrangement, and orientation similar to those of normal blood vessels.
  • Fig. 13 shows the results of CD31 staining of artificial blood vessel tissue in Example 2, and CD31 positive cells were endothelial cells.
  • Fig. 13A is a normal blood vessel
  • Fig. 13B is an artificial blood vessel.
  • adipose-derived mesenchymal stem cells constituting an artificial blood vessel differentiate into endothelial cells at the surface in contact with blood, and a cell morphology and arrangement similar to those of normal blood vessels appear.
  • Figure 14 shows the morphology and blood flow direction of the artificial blood vessel of Example 3.
  • Fig. 14A shows the results of ultrasonic examination, and it can be seen from the figure that the lumen of the artificial blood vessel is unobstructed.
  • Fig. 14B shows the results of color Doppler detection. The results show that the blood flow directions on both sides of the artificial blood vessel are the same, which proves that the blood vessels are unobstructed.
  • FIG. 15A and 15B show the results of immunohistochemical staining of the artificial blood vessel of Example 3.
  • Fig. 15A shows the results of ⁇ -SMA staining, in which the adipose-derived mesenchymal stem cells differentiate into smooth muscle cells, as indicated by the thick arrows in the figure.
  • Figure 15B shows the results of CD31 staining, as indicated by the thin arrows in the figure, in the vascular blood vessels, adipose-derived mesenchymal stem cells Differentiation into endothelial cells.
  • Figure 15C shows the results of Sirius red staining. As shown, the artificial blood vessels form a collagen structure similar to normal blood vessels.
  • 16A and 16B show the results of immunohistochemical staining after the artificial blood vessel precursor was implanted into the rhesus monkey for 14 days in Example 4.
  • Fig. 16A shows the results of ⁇ -SMA staining. As indicated by the thick arrows in the figure, adipose-derived mesenchymal stem cells differentiate into smooth muscle cells in artificial blood vessels.
  • Fig. 16B shows the results of CD31 staining, in which the adipose-derived mesenchymal stem cells differentiate into endothelial cells in the artificial blood vessels as indicated by the thin arrows.
  • 17A-17C are cross-sectional views of an artificial blood vessel obtained by implanting an artificial blood vessel precursor into a rhesus monkey for 14 days, and a result of immunohistochemical staining of an artificial blood vessel, respectively, in Example 6.
  • Fig. 17A is a cross-sectional view showing an artificial blood vessel obtained by implanting an artificial blood vessel precursor into a rhesus monkey for 14 days.
  • Fig. 17B shows the results of ⁇ -SMA staining. As indicated by the thick arrows in the figure, adipose-derived mesenchymal stem cells differentiate into smooth muscle cells in artificial blood vessels.
  • Fig. 17C shows the results of CD31 staining, in which the adipose-derived mesenchymal stem cells differentiate into endothelial cells in the artificial blood vessels as indicated by the thin arrows.
  • Figure 18 shows the process of preparing an artificial blood vessel precursor of Example 7.
  • Figure 18A Printing bio-bricks on a rotating rod to form a tubular bio-construct
  • Figure 18B and 18C placing artificial blood vessels from left to right over a tubular bio-construct
  • Figure 18D Artificial blood vessels and tubular bio-constructs Bonded together to form an artificial blood vessel precursor.
  • Figure 19 depicts the process of preparing an artificial blood vessel precursor using the degradable polylactic acid tubular support and the bio-brick prepared in Example 1 in Example 8.
  • 19A and 19B show a tubular solid support produced by electrospinning using polylactic acid as a basic material.
  • Fig. 19C shows the operation of cutting the polylactic acid tubular support, then dropping the medical glue on one side of the tubular support, and placing the bio-brick on the other side.
  • Figure 19D shows that the medical glue can penetrate through the tube wall, allowing the bio-brick to adhere to the inner wall to obtain an artificial blood vessel precursor.
  • the medical glue is added to the outer wall of the polylactic acid tubular solid support to infiltrate the medical glue to the inner wall. Since the medical glue can penetrate the electrospun polylactic acid tube wall, the bio-brick is fixed.
  • polylactic acid can be used as a solid support
  • electrospinning The solid support to which the pore structure is permeable, the medical glue can penetrate through the tube wall, the medical glue can be added on one side of the solid support, and the biological brick can be placed on the other side, thereby fixing the biological brick and obtaining the artificial tissue. body.
  • Figure 20 is a diagram showing the experimental steps and experimental results of preparing a tubular three-dimensional construct using biobrick, fibrinogen and thrombin in Example 9, wherein Fig. 20A shows the attachment/assembly of fibrinogen on the surface of the biobrick. Fig. 20B shows that the annular auxiliary structure is constructed with an auxiliary material (optional step); Fig. 20C shows that the second reagent is dropped along the annular auxiliary structure to draw a circular pattern; Fig. 20D shows that The assembly unit is placed along the circular pattern to form a ring structure; FIG.
  • FIG. 20E shows that the annular pattern is drawn with the second reagent on the upper surface of the annular structure, and then the assembly unit is placed along the circular pattern (optionally, This step is repeated one or more times to construct a construct containing a multilayer structure;
  • Figure 20F shows the constructed tubular structure;
  • Figure 20G shows the removal of the auxiliary structure (optional step).
  • Experimental results show that the method of the present invention can be used to construct tubular three-dimensional constructs in a fast, multi-directional, and precise manner.
  • Figure 21 shows the results of microscopic observation of the tubular structure (Fig. 21A) and the cultured tubular structure (Fig. 21B) which were just prepared in Example 9. The results show that in the tubular structure just prepared, the bio-bricks have not yet merged with each other, and the cells are evenly distributed in the respective bio-bricks. In the cultured tubular structure, the bio-bricks are completely fused and closely connected. A complete biological construct is formed.
  • Fig. 22 shows the results of observation and detection of the tissue structure of the vascular implant using HE staining in Example 10, and the scales in the figure were both 200 ⁇ m.
  • the results showed that after 4 hours of implantation, there were still gaps between the bio-bricks, which were not connected to each other independently; after 8 hours to 24 hours, the bio-bricks gradually merged into one another; as the implantation time increased, the bio-bricks The fused artificial blood vessels gradually form a histological structure similar to that of normal blood vessels.
  • Figures 23 and 24 show the results of detecting the expression of CD31 in a vascular implant using immunohistochemical staining in Example 10.
  • Fig. 23 is a result of magnifying 100 times, and the scales in the figure are both 200 ⁇ m.
  • Fig. 24 shows the result of a magnification of 400 times, and the scales in the figure are both 50 ⁇ m.
  • the results showed that after 5 days of implantation, endothelial cells appeared in the luminal surface of the vascular implant and the blood; as the implantation time increased, the endothelial cells increased continuously, and at 28 days, a more complete endothelial cell similar to normal blood vessels was formed.
  • Floor shows that after 5 days of implantation, endothelial cells appeared in the luminal surface of the vascular implant and the blood; as the implantation time increased, the endothelial cells increased continuously, and at 28 days, a more complete endothelial cell similar to normal blood vessels was formed.
  • Floor shows that after 5 days of implantation
  • Fig. 25 shows the results of detecting the expression of ⁇ -SMA in a vascular implant using the immunohistochemical staining method in Example 10, and the scales in the figure were both 200 ⁇ m.
  • the results showed that after 8 hours of implantation, the adipose-derived mesenchymal stem cells encapsulated in bio-brick began to differentiate into smooth muscle cells and expressed ⁇ -SMA; after 3 days of implantation, the morphology of adipose-derived mesenchymal stem cells gradually changed to smooth muscle cells and ⁇ The expression of SMA is further increased; as the implantation time increases, the amount of smooth muscle cells gradually increases and forms a smooth muscle cell layer similar to normal blood vessels.
  • Figure 26 shows the attachment of rhesus autologous blood vessels to vascular implants using HE staining in Example 11. The results of observation of the tissue structure and the results of detection of the expression of CD31 and ⁇ -SMA by immunohistochemical staining, respectively.
  • the first line of pictures is the result of HE staining, the scale in the figure is 200 ⁇ m; the second line is the result of CD31, the scale in the figure is 50 ⁇ m; the third line is the detection result of ⁇ -SMA, in the figure
  • the scale is 200 ⁇ m.
  • the thick arrows in the figure indicate autologous blood vessels, and the thin arrows indicate blood vessel implants.
  • Fig. 27 shows the results of staining vascular collagen using the Sirius red staining method in Example 12, and the scale in the figure was 100 ⁇ m. The results showed that after 5 days of implantation of the vascular implant, collagen expression began to appear; as the implantation time increased, the expressed collagen gradually increased and began to stratify, forming a collagen structure similar to normal blood vessels.
  • Fig. 28 shows the results of ultrasonic examination of the vascular implant (first line picture) and color Doppler detection result (second line picture) in the embodiment 13. The results showed that the blood vessels were vascularized and the blood flow was continuous, the inner surface of the lumen was smooth and no thrombosis, no abnormal hyperplasia, and there was no stenosis at the junction with the normal blood vessels.
  • Figure 29 shows the results of enhanced CT of a vascular implant in Example 14. The results showed that the vascular implants had smooth blood flow and no blockage.
  • Figures 30A and 30B show, respectively, the expanded polytetrafluoroethylene sheet-like solid support of Example 15, and the vascular patch precursor formed thereon by 3D printing with bio-bricks.
  • Figures 30C and 30D show, respectively, the polycaprolactone sheet-like solid support of Example 16, and the vascular patch precursor formed thereon by microcapsules by 3D printing.
  • FIG. 31A and 31B show, respectively, in Example 17, in which a vascular defect was made on the abdominal aorta of a rhesus monkey, and the vascular patch precursor was sutured to the defect site.
  • the thick arrow indicates the precursor of the blood vessel patch containing the biobrick prepared in Example 15
  • the thin arrow indicates the blood vessel patch precursor containing the microcapsule prepared in Example 16.
  • 32A and 32B respectively show vascular tissue formed from a blood vessel patch precursor containing a biobrick and a blood vessel patch precursor containing microcapsules after 7 days of implantation in Example 17. As shown in the figure, the bio-bricks or microcapsules in the patch are integrated to form a complete inner membrane.
  • Figure 33 shows the results of immunohistochemical staining of vascular tissue for CD31 and ⁇ -SMA in Example 17.
  • Figures 33A and 33B show the results of detection of vascular tissue formed from a vascular patch precursor containing biobricks. Result It was shown that bio-brick adipose-derived mesenchymal stem cells differentiated into endothelial cells (Fig. 33A) and smooth muscle cells (Fig. 33B) after 7 days of in vivo implantation.
  • Figures 33C and 33D show the results of detection of vascular tissue formed from a vascular patch precursor containing microcapsules. The results showed that after 7 days of in vivo implantation, the adipose-derived adipose-derived mesenchymal stem cells differentiated into endothelial cells (Fig. 33C) and smooth muscle cells (Fig. 33D).
  • Figure 34 is a view showing the state of the biological brick in the elastic modulus test in Example 18.
  • 35 is a stress-strain curve of the bio-brick prepared in Example 18.
  • the effective Young's modulus value of the bio-brick is 24.77 kPa.
  • Fig. 36 schematically shows the main structure of the 3D bioprinter used in the embodiment 19.
  • Figure 37 shows a tubular biological construct formed on the rotating rod of the spinner in Example 19 having a length of 20 mm and a thickness of about 1 mm.
  • Figure 38 shows an artificial blood vessel precursor prepared in Example 19.
  • Figure 39 shows an artificial blood vessel precursor implanted in a rhesus monkey in Example 19.
  • Figure 40 is a fluorescence micrograph of a vascular implant in Example 19, in which vascular endothelial cells were fluorescently labeled using green fluorescence, and the scale was 200 ⁇ m in the figure. As shown, the vascular implant forms a complete endothelial cell layer.
  • Figure 41 is a fluorescence micrograph of a vascular implant in Example 19, in which vascular smooth muscle cells were fluorescently labeled using red fluorescence, and the scale was 200 ⁇ m in the figure. As shown, the vascular implant forms a complete layer of smooth muscle cells.
  • microcapsule refers to a microstructure (eg, a micron to millimeter-scale structure) containing cells and a biocompatible material, wherein the cells are encapsulated in the biocompatibility. Inside the material. Microcapsules of the invention It has a stable structure under physiological conditions (for example, 4-37 ° C, for example, pH between 6-8, such as under physiological fluid shear forces). In certain preferred embodiments, the microcapsules have a mechanical strength that does not cause microcapsule breakage during aspiration or extrusion.
  • tissue refers to a collection of cells composed of a population of cells of identical or similar morphology, and which typically also comprises a non-cellular form of matter (referred to as intercellular substance, such as matrix, fiber, etc.). ).
  • the tissue can include one or more cells.
  • organ refers to a structure composed of different cells and tissues that is used to achieve a particular function or functions.
  • the organ can include one or more tissues.
  • artificial tissue refers to a tissue that is not formed by natural tissue production or development processes.
  • the artificial tissue may be an artificially manufactured tissue, for example, a tissue obtained by culturing an artificial tissue precursor.
  • the term “artificial tissue precursor” refers to an object comprising a solid support and a plurality of microcapsules of the invention, wherein at least one microcapsule is attached to a solid support.
  • the artificial tissue precursor comprises a solid support and a biological construct constructed from the microcapsules.
  • the artificial tissue precursors of the present invention are capable of forming artificial tissue after the steps of cultivation, induction, and the like.
  • biological construct refers to an object constructed using the microcapsule of the present invention, which may have a two-dimensional or three-dimensional structure, and may be used to prepare an artificial tissue precursor.
  • a microcapsule or biological construct that is attached to a solid support means that the microcapsule or biological construct is bound to a solid support.
  • solid support refers to an object of a certain shape in the artificial tissue precursor of the present invention that conforms to a microcapsule or a biological construct composed of microcapsules.
  • the solid support is capable of providing a corresponding region upon which the biological construct is fully conformed.
  • luminal refers to an organ that is tubular in shape and has a hollow lumen, such as a circulatory lumen, a digestive lumen, a snorkel lumen, a urinary lumen, or a genital lumen, such as a blood vessel, an esophagus, Trachea, stomach, bile duct, intestine (including small intestine and large intestine, such as duodenum, jejunum, ileum, cecum (including appendix), ascending colon, colon right circumflex, transverse colon, colon left koji, descending colon, sigmoid colon, rectum) , fallopian tubes, vas deferens, ureters, bladder or lymphatic vessels).
  • a circulatory lumen such as a circulatory lumen, a digestive lumen, a snorkel lumen, a urinary lumen, or a genital lumen
  • a blood vessel such as a circulatory lumen, a digestive lumen, a snorkel lumen, a urinar
  • artificial lumen includes lumens that are not formed by natural tissue formation or development processes, as well as flaky artificial tissue that is capable of forming lumens with native tissue.
  • the artificial lumen can be obtained by culturing the artificial tissue precursor of the present invention.
  • an artificial blood vessel refers to an artificially manufactured vascular substitute that is generally tubular.
  • an artificial blood vessel is used to reconstruct or repair a vessel that is stenotic, occluded, dilated, damaged, or deformed.
  • the artificial blood vessel is obtained by culturing a tubular artificial tissue precursor of the present invention (e.g., in vitro culture, or implanted in vivo).
  • vascular patch refers to an object used to repair a damaged blood vessel, which is typically in the form of a sheet.
  • Vascular patches can be used to repair the sputum of blood vessels caused by hemangioma, vascular stenosis, etc., and are used in large blood vessels such as aorta. Often, vascular patches require easy suturing and hemostasis.
  • Vascular patches can be used in patients with a defect in the vessel wall but without the need for an entire vascular resection.
  • the vascular patch is obtained by culturing the flaky artificial tissue precursor of the present invention (e.g., in vitro culture, or implanted in vivo).
  • the term "luminal implant” refers to an object that can be implanted in a subject for replacement, reconstruction or repair of a lumen of a subject, comprising one or more of the present invention.
  • Artificial tissue precursors eg tubular artificial tissue precursors or flaky artificial tissue precursors
  • the luminal implant of the present invention comprises a plurality of tubular artificial tissue precursors (or artificial lumens) in fluid communication between the tubular artificial tissue precursors (or artificial lumens).
  • the luminal implant of the present invention may be a linear tubular structure or a branched tubular structure such as an X-shaped tubular shape, a Y-shaped tubular shape or a T-shaped tubular shape.
  • the luminal implant is a vascular implant.
  • the luminal implant further comprises a pharmaceutically active ingredient, an inductive device, and/or an adjustment device.
  • mechanical protection means that the microcapsules have a certain mechanical strength (for example, mechanical strength that does not cause microcapsule breakage during suction or extrusion), thereby reducing or avoiding encapsulation therein.
  • the cells are subject to external mechanical damage/mechanical damage.
  • the microcapsules can cause the cells encapsulated to be washed away by the fluid in the lumen, which is beneficial to the artificial tissue precursor to normal tissues. Conversion.
  • microcapsules eg, biobricks
  • microcapsules can protect cells encapsulated therein from manipulation (eg, during 3D bioprinting) without mechanical damage (including shearing) Shear and squeezing force).
  • the use of microcapsules to construct artificial tissue precursors (such as artificial blood vessel precursors), after implantation in the body, can protect the cells encapsulated in the microcapsules from being washed away by flowing body fluids (such as blood flow), which is beneficial to artificial tissues to normal tissues. Conversion.
  • biocompatible material refers to a material that (and its degradation products) is non-toxic to cells and that is compatible with the host after implantation into a host, such as a human body, Does not cause significant or serious side effects, for example, does not cause toxic effects on the host (such as human tissue), does not cause host immune rejection, allergic reactions or inflammatory reactions.
  • biodegradable material refers to a material that is capable of being degraded and absorbed by a cell or organism and whose degradation products are biocompatible. Such materials may be of natural origin (eg, derived from animals and plants) or synthetic.
  • bio-material refers to a natural or man-made material that can be used to diagnose, repair, or enhance the function of human tissues and organs, which can be used to replace, repair, and alive tissue. Ability to perform, enhance, or replace a tissue's loss of function due to illness, injury, etc.
  • Biomaterials mainly include metal materials (such as alkali metals and their alloys), inorganic materials (such as bioactive ceramics, hydroxyapatite, etc.) and organic materials.
  • Organic materials mainly contain polymer materials. Biomaterials can be further classified into bioinert, bioactive or biodegradable materials depending on the use of the material.
  • viscosity refers to a measure of the viscosity of a fluid and is an indication of the phenomenon of internal friction of fluid flow forces.
  • biological printing refers to the utilization of biological materials (including but not limited to, biomolecules such as proteins, lipids, nucleic acids and metabolites; cells such as cell solutions, cell-containing gels, cell suspensions) , cell concentrates, multicellular aggregates and multicellular bodies; subcellular structures such as organelles and cell membranes; printing of molecules associated with biomolecules such as synthetic biomolecules or analogs of biomolecules.
  • biomolecules such as proteins, lipids, nucleic acids and metabolites
  • cells such as cell solutions, cell-containing gels, cell suspensions
  • subcellular structures such as organelles and cell membranes
  • printing refers to the process of depositing material in a predetermined pattern.
  • the microcapsules are printed by extrusion printing or modular printing.
  • the microcapsules are printed using a modular printing method.
  • module printing method refers to a method of printing by sucking/picking a module (such as a microcapsule of the present invention, such as a bio-brick) and accurately positioning/arranging it. Since the microcapsules used in the present invention contain cells, such modular printing methods are also referred to herein as "modular bioprinting methods".
  • bioprinting is preferably accomplished by a method that matches an automated or semi-automated, computer-aided, three-dimensional prototype device, such as a bio-printer.
  • printing eg, bio-printing
  • a printer such as a 3D printer or a bio-printer
  • an automated or non-automated mechanical process and Printing without a printer
  • printing by hand or by hand for example, using a pipette
  • alginic acid refers to a type of polysaccharide extracted from brown algae, which is ⁇ -1,4-D-mannuronic acid (M unit) and ⁇ -1,4-L-gulose.
  • M unit ⁇ -1,4-D-mannuronic acid
  • G unit A random block copolymer of aldehyde acid
  • the M and G units in alginic acid are linked to form a block copolymer by a 1,4 glycosidic bond in a combination of MM, GG or MG.
  • the experimental formula for alginic acid is (C 6 H 8 O 6 ) n , and its molecular weight is usually from 4 kDa to 1500 kDa.
  • alginate refers to a salt formed from alginic acid including, but not limited to, sodium alginate, calcium alginate, strontium alginate, strontium alginate, and the like.
  • oxidized alginate refers to a product formed by the oxidation reaction of an alginate such as sodium alginate. Typically, the oxidation reaction will cause the hydroxyl group of a portion of the uronic acid unit in the alginate (e.g., sodium alginate) to be oxidized to an aldehyde group.
  • degree of oxidation refers to the mole fraction of oxidized uronic acid units to the total uronic acid units of alginic acid or alginate.
  • sticking agent refers to an agent used to adjust the viscosity of a liquid or semi-solid (eg, a gel).
  • the second agent of the present invention preferably has a viscosity suitable for drawing a pattern or for coating.
  • the viscosity of the second agent can be conveniently adjusted by the use of a viscous agent.
  • biobrick refers to a basic unit that can be used for biological printing, etc., comprising: a cell, a core layer encasing the cell, and a shell layer encapsulating the core layer, wherein the core layer
  • the shell and the shell are each independently made of a biodegradable material.
  • the biodegradable material in the core layer and the shell layer is capable of reducing or avoiding mechanical damage to cells within the bio-brick during operation and is capable of providing a substance (eg, nutrients, Controlled release of extracellular matrices, cytokines, pharmaceutically active ingredients, etc. to promote cellular activity and function (proliferation, differentiation, migration, secretion or metabolism) or to maintain cell dryness.
  • the shell of the bio-brick or bio-brick has a certain mechanical strength to enable steric stacking.
  • the bio-brick and its shell have suitable mechanical protection properties (for example, having a suitable hardness and/or modulus of elasticity).
  • the shell layer is also capable of providing a microenvironment, such as nutrients, for the life activities of the cells.
  • biological ink refers to a liquid, semi-solid (eg, gel) or solid composition comprising one or more microcapsules (eg, biobricks) of the invention.
  • the bio-ink of the present invention may be a solution, suspension, gel, or concentrate comprising microcapsules (eg, bio-bricks).
  • bio-ink can be used for bioprinting to create a particular geometry; and preferably, the resulting geometry can be further stacked to form a biological construct having a particular shape and structure.
  • cells within the microcapsules (eg, biobricks) in the bio-ink can perform various desired life activities before, during, and/or after bio-printing.
  • cells within a microcapsule are dormant prior to bioprinting, while growth and proliferation are performed after bioprinting to form a robust bioconstruct.
  • the bioink is an extrudable composition.
  • extrudable means that the composition is capable of being forced (eg under pressure) Formed through a nozzle or orifice.
  • the present application relates to an artificial tissue precursor comprising a solid support and a plurality of microcapsules, wherein at least one microcapsule is attached to a solid support comprising cells and biological cells encapsulating the cells Capacitive material.
  • Fig. 1 schematically depicts an exemplary structure of a microcapsule of the present invention.
  • the cells may be uniformly dispersed in the microcapsules or may be brought together and located inside the microcapsules.
  • the artificial tissue precursor is a lumen (eg, a circulating lumen, a digestive lumen, a sinus lumen, a urinary lumen, or a genital lumen) precursor.
  • a lumen eg, a circulating lumen, a digestive lumen, a sinus lumen, a urinary lumen, or a genital lumen
  • the lumen is a lumen comprising epithelial cells (eg, blood vessels, esophagus, trachea, stomach, bile duct, intestinal tract (including the small and large intestines, eg, duodenum, jejunum, ileum, The cecum (including the appendix), the ascending colon, the right colon of the colon, the transverse colon, the left collateral, the descending colon, the sigmoid colon, the rectum, the fallopian tube, the vas deferens, the ureter, the bladder or the lymphatic vessels).
  • epithelial cells eg, blood vessels, esophagus, trachea, stomach, bile duct, intestinal tract (including the small and large intestines, eg, duodenum, jejunum, ileum, The cecum (including the appendix), the ascending colon, the right colon of the colon, the transverse colon, the left collateral, the descending colon, the sigmoid colon,
  • the artificial tissue precursor is tubular (eg, a tubular opening or a tubular opening).
  • a tubular artificial tissue precursor that does not open the sidewall can be used to replace a narrow, occluded, dilated, damaged or deformed lumen, or to construct a lumen bypass (eg, a vascular bypass).
  • a tubular artificial tissue precursor with open sidewalls can be used to repair the damaged lumen.
  • the artificial tissue precursor is in the form of a sheet (eg, a planar sheet or a curved sheet).
  • the flaky artificial tissue precursor can be used to repair damaged lumens.
  • a plurality of said microcapsules constitute one or more biological constructs.
  • one or more biological constructs are attached to a solid support.
  • the microcapsules have a stable structure under physiological conditions (eg, 4-37 ° C, such as a pH between 6-8, such as under physiological fluid shear forces). In certain preferred embodiments, the microcapsules have mechanical strength that does not cause microcapsule breakage during aspiration or extrusion. In certain preferred embodiments, the microcapsules provide mechanical protection to the encapsulated cells.
  • the microcapsules are capable of reducing or avoiding mechanical damage to cells within the microcapsules during handling (eg, bioprinting). In certain preferred embodiments, the microcapsules of the invention are capable of reducing mechanical damage to cells during bioprinting.
  • the microcapsules of the present invention can be used in comparison to the use of cells directly for bioprinting, using the same bioprinter and the same printing conditions. It is sufficient to reduce the mechanical damage to the cells by at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 70%, 80%, or 90%.
  • the microcapsules of the invention are capable of retaining the biological activity (e.g., proliferation, differentiation, migration, secretion, and/or metabolism) of cells within the microcapsules during bioprinting.
  • the biological activity e.g., proliferation, differentiation, migration, secretion, and/or metabolism
  • at least 90% of the cells in the microcapsule survive at least 3 hours, 6 hours, 12 hours, 1 day, 2 days, 4 days, or 7 days after bioprinting.
  • At least 80%, 85%, 87.5%, 90%, 92.5%, 95%, or 98% of the cells within the microcapsule are capable of proliferating and/or differentizing after 24 hours of bioprinting. In certain preferred embodiments, at least 80%, 85%, 87.5%, 90%, 92.5%, 95%, or 98% of the cells within the microcapsules have normal metabolism after 24 hours of bioprinting. In certain preferred embodiments, at least 80%, 85%, 87.5%, 90%, 92.5%, 95%, or 98% of the cells within the microcapsule are capable of migrating after 24 hours of bioprinting. In certain preferred embodiments, at least 80%, 85%, 87.5%, 90%, 92.5%, 95%, or 98% of the cells in the microcapsules are capable of being secreted after 24 hours of bioprinting.
  • the microcapsules provide a microenvironment for the life activities of the cells.
  • the microcapsules provide a steric structure and microenvironment suitable for cell adhesion and expansion such that cells can normally proliferate, differentiate, migrate, secrete, or metabolize within the structure.
  • the microenvironment refers to the environment in which the cells grow, and the elements included include physical factors such as spatial structure, mechanical strength, temperature, humidity, osmotic pressure, etc.; chemical factors such as pH, ion concentration, etc.; biological factors, including cells, Cytokines, etc. These elements together constitute the environment of cellular life activities, and dynamically regulate the proliferation, differentiation, migration, secretion and metabolism of cells grown in this environment.
  • the microcapsules are capable of providing nutrients to the life activities of the cells.
  • the microcapsules are biobricks.
  • the biobrick of the present invention comprises: a cell, a core layer encapsulating the cell, and a shell layer encapsulating the core layer, wherein the core layer and the shell layer are each independently made of a biodegradable material.
  • the biodegradable material in the core layer and the shell layer is capable of reducing or avoiding mechanical damage to cells within the bio-brick during operation and is capable of providing a substance (eg, nutrients, Controlled release of extracellular matrices, cytokines, pharmaceutically active ingredients, etc. to promote cellular activity and function (proliferation, differentiation, migration, secretion or metabolism) or to maintain cell dryness.
  • the core layer of the biobrick provides a spatial structure and microenvironment suitable for cell adhesion and expansion such that cells can normally proliferate, differentiate, migrate, secrete, or metabolize within the structure.
  • the microenvironment refers to the environment in which the cells grow, and the elements included include physical factors such as spatial structure, mechanical strength, temperature, humidity, osmotic pressure, etc.; chemical factors such as pH, ion concentration, etc.; biological factors, including cells, Cytokines, etc. These elements together constitute the environment of cellular life activities, and dynamically regulate the proliferation, differentiation, migration, secretion and metabolism of cells grown in this environment.
  • the core layer is capable of providing a microenvironment for the life activities of the cells, such as spatial structures, nutrients, and the like.
  • the shell of the biobrick provides mechanical protection to the wrapped cells.
  • the shell of the bio-brick or bio-brick has a certain degree of mechanical strength to enable steric stacking.
  • the bio-brick and its shell have suitable mechanical protection properties (for example, having a suitable hardness and/or modulus of elasticity).
  • suitable mechanical protection properties for example, having a suitable hardness and/or modulus of elasticity.
  • cells in a bio-brick are susceptible to damage or death during operation due to damage from external pressure or shear forces.
  • FIG. 2A-E schematically depict an exemplary structure of a biobrick of the present invention comprising: a cell, a core layer encasing the cell, and a shell layer encapsulating the core layer.
  • FIG. 2A schematically depicts a structure of a biobrick of the present invention comprising a core layer and a shell layer, wherein the core layer is coated with cells and the shell layer is located at the core layer Outside, and encapsulating the core layer.
  • FIG. 2B schematically depicts a structure of a biobrick of the present invention comprising, from the inside to the outside, a core layer encasing cells, a first shell encapsulating the core layer, and a second shell surrounding the first shell layer.
  • Floor schematically depicts a structure of a biobrick of the present invention comprising, from the inside to the outside, a core layer encasing cells, a first shell encapsulating the core layer, and a second shell surrounding the first shell layer.
  • Floor schematically depicts a structure of a biobrick of the present invention comprising, from the inside to the outside, a core layer encasing cells, a first shell encapsulating the core layer, and a second shell surrounding the first shell layer.
  • 2C schematically depicts a structure of a biobrick of the present invention comprising, from the inside to the outside, a first core layer encasing cells, a second core layer enclosing cells outside the first core layer, and encapsulating said a first shell layer of the first core layer and the second core layer.
  • 2D schematically illustrates a structure of the bio-brick of the present invention, which comprises, in order from the inside to the outside, a first nuclear layer enclosing the cells, a second nuclear layer enclosing the cells outside the first nuclear layer, and the encapsulation.
  • One core layer and second core layer a first shell layer, and a second shell layer surrounding the first shell layer.
  • 2E schematically depicts a structure of a biobrick of the present invention comprising, from the inside to the outside, a first core layer encapsulating cells, a first shell layer encapsulating the first core layer, and a second core encapsulating cells a layer, and a second shell encapsulating the second core layer.
  • the size of the microcapsules of the present invention can be selected according to actual needs without particular limitation.
  • the size of the spherical microcapsules can usually be clearly defined by their diameter.
  • the term “diameter” cannot be used to describe a non-spherical structure.
  • the term “diameter” is also used to describe the size of the non-spherical microcapsules.
  • the term “diameter” means the diameter of a spherical microcapsule having the same volume as a non-spherical microcapsule.
  • the diameter of the spherical microcapsules is used to describe the size of the non-spherical microcapsules having the same volume.
  • the size of the microcapsules of the invention may be from 20 to 2000 ⁇ m, such as from 30 to 1900 ⁇ m, from 40 to 1800 ⁇ m, from 50 to 1700 ⁇ m, from 60 to 1600 ⁇ m, 70. - 1500 ⁇ m, 80-1400 ⁇ m, 90-1300 ⁇ m, 100-1200 ⁇ m, 200-1000 ⁇ m, 300-800 ⁇ m, 400-600 ⁇ m, 100-500 ⁇ m.
  • the size of the microcapsules of the invention can be 20-30, 30-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-600, 600-700, 700-800, 800-900, 900-1000, 1000-1500, 1500-2000, 20- 50, 20-100, 100-200, 200-400, 500-600, 600-800, 800-1000, or 1000-2000 ⁇ m.
  • the size of the microcapsules of the invention is at least 20, 30, 50, 100, 120, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1500, or 2000 ⁇ m.
  • the shape of the microcapsule of the present invention can be selected according to actual needs without particular limitation.
  • the microcapsules of the present invention can be spherical, or of any desired shape (eg, cubes, rectangular prisms, hexagonal prisms, cylinders, or irregular shapes).
  • some shapes eg, spheres, cubes, rectangular prisms, hexagonal prisms
  • the microcapsules of the invention are solid or semi-solid. In certain preferred embodiments, the microcapsules of the invention are in a gel state. For example, the core and/or shell layers of the microcapsules of the invention may be in a gel state. In certain preferred embodiments, the microcapsules of the invention comprise a hydrogel. In certain preferred embodiments, the hydrogel comprises alginate, agarose, gelatin, chitosan, or other water soluble or hydrophilic polymer.
  • the microcapsules of the invention are in the form of a mixture. In such embodiments, the microcapsules can be contacted or fused with another microcapsule in the mixture. In certain preferred embodiments, the microcapsules of the invention are isolated microcapsules. For example, in certain embodiments, the microcapsules are not in direct contact with other microcapsules. In certain preferred embodiments, the isolated microcapsules of the invention are provided in a container.
  • microcapsules of the invention can be prepared using a variety of methods.
  • the microcapsules of the present invention can be prepared using methods for making microspheres, such as using a granulator.
  • the microcapsules of the invention are prepared under sterile conditions.
  • the microcapsules of the invention are prepared in a GMP workstation.
  • the microcapsules of the invention are prepared just prior to use.
  • the microcapsules of the invention are stored at 4 ° C after preparation, for example, for 3 hours, 6 hours, 12 hours, 1 day, 2 days, or 3 days.
  • the kind of the cells contained in the microcapsule of the present invention can be selected according to actual needs without particular limitation.
  • the microcapsules comprise epithelial cells, such as endothelial cells (eg, vascular endothelial cells), smooth muscle cells (eg, vascular smooth muscle cells), and/or undifferentiated cells.
  • the cells in the microcapsules are undifferentiated cells, such as stem cells (eg, adipose-derived mesenchymal stem cells, bone marrow mesenchymal stem cells, induced pluripotent stem cells, and embryonic stem cells).
  • stem cells eg, adipose-derived mesenchymal stem cells, bone marrow mesenchymal stem cells, induced pluripotent stem cells, and embryonic stem cells.
  • the undifferentiated cells are capable of differentiating into epithelial cells (eg, endothelial cells) and/or smooth muscle cells.
  • the undifferentiated cells are selected from the group consisting of stem cells (eg, adipose-derived mesenchymal stem cells, bone marrow mesenchymal stem cells, induced pluripotent stem cells, and embryonic stem cells) and progenitor cells (eg, endothelial progenitor cells).
  • stem cells eg, adipose-derived mesenchymal stem cells, bone marrow mesenchymal stem cells, induced pluripotent stem cells, and embryonic stem cells
  • progenitor cells eg, endothelial progenitor cells
  • the source of the cells contained in the microcapsule of the present invention can be selected according to actual needs without particular limitation.
  • the cells are obtained from an animal, such as a mammal, such as a human, a donkey, a monkey, a gorilla, a cow, a pig, a dog, a sheep, and a goat.
  • the cells are derived from a tissue selected from the group consisting of connective tissue (eg, loose connective tissue, dense connective tissue, elastic tissue, reticular connective tissue, and adipose tissue), muscle tissue (eg, , skeletal muscle, smooth muscle and myocardium), genitourinary tissue, gastrointestinal tissue, lung tissue, bone tissue, nerve tissue and epithelial tissue (eg, monolayer epithelium and stratified epithelium), endoderm-derived tissue, mesoderm-derived Tissue and ectoderm-derived tissue.
  • connective tissue eg, loose connective tissue, dense connective tissue, elastic tissue, reticular connective tissue, and adipose tissue
  • muscle tissue eg, , skeletal muscle, smooth muscle and myocardium
  • genitourinary tissue eg, , skeletal muscle, smooth muscle and myocardium
  • genitourinary tissue eg, , skeletal muscle, smooth muscle and myocardium
  • the number of cells contained in the microcapsule of the present invention can be selected according to actual needs without particular limitation.
  • the microcapsules of the present invention the core layer may comprise 1-106 cells, e.g. 10-900,20-800,30-700,40-600,50-500,60-400,70-300,80-200 , 10-100, 10-10 3 , 10-10 4 , 10-10 5 , 10-10 6 cells.
  • the microcapsules of the invention comprise at least 1, 2, 4, 6, 8, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150 , 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10 4 , 2x10 4 , 3x10 4 , 4x10 4 , 5x10 4 , 6x10 4 , 7x10 4 , 8x10 4 , 9x10 4 , 10 5 , 2x10 5 , 3x10 5 , 4x10 5 , 5x10 5 , 6x10 5 , 7x10 5 , 8x10 5 , 9x10 5 , or 10 6 cells.
  • the microcapsules of the invention comprise 1-2, 2-4, 4-6, 6-8, 8-10, 10-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, 150-200, 200-300, 300-400, 400-500, 500- 1000, 1000-2000, 2000-3000, 3000-4000, 4000-5000, 5000-10 4 , 10 4 -2x10 4 , 2x10 4 -3x10 4 , 3x10 4 -4x10 4 , 4x10 4 -5x10 4 , 5x10 4 - 10 5 , 10 5 -2x10 5 , 2x10 5 -3x10 5 , 3x10 5 -4x10 5 , 4x10 5 -5x10 5 , 5x10 5 -10 6 , 1-10 , 2-10 , 2-5 , 5-10 , 10 -20, 20-30, 30-50, 2-25, 25-50, 2-50, 50-100, 100
  • the microencapsulated cells include additional cells in addition to epithelial cells (eg, endothelial cells), smooth muscle cells, and/or undifferentiated cells as described above.
  • the additional cells are derived from a tissue selected from the group consisting of connective tissue (eg, loose connective tissue, dense connective tissue, elastic tissue, reticular connective tissue, and adipose tissue), muscle tissue ( For example, skeletal muscle, smooth muscle and myocardium), genitourinary tissue, gastrointestinal tissue, lung tissue, bone tissue, nerve tissue and epithelial tissue (eg, monolayer epithelium and stratified epithelium), endoderm-derived tissue, mesoderm source Tissue and tissue derived from ectoderm.
  • connective tissue eg, loose connective tissue, dense connective tissue, elastic tissue, reticular connective tissue, and adipose tissue
  • muscle tissue For example, skeletal muscle, smooth muscle and myocardium
  • genitourinary tissue gastrointestinal tissue
  • the additional cells are selected from the group consisting of muscle cells (eg, skeletal muscle cells, cardiomyocytes, smooth muscle cells, and myoblasts), connective tissue cells (eg, bone cells, chondrocytes, fibroblasts) And cells differentiated into osteoblasts, chondrocytes or lymphoid tissues), bone marrow cells, skin cells, epithelial cells, breast cells, vascular cells, blood cells, lymphocytes, nerve cells, Schwann cells, gastrointestinal cells, hepatocytes, Pancreatic cells, lung cells, tracheal cells, corneal cells, urogenital cells, kidney cells, adipocytes, parenchymal cells, pericytes, mesothelial cells, stromal cells, cells derived from endoderm, mesoderm-derived cells, ectodermal sources Cells, cancer-derived cells, cell lines, or any combination thereof.
  • muscle cells eg, skeletal muscle cells, cardiomyocytes, smooth muscle cells, and myoblasts
  • the microcapsules of the invention comprise cells and a nuclear layer that encapsulates the cells.
  • the core layer is capable of providing a microenvironment for the life activities of the cells.
  • the microcapsules provide a spatial structure and microenvironment suitable for cell adhesion and stretching, whereby cells can normally proliferate, differentiate, migrate, secrete or metabolize within the structure, or maintain dryness.
  • the microenvironment refers to the environment in which the cells grow, and the elements included include physical factors such as spatial structure, mechanical strength, temperature, humidity, osmotic pressure, etc.; chemical factors such as pH, ion concentration, etc.; biological factors, including cells, Cytokines, etc.
  • the core layer is capable of providing nutrients to the life activities of the cells.
  • the core layer is made of a biocompatible material.
  • the microcapsules further comprise a shell layer encapsulating the core layer.
  • the shell of the microcapsule provides mechanical protection to the encapsulated cells.
  • the shell of the microcapsule or microcapsule has a certain mechanical strength to enable steric packing.
  • the microcapsules and their shell layers have suitable mechanical protective properties (for example, having suitable hardness and/or modulus of elasticity).
  • cells within the microcapsule are susceptible to damage or death during operation (eg, during 3D printing) due to damage from external pressure or shear forces.
  • the hardness and/or elastic modulus of the microcapsules and their shells are too low, the cell viability in the microcapsules will be significantly reduced after manual operation, which may result in limited application of the microcapsules or a large amount of use. Cell.
  • the hardness and/or modulus of elasticity of the microcapsules and their shells are too high, it will result in the extension and migration of cells within the microcapsules, and the establishment of cell connections between cells of different microcapsules, Not conducive to the construction of organic whole (for example, artificial organization).
  • proper mechanical protection properties not only enable various operations on the microcapsules of the present invention (for example, performing 3D bioprinting, precise arrangement of microcapsules, etc.), but also facilitating cell expansion, migration, and cell establishment in the microcapsules. It is particularly preferred to join and form an organic construct (e.g., artificial tissue).
  • an organic construct e.g., artificial tissue
  • the core and/or shell layers of the microcapsules of the invention are each optionally treated (eg, using a core layer fixative or shell fixative, for example, to improve the core layer or shell) Mechanical properties of the layer).
  • the microcapsules, the core layer of the microcapsules, or the shell layers of the microcapsules each independently have about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, A hardness of 0.15, 0.2, 0.3, or 0.4 GPa.
  • the microcapsules, the core layer of the microcapsules, or the shell layers of the microcapsules each independently have 0.01-0.02, 0.02-0.03, 0.03-0.04, 0.04-0.05, 0.05-0.06, 0.06.
  • the microcapsules, the core layers of the microcapsules, or the shells of the microcapsules are each Independently having a hardness of 0.01-0.1 GPa or 0.01-0.4 GPa.
  • the microcapsules, the core layer of the microcapsules, or the shell of the microcapsules have a hardness of about 0.083 GPa.
  • the microcapsules, the core layer of the microcapsules, or the shell layers of the microcapsules each independently have about 0.01, 0.05, 0.1, 0.5, 0.8, 1, 1.2, 1.4, 1.6, 1.8, 2.
  • the microcapsules, the core layer of the microcapsules, or the shell layers of the microcapsules each independently have 0.01-0.05, 0.05-0.1, 0.1-0.5, 0.5-0.8, 0.8-1, 1 -1.2, 1.2-1.4, 1.4-1.6, 1.6-1.8, 1.8-2, 2-2.4, 2.4-2.8, 2.8-3.2, 3.2-4, 4-10, 10-20, 20-30, 30-40 40-50, 50-80, 80-100, 0.5-4, 0.5-1, 1-1.5, 1.5-2, 2-3, 0.8-1.6, 1.4-2.4, 0.8-3.2, 0.01-100, 1 -100, 10-100, or 0.5-50 MPa modulus of elasticity.
  • the microcapsules, the core layer of the microcapsules, or the shell layers of the microcapsules each independently have an elastic modulus of 0.01-1, 0.01-10, or 0.01-100 MPa.
  • the mechanical protection of the core or shell eg, hardness and modulus of elasticity
  • the shell layer is also capable of providing a microenvironment, such as nutrients, for the life activities of the cells.
  • the shell layer is made of a biocompatible material.
  • the biocompatible materials used to prepare the core layer and the shell layer may be the same or different. However, it is particularly preferred that the core layer and the shell layer have different compositions depending on their intended purpose. Without being bound by theory, it is generally believed that the shell provides the primary mechanical protection, while the core provides the primary nutrients and microenvironments required for cell life activities. Thus, in certain preferred embodiments, the core layer has more nutrients than the shell layer. In certain preferred embodiments, the shell layer has a lower rate of degradation than the core layer, but has a higher hardness and/or modulus of elasticity. In certain preferred embodiments, no cells are included in the shell.
  • the core layer and the shell layer comprise the same biocompatible material in different weight ratios, respectively.
  • the core layer and the shell layer can be made of the same biocompatible material, but contain biodegradable materials in different weight ratios.
  • the shell layer is permeable.
  • the shell is permeable to water, oxygen, and nutrients (saccharides such as glucose, fat, proteins, amino acids, short peptides, minerals, vitamins, cytokines, nucleotides, etc.).
  • a semi-permeable (ie, selectively permeable) shell may be advantageous because it allows nutrients such as water, oxygen, glucose, minerals, and amino acids to pass through the shell and into the core. And provided to the cells, and can prevent substances harmful to the cells (such as antibody proteins from the host immune system) from entering the nuclear layer.
  • a permeable shell is preferred and advantageous.
  • the permeability of the shell makes each Nutrients (including macromolecules and small molecular nutrients such as glucose, fat, proteins, amino acids, short peptides, minerals, vitamins, cytokines, nucleotides, etc.) can be exchanged more easily and smoothly, avoiding localized areas The cells are unable to get enough nutrients.
  • the permeability of the shell layer can promote the exchange of various nutrients, and promote the cells in the microcapsules inside the artificial tissue to obtain sufficient nutrition. substance.
  • the permeable shell facilitates signal transmission and cell connection between cells between different microcapsules.
  • cells secrete a variety of substances (including certain components of the extracellular matrix and a variety of signaling molecules) during growth, signaling and/or material communication with adjacent, even distal, cells. This affects or regulates the life activities of the cells themselves and the life activities of adjacent, even distal, cells. Therefore, if a permeability-selective shell is used, then signal transmission and/or material exchange between cells may be affected/obstructed, such as certain macromolecular signaling substances (eg, cytokine proteins) secreted by the cell. The inability to penetrate the shell may hinder the transmission of cellular signals and the establishment of cell connections between different microcapsules, which is not conducive to the construction of an organic whole (for example, artificial tissue).
  • macromolecular signaling substances eg, cytokine proteins
  • the use of a permeable shell is preferred for the microcapsules of the present invention.
  • the expression "permeability shell” means that various small molecules and macromolecular substances (for example, proteins) can freely pass through the shell layer.
  • the shell layer is transparent to molecules having a molecular weight below 5000 kDa.
  • the shell layer has a molecular weight below 200 kDa or a molecular weight of 200 kDa-300 kDa, 300 kDa-400 kDa, 400 kDa-500 kDa, 500 kDa-800 kDa, 800 kDa-1000 kDa, 1000 kDa-1500 kDa, 1500 kDa-2000 kDa, 2000 kDa.
  • Molecules in the range of -3000 kDa, 3000 kDa-4000 kDa or 4000 kDa-5000 kDa are permeabilized.
  • the shell layer is permeable to immunoglobulins (eg, IgG, IgM, IgA, IgD, IgE).
  • the shell layer has channels or pores for the exchange of material inside and outside the microcapsules.
  • nutrients saccharides such as glucose, fat, proteins, amino acids, short peptides, minerals, vitamins, cytokines, nucleotides, etc.
  • the channel has a diameter of at least 10, 20, 50, 100, 150, 200, 250, 300, 350, 400, or 500 nm.
  • the diameter of the channel is, for example, 1 nm to 5 ⁇ m; 10 nm to 2 ⁇ m; 100 nm to 1 ⁇ m; 200 to 800 nm, and the like.
  • the pores have a diameter of at least 100, 200, 400, 600, 800, 1000, 1500, 2000, 4000, or 5000 nm.
  • the thickness of the shell layer of the microcapsule of the present invention can be selected according to actual needs without particular limitation.
  • the shell layer of the microcapsules of the invention may have a thickness of from 1 to 20 ⁇ m, such as from 5 to 15 ⁇ m, such as from 8 to 12 ⁇ m.
  • the shell layer of the microcapsules of the present invention may have a thickness of about 0.1, 0.5, 1, 2, 5, 10, 15, 20, 25, 30, or 50 ⁇ m.
  • the shell layer of the microcapsules of the present invention may have a thickness of 0.1-0.5, 0.5-1, 1-2, 2-5, 5-10, 10-15, 15-20, 20 -25, 25-30, 30-50, 50-100, 100-200, 200-300, 300-400, 400-500, 0.1-1, 1-5, 1-10, 5-10, 10-20 , 10-30, 5-20, or 1-20 ⁇ m.
  • the shell of the microcapsules of the invention does not comprise cells.
  • the biocompatible materials of the present invention comprise a biodegradable material.
  • biodegradable materials for the preparation of microcapsules is particularly preferred.
  • the use of materials that are not degradable is disadvantageous for the use of microcapsules in the preparation of artificial tissue precursors. This is because, on the one hand, these non-degradable materials will be retained in the obtained artificial tissue, thereby limiting the application of artificial tissue; on the other hand, these non-degradable materials will hinder the establishment of cells between cells of different microcapsules. Connections are not conducive to building an organic whole (for example, artificial tissue).
  • the use of biodegradable materials in the shell layer is particularly advantageous and preferred for the preparation of artificial tissue precursors using microcapsules.
  • the biodegradable material is a biomaterial that is degradable.
  • the biodegradable material used to prepare the microcapsules may be naturally occurring (eg, naturally occurring biodegradable materials derived from plants and animals, such as collagen, fibrin, chitosan, algae) Acid, starch, hyaluronic acid, laminin, agarose, gelatin, dextran, and any combination thereof, synthetic, recombinantly produced, modified, or any combination thereof.
  • naturally occurring biodegradable materials derived from plants and animals such as collagen, fibrin, chitosan, algae
  • Acid starch, hyaluronic acid, laminin, agarose, gelatin, dextran, and any combination thereof, synthetic, recombinantly produced, modified, or any combination thereof.
  • the biodegradable material used to prepare the microcapsules is a naturally occurring biodegradable material.
  • the naturally occurring biodegradable material is selected from the group consisting of collagen, fibrin, chitosan, alginate (eg, sodium alginate or calcium alginate), starch, hyaluronic acid , laminin, agarose, gelatin, dextran, chitin, cellulose (eg bacterial cellulose), silk protein, chondroitin sulfate, heparin, fibrinogen, fibronectin, mucopolysaccharide, mucin, and Any combination thereof.
  • the biodegradable material used to prepare the microcapsules is a modified biodegradable material, such as a modified alginate, such as an alginate (eg, oxidized alginic acid) Sodium), modified gelatin (such as dialdehyde starch DAS cross-linked modified gelatin), modified cellulose (such as carboxymethyl cellulose, oxidized regenerated cellulose), and any combination thereof.
  • a modified alginate such as an alginate (eg, oxidized alginic acid) Sodium
  • modified gelatin such as dialdehyde starch DAS cross-linked modified gelatin
  • modified cellulose such as carboxymethyl cellulose, oxidized regenerated cellulose
  • the biodegradable material used to prepare the microcapsules is a synthetic biodegradable material such as polyphosphazene, polyacrylic acid and derivatives thereof (eg, polymethacrylic acid, acrylic acid, and Copolymer of methacrylic acid), polylactic acid (PLA), polyglycolic acid (PGA), polylactic acid-glycolic acid copolymer (PLGA), polyorthoester (POE), polycaprolactone (PCL), polyhydroxyl Butyrate (PHB), polyamino acid (eg poly-lysine), degradable polyurethane (eg Starch modified polyurethane), polyhydroxyalkanoate (PHAs), polyhydroxyvalerate (PHV), polybutylene succinate (PBS), polyvinyl alcohol, polydioxanone, poly pair Dioxanone, polydioxanone, polybutylene carbonate, and any combination thereof.
  • synthetic biodegradable material such as polyphosphazene, polyacrylic acid and
  • the biodegradable material used to prepare the microcapsules is capable of being degraded by an enzyme, such as an enzyme secreted by a cell.
  • the degradation rates of different biodegradable materials vary widely and can range from one month to several years. In the present invention, however, it is particularly preferred that the biodegradable material for preparing the shell layer degrades in a period of not more than one month, for example, no more than 30 days, no more than 25 days, no more than 20 days, no more than Degradation within 15 days, no more than 10 days, no more than 5 days, no more than 4 days, no more than 3 days, no more than 2 days, or no more than 1 day.
  • the biodegradable material used to prepare the microcapsules can be in 1-2 days, 2-3 days, 3-4 days, 4-5 days, 5-10 days, 10-15 days, 15-20 days, 20 -25 days, or degradation within 25-30 days.
  • the biodegradable material used to prepare the microcapsules degrades in no more than 10 days.
  • the rate of degradation is closely related to the molecular composition, molecular weight size, and molecular arrangement (eg, linear or branched) of the biodegradable material. In general, the higher the molecular weight, the tighter the molecular arrangement, and the longer the degradation time.
  • the rate of degradation of the microcapsules can be controlled by the configuration of the composition and/or content of the shell.
  • low levels eg, less than 0.5%, 1%, 2%, 3%, 4%, or 5%
  • low molecular weight eg, less than 500 Da
  • high levels eg, above 0.5%, 1%, 2%, 3%, 4%, or 5%
  • high molecular weight eg, above 500 Da, 1 kDa
  • the degradation rate of the biodegradable material can also be adjusted by changing the structure of the microcapsules (eg, multilayer encapsulation, surface porosity, porosity, specific surface area, etc.).
  • the rate of degradation of the biodegradable material can also be adjusted by varying the manner in which the material is polymerized and the proportion of the copolymer; alternatively, it can be adjusted by crosslinking the material.
  • the rate of degradation of biodegradable materials used to prepare microcapsules can also be affected by cell life activities.
  • cells within the microcapsules are capable of growing, stretching, proliferating, migrating, and establishing cell connections with cells within other microcapsules to form an organic construct (e.g., artificial tissue).
  • the microcapsules degrade over a relatively short period of time (e.g., no more than 30 days, such as no more than 10 days) to promote cells between different microcapsules.
  • the establishment of the linkage avoids obstructing or affecting the cells between the different microcapsules to establish a mutual cellular connection.
  • the microcapsules are not more than 30 days, no more than 25 days, no more than 20 days, no more than 15 days, no more than 10 days, no more than 5 days, no more than 4 days, no More than 3 days, Degradation in no more than 2 days, or no more than 1 day.
  • the microcapsules can be in 1-2 days, 2-3 days, 3-4 days, 4-5 days, 5-10 days, 10-15 days, 15-20 days, 20-25 days, or 25 Degraded within -30 days.
  • the degradation of the microcapsules can provide a microenvironment, such as a nutrient, that maintains or promotes the life of the cells.
  • the degradation products of the shell layer are small molecule compounds such as organic acids, monosaccharides (eg, glucose), oligosaccharides, amino acids, lipids, and the like. Such degradation products can be involved in the metabolic activities of cells for the synthesis of extracellular matrices or for the energy required for activity.
  • the biodegradable materials used to prepare the microcapsules and degradation products thereof are non-toxic to the cells, and/or non-immunogenic to the host.
  • the biodegradable material used to prepare the microcapsules contains an extracellular matrix or an analog thereof (e.g., elastin).
  • extracellular matrices or analogs thereof e.g., elastin
  • the use of extracellular matrices or analogs thereof can provide benefits similar to in vivo for the life activities of cells within the microcapsules, particularly cell growth, adhesion, stretching, and establishment of intercellular connections.
  • the microenvironment is thus preferred.
  • the biodegradable material used to prepare the microcapsules is selected from the group consisting of collagen (eg, type I, type II, type III collagen), fibrin, chitosan, alginate (eg, algae) Sodium or calcium alginate), oxidized alginate (eg oxidized sodium alginate), starch, hyaluronic acid, laminin, elastin, gelatin, dextran, polyamino acids (eg polylysine), agar Sugar, or any combination thereof.
  • the microcapsules comprise an alginate (eg, sodium alginate or calcium alginate), for example comprising calcium alginate and gelatin, optionally further comprising elastin.
  • the microcapsules comprise alginate (eg, sodium alginate or calcium alginate) and gelatin.
  • the microcapsules comprise an alginate (eg, sodium alginate or calcium alginate), for example comprising calcium alginate and gelatin, optionally further comprising elastin.
  • the microcapsules comprise oxidized alginate (eg, oxidized sodium alginate).
  • the microcapsules comprise alginate (eg, sodium alginate or calcium alginate) and agarose.
  • oxidized alginate eg, oxidized sodium alginate and oxidized calcium alginate
  • the rate of degradation can be adjusted by controlling the degree of oxidation of the alginate.
  • the rate of degradation of the microcapsules is matched to the rate of growth of the cells encased therein.
  • the microcapsules comprise a core layer and one or more shell layers (eg, 2, 3, 4, or 5).
  • the biodegradable material used to prepare the microcapsule core layer is selected from the group consisting of: collagen (eg, type I, type II, type III collagen), fibrin, chitosan, algae Acid salts (such as sodium alginate or calcium alginate), hyaluronic acid, agarose, gelatin, starch, dextran, polyphosphazene, polyacrylic acid and its derivatives, polylactic acid (PLA), polyamino acids (such as poly Lysine), degradable polyurethane, and any combination thereof.
  • collagen eg, type I, type II, type III collagen
  • fibrin e.g., fibrin, chitosan, algae Acid salts (such as sodium alginate or calcium alginate), hyaluronic acid, agarose, gelatin, starch, dextran, polyphosphazene, polyacrylic acid and its derivatives
  • PHA polylactic acid
  • polyamino acids such as poly Lysine
  • degradable polyurethane
  • the biodegradable material used to prepare the microcapsule shell layer is selected from the group consisting of: alginate (eg, sodium alginate or calcium alginate), elastin, polyamino acids (eg, polylysine) Acid), oxidized alginate, gelatin, chitosan, and any combination thereof.
  • alginate eg, sodium alginate or calcium alginate
  • elastin elastin
  • polyamino acids eg, polylysine
  • oxidized alginate eg, gelatin, chitosan, and any combination thereof.
  • the biodegradable material used to prepare the microcapsule core layer comprises collagen (eg, type I, type II, type III collagen).
  • the biodegradable material used to prepare the microcapsule shell layer is selected from the group consisting of polyamino acids (eg, polylysine) and alginates (eg, sodium alginate or calcium alginate).
  • polyamino acids eg, polylysine
  • alginates eg, sodium alginate or calcium alginate.
  • the microcapsules further comprise additional agents, such as nutrients, extracellular matrices, cytokines, and/or pharmaceutically active ingredients.
  • the additional agent is capable of modulating (e.g., promoting) proliferation, differentiation, migration, secretion, and/or metabolism of the cell, or the additional agent is capable of maintaining the dryness of the cell.
  • the microcapsules comprise at least one (eg, 1, 2, 3, 4, 5 or more) capable of modulating (eg, promoting) proliferation, differentiation, migration, secretion, and/or cells Or additional reagents for metabolism, or additional reagents that are capable of maintaining cell dryness.
  • the microcapsules are capable of releasing the additional agent in a controlled manner.
  • the nutrient materials include, but are not limited to, nucleotides, amino acids, polypeptides, carbohydrates (eg, monosaccharides, oligosaccharides, polysaccharides), lipids, vitamins, and the like.
  • the extracellular matrix is selected from the group consisting of polysaccharides such as glycosaminoglycans, proteoglycans; structural proteins such as collagen and elastin; adhesion proteins such as fibronectin and laminin.
  • polysaccharides such as glycosaminoglycans, proteoglycans
  • structural proteins such as collagen and elastin
  • adhesion proteins such as fibronectin and laminin.
  • the cytokine can be a cytokine for regulating proliferation, differentiation, migration, secretion, and/or metabolism of cells, including but not limited to:
  • Cytokines associated with cell growth such as insulin, insulin-like growth factors (such as IGF-I, IGF-II), transforming growth factors (such as TGF ⁇ and TGF ⁇ ), vascular endothelial growth factor, epidermal growth factor, fibroblast growth Factor, platelet-derived growth factor, osteosarcoma-derived growth factor, growth hormone release inhibitor, nerve growth factor, white Interleukin (eg, IL-1, IL-11, IL-3), erythrocyte growth factor, colony stimulating factor, cortisol, thyroxine, or any combination thereof;
  • Cytokines associated with cell differentiation such as Oct3/4, Sox2, Klf4, c-Myc, GATA4, TSP1, ⁇ -glycerophosphate, dexamethasone, vitamin C, insulin, IBMX, zinc, platelet-derived Growth factor BB (PDGF-BB), 5-azacytidine, or any combination thereof;
  • cytokines associated with cell migration such as cyclic adenosine monophosphate, phosphatidylinositol triphosphate, stromal cell-derived factor-1, N-cadherin, nuclear factor kappa B, osteonectin, thromboxane A2, Ras, or Any combination thereof; and/or
  • Cytokines related to cell metabolism such as insulin growth factor 1, TRIP-Br2, DKK-1, sRANKL, OPG, TRACP-5b, ALP, SIRT1 (2-7), PGC-1 ⁇ , PGC-1 ⁇ , OPG, IL-3, IL-4, IL-6, TGF- ⁇ , PGE2, G-CSF, TNF- ⁇ , or any combination thereof.
  • the pharmaceutically active ingredient is an agent that is capable of modulating (eg, promoting) proliferation, differentiation, migration, secretion, and/or metabolism of cells, or an agent capable of maintaining cell dryness.
  • the pharmaceutically active ingredient is selected from the group consisting of rhIL-2, rhIL-11, rhEPO, IFN-[alpha], IFN-[beta], IFN-[gamma], G-CSF, GM-CSF, rHuEPO, sTNF- R1, and rhTNF- ⁇ .
  • the microcapsules comprise a cytokine capable of inducing differentiation of undifferentiated cells into smooth muscle cells or endothelial cells, such as TGF-[alpha]l, PDGF-BB, VEGF or b-FGF.
  • a cytokine capable of inducing differentiation of undifferentiated cells into smooth muscle cells or endothelial cells, such as TGF-[alpha]l, PDGF-BB, VEGF or b-FGF.
  • the microcapsules comprise: adipose-derived mesenchymal stem cells and a nuclear layer encasing the adipose-derived mesenchymal stem cells, preferably the core layer is made of a biodegradable material; preferably The core layer provides a microenvironment for maintaining the dryness of adipose-derived mesenchymal stem cells (eg, the core layer comprises an additional agent that maintains the adipose-derived mesenchymal stem cell dryness); preferably, the nuclear layer provides induced adipose charge A microenvironment in which a stem cell differentiates into an endothelial cell or a smooth muscle cell (for example, the nuclear layer contains an inducing factor that induces differentiation of adipose-derived mesenchymal stem cells into endothelial cells or smooth muscle cells).
  • the inducing factor that induces differentiation of adipose-derived mesenchymal stem cells into smooth muscle cells is selected from the group consisting of TGF- ⁇ 1 and PDGF-BB. In certain preferred embodiments, the inducing factor that induces differentiation of adipose-derived mesenchymal stem cells into endothelial cells is selected from the group consisting of VEGF and b-FGF.
  • the microcapsules comprise: adipose-derived mesenchymal stem cells, a core layer encapsulating the adipose-derived mesenchymal stem cell cells, and a shell layer encapsulating the core layer; preferably, the The core layer and the shell layer are each independently made of a biodegradable material; preferably, the core layer provides a microenvironment that maintains the adipose-derived mesenchymal stem cells dry (eg, the core layer comprises adipose-derived mesenchymal stem cells) sexual additional reagent); preferably, the nuclear layer provides lure A microenvironment that induces differentiation of adipose-derived mesenchymal stem cells into endothelial cells or smooth muscle cells (for example, the nuclear layer comprises an inducing factor that induces differentiation of adipose-derived mesenchymal stem cells into endothelial cells or smooth muscle cells).
  • the shell of such microcapsules also provides a microenvironment that induces differentiation of adipose-derived mesenchymal stem cells into endothelial cells or smooth muscle cells (eg, the shell comprises induced adipose-derived mesenchymal stem cells to endothelial cells) Or an inducer of smooth muscle differentiation).
  • the inducing factor that induces differentiation of adipose-derived mesenchymal stem cells into smooth muscle cells is selected from the group consisting of TGF- ⁇ 1 and PDGF-BB.
  • the inducing factor that induces differentiation of adipose-derived mesenchymal stem cells into endothelial cells is selected from the group consisting of VEGF and b-FGF.
  • the solid support is made of a biocompatible material.
  • the biocompatible material comprises a biodegradable material.
  • the use of the biodegradable material to prepare the solid support can cause the artificial tissue precursor to gradually degrade during the continuous growth process after implantation in the subject, and finally the artificial tissue and the implanted person The autologous tissue is fully integrated into one.
  • the biodegradable material is a biomaterial that is degradable.
  • the biodegradable material is selected from naturally occurring biodegradable materials (eg, collagen, gelatin, chitosan, polyhydroxybutyrate (PHB), chitin, alginate ( For example, sodium alginate), starch-based biomaterials (such as polysaccharide starch), cellulose (such as bacterial cellulose), silk fibroin, and any combination thereof).
  • naturally occurring biodegradable materials eg, collagen, gelatin, chitosan, polyhydroxybutyrate (PHB), chitin, alginate ( For example, sodium alginate), starch-based biomaterials (such as polysaccharide starch), cellulose (such as bacterial cellulose), silk fibroin, and any combination thereof).
  • the naturally occurring biodegradable material is a starch.
  • the biodegradable material is selected from modified biodegradable materials (eg, modified alginate, such as oxidized alginate (eg, oxidized sodium alginate), modified Gelatin (eg, dialdehyde starch DAS cross-linked modified gelatin), modified cellulose (eg, carboxymethyl cellulose, oxidized regenerated cellulose), and any combination thereof.
  • modified biodegradable materials eg, modified alginate, such as oxidized alginate (eg, oxidized sodium alginate), modified Gelatin (eg, dialdehyde starch DAS cross-linked modified gelatin), modified cellulose (eg, carboxymethyl cellulose, oxidized regenerated cellulose), and any combination thereof.
  • the biodegradable material is selected from the group consisting of synthetically degradable materials (eg, aliphatic polyesters (eg, polylactic acid (PLA), polycaprolactone (PCL), polyhydroxyalkanoates) (PHAs), polyhydroxyvalerate (PHV), polyhydroxybutyrate (PHB), polybutylene succinate (PBS), polyglycolic acid (PGA), polylactic acid-glycolic acid copolymer ( PLGA), polyorthoester (POE), degradable polyurethane (eg starch modified polyurethane), polyvinyl alcohol, polydioxanone, polydioxanone, polydioxole Alkanone, polybutylene carbonate, polyphosphazene, and any combination thereof).
  • synthetically degradable materials eg, aliphatic polyesters (eg, polylactic acid (PLA), polycaprolactone (PCL), polyhydroxyalkanoates) (PHAs), polyhydroxyvalerate (PH
  • the synthetic degradable material is selected from the group consisting of polycaprolactone (PCL), polylactic acid (PLA), polylactic acid-glycolic acid copolymer (PLGA), and polyglycolic acid (PGA). , degradable polyurethane.
  • PCL polycaprolactone
  • PLA polylactic acid
  • PLGA polylactic acid-glycolic acid copolymer
  • PGA polyglycolic acid
  • the biodegradable material is capable of being degraded by an enzyme, such as an enzyme secreted by a cell;
  • the biodegradable material has a degradation time in the body of from 1 to 12 months.
  • the biocompatible material further comprises a biodegradable material (eg, nylon, polyester, polypropylene, polyethylene, polytetrafluoroethylene, silicone rubber, fluorosilicone rubber, natural rubber, poly Acrylate, aromatic polyester (eg polyethylene terephthalate (PET)), non-degradable polyurethane, polyetheretherketone, polyacrylonitrile, polysiloxane, polyoxymethylene, polyvinyl chloride, and Any combination thereof).
  • a biodegradable material eg, nylon, polyester, polypropylene, polyethylene, polytetrafluoroethylene, silicone rubber, fluorosilicone rubber, natural rubber, poly Acrylate, aromatic polyester (eg polyethylene terephthalate (PET)), non-degradable polyurethane, polyetheretherketone, polyacrylonitrile, polysiloxane, polyoxymethylene, polyvinyl chloride, and Any combination thereof).
  • the biocompatible material comprises a biodegradable material (eg, nylon, polyester, polypropylene, polyethylene, polytetrafluoroethylene, silicone rubber, fluorosilicone rubber, natural rubber, polyacrylic acid) Ester, aromatic polyester (such as polyethylene terephthalate (PET)), non-degradable polyurethane, polyetheretherketone, polyacrylonitrile, polysiloxane, polyoxymethylene, polyvinyl chloride, and Any combination).
  • a biodegradable material eg, nylon, polyester, polypropylene, polyethylene, polytetrafluoroethylene, silicone rubber, fluorosilicone rubber, natural rubber, polyacrylic acid
  • aromatic polyester such as polyethylene terephthalate (PET)
  • PET polyethylene terephthalate
  • non-degradable polyurethane such as polyethylene terephthalate (PET)
  • PET polyethylene terephthalate
  • non-degradable polyurethane such as polyethylene terephthalate
  • the biodegradable material is biologically inert.
  • the solid support is a tubular solid support or a sheet solid support.
  • the solid support is made by die impregnation, electrospinning, extrusion forging, 3D printing, or spray coating.
  • the solid support is obtained by a die impregnation process.
  • the mold impregnation method comprises the following steps:
  • a solid support for example, a biodegradable material
  • a suitable solvent for example, an organic solvent such as chloroform, tetrahydrofuran or N,N-dimethylacetamide
  • step (3) repeating step (2) a plurality of times to obtain a solid support
  • the method further comprises the steps of:
  • the solid support is dried, sheared and/or sterilized.
  • the concentration of the material used to prepare the solid support in the preparation solution is from 0.5 wt% to 5 wt%, such as from 0.5 wt% to 1 wt%, from 1 wt% to 1.5 wt%, 1.5 wt%.
  • the solid support is made by an electrospinning process.
  • the method of electrospinning comprises the steps of:
  • a material for example, a biodegradable material
  • a suitable solvent for example, an organic solvent such as chloroform
  • the solid support is made on the surface of a biological construct.
  • the solid support is made by 3D printing or spraying on the surface of the biological construct.
  • the artificial tissue precursor can be formed into any shape as needed.
  • microcapsules are used to prepare biological constructs of any shape, in combination with solid supports of any shape.
  • microcapsules are used to prepare biological constructs of any shape on solid supports of any shape.
  • microcapsules are used to prepare biological constructs of any shape, and solid supports of any shape are prepared on biological constructs.
  • the artificial tissue precursor comprises one or more biological constructs of any shape.
  • the artificial tissue precursor is tubular (eg, a circular tube, such as a tubular wall with or without opening), the solid support being tubular (eg, a circular tube, such as a side wall) An open or non-opening tubular) solid support, the plurality of microcapsules forming one or more tubular (eg, circular tubular, eg, open-ended or non-open tubular) biological constructs, and at least one tubular biological construct
  • the outer wall is attached to the inner wall of the solid support.
  • the artificial tissue precursor comprises a tubular solid support and a tubular biological construct having a side wall that is not open, the outer wall of the tubular biological construct being affixed to the inner wall of the tubular solid support Hehe.
  • the artificial tissue precursor comprises a plurality of tubular biological constructs.
  • 3A-E exemplarily depict the structure of a tubular artificial tissue precursor comprising a plurality of tubular biological constructs.
  • FIG. 3A is a side view of a tubular artificial tissue precursor comprising a tubular solid support and a tubular biological construct with a plurality of sidewalls that are not open, the tubular biostructures of the plurality of sidewalls that are not open Body in the tubular solid
  • the interior of the body support is aligned along the axial direction of the tubular solid support, and the outer walls of the tubular biological construct in which the plurality of side walls are not open are bonded to the inner wall of the tubular solid support.
  • 3B is a top plan view of a tubular artificial tissue precursor comprising a tubular solid support and a plurality of tubular biological constructs that are not open to the side walls, the plurality of laterally open tubular biological constructs Inside the tubular solid support, an outer wall of the tubular biological construct that is disposed coaxially with the tubular solid support and only the outermost sidewall is not open is attached to the inner wall of the tubular solid support.
  • 3C is a side view of a tubular artificial tissue precursor comprising a tubular solid support and a plurality of sidewall open tubular biological constructs, the plurality of laterally open tubular biological constructs
  • the inside of the tubular solid support is aligned along the axial direction of the tubular solid support, and the outer walls of the plurality of side wall open tubular biological constructs are bonded to the inner wall of the tubular solid support;
  • 3D is a top plan view of a tubular artificial tissue precursor comprising a tubular solid support and a plurality of sidewall open tubular biological constructs, the plurality of laterally open tubular biological constructs in the Inside the tubular solid support, disposed coaxially with the tubular solid support and arranged in a radial direction, and an outer wall of the tubular biological construct located only at the outermost side wall is attached to the inner wall of the tubular support;
  • 3E is a top plan view of a tubular artificial tissue precursor comprising a tubular solid support and a plurality of sidewall open tubular biological constructs, the plurality of laterally open tubular biological constructs in the The inside of the tubular solid support is disposed coaxially with the tubular solid support, and the outer walls of the plurality of side wall open tubular biological constructs are bonded to the inner wall of the tubular solid support.
  • the artificial tissue precursor comprises a tubular solid support, a tubular biologic construct with open sidewalls, and a tubular biologic construct with open sidewalls.
  • tubular artificial tissue precursor and the tubular biologic construct and tubular solid support contained therein are sized as needed.
  • the artificial tissue precursor has a length of from 1 cm to 40 cm.
  • the artificial tissue precursor has an inner diameter of from 1 mm to 3 cm (eg, 1-6 mm, 6-8 mm, 8-10 mm, 10-12 mm, 12 mm-3 cm).
  • the artificial tissue precursor has a uniform or non-uniform thickness.
  • a portion of the inner wall of the tubular solid support conforms to the tubular biological construct while the other portions do not conform to the tubular biological construct.
  • different portions of the inner wall of the tubular solid support are fitted with different tubular biological constructs.
  • the tubular solid support has a length of from 1 cm to 40 cm (eg, 1-10 cm, 10-20 cm, 20-30 cm, or 30-40 cm).
  • the tubular solid support has an inner diameter of from 1 mm to 3 cm (eg, 1-6 mm, 6-8 mm, 8-10 mm, 10-12 mm, 12 mm-3 cm).
  • the tubular solid support has a thickness of from 200 ⁇ m to 1 mm (eg, from 200 ⁇ m to 400 ⁇ m, from 400 ⁇ m to 600 ⁇ m, from 600 ⁇ m to 800 ⁇ m, or from 800 ⁇ m to 1 mm).
  • the tubular solid support is a circular tubular opening in the side wall that extends through the ends of the tubular solid support in an axial direction, the tubular solid support in a radial section a fan ring; in certain preferred embodiments, the fan ring has a central angle greater than zero and less than 360°, such as greater than zero and less than 30°, 30°-60°, 60°-90°, 90°- 120°, 120°-150°, 150°-180°, 180°-210°, 210°-240°, 240°-270°, 270°-300°, 300°-330°, or greater than 330° and Less than 360°.
  • the tubular biological construct has a length of from 1 cm to 40 cm (eg, 1-10 cm, 10-20 cm, 20-30 cm, or 30-40 cm).
  • the tubular biologic construct has an inner diameter of from 1 mm to 3 cm (eg, 1-6 mm, 6-8 mm, 8-10 mm, 10-12 mm, 12 mm-3 cm).
  • the tubular biologic construct has a thickness of from 200 [mu]m to 1 mm (eg, from 200 [mu]m to 400 [mu]m, from 400 [mu]m to 600 [mu]m, from 600 [mu]m to 800 [mu]m or from 800 [mu]m to 1 mm).
  • the tubular biologic construct is a circular tubular opening in the side wall, the opening extending through the ends of the tubular biological construct in an axial direction, the tubular biologic construct being radially intersected It is a fan ring; in certain preferred embodiments, the fan ring has a central angle greater than zero and less than 360°.
  • the artificial tissue precursor is in the form of a sheet
  • the solid support is a sheet-like solid support
  • the plurality of microcapsules constitute one or more sheet-like biological constructs, and at least A sheet-like biological construct is attached to the sheet-like solid support.
  • the sheet-like solid support is a flat sheet or a curved sheet.
  • the sheetlike biological construct is in the form of a flat sheet or a curved sheet.
  • the artificial tissue precursor comprises a sheet-like solid support and a sheet-like biological construct, one side of which conforms to one side of the sheet-like solid support.
  • the artificial tissue precursor comprises a flaky solid support and a plurality of flaky bio-structures
  • a plurality of the sheet-like biological constructs are located on one side of the sheet-like solid support, and one side of each of the sheet-like biological constructs is attached to one side of the sheet-like solid support.
  • the artificial tissue precursor comprises a sheet-like solid support and a plurality of sheet-like biological constructs, a plurality of the sheet-like biological constructs being stacked on one side of the sheet-like solid support The cloth, and one side of at least one of the sheet-like biological constructs is attached to one side of the sheet-like solid support.
  • the size of the sheet-like artificial tissue precursor and the sheet-like biological construct and the sheet-like solid support contained therein are set as needed.
  • the artificial tissue precursor is a circular sheet, an elliptical sheet, a parallelogram (e.g., rectangular) sheet, a fan-shaped sheet, or an irregular sheet.
  • the artificial tissue precursor has a thickness of from 0.5 mm to 3 mm (eg, 0.5 mm to 1 mm, 1 mm to 2 mm, or 2 mm to 3 mm).
  • the artificial tissue area of the precursor was 0.5cm 2 -5cm 2 (e.g. 0.5cm 2 -1cm 2, 1cm 2 -1.5cm 2, 1.5cm 2 -2.5cm 2, 2.5cm 2 - 2.5 cm 2 or 3.5 cm 2 - 5 cm 2 ).
  • the artificial tissue precursor has a uniform or non-uniform thickness.
  • a portion of the sheet-like solid support is affixed with a sheet-like biological construct, while other portions are not bonded to a sheet-like biological construct.
  • different portions of the sheet-like solid support are affixed with different flaky biological constructs.
  • the sheet-like solid support is a circular sheet, an elliptical sheet, a parallelogram (eg, rectangular) sheet, a fan-shaped sheet, or an irregular sheet, or a circle , elliptical, parallelogram (such as rectangular) or fan-shaped sheet.
  • the sheet-like solid support has a thickness of from 0.5 mm to 3 mm (eg, from 0.5 mm to 1 mm, from 1 mm to 2 mm, or from 2 mm to 3 mm).
  • the sheet-like solid support area of 0.5cm 2 -5cm 2 (e.g. 0.5cm 2 -1cm 2, 1cm 2 -1.5cm 2, 1.5cm 2 -2.5cm 2, 2.5 cm 2 -2.5cm 2 or 3.5cm 2 -5cm 2).
  • the sheet-like biological construct is a circular sheet shape, an elliptical sheet shape, a parallelogram shape (for example, a rectangular shape), a fan-shaped sheet shape or an irregular sheet shape, or an approximate circular shape, an elliptical shape, a parallelogram shape (for example, a rectangular shape). ) or a fan-shaped sheet.
  • the sheet-like biological construct has a thickness of from 20 ⁇ m to 3 mm (eg, from 20 ⁇ m to 100 ⁇ m, from 100 ⁇ m to 500 ⁇ m, from 500 ⁇ m to 1 mm, from 1 mm to 2 mm, or from 2 mm to 3 mm).
  • the sheet-like body of the biological construct area 0.5cm 2 -5cm 2 (e.g. 0.5 cm 2 -1cm 2, 1cm 2 -1.5cm 2, 1.5cm 2 -2.5cm 2, 2.5 cm 2 -2.5cm 2 or 3.5cm 2 -5cm 2).
  • At least one microcapsule or at least one biological construct of the artificial tissue precursor of the invention is immobilized with the solid support.
  • At least one microcapsule or at least one biological construct is chemically bonded to the solid support.
  • At least one biological construct is bonded to the solid support by a binder
  • the adhesive is a medical glue.
  • Medical glues useful in the present invention include, but are not limited to:
  • Soft tissue medical adhesives for example: tissue adhesives based on octyl 2-cyanoacrylate; fibrin adhesives (FS, mainly comprising fibrinogen + thrombin, Ca 2+ , factor VIII);
  • Hard tissue medical adhesives for example: synthetic resin adhesives for dentistry, such as (1) methacrylates: 4-EMTA (4-methacryloyloxyethyl trimellitic anhydride), phenyp (A) Bisacryloyloxyethyl phenyl phosphate), Bis-GMA (glycidyl methacrylate) with phosphate ester, etc., mostly used for caries filling and dentin bonding, (2) polycarboxylic acid systems, such as poly Acrylate + zinc oxide or special glass filler, used for filling the tooth cavity and bonding plexiglass repair; bone cement (commonly known as bone cement, mainly composed of acrylic cement, polymethyl methacrylate (polymethyl methacrylic, PMMA) and the like.
  • synthetic resin adhesives for dentistry such as (1) methacrylates: 4-EMTA (4-methacryloyloxyethyl trimellitic anhydride), phenyp (A) Bisacryloyloxyethyl phenyl phosphate),
  • the medical glue comprises ⁇ -cyanoacrylate (for example, ⁇ -cyanoacrylate, ⁇ -cyanoacrylate, ⁇ -cyanoacrylate, iso- ⁇ -cyanoacrylate, ⁇ - octyl cyanoacrylate, such as n-octyl cyanoacrylate).
  • ⁇ -cyanoacrylate for example, ⁇ -cyanoacrylate, ⁇ -cyanoacrylate, ⁇ -cyanoacrylate, iso- ⁇ -cyanoacrylate, ⁇ - octyl cyanoacrylate, such as n-octyl cyanoacrylate.
  • the binder is present in a commercially available medical gel, such as a Baiyun medical gel EC type (main component: ⁇ -cyanoacrylate n-octyl ester (508) as a main gel, adding additional Agent (medical grade polymethyl methacrylate)); or Fule® medical glue (ingredient: 99% ⁇ -cyanoacrylate n-butyl ester (NBCA/504) and 1% ⁇ -cyanoacrylate n-octyl ester (NOCA/508)).
  • a Baiyun medical gel EC type main component: ⁇ -cyanoacrylate n-octyl ester (508) as a main gel, adding additional Agent (medical grade polymethyl methacrylate)
  • Fule® medical glue (ingredient: 99% ⁇ -cyanoacrylate n-butyl ester (NBCA/504) and 1% ⁇ -cyanoacrylate n-octyl ester (NOCA/508)).
  • the concentration of the medical glue can be adjusted to adjust the setting time of the medical glue to achieve a good adhesion.
  • the medical glue can be diluted with a suitable solvent, for example, the medical glue is diluted with ethyl acetate.
  • the solvent can be selected from medical grade ester solvents such as medical grade ethyl acetate, medical grade polymethyl methacrylate.
  • the present application provides the following various methods of preparing artificial tissue precursors as described above.
  • Method 1 The artificial tissue precursor is tubular, and the method for preparing the artificial tissue precursor comprises the following steps:
  • the preparation of the tubular biological construct is carried out by a method comprising the steps of:
  • microcapsules having a first component attached to all or part of its surface; preferably, the first component is contained in the first reagent;
  • the temporary support is tubular or columnar (for example, a circular tubular shape in which the side wall is not open, a circular tubular shape in which the side wall is open, a cylindrical shape or a columnar shape disposed along a partial circumference), and the predetermined area is temporarily supported. a curved surface of the object; optionally, coating the substrate material on a predetermined area of the surface of the temporary support prior to coating the second reagent;
  • step (1) placing all or part of the microcapsules to which the first component is attached in the step (1) on a predetermined region coated with the second reagent, so that the first component on the surface of the microcapsule is pre- Contacting the second component on the region to produce a blocking effect, thereby assembling (bonding) the microcapsule into a first layer structure, the first layer structure being a tubular structure;
  • the method further comprises the steps of:
  • repeating steps (4) and (5) one or more times; for example at least 1 time, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 15 times, At least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 100 times, at least 200 times, at least 500 times, or more;
  • the method further comprises: bonding the open tubular biological construct with the side walls open to obtain a circular tubular biological construct with the side walls not open.
  • the method further comprises: separating the tubular biological construct from the temporary support.
  • the temporary support is a curved print platform, such as a rotating rod of a 3D printer.
  • the substrate material is a temperature sensitive material such as gelatin, poly N-isopropylpropyl Enamide, poly N-isopropylacrylamide-polyethylene glycol block copolymer, polyethylene glycol copolymer (for example, polyvinyl alcohol-polyethylene glycol copolymer), polyhydroxyethyl acrylate, agarose , Matrigel, chitosan/sodium glycerophosphate system or Pluronic F127.
  • a temperature sensitive material such as gelatin, poly N-isopropylpropyl Enamide, poly N-isopropylacrylamide-polyethylene glycol block copolymer, polyethylene glycol copolymer (for example, polyvinyl alcohol-polyethylene glycol copolymer), polyhydroxyethyl acrylate, agarose , Matrigel, chitosan/sodium glycerophosphate system or Pluronic F127.
  • the temporary support is a temperature sensitive material (eg, gelatin, poly N-isopropyl acrylamide, poly N-isopropyl acrylamide - polyethylene glycol block copolymer, A cylindrical or round tube made of polyethylene glycol copolymer, polyhydroxyethyl acrylate, agarose, Matrigel, chitosan/sodium glycerophosphate system or Pluronic F127.
  • a temperature sensitive material eg, gelatin, poly N-isopropyl acrylamide, poly N-isopropyl acrylamide - polyethylene glycol block copolymer, A cylindrical or round tube made of polyethylene glycol copolymer, polyhydroxyethyl acrylate, agarose, Matrigel, chitosan/sodium glycerophosphate system or Pluronic F127.
  • the temporary support is a cylinder.
  • Figure 4A exemplifies a cylinder as a temporary support.
  • the temporary support is a cylinder
  • the predetermined area is the entire side of the cylinder, as shown in FIG. 4B, thereby obtaining the first layer structure obtained in the step (3). It is a circular tubular structure in which the side walls are not open.
  • the temporary support is a cylinder
  • the predetermined area is a rectangle on the side of the unfolded cylinder
  • the predetermined area is along the axial direction of the cylinder
  • the side surface of the cylindrical portion is formed as shown in FIG. 4C, so that the first layer structure obtained in the step (3) is a circular tubular structure in which the side wall is not opened.
  • the temporary support is a cylinder
  • the predetermined area is a rectangle on the side of the unfolded cylinder
  • the predetermined area is along the circumference of the cylinder
  • the side of the pillar is penetrated as shown in FIG. 4D, so that the first layer structure obtained in the step (3) is a circular tubular structure in which the side wall is not open.
  • the temporary support is a cylinder
  • the predetermined area is a rectangle on the side of the unfolded cylinder and does not penetrate in the circumferential direction and the axial direction of the cylinder.
  • the side of the cylinder is as shown in Fig. 4E, so that the first layer structure obtained in the step (3) is a circular tubular structure with a side wall opening.
  • step (3) after all or part of the microcapsules to which the first component is attached are placed in the preset region coated with the second reagent of step (2), Set 0.1-60s; (eg 0.1-1s, 1-5s, 5-10s, 10-15s, 15-20s, 20-25s, 25-30s, 30-35s, 35-40s, 40-45s, 45-50s) , 50-55s, or 55-60s).
  • the standing step facilitates sufficient contact between the first component on the surface of the microcapsule and the second component on the predetermined area, and interaction occurs to assemble (bond) the microcapsule into the first layer structure.
  • the method of making a tubular biological construct is carried out by bioprinting.
  • a bioprinter is performed using a printer (eg, a 3D bioprinter); or, using an automated or non-automated mechanical process for bioprinting; or, by using manual placement or manual deposition (eg, Use a pipette) for bioprinting.
  • a printer eg, a 3D bioprinter
  • an automated or non-automated mechanical process for bioprinting or, by using manual placement or manual deposition (eg, Use a pipette) for bioprinting.
  • the microcapsules are printed by extrusion printing or modular printing.
  • the second reagent is printed using a modular printing process, an extrusion printing process, or an inkjet printing process.
  • the auxiliary material is printed using a modular printing process, an extrusion printing process, or an ink jet printing process.
  • a 3D bioprinter is used to prepare the biological construct.
  • the 3D bioprinter includes: a first ink cartridge for providing a microcapsule; a second ink cartridge for providing a second reagent; a first printhead; and, coupled to the second The second print head of the ink cartridge.
  • the 3D bioprinter further includes: a third ink cartridge for providing a substrate material; and a third printhead.
  • the 3D bioprinter further comprises: a fourth ink cartridge for providing a first reagent.
  • the method comprises the steps of:
  • step (3) printing the microcapsules in step (1) onto the predetermined area of the second reagent printed by step (2) through the first print head of the 3D bioprinter, so that the first surface on the surface of the microcapsule Contacting the component with the second component on the predetermined area to produce a blocking effect, thereby assembling (bonding) the microcapsule into a first layer structure;
  • the method further comprises the steps of:
  • step (1) printing, by the first print head, the microcapsules in step (1) onto the structure produced in the previous step, so that the first component on the surface of the microcapsule and the second component on the structure Contacting the components to produce a blocking effect, thereby assembling (bonding) the microcapsules into another layer structure in the structure produced in the previous step;
  • repeating steps (4) and (5) one or more times; for example at least 1 time, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 15 times, At least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 100 times, at least 200 times, at least 500 times, or more;
  • Method 2 The artificial tissue precursor is tubular, and the method for preparing the artificial tissue precursor comprises the following steps:
  • tubular biological construct is attached to the inner wall of the tubular solid support.
  • the preparation of the tubular biological construct is carried out by a method comprising the steps of:
  • microcapsules having a first component attached to all or part of its surface; preferably, the first component is contained in the first reagent;
  • the temporary support has at least one plane, the annular pattern being located in a plane of the temporary support;
  • step (1) placing all or part of the microcapsules to which the first component is attached in the step (1) on a predetermined circular pattern drawn by the second reagent, so that the first component on the surface of the microcapsule is Contacting the second component on the annular pattern to produce a blocking effect, thereby assembling (bonding) the microcapsule into a first layer structure, the first layer structure being a ring structure;
  • repeating steps (4) and (5) one or more times; for example at least 1 time, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 15 times, At least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 100 times, at least 200 times, at least 500 times, or more;
  • the method further comprises: bonding a circular tubular biological construct having a side wall opening to obtain a circular tubular biological construct having a side wall that is not open;
  • the temporary support is a printing platform of a 3D printer
  • step (3) after placing all or part of the microcapsules to which the first component is attached, on the drawn predetermined circular pattern of the step (2), the reaction is allowed to stand 0.1. -60s; (eg 0.1-1s, 1-5s, 5-10s, 10-15s, 15-20s, 20-25s, 25-30s, 30-35s, 35-40s, 40-45s, 45-50s, 50 -55s, or 55-60s).
  • the standing step facilitates charging of the first component on the surface of the microcapsule and the second component on the predetermined area
  • the contacts are contacted and interacted to assemble (bond) the microcapsules into a first layer structure.
  • the method of making a tubular biological construct is carried out by bioprinting.
  • a bioprinter is performed using a printer (eg, a 3D bioprinter); or, using an automated or non-automated mechanical process for bioprinting; or, by using manual placement or manual deposition (eg, Use a pipette) for bioprinting.
  • a printer eg, a 3D bioprinter
  • an automated or non-automated mechanical process for bioprinting or, by using manual placement or manual deposition (eg, Use a pipette) for bioprinting.
  • the microcapsules are printed by extrusion printing or modular printing.
  • the second reagent is printed using a modular printing process, an extrusion printing process, or an inkjet printing process.
  • the auxiliary material is printed using a modular printing process, an extrusion printing process, or an ink jet printing process.
  • the 3D bioprinter includes: a first ink cartridge for providing a microcapsule; a second ink cartridge for providing a second reagent; a first printhead; and, coupled to the second The second print head of the ink cartridge.
  • the 3D bioprinter further includes: a third ink cartridge for providing an auxiliary material; and a third printhead.
  • the 3D bioprinter further comprises: a fourth ink cartridge for providing a first reagent.
  • the method comprises the steps of:
  • step (3) printing the microcapsules in step (1) onto the circular pattern drawn in step (2) through the first print head of the 3D bioprinter, so that the first component on the surface of the microcapsule and the ring Contacting the second component on the pattern to create a blocking effect, thereby assembling (bonding) the microcapsule into a first layer structure;
  • the method further comprises the steps of:
  • step (1) printing, by the first print head, the microcapsules in step (1) onto the structure produced in the previous step, so that the first component on the surface of the microcapsule and the second component on the structure Contacting the components to produce a blocking effect, thereby assembling (bonding) the microcapsules into another layer structure in the structure produced in the previous step;
  • repeating steps (4) and (5) one or more times; for example at least 1 time, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 15 times, At least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 100 times, at least 200 times, at least 500 times, or more;
  • Exemplary ring and fan ring patterns are shown in Figure 5A.
  • the artificial tissue precursor is in the form of a sheet, and the method for preparing the artificial tissue precursor comprises the following steps:
  • the preparation of the flaky biological construct is carried out by a method comprising the steps of:
  • microcapsules having a first component attached to all or part of its surface; preferably, the first component is contained in the first reagent;
  • step (1) placing all or part of the microcapsules to which the first component is attached in the step (1) on a predetermined region coated with the second reagent, so that the first component on the surface of the microcapsule is pre- Contacting the second component on the region to produce a blocking effect, thereby assembling (bonding) the microcapsule into a first layer structure, the first layer structure being a planar sheet-like structure;
  • the method further comprises the steps of:
  • repeating steps (4) and (5) one or more times; for example at least 1 time, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 15 times, At least 20 times, at least 30 times, at least 40 Times, at least 50 times, at least 100 times, at least 200 times, at least 500 times, or more;
  • the method further comprises bending the planar sheet-like biological construct to obtain a curved sheet-like biological construct.
  • the predetermined area is a parallelogram (eg, rectangular) area, a circular area, an elliptical area, a sectored area, or an irregular shaped area.
  • the temporary support is a printing platform for a 3D printer.
  • step (3) after all or part of the microcapsules to which the first component is attached are placed in the preset region coated with the second reagent of step (2), Set 0.1-60s.
  • step (3) after all or part of the microcapsules to which the first component is attached are placed in the preset region coated with the second reagent of step (2), Set 0.1-60s; (eg 0.1-1s, 1-5s, 5-10s, 10-15s, 15-20s, 20-25s, 25-30s, 30-35s, 35-40s, 40-45s, 45-50s) , 50-55s, or 55-60s).
  • the standing step facilitates sufficient contact between the first component on the surface of the microcapsule and the second component on the predetermined area, and interaction occurs to assemble (bond) the microcapsule into the first layer structure.
  • the method of making a tubular biological construct is carried out by bioprinting.
  • a bioprinter is performed using a printer (eg, a 3D bioprinter); or, using an automated or non-automated mechanical process for bioprinting; or, by using manual placement or manual deposition (eg, Use a pipette) for bioprinting.
  • a printer eg, a 3D bioprinter
  • an automated or non-automated mechanical process for bioprinting or, by using manual placement or manual deposition (eg, Use a pipette) for bioprinting.
  • the microcapsules are printed by extrusion printing or modular printing.
  • the second reagent is printed using a modular printing process, an extrusion printing process, or an inkjet printing process.
  • the auxiliary material is printed using a modular printing process, an extrusion printing process, or an ink jet printing process.
  • a 3D bioprinter is used to prepare the biological construct
  • the 3D bioprinter includes: a first ink cartridge for providing a microcapsule; a second ink cartridge for providing a second reagent; a first printhead; and, coupled to the second The second print head of the ink cartridge.
  • the 3D bioprinter further comprises: a third ink cartridge for providing a first reagent.
  • the method comprises the steps of:
  • step (3) printing the microcapsules in step (1) onto the predetermined area of the second reagent printed by step (2) through the first print head of the 3D bioprinter, so that the first surface on the surface of the microcapsule Contacting the component with the second component on the predetermined area to produce a blocking effect, thereby assembling (bonding) the microcapsule into a first layer structure;
  • the method further comprises the steps of:
  • step (1) printing, by the first print head, the microcapsules in step (1) onto the structure produced in the previous step, so that the first component on the surface of the microcapsule and the second component on the structure Contacting the components to produce a blocking effect, thereby assembling (bonding) the microcapsules into another layer structure in the structure produced in the previous step;
  • repeating steps (4) and (5) one or more times; for example at least 1 time, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 15 times, At least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 100 times, at least 200 times, at least 500 times, or more;
  • the method further comprises bending the planar sheet-like biological construct to obtain a curved sheet-like biological construct
  • the predetermined area is a parallelogram (e.g., rectangular) area, a circular area, or an elliptical area.
  • the temporary support is a printing platform for a 3D printer.
  • step (3) after all or part of the microcapsules to which the first component is attached are placed in the preset region coated with the second reagent of step (2), Set 0.1-60s.
  • a 3D bio-printer is used to prepare a sheet-like biological construct.
  • the 3D bioprinter includes: a first ink cartridge for providing a microcapsule; a second ink cartridge for providing a second reagent; a first printhead; and, coupled to the second The second print head of the ink cartridge.
  • the 3D bioprinter further comprises: a third ink cartridge for providing a first reagent.
  • the method comprises the steps of:
  • step (3) printing the microcapsules in step (1) onto the predetermined area of the second reagent printed by step (2) through the first print head of the 3D bioprinter, so that the first surface on the surface of the microcapsule Contacting the component with the second component on the predetermined area to produce a blocking effect, thereby assembling (bonding) the microcapsule into a first layer structure;
  • the method further comprises the steps of:
  • step (1) printing, by the first print head, the microcapsules in step (1) onto the structure produced in the previous step, so that the first component on the surface of the microcapsule and the second component on the structure Contacting the components to produce a blocking effect, thereby assembling (bonding) the microcapsules into another layer structure in the structure produced in the previous step;
  • repeating steps (4) and (5) one or more times; for example at least 1 time, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 15 times, At least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 100 times, at least 200 times, at least 500 times, or more;
  • the artificial tissue precursor is in the form of a sheet, and the method for preparing the artificial tissue precursor comprises the following steps:
  • a material e.g., a biocompatible material for preparing a solid support is provided, and a sheet-like solid support is prepared on the sheet-like biological construct.
  • the sheet-like solid support is prepared by 3D printing or spraying.
  • Method 5 The artificial tissue precursor is tubular, and the method for preparing the artificial tissue precursor comprises the following steps:
  • tubular biological construct is attached to the inner wall of the tubular solid support.
  • the artificial tissue precursor is tubular, and the method for preparing the artificial tissue precursor comprises the following steps:
  • a sheet-like biological construct is prepared by the method of preparing a sheet-like biological construct in Method 3; thereafter, the obtained sheet-like biological construct is bent, and/or the edge of the sheet-like biological construct is subjected to Bonding to obtain a tubular biological construct;
  • the tubular solid support is prepared by 3D printing or spraying.
  • a styling agent e.g., a commercially available alpha-cyanoacrylate containing medical gel
  • a styling agent e.g., a commercially available alpha-cyanoacrylate containing medical gel
  • the method of sizing a biological construct comprises the steps of:
  • steps 2) and 3) are repeated.
  • the medical glue layer can be formed on the surface of the biological construct by the above method to make the biological construct stable and firm.
  • the method can also be used to adjust the thickness of the biological construct to facilitate matching to the solid support.
  • the artificial tissue precursor is tubular or flaky, and the method for preparing the artificial tissue precursor comprises the following steps:
  • the first component is included in the first reagent
  • step (1) placing all or part of the microcapsules to which the first component is attached in the step (1) on a predetermined region coated with the second reagent, so that the first component on the surface of the microcapsule is pre- Contacting the second component on the region to produce a blocking effect, thereby assembling (bonding) the microcapsule into a first layer structure on the surface of the solid support;
  • the method further comprises the steps of:
  • repeating steps (4) and (5) one or more times; for example at least 1 time, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 15 times, At least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 100 times, at least 200 times, at least 500 times, or more;
  • the solid support is tubular or flaky
  • the solid support is a tubular solid support, the predetermined region being located on an inner wall of the solid support;
  • step (3) after all or part of the microcapsules to which the first component is attached are placed in the preset region coated with the second reagent of step (2), Set 0.1-60s; (eg 0.1-1s, 1-5s, 5-10s, 10-15s, 15-20s, 20-25s, 25-30s, 30-35s, 35-40s, 40-45s, 45-50s) , 50-55s, or 55-60s).
  • the standing step facilitates sufficient contact between the first component on the surface of the microcapsule and the second component on the predetermined area, and interaction occurs to assemble (bond) the microcapsule into the first layer structure.
  • a 3D bioprinter is used to prepare an artificial tissue precursor.
  • the 3D bioprinter includes: a first ink cartridge for providing a microcapsule; a second ink cartridge for providing a second reagent; a first printhead; and, coupled to the second The second print head of the ink cartridge.
  • the 3D bioprinter further comprises: a third ink cartridge for providing a first reagent.
  • the method comprises the steps of:
  • step (3) printing the microcapsules in step (1) onto the predetermined area of the second reagent printed by step (2) through the first print head of the 3D bioprinter, so that the first surface on the surface of the microcapsule Contacting the component with the second component on the predetermined area to produce a blocking effect, thereby assembling (bonding) the microcapsule into a first layer structure;
  • the method further comprises the steps of:
  • step (1) printing, by the first print head, the microcapsules in step (1) onto the structure produced in the previous step, so that the first component on the surface of the microcapsule and the second component on the structure Contacting the components to produce a blocking effect, thereby assembling (bonding) the microcapsules into another layer structure in the structure produced in the previous step;
  • repeating steps (4) and (5) one or more times; for example at least 1 time, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 15 times, At least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 100 times, at least 200 times, at least 500 times, or more;
  • the first component and/or the second component is a biocompatible material.
  • the first component and/or the second component are biologically derived materials.
  • the first component and/or the second component are biodegradable materials.
  • the first component and the second component are capable of undergoing a strong interaction (e.g., capable of undergoing a chemical reaction) upon contact, and produce a blocking effect to effect adhesion.
  • a strong interaction e.g., capable of undergoing a chemical reaction
  • Such adhesion not only enables cell-to-cell, cell-to-tissue, tissue-to-tissue adhesion, but also adhesion between cells/tissues and foreign substances.
  • such adhesion has at least one property selected from the group consisting of: (1) it is safe, reliable, non-toxic, non-carcinogenic, non-teratogenic, non-mutagenic; (2) it has a good biological phase Capacity, does not hinder the body's own healing; (3) it can be used under conditions of blood and tissue fluid; (4) it can achieve rapid adhesion at normal temperature and pressure; (5) it has good adhesion Strength and durability, the bonded part has certain elasticity and toughness; (6) It is not irritating to the body tissues during use; (7) After the bonding effect is achieved, the relevant components can be gradually degraded and Absorption; and, (8) the binding site can be migrated through the cells.
  • the blocking effect produced by contacting the first component with the second component can be used to bond the two microcapsules together to form a biological construct; and the resulting biological construct
  • the tensile modulus of the body is not less than 10 Pa, such as not less than 20 Pa, not less than 30 Pa, not less than 40 Pa, not less than 50 Pa, not less than 60 Pa, not less than 70 Pa, not less than 80 Pa, not less than 90Pa, no less than 100Pa, no less than 200Pa, no less than 300 Pa, not less than 400 Pa, not less than 500 Pa, not less than 600 Pa, not less than 700 Pa, not less than 800 Pa, not less than 900 Pa or not less than 1000 Pa.
  • the obtained construct has a tensile modulus of from 1 KPa to 10 MPa, such as from 1 to 5 KPa, from 5 to 10 KPa, from 10 to 50 KPa, from 50 to 100 KPa, from 100 to 500 KPa, from 500 to 1000 KPa, -5 MPa, or 5-10 MPa.
  • cells in the microcapsules are capable of migrating through the binding site into adjacent microcapsules or more distal microcapsules. Thereby, the cells in the microcapsules are capable of growth, migration, differentiation and proliferation throughout the construct.
  • the first component and the second component are selected from the group consisting of:
  • Alginate such as sodium alginate
  • oxidized alginate such as oxidized sodium alginate
  • substances containing Ca 2+ , Mg 2+ , Ba 2+ , Sr 2+ , or Fe 3+ eg, a solution containing a Ca 2+ , Mg 2+ , Ba 2+ , Sr 2+ , or Fe 3+ or a semi-solid (eg, a gel)
  • a semi-solid eg, a gel
  • a molecule containing a maleimide group for example, a polyethylene glycol (MAL-PEG) containing a maleimide group
  • a molecule containing a free thiol group for example, a polyethylene glycol containing a free thiol group (PEG-SH)
  • An anion-containing substance for example, an anion-containing solution or a semi-solid (e.g., a gel)
  • an ⁇ -cyanoacrylate e.g., ⁇ -cyanoacrylate, ⁇ -cyanoacrylate, ⁇ -cyanide
  • Fibrinogen and ⁇ -cyanoacrylate for example, ⁇ -cyanoacrylate, ⁇ -cyanoacrylate, ⁇ -cyanoacrylate, iso- ⁇ -cyanoacrylate, ⁇ - N-octyl cyanoacrylate;
  • serum albumin for example, bovine serum albumin
  • glutaraldehyde glutaraldehyde
  • an embodiment of the present invention can be used as long as the first component is in contact with the second component to produce a blocking effect and achieve adhesion.
  • the first component and the second component of the present invention are not limited to the specific combinations described above.
  • the first component may be any member of the combination, and the second component is another member of the combination.
  • the first component may be fibrinogen (when the second component is thrombin) or may be thrombin (when the second component is fibrinogen).
  • the first component is fibrinogen and the second component is thrombin.
  • the first component is an alginate (eg, sodium alginate) or an oxidized alginate (eg, oxidized sodium alginate) and the second component is Ca2 +-containing.
  • a substance such as Mg 2+ , Ba 2+ , Sr 2+ , or Fe 3+ , such as a solution containing a Ca 2+ , Mg 2+ , Ba 2+ , Sr 2+ , or Fe 3+ or a semi-solid (eg, coagulation) gum).
  • the first component is a maleimide group-containing molecule (eg, a maleimide group-containing polyethylene glycol (MAL-PEG)), and
  • the two components are molecules containing a free sulfhydryl group (for example, polyethylene glycol (PEG-SH) containing a free sulfhydryl group).
  • the first component is an anion-containing material, such as an anion containing solution or semi-solid (eg, a gel)
  • the second component is an alpha-cyanoacrylate (eg, alpha) Methyl cyanoacrylate, ethyl ⁇ -cyanoacrylate, isobutyl ⁇ -cyanoacrylate, iso- ⁇ -cyanoacrylate, n-octyl ⁇ -cyanoacrylate).
  • the first component is fibrinogen and the second component is alpha-cyanoacrylate (eg, alpha-cyanoacrylate, alpha-cyanoacrylate, Isobutyl isocyanoacrylate, isohexyl cyanoacrylate, n-octyl ⁇ -cyanoacrylate).
  • the first component is serum albumin (eg, bovine serum albumin) and the second component is glutaraldehyde.
  • the first component is a urethane-containing group (-NHCOO-) or an isocyanate group (-NCO)-containing molecule (eg, a urethane group-containing polyethylene group) A diol or an isocyanate group-containing polyethylene glycol), and the second component is an active hydrogen-containing molecule (for example, a carboxyl group-containing polyethylene glycol).
  • the first component is gelatin-resorcinol and the second component is glutaraldehyde.
  • the first component is carbodiimide crosslinked gelatin and the second component is poly L-glutamic acid (PLGA).
  • the first component is an aminated gelatin and the second component is an aldehydelated polysaccharide.
  • the concentration of the first component in the first reagent is from 0.01 to 50% by weight.
  • the concentration of the first component is from 0.01 to 0.05 wt%, from 0.05 to 0.1 wt%, from 0.1 to 0.5 wt%, from 0.5 to 1 wt%, from 1 to 5 wt%, 5- 10 wt%, 10-15 wt%, 15-20 wt%, 20-25 wt%, 25-30 wt%, 30-35 wt%, 35-40 wt%, 40-45 wt%, or 45-50 wt%.
  • the concentration of the second component in the second reagent is from 0.01 to 50% by weight.
  • the concentration of the second component is from 0.01 to 0.05 wt%, from 0.05 to 0.1 wt%, from 0.1 to 0.5 wt%, from 0.5 to 1 wt%, from 1 to 5 wt%, 5- 10 wt%, 10-15 wt%, 15-20 wt%, 20-25 wt%, 25-30 wt%, 30-35 wt%, 35-40 wt%, 40-45 wt%, or 45-50 wt%.
  • the strength and/or duration of adhesion can be controlled by selecting the class and/or concentration of the first component and the second component.
  • fibrinogen and thrombin can be used as the first component and the second component, and such agents are particularly suitable for use in constructing tissues of lesser mechanical strength, such as elasticity.
  • ⁇ -cyanoacrylate can undergo a strong polymerization reaction with an anion-containing solution to form a polymer having a relatively high mechanical strength.
  • an anionic containing material and alpha-cyanoacrylate can be used as the first component and the second component, and such kits are particularly suitable for use in constructing mechanical strengths.
  • Large tissues such as tissues with a modulus of elasticity greater than 10 MPa.
  • the second agent is a liquid or semi-solid (e.g., a gel).
  • the second reagent is used to draw a predetermined pattern or to be applied to a predetermined area. Therefore, it is particularly preferred that the second agent has a suitable viscosity so that it can stably maintain the shape/pattern/profile of the pattern or region when used for drawing a pattern without arbitrarily flowing.
  • the second agent has a viscosity of from 1 to 1000 Pa.s, such as from 30 to 160 Pa.s.
  • the viscosity of the second agent is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 50, 80, 100, 200, 300, 400, 500, 800, or 1000 Pa ⁇ s.
  • the viscosity of the second agent is 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9 -10, 10-12, 12-14, 14-16, 16-18, 18-20, 20-25, 25-30, 30-50, 50-80, 80-100, 100-200, 200-300 , 300-400, 400-500, 500-800, or 800-1000, 1-3, 3-8, 8-16, 3-10, 10-20, 20-50, 50-160Pa ⁇ s, or 30 -160Pa ⁇ s.
  • the second agent further comprises a third component, the third component being a viscous agent.
  • the viscosity of the second agent can be conveniently adjusted by adjusting the content of the third component (viscous agent) so that the second agent can maintain a specific shape, which is suitable for drawing a pattern or coating.
  • the third component is a biocompatible material.
  • the third component is a biologically derived material.
  • the third component is a biodegradable material.
  • the third component is a temperature sensitive material. In certain preferred embodiments, the temperature sensitive material has a different morphology at different temperatures.
  • the temperature sensitive material e.g., gelatin
  • the temperature sensitive material is solid or semi-solid at lower temperatures and liquid at higher temperatures.
  • the temperature sensitive material has a phase transition temperature between 5 and 40 ° C, such as 5-10 ° C, 10-15 ° C, 15-20 ° C, 20-25 ° C, 25- 30 ° C, 30-35 ° C, 35-40 ° C.
  • the temperature sensitive material is selected from the group consisting of gelatin, poly N-isopropyl acrylamide-polyethylene glycol block copolymer, polyethylene glycol copolymer (eg, polyvinyl alcohol-poly Ethylene glycol copolymer), agarose, Matrigel, chitosan/sodium glycerophosphate system, Pluronic F127, and poly N-isopropyl acrylamide (PNIPAAm) hydrogel.
  • gelatin poly N-isopropyl acrylamide-polyethylene glycol block copolymer
  • polyethylene glycol copolymer eg, polyvinyl alcohol-poly Ethylene glycol copolymer
  • agarose e.g, polyvinyl alcohol-poly Ethylene glycol copolymer
  • Matrigel chitosan/sodium glycerophosphate system
  • Pluronic F127 Pluronic F127
  • PNIPAAm poly N-isopropy
  • the third component is selected from the group consisting of gelatin, block polymer F-127, agarose, polyethylene glycol, guar gum, polyvinyl alcohol, chitosan , collagen, hyaluronic acid, chitin, cellulose and its derivatives (such as hydroxypropyl cellulose), polyamino acids, poly N-isopropyl acrylamide-polyethylene glycol block copolymer, polyethylene glycol Copolymers (such as polyvinyl alcohol-polyethylene glycol copolymers), alginates (such as sodium alginate), modified alginates (such as oxidized alginates, such as oxidized sodium alginate), Matrigel, Chitosan/sodium glycerophosphate system, and poly N-isopropyl acrylamide (PNIPAAm) hydrogel.
  • PNIPAAm poly N-isopropyl acrylamide
  • the third component is gelatin.
  • the concentration of the third component in the second reagent is from 0.01 to 50% by weight.
  • the concentration of the third component is from 0.01 to 0.05 wt%, from 0.05 to 0.1 wt%, from 0.1 to 0.5 wt%, from 0.5 to 1 wt%, from 1 to 5 wt%, 5- 10 wt%, 10-15 wt%, 15-20 wt%, 20-25 wt%, 25-30 wt%, 30-35 wt%, 35-40 wt%, 40-45 wt%, or 45-50 wt%.
  • step (1) all or part of the microcapsules to which the first component is attached are coated by applying a first reagent comprising the first component to the microcapsules. Obtained from the surface. Accordingly, in certain preferred embodiments, step (1) comprises applying the first component to all or a portion of the surface of the microcapsule to provide all or a portion of the surface to which the first agent is attached. Microcapsules.
  • step (1) all or a portion of the microcapsules to which the first component is attached is by impregnating the microcapsules in a first reagent comprising the first component. acquired.
  • the microcapsules are immersed in the first reagent for 1-30 min, such as 1-5 min, 5-10 min, 10-15 min, 15-20 min, 20-25 min, or 25-30 min.
  • the microcapsules are impregnated in a first reagent under shaking or shaking conditions. Shaking or shaking conditions can be used to promote attachment of the first reagent to the surface of the microcapsules.
  • step (1) is carried out under room temperature conditions (e.g., 15-37 ° C). In certain preferred embodiments, step (1) is carried out under low temperature conditions (e.g., 4-15 ° C).
  • step (1) further comprises washing the microcapsules after immersion in the first reagent.
  • the microcapsules are washed using a buffer (eg, a physiological buffer solution) or a medium solution.
  • the microcapsules are washed by immersing the microcapsules in a buffer (eg, a physiological buffer solution) or a medium solution after immersion in the first reagent.
  • the washing step can be used to remove the surface of the microcapsule Excessive first reagent attached.
  • the washing step can be carried out for 1-5 min or 5-10 min.
  • the washing step can be carried out under room temperature conditions (e.g., 15-37 ° C) or under low temperature conditions (e.g., 4-15 ° C).
  • step (3) is carried out under room temperature conditions (e.g., 15-37 ° C) or under low temperature conditions (e.g., 4-15 ° C).
  • step (5) after placing all or part of the microcapsules to which the first component is attached, on the structure produced in the previous step, it is allowed to stand for 0.1-60 s (for example, 0.1- 1s, 1-5s, 5-10s, 10-15s, 15-20s, 20-25s, 25-30s, 30-35s, 35-40s, 40-45s, 45-50s, 50-55s, or 55-60s ).
  • the standing step facilitates sufficient contact of the first reagent on the surface of the microcapsule with the second reagent on the structure and interaction, thereby assembling the microcapsules in the structure produced in the previous step ( Bonding into a new structural layer.
  • step (6) is carried out under room temperature conditions (e.g., 15-37 ° C) or under low temperature conditions (e.g., 4-15 ° C).
  • an auxiliary material e.g., an auxiliary material for forming a stent
  • an auxiliary material for support is also added inside or outside the resulting structure during the steps (2) - (6).
  • the auxiliary material does not comprise cells.
  • the addition/use of such ancillary materials can help define the shape of the artificial tissue precursor produced, and/or help maintain or enhance the stability of the resulting artificial tissue precursor.
  • the auxiliary material is included in the artificial tissue precursor prepared by the method of the invention.
  • the auxiliary material is included in the artificial tissue precursor prepared by the method of the invention, and which can subsequently be degraded.
  • the auxiliary material only temporarily forms part of the artificial tissue precursor.
  • the auxiliary material is included in the artificial tissue precursor prepared by the method of the invention and is non-degradable.
  • the ancillary material directly (stablely) forms part of the artificial tissue precursor.
  • such ancillary materials are biocompatible and/or biodegradable.
  • the auxiliary material is a temperature sensitive material.
  • the temperature sensitive material has a different morphology at different temperatures.
  • the temperature sensitive material e.g., gelatin
  • the temperature sensitive material is solid or semi-solid at lower temperatures and liquid at higher temperatures.
  • the temperature sensitive material has a phase transition temperature between 5 and 40 ° C, such as 5-10 ° C, 10-15 ° C, 15-20 ° C, 20-25 ° C, 25- 30 ° C, 30-35 ° C, 35-40 ° C.
  • the temperature sensitive material is selected from the group consisting of gelatin, poly N-isopropylacrylamide-polyethylene glycol block copolymer, polyethylene glycol copolymer (eg, polyvinyl alcohol-polyethyl b Glycol Copolymer), Polyhydroxyethyl Acrylate, Agarose, Matrigel, Chitosan / Sodium Glycerate System, Pluronic F127, and poly N-isopropyl acrylamide (PNIPAAm) hydrogel.
  • gelatin poly N-isopropylacrylamide-polyethylene glycol block copolymer
  • polyethylene glycol copolymer eg, polyvinyl alcohol-polyethyl b Glycol Copolymer
  • Polyhydroxyethyl Acrylate Agarose
  • Matrigel Chitosan / Sodium Glycerate System
  • Pluronic F127 Pluronic F127
  • PNIPAAm poly N-isopropyl acrylamide
  • the auxiliary material can have any desired size.
  • the auxiliary material has a size on the order of micrometers to centimeters, for example, 1 ⁇ m to 10 cm, for example, 1 ⁇ m to 2 ⁇ m, 2 ⁇ m to 3 ⁇ m, 3 ⁇ m to 4 ⁇ m, 4 ⁇ m to 5 ⁇ m, 5 ⁇ m to 6 ⁇ m, 6 ⁇ m- 7 ⁇ m, 7 ⁇ m-8 ⁇ m, 8 ⁇ m-9 ⁇ m, 9 ⁇ m-10 ⁇ m, 10 ⁇ m-20 ⁇ m, 20 ⁇ m-30 ⁇ m, 30 ⁇ m-40 ⁇ m, 40 ⁇ m-50 ⁇ m, 50 ⁇ m-60 ⁇ m, 60 ⁇ m-70 ⁇ m, 70 ⁇ m-80 ⁇ m, 80 ⁇ m-90 ⁇ m, 90 ⁇ m-100 ⁇ m, 100 ⁇ m-200 ⁇ m, 200 ⁇ m-300 ⁇ m, 300 ⁇ m-400
  • the auxiliary material can have any desired shape.
  • the auxiliary material may be a sheet-like structure (for example, a rectangular, square, circular, elliptical, hexagonal or irregularly shaped sheet-like structure), or a hollow tubular structure, or a hollow three-dimensional structure (for example, a hollow cube, hollow a sphere, a hollow rectangular prism, a hollow cylinder, or a hollow, three-dimensional structure of irregular shape, or a solid three-dimensional structure (such as a solid cube, a solid sphere, a solid rectangular prism, a solid cylinder, or a solid irregular shape) Three-dimensional structure), or any combination thereof.
  • the shape of the auxiliary material mimics the shape of a native tissue or organ.
  • the microcapsules used to prepare the biological construct are present in a bioink.
  • the bio-ink further comprises a carrier.
  • the vector and its degradation products are non-toxic to the cell and/or non-immunogenic to the host.
  • the carrier comprises a biodegradable material.
  • the biodegradable material in the carrier is biocompatible.
  • degradation of the biodegradable material in the carrier can provide a microenvironment, such as a nutrient, that maintains or promotes the life activities of cells within the microcapsules (eg, biobricks).
  • the degradation products are small molecule compounds such as organic acids, monosaccharides (eg, glucose), oligosaccharides, amino acids, lipids, and the like. Such degradation products can be involved in the metabolic activities of the cell (eg, for the synthesis of extracellular matrices), for the synthesis of extracellular matrices or for conversion to the energy required for activity.
  • the biodegradable material in the carrier is naturally occurring (eg, derived from Naturally occurring biodegradable materials of plants and animals, such as collagen, fibrin, chitosan, alginate, starch, hyaluronic acid, laminin, agarose, gelatin, dextran, and any combination thereof) , synthetic, recombinantly produced, modified, or any combination thereof.
  • Naturally occurring biodegradable materials of plants and animals such as collagen, fibrin, chitosan, alginate, starch, hyaluronic acid, laminin, agarose, gelatin, dextran, and any combination thereof
  • the biodegradable material in the carrier is a naturally occurring degradable polymer.
  • the degradable polymer is selected from the group consisting of collagen, fibrin, chitosan, alginate, starch, hyaluronic acid, laminin, gelatin, dextran, elastin, and any combination thereof.
  • the biodegradable material in the carrier is a modified degradable polymer, such as a modified alginate, such as an alginate (eg, oxidized alginate).
  • a modified degradable polymer such as a modified alginate, such as an alginate (eg, oxidized alginate).
  • the biodegradable material in the carrier is a synthetic degradable polymer.
  • degradable polymers include, but are not limited to, polyphosphazenes, polyacrylic acids and derivatives thereof (eg, polymethacrylic acid, copolymers of acrylic acid and methacrylic acid), polylactic acid (PLA), polyglycolic acid (PGA). , polylactic acid-glycolic acid copolymer (PLGA), polyorthoester (POE), polycaprolactone (PCL), polyhydroxybutyrate (PHB), polyamino acid (eg polylysine), degradability Polyurethane, and any combination thereof.
  • the carrier further comprises water, an inorganic salt, a pH buffer, a stabilizer, a preservative, or any combination thereof.
  • the carrier facilitates placement of the microcapsules (e.g., biobricks) on the construct, and/or immobilizes the biobricks on the construct.
  • the microcapsules e.g., biobricks
  • the carrier is a liquid or semi-liquid (e.g., a gel).
  • the carrier has a viscosity of from 1 to 1000 Pas, such as from 30 to 160 Pas.
  • the carrier has a viscosity of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 50, 80, 100, 200, 300, 400, 500, 800, or 1000 Pas.
  • the carrier has a viscosity of 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10 , 10-12, 12-14, 14-16, 16-18, 18-20, 20-25, 25-30, 30-50, 50-80, 80-100, 100-200, 200-300, 300 -400, 400-500, 500-800, or 800-1000, 1-3, 3-8, 8-16, 3-10, 10-20, 20-50, 50-160 Pas, or 30-160 Pas.
  • the microcapsules to which all or part of the surface used in the step (3) is attached with the first component and the microcapsules to which the first component is attached to all or part of the surface used in the step (5) Can be the same or different.
  • the step (5) uses the first component microcapsules to which all or part of the surface different from the step (3) is attached, the different microcapsules may be provided and the microcapsules may be provided before the step (5) is performed.
  • the first component is attached to all or part of the surface. Thereby, all or part of the microcapsules to which the first component is attached to the surface different from the step (3) are provided.
  • the microcapsules used in the step (3) and the step (5) are selected according to the cell distribution pattern of the artificial tissue precursor to be prepared.
  • the tubular biological construct to be prepared contains only one type of cells
  • microcapsules containing the same cells can be used in the steps (3) and (5).
  • two or more microcapsules may be used in step (3) and step (5), each of which contains a different cell or Combination of cells; or two or more microcapsules comprising the same undifferentiated cells, but comprising cells that can induce undifferentiated cells into different adult cells, can be used in steps (3) and (5) Inducing factor.
  • the first component and the second component of the present invention are not limited to a particular combination. Accordingly, the method of the present invention is also not limited to a particular combination of the first component and the second component.
  • steps (2) and (3) constitute a round of drawing/coating, and microcapsule assembly
  • steps (4) and (5) The same/different combination of the first component and the second component can be used to form another round of drawing/coating, and microcapsule assembly.
  • steps (2) and (3) of the method of the invention may use a first combination of the first component and the second component, while steps (4) and (5) may use the same combination or different combinations ( That is, another combination of the first component and the second component).
  • steps (2) and (3) use a first combination of a first component and a second component (eg, an anion containing species and alpha) - a combination of cyanoacrylates); and steps (4) and (5) use a different combination than steps (2) and (3) (for example a combination different from the combination of an anion-containing substance and a-cyanoacrylate) , for example, a combination of fibrinogen and thrombin).
  • a first component and a second component eg, an anion containing species and alpha
  • steps (4) and (5) use a different combination than steps (2) and (3) (for example a combination different from the combination of an anion-containing substance and a-cyanoacrylate) , for example, a combination of fibrinogen and thrombin).
  • each of the repetitions of steps (4) and (5) each constitutes a round of microcapsule assembly.
  • the same or different microcapsules; and/or the same or different combination of the first component and the second component can be used.
  • the microcapsules, the first reagent comprising the first component can be achieved by replacing the ink in the respective ink cartridge, or by providing an additional ink cartridge. Replacement with a second reagent comprising a second component.
  • the bioprinting steps (e.g., steps (2)-(6)) in the methods of the invention are continuous and/or substantially continuous.
  • steps (2)-(6) of the method of the invention the multilayer structure is continuously bioprinted to obtain a biological construct or artificial tissue having a predetermined pattern comprising a multilayer structure Precursor.
  • steps (2)-(6) of the method of the invention the same or different microcapsules can be used for printing for each layer structure.
  • the pattern uses one or more microcapsules to print a multilayer structure.
  • the plurality of segments are continuously bioprinted to obtain a biological construct comprising a plurality of segments having a predetermined pattern or Artificially organize precursors.
  • the same or different microcapsules can be used for printing for each segment.
  • one or more microcapsules are used to print a plurality of segments according to a predetermined pattern.
  • the artificial tissue precursors of the invention are used for tissue transplantation (e.g., lumen transplantation, such as vascular grafting).
  • cell distribution information for a tissue or tissue injury site is obtained prior to performing the methods of the invention.
  • the method of the invention further comprises obtaining cell distribution information of the tissue or tissue injury site, and then preparing the artificial tissue precursor based on the cell distribution information.
  • the cells in the microcapsules used in the methods of the invention are derived from a subject.
  • the cells in the microcapsules used in the methods of the invention are derived from other subjects having similar or identical characteristics (eg, species, age, sex, genetic information, etc.) to the subject.
  • the cells in the microcapsules used in the methods of the invention are derived from allogeneic. In certain preferred embodiments, the cells in the microcapsules used in the methods of the invention are derived from a cell line. In certain preferred embodiments, the methods of making artificial tissue precursors of the invention are performed in vitro.
  • the biological construct is immobilized with a solid support.
  • the biological construct is chemically bound to the solid support.
  • the biological construct is bound to the solid support by a binder.
  • the binder is an alpha-cyanoacrylate (e.g., alpha-cyanoacrylate, alpha-cyanoacrylate, alpha-cyanoacrylate, alpha-) Isohexyl acrylate, octyl ⁇ -cyanoacrylate).
  • alpha-cyanoacrylate e.g., alpha-cyanoacrylate, alpha-cyanoacrylate, alpha-cyanoacrylate, alpha-
  • Isohexyl acrylate octyl ⁇ -cyanoacrylate
  • the present application is also directed to a biological construct made by the method of the prepared biological construct as defined in any one of methods 1, 2 or 3.
  • the present application is also directed to a kit for preparing an artificial tissue precursor, the kit comprising a microcapsule, and a first reagent and a second reagent separated from each other, wherein the microcapsule comprises a cell And enveloping cells a compatibilizing material, the first reagent containing a first component, the second reagent containing a second component, and when the first component is in contact with the second component, capable of producing a blocking effect, achieving adhesion Cooperation.
  • the first component and the second component are capable of undergoing a strong interaction (e.g., capable of undergoing a chemical reaction) upon contact, and produce a blocking effect to effect adhesion.
  • a strong interaction e.g., capable of undergoing a chemical reaction
  • Such adhesion not only enables cell-to-cell, cell-to-tissue, tissue-to-tissue adhesion, but also adhesion between cells/tissues and foreign substances.
  • such adhesion has at least one property selected from the group consisting of: (1) it is safe, reliable, non-toxic, non-carcinogenic, non-teratogenic, non-mutagenic; (2) it has a good biological phase Capacity, does not hinder the body's own healing; (3) it can be used under conditions of blood and tissue fluid; (4) it can achieve rapid adhesion at normal temperature and pressure; (5) it has good adhesion Strength and durability, the bonded part has certain elasticity and toughness; (6) It is not irritating to the body tissues during use; (7) After the bonding effect is achieved, the relevant components can be gradually degraded and Absorption; and, (8) the binding site can be migrated through the cells.
  • the blocking effect produced by contacting the first component with the second component can be used to bond the two microcapsules together to form a construct; and the construct thus obtained
  • the tensile modulus is not less than 10 Pa, for example, not less than 20 Pa, not less than 30 Pa, not less than 40 Pa, not less than 50 Pa, not less than 60 Pa, not less than 70 Pa, not less than 80 Pa, not less than 90 Pa, Not less than 100 Pa, not less than 200 Pa, not less than 300 Pa, not less than 400 Pa, not less than 500 Pa, not less than 600 Pa, not less than 700 Pa, not less than 800 Pa, not less than 900 Pa, not less than 1000 Pa.
  • the obtained construct has a tensile modulus of from 1 KPa to 10 MPa, such as from 1 to 5 KPa, from 5 to 10 KPa, from 10 to 50 KPa, from 50 to 100 KPa, from 100 to 500 KPa, from 500 to 1000 KPa, -5 MPa, or 5-10 MPa.
  • cells in the microcapsules are capable of migrating through the binding site into adjacent microcapsules or more distal microcapsules. Thereby, the cells in the microcapsules are capable of growth, migration, differentiation and proliferation throughout the construct.
  • the first component and the second component are selected from the group consisting of:
  • Alginate such as sodium alginate
  • oxidized alginate such as oxidized sodium alginate
  • substances containing Ca 2+ , Mg 2+ , Ba 2+ , Sr 2+ , or Fe 3+ eg, a solution containing a Ca 2+ , Mg 2+ , Ba 2+ , Sr 2+ , or Fe 3+ or a semi-solid (eg, a gel)
  • a semi-solid eg, a gel
  • a molecule containing a maleimide group for example, a polyethylene glycol (MAL-PEG) containing a maleimide group
  • a molecule containing a free thiol group for example, a polyethylene glycol containing a free thiol group (PEG-SH)
  • substances containing anions for example, solutions containing anions or semi-solids (such as gels)
  • ⁇ -cyano groups Acrylate (for example, methyl ⁇ -cyanoacrylate, ethyl ⁇ -cyanoacrylate, isobutyl ⁇ -cyanoacrylate, iso- ⁇ -cyanoacrylate, n-octyl ⁇ -cyanoacrylate);
  • Fibrinogen and ⁇ -cyanoacrylate for example, ⁇ -cyanoacrylate, ⁇ -cyanoacrylate, ⁇ -cyanoacrylate, iso- ⁇ -cyanoacrylate, ⁇ - N-octyl cyanoacrylate;
  • serum albumin for example, bovine serum albumin
  • glutaraldehyde glutaraldehyde
  • an embodiment of the present invention can be used as long as the first component is in contact with the second component to produce a blocking effect and achieve adhesion.
  • the first component and the second component of the present invention are not limited to the specific combinations described above.
  • the first component may be any member of the combination, and the second component is another member of the combination.
  • the first component may be fibrinogen (when the second component is thrombin) or may be thrombin (when the second component is fibrin) original).
  • the first component is fibrinogen and the second component is thrombin.
  • the first component is an alginate (eg, sodium alginate) or an oxidized alginate (eg, oxidized sodium alginate) and the second component is Ca2 +-containing.
  • a substance such as Mg 2+ , Ba 2+ , Sr 2+ , or Fe 3+ , such as a solution containing a Ca 2+ , Mg 2+ , Ba 2+ , Sr 2+ , or Fe 3+ or a semi-solid (eg, coagulation) gum).
  • the first component is a maleimide group-containing molecule (eg, a maleimide group-containing polyethylene glycol (MAL-PEG)), and
  • the two components are molecules containing a free sulfhydryl group (for example, polyethylene glycol (PEG-SH) containing a free sulfhydryl group).
  • the first component is an anion-containing material, such as an anion containing solution or semi-solid (eg, a gel)
  • the second component is an alpha-cyanoacrylate (eg, alpha) Methyl cyanoacrylate, ethyl ⁇ -cyanoacrylate, isobutyl ⁇ -cyanoacrylate, iso- ⁇ -cyanoacrylate, n-octyl ⁇ -cyanoacrylate).
  • the first component is fibrinogen and the second component is alpha-cyanoacrylate (eg, alpha-cyanoacrylate, alpha-cyanoacrylate, Isobutyl isocyanoacrylate, isohexyl cyanoacrylate, n-octyl ⁇ -cyanoacrylate).
  • the first component is serum albumin (e.g., bovine serum albumin) and the second component is glutaraldehyde.
  • the first component is a urethane-containing group (-NHCOO-) or an isocyanate group (-NCO)-containing molecule (eg, a urethane group-containing polyethylene group) A diol or an isocyanate group-containing polyethylene glycol), and the second component is an active hydrogen-containing molecule (for example, a carboxyl group-containing polyethylene glycol).
  • the first component is gelatin-resorcinol and the second component is glutaraldehyde.
  • the first component is carbodiimide crosslinked gelatin and the second component is poly L-glutamic acid (PLGA).
  • the first component is an aminated gelatin and the second component is an aldehydelated polysaccharide.
  • the concentration of the first component in the first reagent is from 0.01 to 50% by weight.
  • the concentration of the first component is from 0.01 to 0.05 wt%, from 0.05 to 0.1 wt%, from 0.1 to 0.5 wt%, from 0.5 to 1 wt%, from 1 to 5 wt%, 5- 10 wt%, 10-15 wt%, 15-20 wt%, 20-25 wt%, 25-30 wt%, 30-35 wt%, 35-40 wt%, 40-45 wt%, or 45-50 wt%.
  • the concentration of the second component in the second reagent is from 0.01 to 50% by weight.
  • the concentration of the second component is from 0.01 to 0.05 wt%, from 0.05 to 0.1 wt%, from 0.1 to 0.5 wt%, from 0.5 to 1 wt%, from 1 to 5 wt%, 5- 10 wt%, 10-15 wt%, 15-20 wt%, 20-25 wt%, 25-30 wt%, 30-35 wt%, 35-40 wt%, 40-45 wt%, or 45-50 wt%.
  • the second agent further comprises a third component, the third component being a viscous agent.
  • the viscosity of the second agent can be conveniently adjusted by adjusting the content of the third component (viscous agent) so that the second agent can maintain a specific shape, which is suitable for drawing a pattern or coating.
  • the third component is a biocompatible material.
  • the third component is a biologically derived material.
  • the third component is a biodegradable material.
  • the third component is a temperature sensitive material. In certain preferred embodiments, the temperature sensitive material has a different morphology at different temperatures.
  • the temperature sensitive material e.g., gelatin
  • the temperature sensitive material is solid or semi-solid at lower temperatures and liquid at higher temperatures.
  • the temperature sensitive material has a phase transition temperature between 5 and 40 ° C, such as 5-10 ° C, 10-15 ° C, 15-20 ° C, 20-25 ° C, 25- 30 ° C, 30-35 ° C, 35-40 ° C.
  • the temperature sensitive material is selected from the group consisting of gelatin, poly N-isopropyl acrylamide-polyethylene glycol block copolymer, polyethylene glycol copolymer (eg, polyvinyl alcohol-poly Ethylene glycol copolymer), agarose, Matrigel, chitosan/sodium glycerophosphate system, Pluronic F127, and poly N-isopropylacrylamide (PNIPAAm) hydrogel.
  • gelatin poly N-isopropyl acrylamide-polyethylene glycol block copolymer
  • polyethylene glycol copolymer eg, polyvinyl alcohol-poly Ethylene glycol copolymer
  • agarose e.g, polyvinyl alcohol-poly Ethylene glycol copolymer
  • Matrigel chitosan/sodium glycerophosphate system
  • Pluronic F127 Pluronic F127
  • PNIPAAm poly N-isopropyl
  • the third component (viscous agent) is selected from the group consisting of gelatin, block polymer F-127, agarose, polyethylene glycol, guar gum, polyvinyl alcohol, chitosan , collagen, hyaluronic acid, chitin, cellulose and Its derivatives (such as hydroxypropyl cellulose), polyamino acids, poly N-isopropyl acrylamide-polyethylene glycol block copolymers, polyethylene glycol copolymers (such as polyvinyl alcohol-polyethylene glycol copolymerization) , alginate (such as sodium alginate), modified alginate (such as oxidized alginate, such as oxidized sodium alginate), Matrigel, chitosan / sodium glycerophosphate system, and poly N- Isopropyl acrylamide (PNIPAAm) hydrogel.
  • the third component (viscous agent) is gelatin.
  • the concentration of the third component in the second reagent is from 0.01 to 50% by weight.
  • the concentration of the third component is from 0.01 to 0.05 wt%, from 0.05 to 0.1 wt%, from 0.1 to 0.5 wt%, from 0.5 to 1 wt%, from 1 to 5 wt%, 5- 10 wt%, 10-15 wt%, 15-20 wt%, 20-25 wt%, 25-30 wt%, 30-35 wt%, 35-40 wt%, 40-45 wt%, or 45-50 wt%.
  • the microcapsules contained in the kit are microcapsules as defined in any of the above.
  • kits for preparing a tubular biological construct comprising one or more kits as defined above.
  • different kits use the same combination of first reagent and second reagent.
  • different kits use a combination of different first reagents and second reagents.
  • the artificial tissue precursors of the invention can be further cultured. Accordingly, the present application also relates to an artificial tissue obtained by culturing an artificial tissue precursor of the present invention (for example, in vitro culture or in vivo culture).
  • the artificial tissue is an artificial lumen.
  • the lumen is a lumen comprising epithelial cells (eg, blood vessels, esophagus, trachea, stomach, bile duct, intestinal tract (including the small and large intestines, eg, duodenum, jejunum, ileum, The cecum (including the appendix), the ascending colon, the right colon of the colon, the transverse colon, the left collateral, the descending colon, the sigmoid colon, the rectum, the fallopian tube, the vas deferens, the ureter, the bladder or the lymphatic vessels).
  • epithelial cells eg, blood vessels, esophagus, trachea, stomach, bile duct, intestinal tract (including the small and large intestines, eg, duodenum, jejunum, ileum, The cecum (including the appendix), the ascending colon, the right colon of the colon, the transverse colon, the left collateral, the descending colon, the sigmoid colon,
  • the artificial lumen is a tubular artificial lumen or a sheet-like artificial lumen.
  • the artificial lumen is an artificial blood vessel or a blood vessel patch.
  • the artificial tissue precursor is cultured under conditions that permit cell proliferation, differentiation, migration, secretion, and/or metabolism within the microcapsules.
  • the culture conditions depend on the type of cells within the microcapsules, the type of microcapsules used, the structure and shape of the artificial tissue precursors, the purpose of the culture, and the like. Those skilled in the art can select appropriate culture conditions, such as media, pH, temperature, CO 2 level and duration. Typical tissue and cell culture conditions can be found, for example, in Doyle, Alan, and J. Bryan Griffiths, eds. Cell and tissue culture: laboratory procedures in biotechnology. New York: Wiley, 1998.
  • the artificial tissue precursor is cultured for at least 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 25, or 30 days.
  • the artificial tissue precursors 1-3, 3-5, 5-7, 7-10, 10-14, 14-21, 21-28, 1-7, 7-14 are cultured. 1-14, or 14-28 days.
  • the obtained artificial tissue precursor is cultured in a 3D incubator.
  • the obtained artificial tissue precursor is cultured in a bioreactor.
  • the obtained artificial tissue precursor is cultured at 37 ° C, 5% CO 2 .
  • physical stimulation e.g., pressure, shear, illumination, heat, etc.
  • chemical stimulation e.g., hormones, cytokines, chemical agents, etc.
  • the biodegradable material in the microcapsules is degraded during the cultivation process.
  • the degradation products of such biodegradable materials provide nutrients and/or extracellular matrices to cells in the microcapsules.
  • the biodegradable material in the microcapsules is degraded by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.
  • the cells in the artificial tissue precursor secrete secretions during culture and these secretions are integrated into the artificial tissue precursor.
  • the cells within the microcapsules are joined to one another during culture.
  • the cells between the microcapsules are joined to one another during culture.
  • the biological construct has a high cell density (eg, at least 100, 200, 500, 1000, 2000, 5000, 10,000, 20,000, 50,000, or 100,000 cells/mm 3 ) after culture.
  • the cells within the microcapsules proliferate at least 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10,000, 20,000, 50,000, or 100,000 times after culture. .
  • the artificial tissue precursor is implanted into a non-human subject and cultured in the non-human subject.
  • the non-human subject is a mammal, such as a bovine, equine, ovine, porcine, canine, feline, rodent, spiritual Long-lived animals.
  • the present invention provides a luminal implant comprising an artificial tissue precursor of the present invention (eg, a tubular artificial tissue precursor or a flaky artificial tissue precursor) or an artificial tube Cavity.
  • an artificial tissue precursor of the present invention eg, a tubular artificial tissue precursor or a flaky artificial tissue precursor
  • an artificial tube Cavity e.g., a tubular artificial tissue precursor or a flaky artificial tissue precursor
  • the luminal implant comprises one or more (eg, 2, 3, 4, or 5) artificial tissue precursors of the invention (eg, tubular artificial tissue precursors or A flaky artificial tissue precursor), or one or more (eg, 2, 3, 4, or 5) artificial lumens of the present invention (eg, tubular artificial lumens or flaky artificial lumens).
  • artificial tissue precursors of the invention eg, tubular artificial tissue precursors or A flaky artificial tissue precursor
  • artificial lumens of the present invention eg, tubular artificial lumens or flaky artificial lumens.
  • the luminal implant comprises a plurality (eg, 2, 3, 4, or 5) of tubular artificial tissue precursors of the invention, a plurality of said tubular artificial tissue Fluid communication between the bodies.
  • the lumen implant comprises a plurality (eg, 2, 3, 4, or 5) of tubular artificial lumens of the present invention, a plurality of said tubular artificial lumens Interconnected fluid.
  • the lumen implant is a linear tubular structure or a branched tubular structure.
  • the lumen implant is an X-shaped tubular, Y-shaped tubular or T-shaped tubular.
  • the lumen is a lumen comprising epithelial cells (eg, blood vessels, esophagus, trachea, stomach, bile duct, intestinal tract (including the small and large intestines, eg, duodenum, jejunum, ileum, The cecum (including the appendix), the ascending colon, the right colon of the colon, the transverse colon, the left collateral, the descending colon, the sigmoid colon, the rectum, the fallopian tube, the vas deferens, the ureter, the bladder or the lymphatic vessels).
  • epithelial cells eg, blood vessels, esophagus, trachea, stomach, bile duct, intestinal tract (including the small and large intestines, eg, duodenum, jejunum, ileum, The cecum (including the appendix), the ascending colon, the right colon of the colon, the transverse colon, the left collateral, the descending colon, the sigmoid colon,
  • the lumen comprising epithelial cells is a blood vessel.
  • the luminal implant is a vascular implant comprising an artificial blood vessel or a blood vessel patch of the present invention.
  • the luminal implant further comprises a pharmaceutically active ingredient, such as a pharmaceutically active ingredient for preventing thrombosis, calcification, infection, and/or rejection.
  • a pharmaceutically active ingredient such as a pharmaceutically active ingredient for preventing thrombosis, calcification, infection, and/or rejection.
  • the luminal implant further comprises an inductive device for detecting fluid parameters within the lumen.
  • the luminal implant further includes an adjustment device for adjusting fluid parameters within the lumen.
  • the luminal implant is implanted in the body of a subject.
  • the subject has one or more of the following diseases: cardiovascular disease, cerebrovascular disease, peripheral vascular disease, orthopedic disease, urinary system disease, or neoplastic disease;
  • the subject has one or more of the following conditions: coronary heart disease, cerebral ischemic stroke, hemangioma, malignant tumor invasion of blood vessels, thromboangiitis obliterans, blood Orthopedic diseases caused by transport obstruction, chronic renal failure;
  • the subject is a mammal, such as a bovine, equine, ovine, porcine, canine, feline, rodent, primate An animal; among them, a particularly preferred subject is a human.
  • the luminal implant of the present invention can be used to replace a narrow, occluded, dilated, damaged or deformed lumen (e.g., a blood vessel) of a subject, or to construct a lumen bypass, from a function Replacement of a narrow, occluded, dilated, damaged or deformed lumen.
  • the lumen implant of the present invention can be used as a lumen replacement in the event that the subject's autologous lumen supply is insufficient.
  • the vascular implant of the present invention can be used for coronary artery bypass grafting (CABG), administered to a subject suffering from coronary heart disease, or for arteriovenous fistula, for administration to patients suffering from chronic renal failure. Tester.
  • CABG coronary artery bypass grafting
  • the present application provides a method of replacing or repairing a lumen (eg, a blood vessel) of a subject, comprising implanting a luminal implant of the present invention into a subject.
  • a lumen eg, a blood vessel
  • the methods are used for therapeutic purposes.
  • a luminal implant of the invention is used to replace a narrow, occluded, dilated, damaged or deformed lumen of a subject.
  • the methods are used for non-therapeutic purposes.
  • the normal lumen of a non-human subject is replaced with a luminal implant of the invention for medical research.
  • the subject is a mammal, such as a bovine, equine, ovine, porcine, canine, feline, rodent, primate Animal; for example, the subject is a human.
  • the present invention provides a lumen model comprising the artificial lumen of the present invention.
  • the lumen model comprises one or more (eg, 2, 3, 4, or 5) artificial lumens of the invention (eg, tubular artificial lumens, such as artificial blood vessels) .
  • the lumen model comprises a plurality (eg, 2, 3, 4, or 5) of tubular artificial lumens of the present invention, between the plurality of tubular artificial lumens Fluid communication.
  • the lumen model is a linear tubular structure or a branched tubular structure.
  • the lumen model is an X-shaped tubular, Y-shaped tubular or T-shaped tubular.
  • the lumen model further includes an inductive device for detecting fluid parameters within the lumen.
  • the lumen model further includes an adjustment device for adjusting fluid parameters within the lumen.
  • the luminal model is used in medical teaching demonstrations, screening of drugs (eg, drugs for preventing and/or treating vascular diseases, such as active ingredients of drugs), biological studies, or medical studies ( For example, research on vascular fluid mechanics).
  • drugs eg, drugs for preventing and/or treating vascular diseases, such as active ingredients of drugs
  • biological studies e.g., biological studies, or medical studies ( For example, research on vascular fluid mechanics).
  • Figure 5B exemplarily shows the shape of a luminal implant or lumen model of the present invention.
  • a branched tubular artificial tissue precursor (or tubular artificial lumen) can be obtained by joining (eg, bonding or suturing) a linear tubular artificial tissue precursor (or tubular artificial lumen) to obtain a branched A luminal implant or lumen model.
  • the invention provides the use of an artificial tissue precursor of the invention for the preparation of an artificial tissue, a luminal implant or a lumen model.
  • the artificial tissue is an artificial tissue (eg, an artificial lumen) as described in any one of the above.
  • the luminal implant is a luminal implant as described in any one of the preceding claims.
  • the lumen model is a lumen model as described in any one of the above.
  • the invention provides the use of the artificial tissue of the invention for preparing a luminal implant or lumen model.
  • the luminal implant is a luminal implant as described in any one of the preceding claims.
  • the lumen model is a lumen model as described in any one of the above.
  • the technical solution of the present invention has one or more of the following beneficial effects:
  • the number of cells in the microcapsules is generally uniform, and the microcapsules provide a suitable microenvironment for cell differentiation and/or growth, which is favorable for the cells to maintain dryness and maintain their differentiation.
  • the ability to construct a tissue with a uniform distribution of cells is conducive to the formation of structurally and functionally intact tissues.
  • the artificial tissue precursor of the present invention is capable of forming a stable structure such that cells enclosed therein can stay in a specific position.
  • the microcapsules provide mechanical protection to the cells, allowing them to withstand the impact of body fluids in the lumen during the preparation of artificial tissue precursors and after implantation in the body, without being susceptible to damage or shedding.
  • the cells are evenly distributed, and the artificial tissue is easy to form a complete structure and function.
  • adipose-derived mesenchymal stem cells are used to prepare artificial tissue precursors. Adipose-derived mesenchymal stem cells are easy to obtain and have high cell safety. Since adipose-derived mesenchymal stem cells have been used in vitro and in vivo, no reports have been made on the tumorigenicity of adipose-derived mesenchymal stem cells.
  • the artificial tissue precursor of the present invention can be customized according to the needs of the patient to realize personalized preparation.
  • the solid support is closely adhered to the microcapsule or the biological construct composed of the microcapsule, and no relative movement occurs.
  • Reagents, kits or instruments from which the source is not indicated in the examples are conventional products commercially available.
  • the invention is described by way of example, and is not intended to limit the scope of the invention.
  • (1) Collection of adipose-derived mesenchymal stem cells The rhesus monkey was used as an animal model, and the adipose tissue was cut out from the groin into a 50 mL centrifuge tube; the adipose tissue was digested with trypsin, and the adipose-derived mesenchymal stem cells were collected by centrifugation.
  • Adipose-derived mesenchymal stem cells were expanded and cultured using serum-free Lonza medium.
  • a photomicrograph of the primary cultured cells of the fourth generation is shown in FIG. It can be seen from the figure that the cell morphology is uniform and the growth state is good. The cells were collected by centrifugation.
  • the raw material is collagen solid, you can follow the above procedure; if the raw material is collagen solution, it can be used directly or diluted.
  • the collagen solution can be diluted with ultrapure water (filtered through a 0.22 ⁇ m filter) depending on the concentration actually used.
  • Polylysine purchased from Sigma, number average molecular weight (M n ) of 150,000-300,000 was dissolved in DMEM high glucose medium of pH 7.2 to obtain a concentration of 1 wt%. Polylysine solution.
  • Collagen dropping formation of nuclear layer structure: 0.1 ⁇ L of collagen solution containing seed cells is accurately aspirated by an electronic suction device capable of sucking and discharging the nano-upgrading liquid, and the U-bottom superhydrophobic orifice plate prepared in the step 1 is dropped. In the middle, droplets were formed and kept at 37 ° C for 30 min at a constant temperature to form.
  • the optional electronic suction device is Eppendorf Xplorer 0.5-10uL or TransferpetteElectronic0.5-10uL, with a dispensing function of at least 0.1 ⁇ L per dispense, or 1 ⁇ L or 0.5 ⁇ L with SGE autosampler.
  • Formulating a fibrinogen solution (5 wt%) for use as a first reagent weigh 0.1 g of fibrinogen, and dissolve it in 2 mL of physiological saline (if necessary, it can be fully dissolved in a 37 ° C water bath); The fibrinogen solution was passed through a 0.22 ⁇ m filter for filter sterilization; the filtered fibrinogen solution was stored and used.
  • thrombin solution 2000 U/mL
  • thrombin solution 2000 U/mL
  • Second reagent weigh 0.011 g of CaCl 2 , add 2000 U of thrombin (Ca 2+ concentration of 10 mmol/mL), and then fully dissolve by adding 1 mL of physiological saline; Subsequently, the thrombin solution was passed through a 0.22 ⁇ m filter for filter sterilization; stored and set aside.
  • the fibrinogen on the surface of the bio-brick is cross-linked with each other to connect the bio-bricks into one body, forming a circular tubular biological construct with no opening on the side wall.
  • the length is 20mm, the diameter is 6mm, and the wall thickness is 1mm.
  • a layer of medical glue (white cloud medical glue medical EC type) is sprayed on the outer wall of the tubular biological construct.
  • the expanded polytetrafluoroethylene tubular solid support is sleeved on the outer side of the tubular biological construct, and the outer wall of the biological construct is bonded to the inner wall of the expanded polytetrafluoroethylene tubular solid support by a medical glue to obtain an artificial blood vessel.
  • Body its shape is shown in Figure 9.
  • Step (1) Open the abdomen of the rhesus monkey and expose the abdominal aorta.
  • Step (2) The abdominal aorta is cut, and the two broken ends are respectively sutured with the obtained artificial blood vessel precursor.
  • Step (3) sutures the wound of the animal's abdomen.
  • Fig. 10A shows the morphology of the artificial blood vessel
  • Fig. 10B shows the vascular tissue obtained by removing the tubular support
  • Fig. 10C shows the morphology after the longitudinal section of the tissue.
  • HE staining and immunohistochemical staining were performed on the tissues and compared with normal blood vessels. The results are shown in Figure 11-13.
  • Fig. 11 shows the results of HE staining
  • Fig. 11A shows normal blood vessels
  • Fig. 11B shows artificial blood vessels.
  • the artificial blood vessel has a similar cell arrangement to the normal blood vessel, the endothelial cell layer (referred to by the thin arrow) and the smooth muscle cell layer (referred to by the thick arrow).
  • Figure 12 shows the results of ⁇ -SMA staining, and ⁇ -SMA positive cells were smooth muscle cells.
  • Fig. 12A is a normal blood vessel
  • Fig. 12B is an artificial blood vessel.
  • adipose-derived mesenchymal stem cells constituting an artificial blood vessel differentiate into smooth muscle cells, and have a cell morphology, arrangement, and orientation similar to those of normal blood vessels.
  • Figure 13 shows the results of CD31 staining, and CD31 positive cells are endothelial cells.
  • Fig. 13A is a normal blood vessel
  • Fig. 13B is an artificial blood vessel.
  • adipose-derived mesenchymal stem cells constituting an artificial blood vessel differentiate into endothelial cells at the surface in contact with blood, and a cell morphology and arrangement similar to those of normal blood vessels appear.
  • Step (1) Weigh a certain amount of polycaprolactone dissolved in tetrahydrofuran to prepare a preparation solution having a concentration of 2% by weight.
  • Step (2) The artificial blood vessel mold is immersed in the preparation solution, slowly taken out, and the operation is repeated after the solvent is volatilized until a polycaprolactone tubular solid support having a tube wall thickness of 0.5 mm is obtained.
  • Step (3) The polycaprolactone tubular solid support is removed from the mold and washed in ultrapure water.
  • Step (4) The polycaprolactone tubular solid support is dried and cut to a desired length, and subjected to ethylene oxide sterilization for use.
  • Example 2 Using the bio-brick and polycaprolactone tubular solid support containing rhesus adipose-derived mesenchymal stem cells prepared in Example 1, according to the procedure of Example 2, a circular tubular biological construct without sidewalls was prepared using a 3D printer. Body, and using medical glue (white cloud medical glue medical EC type), the polycaprolactone tubular solid support is placed on the outer side of the tubular biological construct) the outer wall of the biological construct and the inner wall of the polycaprolactone tubular solid support Adhesion is performed to obtain an artificial blood vessel precursor.
  • medical glue white cloud medical glue medical EC type
  • the artificial blood vessel precursor prepared in Example 3 was implanted into the rhesus monkey, and the morphology and blood flow direction of the formed artificial blood vessel were detected 6 days after the operation, and the results are shown in Fig. 14.
  • Fig. 14A shows the results of ultrasonic examination, and it can be seen from the figure that the lumen of the artificial blood vessel is unobstructed.
  • Fig. 14B shows the results of color Doppler detection. The results show that the blood flow directions on both sides of the artificial blood vessel are the same, which proves that the blood vessels are unobstructed.
  • the artificial blood vessel was taken out 20 days after the operation, and the artificial blood vessel was detected by immunohistochemistry, and the results are shown in Figs. 15A and 15B (the scales in the figure are both 200 ⁇ m).
  • Fig. 15A shows the results of ⁇ -SMA staining, in which the adipose-derived mesenchymal stem cells differentiate into smooth muscle cells, as indicated by the thick arrows in the figure.
  • Fig. 15B shows the results of CD31 staining, in which the adipose-derived mesenchymal stem cells differentiate into endothelial cells in the vascular blood vessels as indicated by the thin arrows.
  • Figure 15C shows the results of Sirius red staining. As shown, the artificial blood vessels form a collagen structure similar to normal blood vessels.
  • Example 1 The biobrick of Example 1 was immersed in a 5% fibrinogen solution for 5 minutes, followed by removal of the fibrinogen solution, and addition to H-DMEM medium for further 5 minutes.
  • bio-bricks are attached one by one to the inner wall of the expanded polytetrafluoroethylene artificial blood vessel. Under the action of the medical glue, the bio-brick and the expanded polytetrafluoroethylene artificial blood vessel are firmly bonded together to form an artificial blood vessel precursor.
  • the artificial blood vessel precursor was implanted into the rhesus monkey for 14 days, and was detected by immunohistochemical staining, and the results are shown in Figs. 16A and 16B (the scales in the figure are both 50 ⁇ m).
  • Fig. 16A shows the results of ⁇ -SMA staining. As indicated by the thick arrows in the figure, adipose-derived mesenchymal stem cells differentiate into smooth muscle cells in artificial blood vessels.
  • Figure 16B shows the results of CD31 staining. As indicated by the thin arrows in the figure, there are adipose-derived mesenchymal stem cells in the artificial blood vessels. Endothelial cells undergo differentiation.
  • Collagen dropping formation of nuclear layer structure: 0.1 ⁇ L of collagen solution containing seed cells is accurately aspirated by an electronic suction device capable of sucking and discharging the liquid, and the U-bottom superhydrophobic plate prepared in step 1 is dropped. In the middle, droplets were formed and kept at a constant temperature of 37 ° C for 30 min to form a microcapsule containing rhesus adipose-derived mesenchymal stem cells.
  • Example 5 The microcapsule of Example 5 was immersed in a 5% fibrinogen solution for 5 minutes, followed by removal of the fibrinogen solution, and addition to H-DMEM medium for further 5 minutes.
  • microcapsules are attached one by one to the inner wall of the expanded polytetrafluoroethylene artificial blood vessel. Under the action of the medical glue, the microcapsules are firmly bonded with the expanded polytetrafluoroethylene artificial blood vessels to form an artificial blood vessel precursor.
  • Fig. 17A is a sectional view of the obtained artificial blood vessel.
  • the detection was carried out by immunohistochemistry, and the results are shown in Figs. 17B and 17C (the scales in the figure are both 50 ⁇ m).
  • Figure 17B shows the results of ⁇ -SMA staining, as indicated by the thick arrows in the figure, there are adipose-derived mesenchymal stem cells in artificial blood vessels. Differentiation into smooth muscle cells.
  • Fig. 17C shows the results of CD31 staining, in which the adipose-derived mesenchymal stem cells differentiate into endothelial cells in the artificial blood vessels as indicated by the thin arrows.
  • Example 7 Preparation of a reinforced bio-brick-expanded polytetrafluoroethylene artificial blood vessel precursor using a 3D bioprinter
  • Example 1 The biobrick of Example 1 was immersed in a 5% fibrinogen solution for 5 minutes, followed by removal of the fibrinogen solution, and addition to H-DMEM medium for further 5 minutes.
  • a rotating rod having an outer diameter of 4 mm was prepared, and the biological bricks prepared in the step (1) were printed one by one on a rotating rod to form a tubular biological construct.
  • Figure 18 shows the above preparation process.
  • Figure 158 Printing bio-bricks on a rotating rod to form a tubular biological construct;
  • Figures 18B and 18C arranging artificial blood vessels from left to right over a tubular biological construct;
  • Figure 18D Artificial blood vessels and tubular biological constructs Bonded together to form an artificial blood vessel precursor.
  • 19A and 19B show a tubular solid support produced by electrospinning using polylactic acid as a basic material.
  • Fig. 19C shows the operation of cutting the polylactic acid tubular support, then dropping the medical glue on one side of the tubular support, and placing the bio-brick on the other side.
  • Figure 19D shows that the medical glue can penetrate through the tube wall, allowing the bio-brick to adhere to the inner wall to obtain an artificial blood vessel precursor.
  • the medical glue is added to the outer wall of the polylactic acid tubular solid support to infiltrate the medical glue to the inner wall. Because the medical glue can penetrate the wall of the electrospun polylactic acid tube, thus the organism The brick is fixed.
  • polylactic acid can be used as a solid support
  • the solid support obtained by electrospinning because of its pore structure permeability, the medical glue can permeate through the tube wall and can be in the solid support.
  • the bio-brick is placed on one side and the bio-brick is placed on the other side to obtain an artificial tissue precursor.
  • Biobricks are prepared, which comprise murine bone marrow mesenchymal stem cells, and whose core layer comprises collagen, the shell layer comprises sodium alginate; and the preparation method thereof is as described in Chinese Application No. 201610211570.4.
  • biobrick prepared according to the method described in Chinese Patent Application No. 201610211570.4
  • fibrinogen bovine source
  • thrombin bovine source
  • physiological saline medical grade
  • CaCl 2 sterile water
  • gelatin sterile water
  • thrombin solution 2000 U/mL: Weigh 0.011 g of CaCl 2 , add 2000 U of thrombin (Ca 2+ concentration of 10 mmol/mL), and then fully dissolve by adding 1 mL of physiological saline; then, thrombin solution Filter and sterilize through a 0.22 ⁇ m filter; store and set aside.
  • the bio-brick was immersed in the first reagent for 10 min to attach/assemble fibrinogen molecules on its surface (if necessary, it can be gently shaken to facilitate assembly). The bio-brick was then immersed in the cell culture medium for 5 min to wash away the unassembled fibrinogen molecules on the surface, thereby obtaining impregnated biobricks.
  • the coagulation reaction of fibrinogen and thrombin is used to connect and assemble biobricks to form a predetermined three-dimensional structure.
  • the build steps are as follows:
  • step cd as needed to form different layers of ring-shaped structures made of bio-bricks, ie circular tubular structures with no openings on the side walls (if necessary, the tubular structure containing auxiliary materials can be placed at 37 ° C In the environment, wash away the auxiliary materials).
  • Figure 20 shows the experimental steps and experimental results of preparing a tubular three-dimensional construct using bio-brick, fibrinogen and thrombin; wherein Figure 20A shows the attachment/assembly of fibrinogen on the surface of the bio-brick; Figure 20B shows Yes, the annular auxiliary structure is constructed with an auxiliary material (optional step); FIG. 20C shows that the second reagent is dropped along the annular auxiliary structure to draw a circular pattern; FIG. 20D shows that the assembly unit is rounded. The ring pattern is placed to form a ring structure; FIG.
  • FIG. 20E shows that the ring pattern is drawn with the second reagent on the upper surface of the ring structure, and then the assembly unit is placed along the ring pattern (optionally, the step can be repeated once or Multiple times to construct a construct containing a multilayer structure;
  • Figure 20F shows the resulting tubular structure;
  • Figure 20G shows the removal of the auxiliary structure (optional step).
  • the tensile modulus of the obtained biological construct was tested on an electronic tensile tester (Model 5967, Instron) with reference to GB/T228.1-2010, with a load of 10 N and a tensile speed of 20 mm/min. The temperature was 25 ° C and the sample was kept wet during the test. The measurement results showed that the obtained tubular structure had a tensile modulus of 1.25 KPa.
  • Example 10-14 the artificial blood vessel precursor containing the bio-brick and the expanded polytetrafluoroethylene prepared in Example 2 was implanted into the rhesus monkey by using the rhesus monkey as an animal model (ie, used as a blood vessel plant). Inclusion) and evaluation of the condition after implantation.
  • Example 10-14 the steps of vascular anastomosis between the artificial vascular precursor and the rhesus monkey autologous abdominal aorta are referred to The procedure of Example 2 was carried out.
  • Eleven rhesus monkeys were numbered as NO.1-NO.11, and NO.11 was a control group.
  • the artificial blood vessel precursor was anastomosed with the autologous abdominal aorta of NO.1-NO.10 rhesus monkey.
  • vascular implants in each rhesus monkey were taken and tested according to the time after implantation as shown in the table below.
  • the tissue structure of the vascular implant was observed by HE staining, and the results of the test were shown in Fig. 22, and the scales in the figure were both 200 ⁇ m.
  • the results showed that after 4 hours of implantation, there were still gaps between the bio-bricks, which were not connected to each other independently; after 8 hours to 24 hours of implantation, the bio-bricks gradually merged into one another; as the implantation time increased, the bio-brick fusion
  • the artificial blood vessels gradually form a histological structure similar to that of normal blood vessels.
  • Fig. 23 is a result of magnifying 100 times, and the scales in the figure are both 200 ⁇ m.
  • Fig. 24 shows the result of a magnification of 400 times, and the scales in the figure are both 50 ⁇ m.
  • the results showed that after 5 days of implantation, endothelial cells appeared in the luminal surface of the vascular implant and the blood; as the implantation time increased, the endothelial cells increased continuously, and at 28 days, a more complete endothelial cell similar to normal blood vessels was formed.
  • Floor shows that after 5 days of implantation, endothelial cells appeared in the luminal surface of the vascular implant and the blood; as the implantation time increased, the endothelial cells increased continuously, and at 28 days, a more complete endothelial cell similar to normal blood vessels was formed.
  • the artificial blood vessel precursor was anastomosed with the rhesus monkey autologous abdominal aorta.
  • Rhesus monkeys were divided into 4 groups, and the autologous blood vessel junctions of vascular implants and rhesus monkeys were taken at 7 days, 14 days, 21 days and 28 days after implantation.
  • the tissue structure of the junction was observed by HE staining, and the expression of CD31 and ⁇ -SMA was detected by immunohistochemical staining.
  • rhesus monkeys that were not implanted were used as a control group, and normal blood vessels were taken for detection.
  • Figure 26 shows the results of the test.
  • the first line of pictures is the result of HE staining, the scale in the figure is 200 ⁇ m; the second line is the result of CD31, the scale in the figure is 50 ⁇ m; the third line is the detection result of ⁇ -SMA, in the figure
  • the scale is 200 ⁇ m.
  • the thick arrows in the figure indicate autologous blood vessels, and the thin arrows indicate blood vessel implants.
  • the artificial blood vessel precursor was anastomosed with the rhesus monkey autologous abdominal aorta.
  • Rhesus monkeys were divided into 4 groups, and the vascular implants were taken at 5 days, 7 days, 21 days, and 28 days after implantation.
  • the vascular collagen was stained using the Sirius red staining method, and the results are shown in Fig. 27, and the scale in the figure was 100 ⁇ m. The results showed that after 5 days of implantation of the vascular implant, collagen expression began to appear; as the implantation time increased, the expressed collagen gradually increased and began to stratify, forming a collagen structure similar to normal blood vessels.
  • the artificial blood vessel precursor was anastomosed with the rhesus monkey autologous abdominal aorta.
  • the vascular implants were examined using ultrasound at 5, 18, and 61 days after surgery. The results are shown in Figure 28.
  • the first line of images is the result of the ultrasound test, and the second line is the color Doppler test result. .
  • the results showed that the blood vessels were vascularized and the blood flow was continuous, the inner surface of the lumen was smooth and no thrombosis, no abnormal hyperplasia, and there was no stenosis at the junction with the normal blood vessels.
  • the artificial blood vessel precursor was anastomosed with the rhesus monkey autologous abdominal aorta.
  • the test was performed using enhanced CT at 19 days and 62 days after implantation, and the results are shown in FIG. The results showed that the vascular implants had smooth blood flow and no blockage.
  • a commercially available expanded polytetrafluoroethylene artificial blood vessel (wall thickness: 0.56 mm, inner diameter: 8 mm) was cut into a rectangular shape having a curvature of 4 cm and a width of 1 cm and having a certain curvature to obtain a sheet-like solid support. As shown in Figure 30A;
  • the soaked bio-bricks are printed one by one on the surface of the medical glue, and the sheet-like solid support is spread to form a blood vessel patch precursor, as shown in Fig. 30B.
  • Example 3 a flat polycaprolactone sheet-like solid support having a thickness of 0.5 mm was prepared and cut into an approximately rectangular sheet shape of 3.5 cm in length and 1 cm in width, as shown in Fig. 30C;
  • the microcapsules containing the rhesus adipose-derived mesenchymal stem cells prepared in Example 5 were printed one by one on the surface of the medical glue, and covered with a sheet-like solid support to form a vascular patch precursor, such as Figure 30D is shown.
  • the vascular patch precursors prepared in Examples 15 and 16 were implanted in vivo using rhesus monkeys as animal models. After the vascular defect is created on the abdominal aorta of the rhesus monkey, the vascular patch precursor is trimmed into a suitable oblong shape according to the specific vascular defect, and the vascular patch precursor and the defect site are sutured.
  • Figure 31A shows the creation of a vascular defect on the abdominal aorta of the rhesus monkey
  • Figure 31B shows the suture of the vascular patch precursor with the defect site.
  • the thick arrow indicates the precursor of the blood vessel patch containing the biobrick prepared in Example 15
  • the thin arrow indicates the blood vessel patch precursor containing the microcapsule prepared in Example 16.
  • FIGS 32A and 32B show vascular tissue formed from a vascular patch precursor comprising a bio-brick and a vascular patch precursor comprising microcapsules, respectively. As shown in the figure, the bio-bricks or microcapsules in the patch are integrated to form a complete inner membrane.
  • the vascular tissues were subjected to immunohistochemical staining for CD31 and ⁇ -SMA, and the results are shown in FIG.
  • Figures 33A and 33B show the results of detection of vascular tissue formed from a vascular patch precursor containing biobricks. The results showed that bio-brick adipose-derived mesenchymal stem cells differentiated into endothelial cells (Fig. 33A) and smooth muscle cells (Fig. 33B) after 7 days of in vivo implantation.
  • Figures 33C and 33D show the results of detection of vascular tissue formed from a vascular patch precursor containing microcapsules. The results showed that after 7 days of in vivo implantation, the adipose-derived adipose-derived mesenchymal stem cells differentiated into endothelial cells (Fig. 33C) and smooth muscle cells (Fig. 33D).
  • Polylysine (Sigma, number average molecular weight Mn is 150,000-300,000) is dissolved in H-DMEM medium of pH 7.2 to obtain a polylysine solution having a concentration of 1 wt%. .
  • sodium alginate (Sigma) was dissolved in H-DMEM medium of pH 7.2 to obtain a sodium alginate solution having a concentration of 1 wt%.
  • Collagen dropwise addition formation of nuclear layer structure: 0.1 ⁇ L of the type I collagen solution prepared in step 2 is accurately aspirated by an electronic suction device capable of sucking and discharging the nano-upgraded liquid, and the U-bottom superhydrophobic prepared by the step 1 is dropped. In the well plate, droplets were formed and held at 37 ° C for 30 min at a constant temperature to form.
  • the optional electronic suction device is Eppendorf Xplorer 0.5-10uL or TransferpetteElectronic0.5-10uL, with a dispensing function of at least 0.1 ⁇ L per dispense; or SGE auto-injection 1 ⁇ L or 0.5 ⁇ L can achieve 10 ⁇ and 5 times of 0.1 ⁇ L liquid titration respectively; in particular, taper special needle can be used for titration to improve accuracy.
  • step 6 The product of step 6 was washed twice with H-DMEM medium.
  • Adding sodium alginate solution After replacing the tip, 0.5 ⁇ L of the polylysine solution prepared in the step 4 is accurately aspirated, and dropped into the surface of the shell layer formed in the step 6 in the center of the superhydrophobic orifice plate, and the reaction is carried out. 10 min to form the second layer of the bio-brick shell, obtaining a bio-brick with a shell of 2 layers; the prepared bio-brick is about 300 ⁇ m in diameter.
  • the prepared bio-bricks are placed in PBS to form a bio-brick suspension.
  • the elastic modulus of the bio-brick was tested using a Piuma Nanoindenter nanoindenter.
  • Figure 35 is a graph showing the stress and strain of the bio-brick of the present embodiment.
  • the effective Young's modulus value of the biobrick of this example was 24.77 kPa.
  • the collagen solution was prepared using bovine type I collagen as a carrier for the bio-ink.
  • Parameter settings in the computer workstation including: diameter, length, print order, print thickness, temperature of the spinner, nozzle temperature.
  • Fig. 36 schematically shows a 3D bioprinter used in the present embodiment.
  • the size-matched Gore expanded polytetrafluoroethylene artificial blood vessel is used as a tubular solid support, which is placed on the outer surface of the tubular biological construct, assembled, and the two are bonded together by ink B (medical glue).
  • An artificial blood vessel precursor (shown in Figure 38) is formed and removed from the rotating rod.
  • the distance between the two arterial clips is about 3 cm, and the abdominal aorta about 2 cm is removed from the middle;
  • Figure 39 shows the artificial blood vessel precursor after implantation.
  • the vascular implant was taken. Immunofluorescence was used to detect the formation of endothelial cells and smooth muscle cells in the implant.
  • Fluorescent labeling of vascular endothelial cells using green fluorescence showed that the vascular implant formed a complete endothelial cell layer.
  • the fluorescence micrograph is shown in Fig. 40, and the scale is 200 ⁇ m.
  • Fluorescent labeling of vascular smooth muscle cells using red fluorescence showed that the vascular implant formed a complete smooth muscle cell layer.
  • the fluorescence micrograph is shown in Fig. 41, and the scale is 200 ⁇ m.

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Abstract

一种涉及组织工程领域和3D打印领域的发明，具体涉及一种人工组织前体及制备其的方法。具体而言，涉及一种人工组织前体，其包含固体支持物和多个微囊，其中，至少一个微囊与固体支持物贴合，所述微囊包含细胞和包裹细胞的生物相容性材料；制备所述人工组织前体的方法；用于制备人工组织前体的试剂盒和套盒；由所述人工组织前体培养得到的人工组织，例如人工管腔；包含所述人工组织前体或人工管腔的管腔植入体或管腔模型；人工组织前体用于制备人工组织、管腔植入体或管腔模型的用途；以及，人工组织用于制备管腔植入体或管腔模型的用途。

Description

人工组织前体及制备其的方法 技术领域
本发明涉及组织工程领域和3D打印领域。具体而言，本发明涉及一种人工组织前体，其包含固体支持物和多个微囊，其中，至少一个微囊与固体支持物贴合，所述微囊包含细胞和包裹细胞的生物相容性材料；制备所述人工组织前体的方法；用于制备人工组织前体的试剂盒和套盒；由所述人工组织前体培养得到的人工组织，例如人工管腔；包含所述人工组织前体或人工管腔的管腔植入体或管腔模型；人工组织前体用于制备人工组织、管腔植入体或管腔模型的用途；以及，人工组织用于制备管腔植入体或管腔模型的用途。
背景技术
血管移植术和血管补片修补术可用于对狭窄、闭塞、扩张、损伤或畸形的血管进行替换、重建或修补。常见的血管移植物或血管补片的来源为患者自体的动脉或静脉，但是，在患者自体的脉管供给不足的情况下(例如患者患有脉管疾病或先前已实施过血管移植术)，需要使用人工血管(补片)或异源血管(补片)作为替代物。
人工血管的研制开始于20世纪初，用于制造人工血管的材料包括金属、玻璃、聚乙烯、硅橡胶等。大量动物实验发现，这些材料制备的人工血管在短期内会出现腔内血栓，因此无法用于临床。
1952年，Voorhees将用维尼纶制造的人工血管应用到动物试验中并获得成功(参见Voorhees AB Jr,Jaretzki A 3rd,Blakemore AH.The use of tubes constructed from Vinyon“N”cloth in bridging arterial defects.J.Annals of Surgery,1952,135(3):332-336)。20世纪50-70年代，管壁带有网孔的人工血管陆续出现，采用的材料包括涤纶、真丝、膨体聚四氟乙烯等。但是，单纯通过材料的改进还无法解决由血栓和新生内膜增厚所引起的血管再狭窄或堵塞的问题。研究人员进一步尝试了优化人工血管的材料，包括：从1980年开始对人工血管内表面添加材料涂层，如碳涂层、纳米颗粒涂层、蛋白涂层等；从1982年开始使用复合型材料制备人工血管；从1984年开始对人工血管内表面进行材料的改性，如材料内添加肝素或尿激酶等抗凝剂，对内壁材料磺酸化、等离子体化；从1992年开始研发新型生物相容性抗凝材料，如聚氨酯；从1998年开始使用天然生物材料，如脱细胞血管基质材料支 架。这些方法在一定程度上确实对人工血管的性能有所改善，但这些人工血管植入体内后，依然会出现血栓和再狭窄的情况，无法达到与正常血管一致的功能。相关文献也报道了这些现象，例如MacLeod DC,Strauss BH,de Jong M,Escaned J,Umans VA,van Suylen RJ,Verkerk A,de Feyter PJ,Serruys PW.Proliferation and extracellular matrix synthesis of smooth muscle cells cultured from human coronary atherosclerotic and restenotic lesions.J Am Coll Cardiol.1994,23(1):59-65；以及Baumgartner I,Schainfeld R,GrazianiL.Management of Peripheral Vascular Disease.Annual Review of Medicine.2005,56(1):249-272。
正常的血管之所以不出现血栓，是因为其管腔内壁有一层内皮细胞层。因此，要想人工血管达到和正常血管一样的功能，最根本的解决办法就是使人工血管内皮化，也就是在人工血管的内壁表面形成一层完整的内皮细胞层。
1978年，Herring等人首先报道了使用自体内皮细胞种植技术对人工血管进行内皮化的实验研究(参见HerringM,GardnerA,GloverJ.A single-staged technique for seeding vascular grafts with autogenous endothelium.Surgery.1978,84(4):498-504)。该技术将自体来源的内皮细胞在体外培养扩增后，直接种植到人工血管内壁表面，希望经过体外短时间培养并植入体内后，这些内皮细胞能够形成完整的内皮细胞层。该研究为临床内皮细胞种植研究开创了先河。但是，内皮细胞体外培养生长缓慢，难以获得足够数量的细胞，且传代5-8代后迅速发生衰老，直接影响细胞种植后的功能体现。大量的体内外实验证明，这种将内皮细胞直接种植在人工血管内壁的方法，细胞抗血流冲击能力弱，容易脱落，无法形成内皮细胞层，与没有种植内皮细胞的人工血管相比，在抗血栓方面没有明显差异(参见HerringM,SmithJ,DalsingM,GloverJ,ComptonR,EtchbergerK,ZollingerT.Endothelial seeding of polytetrafluoroethylene femoral popliteal bypasses:the failure of low-density seeding to improve patency.J Vasc Surg,1994；20(4):650-655；以及JensenN,LindbladB,BergqvistD.Endothelialcell seeded dacron aortobifurcated grafts:platelet deposition and long-term follow-up.J Cardiovasc Surg(Torino),1994；35(5):425-429)。
1986年，研究人员开始了组织工程血管的研究，随后将干细胞作为种子细胞进行人工血管的构建。干细胞的来源主要包括胚胎干细胞、造血干细胞、间充质干细胞和诱导性多能干细胞(IPS)。将干细胞作为种子细胞构建人工血管的方法包括：制造血管支架材料，将干细胞在体外诱导为血管细胞(包括内皮细胞，平滑肌细胞和成纤维细胞)后，种植在 支架材料中，进行体内植入；或者直接将干细胞种植到血管支架材料中。后者的具体过程是：先制备血管支架，再将培养好的种子细胞以细胞悬液的方式滴加在血管支架表面，经过体外培养，使细胞粘附在支架表面，再植入体内。细胞要进入支架内部，需要经过迁移过程。因此，这两种方法制造的人工血管往往在支架表面有大量的细胞聚集，而内部只有很少量的细胞存在，或者细胞分布不均匀，从而使制造的人工血管难以形成完整的结构和功能。如果是多种细胞种植在支架中，更会出现细胞分布紊乱的现象。因此，使用这两种方法制造的人工血管，其内部血管细胞排列紊乱，难以形成完整的内皮细胞层以及结构规整的平滑肌细胞层，依然无法应用于临床。
发明概述
为了解决上述技术问题，本申请发明人开发了新的用于制备人工组织前体的方法。在某些优选的实施方案中，所述人工组织前体是人工管腔前体，其可以形成人工管腔(例如人工血管)。
在一个方面，本发明涉及一种人工组织前体，其包含固体支持物和多个微囊，其中，至少一个微囊与固体支持物贴合，所述微囊包含细胞和包裹细胞的生物相容性材料。
在某些优选的实施方案中，所述人工组织前体为管腔(例如循环管腔、消化管腔、呼吸管腔、泌尿管腔或生殖管腔)前体。
在某些优选的实施方案中，所述管腔为包含上皮细胞的管腔(例如血管，食管，气管，胃，胆管，肠道(包括小肠和大肠，例如十二指肠、空肠、回肠、盲肠(包括阑尾)、升结肠、结肠右曲、横结肠、结肠左曲、降结肠、乙状结肠、直肠)，输卵管，输精管，输尿管，膀胱或淋巴管)。
在某些优选的实施方案中，所述人工组织前体为管状或片状。
在某些优选的实施方案中，多个所述微囊构成一个或多个生物构建体。
在某些优选的实施方案中，一个或多个生物构建体与固体支持物贴合。
在一个方面，本发明涉及一种制备所述人工组织前体的方法，所述人工组织前体为管状；其包含以下步骤：
(I)制备管状(例如圆形管状；例如侧壁开口或不开口的管状)生物构建体；
(II)将管状生物构建体与管状固体支持物的内壁进行贴合。
在某些优选的实施方案中，制备管状生物构建体通过包含以下步骤的方法进行：
(1)提供一种或多种微囊，其全部或者部分表面附着有第一组分；优选地，所述第一组分包含于第一试剂中；
(2)在临时支持物表面的预设区域上涂布含有第二组分的第二试剂，其中，当所述第一组分与第二组分接触时，能够产生粘连效果，实现粘合作用；所述临时支持物为管状或柱状(例如侧壁不开口的圆形管状、侧壁开口的圆形管状、圆柱状或沿部分圆周设置的柱状)物，所述预设区域位于临时支持物的曲面；任选地，在涂布第二试剂之前，将衬底材料涂布在临时支持物表面的预设区域上；
(3)将步骤(1)中的全部或者部分表面附着有第一组分的微囊放置于涂布了第二试剂的预设区域，使所述微囊表面上的第一组分与预设区域上的第二组分接触，产生粘连效果，从而将所述微囊组装(粘合)成第一层结构，所述第一层结构为管状结构；
任选地，所述方法还包括以下步骤：
(4)在前一步骤产生的结构上涂布第二试剂；
(5)将步骤(1)中的全部或者部分表面附着有第一组分的微囊放置于前一步骤产生的结构上，使所述微囊表面上的第一组分与前一步骤产生的结构上的第二组分接触，产生粘连效果，从而在前一步骤产生的结构上，将所述微囊组装(粘合)成另一层结构；
(6)任选地，重复步骤(4)和(5)一次或多次；
从而得到管状生物构建体。
在一个方面，本发明涉及另一种制备所述人工组织前体的方法，所述人工组织前体为管状，其包含以下步骤：
(I)制备管状(例如圆形管状；例如侧壁开口或不开口的管状)生物构建体；
(II)将管状生物构建体与管状固体支持物的内壁进行贴合。
在某些优选的实施方案中，制备管状生物构建体通过包含以下步骤的方法进行：
(1)提供一种或多种微囊，其全部或者部分表面附着有第一组分；优选地，所述第一组分包含于第一试剂中；
(2)在临时支持物的表面上，用含有第二组分的第二试剂绘制预设的环形(例如圆环或扇环)图案，其中，当所述第一组分与第二组分接触时，能够产生粘连效果，实现 粘合作用；所述临时支持物具有至少一个平面，所述环形图案位于临时支持物的平面；
(3)将步骤(1)中的全部或者部分表面附着有第一组分的微囊放置于第二试剂绘制的预设的环形图案上，使所述微囊表面上的第一组分与所述环形图案上的第二组分接触，产生粘连效果，从而将所述微囊组装(粘合)成第一层结构，所述第一层结构为环形结构；
(4)在环形结构上涂布第二试剂；
(5)将步骤(1)中的全部或者部分表面附着有第一组分的微囊放置于前一步骤产生的结构上，使所述微囊表面上的第一组分与前一步骤产生的结构上的第二组分接触，产生粘连效果，从而在前一步骤产生的结构上，将所述微囊组装(粘合)成另一层结构；
(6)任选地，重复步骤(4)和(5)一次或多次；
从而得到管状生物构建体。
在一个方面，本发明涉及另一种制备所述人工组织前体的方法，所述人工组织前体为片状，其包含以下步骤：
(I)制备片状(例如平面的片状或弯曲的片状)生物构建体；
(II)将片状生物构建体与片状固体支持物进行贴合。
在某些优选的实施方案中，制备片状生物构建体通过包含以下步骤的方法进行：
(1)提供一种或多种微囊，其全部或者部分表面附着有第一组分；优选地，所述第一组分包含于第一试剂中；
(2)在临时支持物表面的预设区域上涂布含有第二组分的第二试剂，其中，当所述第一组分与第二组分接触时，能够产生粘连效果，实现粘合作用；所述临时支持物具有至少一个平面，所述预设的图案位于临时支持物的平面；
(3)将步骤(1)中的全部或者部分表面附着有第一组分的微囊放置于涂布了第二试剂的预设区域，使所述微囊表面上的第一组分与预设区域上的第二组分接触，产生粘连效果，从而将所述微囊组装(粘合)成第一层结构，所述第一层结构为平面的片状结构；
任选地，所述方法还包括以下步骤：
(4)在前一步骤产生的结构上涂布第二试剂；
(5)将步骤(1)中的全部或者部分表面附着有第一组分的微囊放置于前一步骤产 生的结构上，使所述微囊表面上的第一组分与前一步骤产生的结构上的第二组分接触，产生粘连效果，从而在前一步骤产生的结构上，将所述微囊组装(粘合)成另一层结构；
(6)任选地，重复步骤(4)和(5)一次或多次，得到平面的片状生物构建体；
任选地，所述方法还包括，对平面的片状生物构建体进行弯折，得到弯曲的片状生物构建体。
在一个方面，本发明涉及另一种制备所述人工组织前体的方法，所述人工组织前体为片状，其包含以下步骤：
(I)由上述制备片状生物构建体的方法，制备片状生物构建体；
(II)提供制备固体支持物的材料(例如生物相容性材料)，在片状生物构建体上，制备片状固体支持物。
在某些优选的实施方案中，通过3D打印或喷涂的方法制备片状固体支持物。
在一个方面，本发明涉及另一种制备所述人工组织前体的方法，所述人工组织前体为管状，其包含以下步骤：
(I)通过上述制备片状生物构建体的方法，制备片状生物构建体；
(II)将步骤(I)制得的片状生物构建体进行弯折，和/或将片状生物构建体的边缘进行粘合，得到管状生物构建体；
(III)将管状生物构建体与管状固体支持物的内壁进行贴合。
在一个方面，本发明涉及另一种制备所述人工组织前体的方法，所述人工组织前体为管状，其包含以下步骤：
(I)由上述任一项定义的制备管状生物构建体的方法，制备管状生物构建体；
或者，由如上定义的制备片状生物构建体的方法，制备片状生物构建体；之后，对制得的片状生物构建体进行弯折，和/或将片状生物构建体的边缘进行粘合，得到管状生物构建体；
(II)提供制备固体支持物的材料(例如生物相容性材料)，在管状生物构建体的外壁上，制备管状固体支持物。
在某些优选的实施方案中，通过3D打印或喷涂的方法制备管状固体支持物。
在一个方面，本发明涉及另一种制备所述人工组织前体的方法，其包含以下步骤：
(1)提供一种或多种微囊，其全部或者部分表面附着有第一组分；优选地，所述第一组分包含于第一试剂中；
(2)提供固体支持物，在所述固体支持物表面的预设区域上涂布含有第二组分的第二试剂，其中，当所述第一组分与第二组分接触时，能够产生粘连效果，实现粘合作用；
(3)将步骤(1)中的全部或者部分表面附着有第一组分的微囊放置于涂布了第二试剂的预设区域，使所述微囊表面上的第一组分与预设区域上的第二组分接触，产生粘连效果，从而在固体支持物表面，将所述微囊组装(粘合)成第一层结构；
任选地，所述方法还包括以下步骤：
(4)在前一步骤产生的结构上涂布第二试剂；
(5)将步骤(1)中的全部或者部分表面附着有第一组分的微囊放置于前一步骤产生的结构上，使所述微囊表面上的第一组分与前一步骤产生的结构上的第二组分接触，产生粘连效果，从而在前一步骤产生的结构上，将所述微囊组装(粘合)成另一层结构；
(6)任选地，重复步骤(4)和(5)一次或多次；
从而得到人工组织前体。
在某些优选的实施方案中，所述固体支持物为管状或片状。
在某些优选的实施方案中，所述固体支持物为管状固体支持物，所述预设区域位于固体支持物的内壁。
在本发明的制备人工组织前体的方法中，优选地，所述第一组分和/或第二组分为生物相容性材料，为来源于生物的材料，和/或为生物可降解材料。
在某些优选的实施方案中，所述第一组分与第二组分接触而产生的粘连效果可用于将两个微囊粘合在一起，形成生物构建体；并且由此所获得的生物构建体的拉伸模量不低于10Pa，例如不低于20Pa，不低于30Pa，不低于40Pa，不低于50Pa，不低于60Pa，不低于70Pa，不低于80Pa，不低于90Pa，不低于100Pa，不低于200Pa，不低于300Pa，不低于400Pa，不低于500Pa，不低于600Pa，不低于700Pa，不低于800Pa，不低于900Pa或不低于1000Pa。
在某些优选的实施方案中，所述第一组分和第二组分是选自下列的组合：
(1)纤维蛋白原和凝血酶；
(2)海藻酸盐(例如海藻酸钠)或氧化的海藻酸盐(例如氧化的海藻酸钠)和含有Ca2+、Mg2+、Ba2+、Sr2+、或Fe3+的物质(例如含有Ca2+、Mg2+、Ba2+、Sr2+、或Fe3+的溶液或半固体(例如凝胶))；
(3)含马来酰亚胺基团的分子(例如含有马来酰亚胺基团的聚乙二醇(MAL-PEG))和含自由巯基的分子(例如含有自由巯基的聚乙二醇(PEG-SH))；
(4)含有阴离子的物质(例如含有阴离子的溶液或半固体(例如凝胶))和α-氰基丙烯酸酯(例如α-氰基丙烯酸甲酯，α-氰基丙烯酸乙酯，α-氰基丙烯酸异丁酯，α-氰基丙烯酸异己酯，α-氰基丙烯酸正辛酯)；
(5)纤维蛋白原和α-氰基丙烯酸酯(例如α-氰基丙烯酸甲酯，α-氰基丙烯酸乙酯，α-氰基丙烯酸异丁酯，α-氰基丙烯酸异己酯，α-氰基丙烯酸正辛酯)；
(6)血清白蛋白(例如，牛血清白蛋白)和戊二醛；
(7)含氨基甲酸酯基团(-NHCOO-)或异氰酸酯基团(-NCO)的分子(例如含氨基甲酸酯基团的聚乙二醇或含异氰酸酯基团的聚乙二醇)和含活泼氢的分子(例如含羧基的聚乙二醇)；
(8)明胶-间苯二酚和戊二醛；
(9)碳化二亚胺交联明胶和聚L-谷氨酸(PLGA)；和
(10)胺基化明胶和醛基化多糖。
在一个方面，本发明涉及一种生物构建体，由如上任一项定义的制备生物构建体的方法得到。
在一个方面，本发明涉及一种用于制备人工组织前体的试剂盒，所述试剂盒包含微囊，以及彼此分离的第一试剂和第二试剂，其中，所述微囊包含细胞和包裹细胞的生物相容性材料，所述第一试剂含有第一组分，所述第二试剂含有第二组分，并且当所述第一组分与第二组分接触时，能够产生粘连效果，实现粘合作用。
在某些优选的实施方案中，所述第一组分与第二组分接触而产生的粘连效果可用于将两个微囊粘合在一起，形成生物构建体；并且由此所获得的生物构建体的拉伸模量不低于10Pa(例如不低于100Pa)。
在某些优选的实施方案中，所述第一组分和/或第二组分为生物相容性材料；为来源于生物的材料；和/或，为生物可降解材料。
在某些优选的实施方案中，所述第一组分和第二组分是选自下列的组合：
(1)纤维蛋白原和凝血酶；
(2)海藻酸盐(例如海藻酸钠)或氧化的海藻酸盐(例如氧化的海藻酸钠)和含有Ca2+、Mg2+、Ba2+、Sr2+、或Fe3+的物质(例如含有Ca2+、Mg2+、Ba2+、Sr2+、或Fe3+的溶液或半固体(例如凝胶))；
(3)含马来酰亚胺基团的分子(例如含有马来酰亚胺基团的聚乙二醇(MAL-PEG))和含自由巯基的分子(例如含有自由巯基的聚乙二醇(PEG-SH))；
(4)含有阴离子的物质(例如含有阴离子的溶液或半固体(例如凝胶))和α-氰基丙烯酸酯(例如α-氰基丙烯酸甲酯，α-氰基丙烯酸乙酯，α-氰基丙烯酸异丁酯，α-氰基丙烯酸异己酯，α-氰基丙烯酸正辛酯)；
(5)纤维蛋白原和α-氰基丙烯酸酯(例如α-氰基丙烯酸甲酯，α-氰基丙烯酸乙酯，α-氰基丙烯酸异丁酯，α-氰基丙烯酸异己酯，α-氰基丙烯酸正辛酯)；
(6)血清白蛋白(例如，牛血清白蛋白)和戊二醛；
(7)含氨基甲酸酯基团(-NHCOO-)或异氰酸酯基团(-NCO)的分子(例如含氨基甲酸酯基团的聚乙二醇或含异氰酸酯基团的聚乙二醇)和含活泼氢的分子(例如含羧基的聚乙二醇)；
(8)明胶-间苯二酚和戊二醛；
(9)碳化二亚胺交联明胶和聚L-谷氨酸(PLGA)；和
(10)胺基化明胶和醛基化多糖。
在一个方面，本申请涉及一种用于制备人工组织前体的套盒，其包含一个或多个本发明的试剂盒。
在一个方面，本申请涉及一种人工组织，所述人工组织通过对本发明的人工组织前体进行培养(例如体外培养或体内培养)得到。
在某些优选的实施方案中，所述人工组织为人工管腔。
在某些优选的实施方案中，所述管腔为包含上皮细胞的管腔(例如血管，食管，气 管，胃，胆管，肠道(包括小肠和大肠，例如十二指肠、空肠、回肠、盲肠(包括阑尾)、升结肠、结肠右曲、横结肠、结肠左曲、降结肠、乙状结肠、直肠)，输卵管，输精管，输尿管，膀胱或淋巴管)。
在某些优选的实施方案中，所述人工管腔为管状人工管腔或片状人工管腔。
在某些优选的实施方案中，所述人工管腔为人工血管或血管补片。
在一个方面，本申请涉及一种管腔植入体，所述管腔植入体包含本发明的人工组织前体(例如管状人工组织前体或片状人工组织前体)或人工管腔。
在某些优选的实施方案中，所述管腔植入体包含一个或多个本发明的人工组织前体(例如管状人工组织前体或片状人工组织前体)，或一个或多个本发明的人工管腔(例如管状人工管腔或片状人工管腔)。
在某些优选的实施方案中，所述管腔植入体为线性的管状结构，或者为枝化的管状结构。
在某些优选的实施方案中，所述管腔植入体为X形管状、Y形管状或T形管状。
在某些优选的实施方案中，所述管腔为包含上皮细胞的管腔，例如血管。
在某些优选的实施方案中，所述管腔植入体为血管植入体，其包含本发明的人工血管或血管补片。
在一个方面，本申请涉及一种管腔(例如血管)模型，所述管腔模型包含本发明的人工管腔(例如人工血管)。
在某些优选的实施方案中，所述管腔模型包含一个或多个本发明的人工管腔(例如人工血管)。
在一个方面，本申请涉及本发明的人工组织前体用于制备人工组织、管腔植入体或管腔模型的用途。
在一个方面，本申请涉及本发明的人工组织用于制备管腔植入体或管腔模型的用途。
下面将结合附图和发明详述来对本发明的实施方案进行详细阐释。但是，本领域技 术人员将理解，下列附图和发明详述仅用于说明本发明，而不是对本发明的范围的限定。根据附图和发明详述的详细公开内容，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。
附图说明
图1示意性描述了本发明的微囊的示例性结构，其包括：细胞和包裹细胞的生物相容性材料；在优选的实施方案中，细胞可均匀分散于微囊中，或者可以聚集在一起，位于微囊内部。
图2A-E示意性描述了本发明的生物砖的示例性结构，其包括：细胞，包裹细胞的核层和封装核层的壳层。
特别地，图2A示意性描述了本发明生物砖的一种结构，其包括一个核层和一个壳层，其中，所述核层包裹有细胞，并且所述壳层位于核层外侧，且封装核层。
图2B示意性描述了本发明生物砖的一种结构，其由内到外依次包含：包裹细胞的核层、封装所述核层的第一壳层、和围绕第一壳层的第二壳层。
图2C示意性描述了本发明生物砖的一种结构，其由内到外依次包含：包裹细胞的第一核层、位于第一核层外侧的包裹细胞的第二核层、和封装所述第一核层和第二核层的第一壳层。
图2D示意性描述了本发明生物砖的一种结构，其由内到外依次包含：包裹细胞的第一核层、位于第一核层外侧的包裹细胞的第二核层、封装所述第一核层和第二核层的第一壳层、和围绕第一壳层的第二壳层。
图2E示意性描述了本发明生物砖的一种结构，其由内到外依次包含：包裹细胞的第一核层、封装所述第一核层的第一壳层、包裹细胞的第二核层、和封装所述第二核层的第二壳层。
图3A-E示例性描述了本发明的包含多个管状生物构建体的管状人工组织前体的结构。
图3A为一种管状人工组织前体的侧视图，所述人工组织前体包含管状固体支持物和多个侧壁不开口的管状生物构建体，所述多个侧壁不开口的管状生物构建体在所述管状固体支持物的内部，并沿管状固体支持物的轴向排列，并且所述多个侧壁不开口的管状生物构建体的外壁均与所述管状固体支持物的内壁贴合。
图3B为一种管状人工组织前体的俯视图，所述人工组织前体包含管状固体支持物和多个侧壁不开口的管状生物构建体，所述多个侧壁不开口的管状生物构建体在所述管状固体支持物的内部，与管状固体支持物同轴设置，并且仅最外侧的侧壁不开口的管状生物构建体的外壁与所述管状固体支持物的内壁贴合。
图3C为一种管状人工组织前体的侧视图，所述人工组织前体包含管状固体支持物和多个侧壁开口的管状生物构建体，所述多个侧壁开口的管状生物构建体在所述管状固体支持物的内部，并沿管状固体支持物的轴向排列，并且所述多个侧壁开口的管状生物构建体的外壁均与所述管状固体支持物的内壁贴合。
图3D为一种管状人工组织前体的俯视图，所述人工组织前体包含管状固体支持物和多个侧壁开口的管状生物构建体，所述多个侧壁开口的管状生物构建体在所述管状固体支持物内部，与管状固体支持物同轴设置并沿径向排列，并且位于仅最外侧的侧壁开口的管状生物构建体的外壁与所述管状支持物的内壁贴合。
图3E为一种管状人工组织前体的俯视图，所述人工组织前体包含管状固体支持物和多个侧壁开口的管状生物构建体，所述多个侧壁开口的管状生物构建体在所述管状固体支持物内部，并与管状固体支持物同轴设置，并且所述多个侧壁开口的管状生物构建体的外壁均与所述管状固体支持物的内壁贴合。
图4A-E示例性描述了本发明的制备管状生物构建体的方法中，作为临时支持物的柱状物，柱状物的展开的侧面和侧面上的预设区域。
图4A示例性描述了本发明的制备管状生物构建体的方法中，作为临时支持物的圆柱状物。
图4B示例性描述了图4A的圆柱状物的展开的侧面和侧面上的预设区域，其中，所述预设区域为圆柱状物的整个侧面。
图4C示例性描述了图4A的圆柱状物的展开的侧面和侧面上的预设区域，其中，所述预设区域在展开的圆柱状物的侧面上为一矩形，并且所述预设区域沿圆柱状物的轴向贯通圆柱状物的侧面。
图4D示例性描述了图4A的圆柱状物的展开的侧面和侧面上的预设区域，其中，所述预设区域在展开的圆柱状物的侧面上为一矩形，并且所述预设区域沿圆柱状物的圆周方向贯通圆柱状物的侧面。
图4E示例性描述了图4A的圆柱状物的展开的侧面和侧面上的预设区域，其中，所 述预设区域在展开的圆柱状物侧面上为一矩形，并且未沿圆柱状物的圆周方向和轴向贯通圆柱状物的侧面。
图5A示例性描述了本发明的制备人工组织前体的方法2中，作为预设区域的圆环图案和扇环图案。
图5B示例性地显示了本发明的管腔植入体或管腔模型的形状。
图6为实施例1中，原代培养第四代的脂肪间充质干细胞的显微照片。从图中可见，细胞形态均一，生长状态良好。
图7为实施例1中，包含恒河猴脂肪间充质干细胞的生物砖的形态。
图8为实施例1中，激光共聚焦显微镜拍摄的包含恒河猴脂肪间充质干细胞的生物砖的照片，其中绿色荧光为微囊的壳，红色荧光为脂肪间充质干细胞。
图9为实施例2得到的人工血管前体的照片。
图10A-C为将实施例2制得的人工血管前体植入恒河猴，术后5天得到的人工血管。图10A为人工血管的整体形态。图10B为去除管状支持物得到的组织，图10C为将组织纵向剖开后的形态。从图可以看出，管状支持物的表面已经形成了内皮组织。
图11为实施例2中，人工血管组织的HE染色结果，图11A为正常血管，图11B为人工血管。如图所示，人工血管与正常血管有相似的细胞排列、内皮细胞层(细箭头所指)和平滑肌细胞层(粗箭头所指)。
图12为实施例2中，人工血管组织的α-SMA染色结果，α-SMA阳性细胞为平滑肌细胞。图12A为正常血管，图12B为人工血管。如图所示，构成人工血管的脂肪间充质干细胞向平滑肌细胞分化，并出现与正常血管相似的细胞形态、排列及方向性。
图13为实施例2中，人工血管组织的CD31染色结果，CD31阳性细胞为内皮细胞。图13A为正常血管，图13B为人工血管。如图所示，在与血液接触面，构成人工血管的脂肪间充质干细胞向内皮细胞分化，并出现与正常血管相似的细胞形态及排列。
图14显示了实施例3的人工血管的形态和血流方向。图14A为超声检测结果，从图中可以看出，人工血管的管腔通畅。图14B为彩色多普勒检测结果，结果显示，人工血管两侧血流方向一致，证明血管通畅。
图15A和15B为实施例3的人工血管的免疫组化法染色结果。图15A显示了α-SMA染色结果，如图中粗箭头所指，人工血管中，脂肪间充质干细胞向平滑肌细胞进行分化。图15B显示了CD31染色结果，如图中细箭头所指，人工血管血管中，脂肪间充质干细胞 向内皮细胞进行分化。图15C为天狼星红染色结果，如图所示，人工血管形成与正常血管相似的胶原结构。
图16A和16B为实施例4中，将人工血管前体植入恒河猴体内14天后取材，使用免疫组化染色进行检测的结果。
图16A为α-SMA染色结果，如图中粗箭头所指，人工血管中有脂肪间充质干细胞向平滑肌细胞进行分化。
图16B为CD31染色结果，如图中细箭头所指，人工血管中有脂肪间充质干细胞向内皮细胞进行分化。
图17A-17C分别为实施例6中，将人工血管前体植入恒河猴体内14天后，得到的人工血管的剖面图，和对人工血管进行免疫组化染色的检测结果。
图17A为将人工血管前体植入恒河猴体内14天后，得到的人工血管的剖面图。
图17B为α-SMA染色结果，如图中粗箭头所指，人工血管中有脂肪间充质干细胞向平滑肌细胞进行分化。
图17C为CD31染色结果，如图中细箭头所指，人工血管中有脂肪间充质干细胞向内皮细胞进行分化。
图18显示了实施例7的制备人工血管前体的过程。图18A：将生物砖打印在旋转杆上，形成管状生物构建体；图18B和18C：将人工血管由左至右，套在管状生物构建体上；图18D：将人工血管与管状生物构建体粘合在一起，形成人工血管前体。
图19描述了实施例8中，使用可降解的聚乳酸管状支持物，以及实施例1制得的生物砖，制备人工血管前体的过程。
图19A和19B显示了以聚乳酸为基本材料，采用电纺丝的方法制造出的管状固体支持物。
图19C显示了以下操作：将聚乳酸管状支持物剪开，然后在管状支持物的一面滴加医用胶，另一面对应位置放置生物砖。
图19D显示了，医用胶可以渗透通过管壁，使得生物砖与内壁相粘合在一起，得到人工血管前体。
上述操作仅为了方便观察和拍照。在实际制备中，是在聚乳酸管状固体支持物的外壁滴加医用胶，使医用胶渗透至内壁。由于医用胶可以渗透过电纺聚乳酸管壁，从而将生物砖固定。上述结果一方面说明，聚乳酸可以作为固体支持物使用，另一方面说明，电纺得 到的固体支持物，由于其孔结构具有渗透性，医用胶可以渗透通过管壁，可以在固体支持物的一面滴加医用胶，在另一面放置生物砖，从而固定生物砖，得到人工组织前体。图20展示了实施例9中，利用生物砖、纤维蛋白原和凝血酶来制备管状三维构建体的实验步骤和实验结果；其中，图20A显示的是，在生物砖表面附着/组装纤维蛋白原；图20B显示的是，用辅助材料构建环状辅助结构(任选的步骤)；图20C显示的是，沿环状辅助结构滴加第二试剂，绘制圆环图案；图20D显示的是，将组装单元沿圆环图案放置形成环状结构；图20E显示的是，在环状结构的上表面用第二试剂绘制圆环图案，然后将组装单元沿圆环图案放置(任选地，可以重复该步骤一次或多次，以构建含有多层结构的构建体)；图20F显示的是，构建得到的管状结构；图20G显示的是，去除辅助结构(任选的步骤)。实验结果显示，本发明的方法可用于快速、多向、精确地构建管状三维构建体。
图21展示了实施例9中刚刚制备获得的管状结构(图21A)以及经培养的管状结构(图21B)的显微镜观察结果。结果显示，在刚刚制备获得的管状结构中，生物砖尚未发生相互融合，细胞在各自的生物砖中均匀分布；而在经培养的管状结构中，生物砖已完全相互融合，紧密连接在一起，形成了完整的生物构建体。
图22显示了实施例10中，使用HE染色法对血管植入体的组织结构进行观察和检测的结果，图中的标尺均为200μm。结果显示：植入4小时后，生物砖之间仍存在缝隙，彼此独立不相连；植入8小时-24小时h后，生物砖逐渐相互融合成一体；随着植入时间的增加，生物砖融合成的人工血管逐渐形成与正常血管相类似的组织学结构。
图23和图24显示了实施例10中，使用免疫组化染色方法对血管植入体中CD31的表达进行检测的结果。图23为放大100倍的结果，图中的标尺均为200μm。图24为放大400倍的结果，图中的标尺均为50μm。结果显示：植入5天后，血管植入体与血液接触的腔面出现内皮细胞；随着植入时间的增加，内皮细胞不断增加，在28天时，形成与正常血管相似的较完整的内皮细胞层。
图25显示了实施例10中，使用免疫组化染色方法对血管植入体中，α-SMA的表达进行检测的结果，图中的标尺均为200μm。结果显示：植入8小时后，生物砖内包裹的脂肪间充质干细胞开始向平滑肌细胞进行分化并表达α-SMA；植入3天后，脂肪间充质干细胞的形态逐渐向平滑肌细胞转变且α-SMA的表达进一步增加；随着植入时间的增加，平滑肌细胞量逐渐增加并形成与正常血管相似的平滑肌细胞层。
图26显示了实施例11中，使用HE染色法对恒河猴自体血管与血管植入体连接处的 组织结构进行观察的结果，以及使用免疫组织化学染色法分别对CD31和α-SMA的表达进行检测的结果。
第一行图片为HE染色法的检测结果，图中的标尺为200μm；第二行图片为CD31的检测结果，图中的标尺为50μm；第三行图片为α-SMA的检测结果，图中的标尺为200μm。图中的粗箭头表示自体血管，细箭头表示血管植入体。
结果显示：在植入7天时，血管植入体与恒河猴自体血管相互连接，但彼此组织结构有显著差别，内皮细胞层连续但不完整，平滑肌细胞层不连续；随着植入时间的增加，血管植入体与恒河猴自体血管不断相互融合；在植入28天时，血管植入体与恒河猴自体血管融合成一体，内皮细胞层及平滑肌细胞层连续完整，形成与正常血管相似的组织结构。
图27为实施例12中，使用天狼猩红染色法对血管胶原进行染色的结果，图中的标尺为100μm。结果显示：血管植入体植入5天后，开始出现胶原的表达；随着植入时间的增加，表达的胶原逐渐增多并开始分层，形成与正常血管相似的胶原结构。
图28显示了实施例13中，对血管植入体进行超声检测的结果(第一行图片)和彩色多普勒检测结果(第二行图片)。结果显示，血管植入体内血管通畅，血流连续，管腔内表面光滑无血栓形成，无异常增生，与正常血管连接处无狭窄。
图29显示了实施例14中，对血管植入体进行增强CT的结果。结果显示，血管植入体血流通畅，无堵塞。
图30A和30B分别显示了实施例15中，膨体聚四氟乙烯片状固体支持物，以及用生物砖通过3D打印在其上形成的血管补片前体。
图30C和30D分别显示了实施例16中，聚己内酯片状固体支持物，以及用微囊通过3D打印在其上形成的血管补片前体。
图31A和31B分别显示了实施例17中，在恒河猴腹主动脉上制造血管缺损，以及将血管补片前体与缺损部位进行缝合。图31B图中，粗箭头所指为实施例15制得的包含生物砖的血管补片前体，细箭头所指为实施例16制得的包含微囊的血管补片前体。
图32A和32B分别显示了实施例17中，植入7天后，由包含生物砖的血管补片前体和包含微囊的血管补片前体形成的血管组织。如图所示，补片内生物砖或微囊融合成一体，形成完整的内膜。
图33显示了实施例17中，对血管组织进行CD31和α-SMA免疫组化染色的结果。
图33A和33B显示了由包含生物砖的血管补片前体形成的血管组织的检测结果。结果 显示，体内植入7天后，生物砖内脂肪间充质干细胞向内皮细胞(图33A)和平滑肌细胞(图33B)分化。
图33C和33D显示了由包含微囊的血管补片前体形成的血管组织的检测结果。结果显示，体内植入7天后，微囊内脂肪间充质干细胞向内皮细胞(图33C)和平滑肌细胞(图33D)分化。
图34显示了实施例18中，生物砖在进行弹性模量测试时的状态。
图35为实施例18制得的生物砖的应力应变曲线，生物砖的有效杨氏模量数值为24.77kPa。
图36示意性地展示了实施例19所使用的3D生物打印机的主要结构。
图37显示了实施例19中，在旋生仪的旋转杆上形成的管状生物构建体，其长度为20mm，厚度约1mm。
图38显示了实施例19制得的人工血管前体。
图39显示了实施例19中，植入恒河猴体内的人工血管前体。
图40为实施例19中血管植入体的荧光显微照片，其中，使用绿色荧光对血管内皮细胞进行荧光标记，图中标尺为200μm。如图所示，血管植入体形成了完整的内皮细胞层。
图41为实施例19中血管植入体的荧光显微照片，其中，使用红色荧光对血管平滑肌细胞进行荧光标记，图中标尺为200μm。如图所示，血管植入体形成了完整的平滑肌细胞层。
发明详述
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本说明书和所附权利要求书中使用的，单数形式“一个”、“一种”和“该/所述”包括复数的指示物，除非上下文另有明确规定。此外，本文中任何提及的“或”意在包括“和/或”，除非另有说明。
如本文中所使用的，术语“微囊”是指，含有细胞和生物相容性材料的微结构(例如，微米级至毫米级的结构)，其中，细胞被包裹在所述生物相容性材料内。本发明的微囊 在生理环境下(例如4-37℃，例如pH在6-8之间，例如在生理环境的流体剪切力下)具有稳定的结构。在某些优选的实施方案中，微囊具有在吸取或挤压中不会造成微囊破碎的力学强度。
如本文中使用的，术语“组织”是指由形态相同或类似、机能相同的细胞群构成的细胞集合体，并且通常还包含非细胞形态的物质(称为细胞间质，例如基质、纤维等)。组织可包括一种或多种细胞。
如本文中使用的，术语“器官”是指由不同的细胞和组织构成的、用于实现某一或某些特定功能的结构。器官可包括一种或多种组织。
如本文中使用的，术语“人工组织”是指，不是通过天然组织生成或发育过程而形成的组织。人工组织可以是人为制造的组织，例如是对人工组织前体进行培养得到的组织。
如本文中使用的，术语“人工组织前体”是指包含固体支持物以及多个本发明的微囊的物体，其中，至少一个微囊与固体支持物贴合。在某些实施方案中，人工组织前体包含固体支持物以及由微囊构建的生物构建体。在某些实施方案中，本发明的人工组织前体在培养、诱导等操作步骤后，能够形成人工组织。
在本发明中，术语“生物构建体”是指使用本发明的微囊构建的物体，其可以具有二维或三维的结构，可以用于制备人工组织前体。
如本文中使用的，术语“贴合”是指不发生相对位移。在某些实施方案中，微囊或生物构建体与固体支持物贴合，是指微囊或生物构建体结合在固体支持物上。
如本文中使用的，术语“固体支持物”是指本发明的人工组织前体中，与微囊或由微囊构成的生物构建体贴合的，具有一定形状的物体。固体支持物能够提供相应的区域，使生物构建体完全贴合在其上。
如本文中使用的，术语“管腔”是指形状为管状、具有中空内腔的器官，例如循环管腔、消化管腔、呼吸管腔、泌尿管腔或生殖管腔，例如血管，食管，气管，胃，胆管，肠道(包括小肠和大肠，例如十二指肠、空肠、回肠、盲肠(包括阑尾)、升结肠、结肠右曲、横结肠、结肠左曲、降结肠、乙状结肠、直肠)，输卵管，输精管，输尿管，膀胱或淋巴管)。
如本文中使用的，术语“人工管腔”包括不是通过天然组织生成或发育过程而形成的管腔，也包括能够与天然组织共同形成管腔的片状人工组织。人工管腔可以是对本发明的人工组织前体进行培养得到的。
如本文中使用的，术语“人工血管”是指人为制造的血管替代物，其通常是管状。在某些实施方案中，人工血管用于对狭窄、闭塞、扩张、损伤或畸形的血管进行重建或修补。在某些实施方案中，人工血管是对本发明的管状人工组织前体进行培养(例如体外培养，或植入体内培养)得到的。
如本文中使用的，术语“血管补片”是指用于对破损血管进行修补的物体，其通常是片状。血管补片可以用来修补由于血管瘤、血管狭窄等原因造成的血管的瘘口，在主动脉等大血管上的运用较多。通常，血管补片要求易于缝合和止血。血管补片可适用于血管壁有缺损，但无需整个血管切除的患者。在某些实施方案中，血管补片是对本发明的片状人工组织前体进行培养(例如体外培养，或植入体内培养)得到的。
如本文中使用的，术语“管腔植入体”是指可以植入受试者体内，用于对受试者的管腔进行替换、重建或修补的物体，其包含一个或多个本发明的人工组织前体(例如管状人工组织前体或片状人工组织前体)或一个或多个人工管腔。在某些实施方案中，本发明的管腔植入体包括多个管状人工组织前体(或人工管腔)，这些管状人工组织前体(或人工管腔)之间流体连通。本发明的管腔植入体可以是线性的管状结构，也可以是枝化的管状结构，例如为X形管状、Y形管状或T形管状。在某些实施方案中，所述管腔植入体为血管植入体。在某些实施方案中，管腔植入体还包含药物活性成分、感应装置和/或调节装置。如本文中所使用的，术语“力学保护”是指，微囊具有一定的力学强度(例如，在吸取或挤压中不会造成微囊破碎的力学强度)，从而能够减少或避免其内封装的细胞遭受外界的机械损伤/力学损伤。此外，当微囊用于制备用于管腔移植或修补的人工组织前体时，微囊可以使其包裹的细胞不被管腔内的流体冲走，有利于人工组织前体向正常组织的转化。
如本文中所使用的，术语“力学保护”是指，微囊(例如生物砖)可以保护其内包裹的细胞在操作中(例如3D生物打印过程中)，不受到机械力的损伤(包括剪切力和挤压力)。使用微囊构建人工组织前体(例如人工血管前体)，在植入体内后，可以保护微囊内包裹的细胞不被流动的体液(例如血流)冲走，有利于人工组织向正常组织的转化。
如本文中所使用的，术语“生物相容性材料”是指这样的材料，其(以及其降解产物)对于细胞是无毒性的，并且在植入宿主(例如人体)后与宿主相容，不会造成显著的或者严重的副作用，例如，不会对宿主(例如人体组织)造成毒害作用，不会引起宿主的免疫排斥反应、过敏反应或炎症反应等等。
如本文中所使用的，术语“生物可降解材料”是指这样的材料，其能够被细胞或生物体降解和吸收，并且其降解产物是生物相容性的。此类材料可以是天然来源的(例如来源于动植物)，也可以是人工合成的。
如本文中所使用的，术语“生物材料(bio-material)”是指能够用于人体组织和器官的诊断、修复或增进其功能的天然或人造材料，其可以用于取代、修复活组织，能够执行、增进或替换组织因疾病、损伤等失去的某种功能。生物材料主要包括金属材料(例如碱金属及其合金等)、无机材料(例如生物活性陶瓷，羟基磷灰石等)和有机材料三大类，有机材料主要包含高分子材料。根据材料的用途，生物材料又可以分为生物惰性(bioinert)、生物活性(bioactive)或生物可降解(biodegradable)材料。
如本文中使用的，“粘度”是指流体粘滞性的一种量度，是流体流动力对其内部摩擦现象的一种表示。将两块面积为1㎡的板浸于液体中,两板距离为1米,若在某一块板上加1N的切应力,使两板之间的相对速率为1m/s,则此液体的粘度为1Pas。
如本文中使用的，术语“生物打印”是指：利用生物材料(包括但不限于，生物分子例如蛋白质，脂质，核酸和代谢产物；细胞例如细胞溶液、含细胞的凝胶、细胞悬浮液、细胞浓缩物、多细胞聚集体和多细胞体；亚细胞结构例如细胞器和细胞膜；与生物分子相关的分子例如合成的生物分子或生物分子的类似物)的打印。如本文中使用的，术语“打印”是指，按照预定的模式沉积材料的过程。在某些优选的实施方案中，通过挤出式打印法或模块化打印法来对微囊进行打印。优选地，使用模块化打印法来对微囊进行打印。如本文中所使用的，“模块化打印法”是指，通过吸取/抓取模块(例如本发明的微囊，例如生物砖)，并将其精确定位/排布来进行打印的方法。由于本发明所使用的微囊包含细胞，因此，在本文中，此类模块化打印法也被称为“模块化生物打印法”。在本发明中，生物打印优选地通过与自动的或半自动的、计算机辅助的三维原型装置(例如生物打印机)相匹配的方法来实现。然而，在本发明中，“打印”(例如生物打印)可通过各种方法来进行，包括但不限于，使用打印机(例如3D打印机或生物打印机)进行打印；使用自动化或非自动化机械过程(而非打印机)进行打印；通过手工放置或手工沉积(例如使用移液器)进行打印。
如本文中使用的，“海藻酸”是指一类由褐藻提取的多糖,其为β-1,4-D-甘露糖醛酸(M单元)和α-1,4-L-古洛糖醛酸(G单元)的无规嵌段共聚物。通常，海藻酸中的M和G单元以M-M，G-G或M-G的组合方式通过1,4糖苷键相连成为嵌段共聚物。海藻酸的实验 式为(C6H8O6)n，其分子量通常为4kDa-1500kDa。如本文中使用的，“海藻酸盐”是指由海藻酸形成的盐，包括但不限于，海藻酸钠，海藻酸钙，海藻酸锶，海藻酸钡等。
如本文中使用的，“氧化的海藻酸盐”是指，对海藻酸盐(例如海藻酸钠)进行氧化反应后形成的产物。通常情况下，氧化反应将使得海藻酸盐(例如海藻酸钠)中的部分糖醛酸单元的羟基被氧化为醛基。
如本文中使用的，“氧化度”是指被氧化的糖醛酸单元占海藻酸或海藻酸盐的总糖醛酸单元的摩尔分数。
如本文中使用的，“粘性剂”是指用于调节液体或半固体(例如凝胶)的粘度的试剂。如本文中所描述的，本发明的第二试剂优选地具有适于绘制图案或进行涂布的粘度。因此，在某些优选实施方案中，可通过使用粘性剂来方便地调节第二试剂的粘度。
如本文中使用的，“生物砖”是指一种可用于生物打印等用途的基础单元，其包括：细胞，包裹细胞的核层，和，封装核层的壳层，其中，所述核层和壳层各自独立地由生物可降解材料制成。在本发明的某些优选实施方案中，所述核层和壳层中的生物可降解材料能够减少或避免生物砖内的细胞在操作过程中遭受机械损伤，并且能够提供物质(例如营养物质，细胞外基质，细胞因子，药物活性成分等)的可控释放，以促进细胞活性和功能(增殖、分化、迁移、分泌或新陈代谢)或维持细胞的干性。在本发明的某些优选实施方案中，所述生物砖或生物砖的壳层具有一定的力学强度，从而能够实现立体堆积。在本发明中，特别优选地，生物砖及其壳层具有适当的力学保护性能(例如，具有合适的硬度和/或弹性模量)。在某些优选的实施方案中，所述壳层也能够为细胞的生命活动提供微环境，例如营养物质。
如本文中使用的，“生物墨汁”是指，包含一种或多种本发明的微囊(例如生物砖)的液体，半固体(例如凝胶)或固体组合物。例如，本发明的生物墨汁可以是包含微囊(例如生物砖)的溶液，悬浮液，凝胶，或浓缩物。在本发明中，生物墨汁能够被用于生物打印，以产生特定的几何形状；并且优选地，所产生的几何形状能够进一步堆叠，从而形成具有特定形状和结构的生物构建体。此外，在生物打印之前，期间和/或之后，生物墨汁中的微囊(例如生物砖)内的细胞能够进行各种期望的生命活动。在优选的实施方案中，微囊(例如生物砖)内的细胞在生物打印之前处于休眠状态，而在生物打印之后进行生长和增殖，从而形成稳固的生物构建体。在优选的实施方案中，生物墨汁是可挤出的组合物。如本文中所使用的，“可挤出的”是指，组合物能够通过被迫(例如在压力下) 穿过喷嘴或孔口而成形。
在一个方面，本申请涉及一种人工组织前体，其包含固体支持物和多个微囊，其中，至少一个微囊与固体支持物贴合，所述微囊包含细胞和包裹细胞的生物相容性材料。图1示意性描述了本发明的微囊的示例性结构。在某些优选的实施方案中，细胞可均匀分散于微囊中，或者可以聚集在一起，位于微囊内部。
在某些优选的实施方案中，所述人工组织前体为管腔(例如循环管腔、消化管腔、呼吸管腔、泌尿管腔或生殖管腔)前体。
在某些优选的实施方案中，所述管腔为包含上皮细胞的管腔(例如血管，食管，气管，胃，胆管，肠道(包括小肠和大肠，例如十二指肠、空肠、回肠、盲肠(包括阑尾)、升结肠、结肠右曲、横结肠、结肠左曲、降结肠、乙状结肠、直肠)，输卵管，输精管，输尿管，膀胱或淋巴管)。
在某些优选的实施方案中，所述人工组织前体为管状(例如侧壁开口或不开口的管状)。侧壁不开口的管状人工组织前体可用于对狭窄、闭塞、扩张、损伤或畸形的管腔进行替换，或用于构建管腔旁路(例如血管旁路)。侧壁开口的管状人工组织前体可用于对破损的管腔进行修补。
在某些优选的实施方案中，所述人工组织前体为片状(例如平面的片状或弯曲的片状)。片状人工组织前体可用于对破损的管腔进行修补。
在某些优选的实施方案中，多个所述微囊构成一个或多个生物构建体。
在某些优选的实施方案中，一个或多个生物构建体与固体支持物贴合。
在某些优选的实施方案中，所述微囊在生理环境下(例如4-37℃，例如pH在6-8之间，例如在生理环境的流体剪切力下)具有稳定的结构。在某些优选的实施方案中，所述微囊具有在吸取或挤压中不会造成微囊破碎的力学强度。在某些优选的实施方案中，所述微囊为包裹的细胞提供了力学保护。
在本发明的某些优选实施方案中，所述微囊能够减少或避免微囊内的细胞在操作(例如生物打印)过程中遭受机械损伤。在某些优选的实施方案中，本发明的微囊能够减少细胞在生物打印过程中受到的机械损伤。例如，在某些优选的实施方案中，在使用相同生物打印机和相同打印条件的情况下，与将细胞直接用于生物打印相比，本发明的微囊能 够减少细胞受到的机械损伤至少5％、10％、15％、20％、25％、30％、40％、50％、70％、80％、或90％。在某些优选的实施方案中，本发明的微囊能够在生物打印过程中保留微囊内的细胞的生物活性(例如，增殖、分化、迁移、分泌和/或新陈代谢)。在某些优选的实施方案中，微囊内至少80％、85％、87.5％、90％、92.5％、95％、或98％的细胞在生物打印后存活至少24小时。在某些优选的实施方案中，微囊内至少90％的细胞在生物打印后存活至少3小时、6小时、12小时、1天、2天、4天、或7天。在某些优选的实施方案中，微囊内至少80％、85％、87.5％、90％、92.5％、95％、或98％的细胞在生物打印24小时后能够增殖和/或分化。在某些优选的实施方案中，微囊内至少80％、85％、87.5％、90％、92.5％、95％、或98％的细胞在生物打印24小时后具有正常的新陈代谢。在某些优选的实施方案中，微囊内至少80％、85％、87.5％、90％、92.5％、95％、或98％的细胞在生物打印24小时后能够迁移。在某些优选的实施方案中，微囊内至少80％、85％、87.5％、90％、92.5％、95％、或98％的细胞在生物打印24小时后能够分泌。
在某些优选的实施方案中，所述微囊为细胞的生命活动提供微环境。在某些优选的实施方案中，所述微囊提供了适合细胞粘附和伸展的空间结构和微环境，从而细胞在该结构内能够正常进行增殖、分化、迁移、分泌或新陈代谢。所述微环境指细胞所生长的环境，其包含的要素包括物理因素，比如空间结构、力学强度、温度、湿度、渗透压等；化学因素，比如酸碱度、离子浓度等；生物因素，包括细胞、细胞因子等。这些要素共同构成细胞生命活动的环境，并对在这个环境中生长的细胞的增殖、分化、迁移、分泌和新陈代谢进行动态调控。在某些优选的实施方案中，所述微囊能够为细胞的生命活动提供营养物质。
在本发明的某些实施方案中，所述微囊为生物砖。
本发明的生物砖包括：细胞，包裹细胞的核层，和，封装核层的壳层，其中所述核层和壳层各自独立地由生物可降解材料制成。在本发明的某些优选实施方案中，所述核层和壳层中的生物可降解材料能够减少或避免生物砖内的细胞在操作过程中遭受机械损伤，并且能够提供物质(例如营养物质，细胞外基质，细胞因子，药物活性成分等)的可控释放，以促进细胞活性和功能(增殖、分化、迁移、分泌或新陈代谢)或维持细胞的干性。
在某些优选的实施方案中，生物砖的核层提供了适合细胞粘附和伸展的空间结构和微环境，从而细胞在该结构内能够正常进行增殖、分化、迁移、分泌或新陈代谢。所述微环境指细胞所生长的环境，其包含的要素包括物理因素，比如空间结构、力学强度、温度、湿度、渗透压等；化学因素，比如酸碱度、离子浓度等；生物因素，包括细胞、细胞因子等。这些要素共同构成细胞生命活动的环境，并对在这个环境中生长的细胞的增殖、分化、迁移、分泌和新陈代谢进行动态调控。在某些实施方案中，所述核层能够为细胞的生命活动提供微环境，例如空间结构、营养物质等。
在某些优选的实施方案中，生物砖的壳层为包裹的细胞提供了力学保护。在某些优选的实施方案中，所述生物砖或生物砖的壳层具有一定的力学强度，从而能够实现立体堆积。在本发明中，特别优选地，生物砖及其壳层具有适当的力学保护性能(例如，具有合适的硬度和/或弹性模量)。一方面，生物砖内的细胞在操作过程中易于因外界压力或剪切力的伤害而受损或死亡。因此，如果生物砖及其壳层的硬度和/或弹性模量太低，那么将导致生物砖内的细胞存活率在人工操作后显著下降，进而导致生物砖的应用受到限制，或者需要使用大量的细胞。另一方面，如果生物砖及其壳层的硬度和/或弹性模量太高，那么将导致生物砖内的细胞的伸展、迁移受到限制，并且阻碍不同生物砖的细胞之间建立细胞连接，不利于构建有机的整体(例如，人工组织)。因此，适当的力学保护性能不仅使得能够对本发明的生物砖进行各种操作，而且有利于生物砖内的细胞伸展、迁移、建立细胞连接，并形成有机的构建体(例如人工组织)，因此，是特别优选的。
图2A-E示意性描述了本发明的生物砖的示例性结构，其包括：细胞，包裹细胞的核层和封装核层的壳层。
图2A-2E特别地，图2A示意性描述了本发明生物砖的一种结构，其包括一个核层和一个壳层，其中，所述核层包裹有细胞，并且所述壳层位于核层外侧，且封装核层。
图2B示意性描述了本发明生物砖的一种结构，其由内到外依次包含：包裹细胞的核层、封装所述核层的第一壳层、和围绕第一壳层的第二壳层。
图2C示意性描述了本发明生物砖的一种结构，其由内到外依次包含：包裹细胞的第一核层、位于第一核层外侧的包裹细胞的第二核层、和封装所述第一核层和第二核层的第一壳层。
图2D示意性描述了本发明生物砖的一种结构，其由内到外依次包含：包裹细胞的第一核层、位于第一核层外侧的包裹细胞的第二核层、封装所述第一核层和第二核层的 第一壳层、和围绕第一壳层的第二壳层。
图2E示意性描述了本发明生物砖的一种结构，其由内到外依次包含：包裹细胞的第一核层、封装所述第一核层的第一壳层、包裹细胞的第二核层、和封装所述第二核层的第二壳层。
有关生物砖的详细描述可参见例如，中国专利申请201610211570.4和PCT国际申请PCT/CN2016/078678，其各自以其全文通过引用并入本文。
本发明的微囊的尺寸可以根据实际需要进行选择，而不受特别限制。球形微囊的尺寸通常可以通过其直径来进行明确定义。在严格定义的情况下，术语“直径”不能用于描述非球形的结构。然而，在本发明中，也使用术语“直径”来描述非球形的微囊的尺寸。在此情况下，术语“直径”表示，与非球形的微囊具有相同体积的球形微囊的直径。换言之，在本发明中，使用球形微囊的直径来描述具有相同体积的非球形的微囊的尺寸。因此，在某些优选的实施方案中，本发明微囊的尺寸(即，本文所定义的直径)可以为20-2000μm，例如30-1900μm，40-1800μm，50-1700μm，60-1600μm，70-1500μm，80-1400μm，90-1300μm，100-1200μm，200-1000μm，300-800μm，400-600μm，100-500μm。在某些优选的实施方案中，本发明微囊的尺寸(即，本文所定义的直径)可以为20-30、30-50、50-100、100-150、150-200、200-250、250-300、300-350、350-400、400-450、450-500、500-600、600-700、700-800、800-900、900-1000、1000-1500、1500-2000、20-50、20-100、100-200、200-400、500-600、600-800、800-1000、或1000-2000μm。在某些优选的实施方案中，本发明微囊的尺寸(即，本文所定义的直径)为至少20、30、50、100、120、150、200、250、300、350、400、450、500、600、700、800、900、1000、1500、或2000μm。
本发明的微囊的形状可以根据实际需要进行选择，而不受特别限制。例如，本发明微囊可以是球形，或者任何期望的形状(例如立方体，矩形棱柱，六棱柱，圆柱，或不规则的形状)。例如，一些形状(例如球形，立方体，矩形棱柱，六棱柱)可用于实现微囊在构建体中的紧密堆积。
在某些优选的实施方案中，本发明的微囊为固体或半固体。在某些优选的实施方案中，本发明的微囊为凝胶态。例如，本发明的微囊的核层和/或壳层可以为凝胶态。在某些优选的实施方案中，本发明的微囊包含水凝胶。在某些优选的实施方案中，所述水凝胶包含海藻酸盐，琼脂糖，明胶，壳聚糖，或其它水溶性或亲水性聚合物。
在某些优选的实施方案中，本发明的微囊以混合物的形式存在。在此类实施方案中，微囊可以与混合物中的另一微囊接触或融合。在某些优选的实施方案中，本发明的微囊是分离的微囊。例如，在某些实施方案中，微囊不与其他的微囊直接接触。在某些优选的实施方案中，本发明的分离的微囊提供于容器中。
本发明的微囊可使用各种方法来制备。例如，在某些优选的实施方案中，可使用用于制造微球体的方法来制备本发明的微囊，例如使用造粒仪来进行制备。在某些优选的实施方案中，本发明的微囊是在无菌条件下制备的。某些优选的实施方案中，本发明的微囊是在GMP工作间中制备的。在某些优选的实施方案中，本发明的微囊在即将使用前被制备。在某些优选的实施方案中，本发明的微囊在制备后贮存于4℃，例如贮存3小时、6小时、12小时、1天、2天、或3天。
本发明微囊包含的细胞的种类可以根据实际需要进行选择，而不受特别限制。在某些优选的实施方案中，所述微囊中包含上皮细胞，例如内皮细胞(例如血管内皮细胞)、平滑肌细胞(例如血管平滑肌细胞)和/或未分化的细胞。
在某些优选的实施方案中，所述微囊中的细胞为未分化的细胞，例如干细胞(例如脂肪间充质干细胞、骨髓间充质干细胞、诱导多能干细胞和胚胎干细胞)。
在某些优选的实施方案中，所述未分化的细胞能够分化为上皮细胞(例如内皮细胞)和/或平滑肌细胞。
在某些优选的实施方案中，所述未分化的细胞选自干细胞(例如脂肪间充质干细胞、骨髓间充质干细胞、诱导多能干细胞和胚胎干细胞)和祖细胞(例如内皮祖细胞)中的一种或多种。
本发明微囊包含的细胞的来源可以根据实际需要进行选择，而不受特别限制。在某些优选的实施方案中，所述细胞获自动物，例如哺乳动物，例如人、猿、猴、大猩猩、牛、猪、犬、绵羊和山羊。
在某些优选的实施方案中，所述细胞来源于选自下述的组织：结缔组织(例如，疏松结缔组织、致密结缔组织、弹性组织、网状结缔组织和脂肪组织)、肌肉组织(例如，骨骼肌、平滑肌和心肌)、泌尿生殖组织、胃肠组织、肺组织、骨组织、神经组织和上皮组织(例如，单层上皮和复层上皮)、内胚层来源的组织、中胚层来源的组织和外胚层来源的组织。
本发明微囊包含的细胞的数量可以根据实际需要进行选择，而不受特别限制。例如， 本发明微囊的核层可以包含1-106个细胞，例如10-900、20-800、30-700、40-600、50-500、60-400、70-300、80-200、10-100个、10-103个、10-104个、10-105个、10-106个细胞。在某些优选的实施方案中，本发明微囊包含至少1、2、4、6、8、10、15、20、25、30、40、50、60、70、80、90、100、150、200、300、400、500、600、700、800、900、1000、2000、3000、4000、5000、6000、7000、8000、9000、104、2x104、3x104、4x104、5x104、6x104、7x104、8x104、9x104、105、2x105、3x105、4x105、5x105、6x105、7x105、8x105、9x105、或106个细胞。在某些优选的实施方案中，本发明微囊包含1-2、2-4、4-6、6-8、8-10、10-15、15-20、20-25、25-30、30-40、40-50、50-60、60-70、70-80、80-90、90-100、100-150、150-200、200-300、300-400、400-500、500-1000、1000-2000、2000-3000、3000-4000、4000-5000、5000-104、104-2x104、2x104-3x104、3x104-4x104、4x104-5x104、5x104-105、105-2x105、2x105-3x105、3x105-4x105、4x105-5x105、5x105-106、1-10、2-10、2-5、5-10、10-20、20-30、30-50、2-25、25-50、2-50、50-100、100-200、50-250、250-500、500-2000、2-100、2-500、或2-2000个细胞。
在某些优选的实施方案中，除了如上描述的上皮细胞(例如内皮细胞)、平滑肌细胞和/或未分化的细胞之外，所述微囊包裹的细胞还包括额外细胞。在某些优选的实施方案中，所述额外细胞来源于选自下述的组织：结缔组织(例如，疏松结缔组织、致密结缔组织、弹性组织、网状结缔组织和脂肪组织)、肌肉组织(例如，骨骼肌、平滑肌和心肌)、泌尿生殖组织、胃肠组织、肺组织、骨组织、神经组织和上皮组织(例如，单层上皮和复层上皮)、内胚层来源的组织、中胚层来源的组织和外胚层来源的组织。在某些优选的实施方案中，所述额外细胞选自肌肉细胞(例如，骨骼肌细胞、心肌细胞、平滑肌细胞和成肌细胞)、结缔组织细胞(例如，骨细胞、软骨细胞、成纤维细胞以及分化为成骨细胞、软骨细胞或淋巴组织的细胞)、骨髓细胞、皮肤细胞、上皮细胞、乳腺细胞、血管细胞、血细胞、淋巴细胞、神经细胞、许旺细胞、胃肠细胞、肝细胞、胰细胞、肺细胞、气管细胞、角膜细胞、泌尿生殖细胞、肾细胞、脂肪细胞、实质细胞、周细胞、间皮细胞、基质细胞、内胚层来源的细胞、中胚层来源的细胞、外胚层来源的细胞、癌来源的细胞、细胞系、或其任何组合。
在某些优选的实施方案中，本发明的微囊包含细胞和包裹所述细胞的核层。在某些优选的实施方案中，所述核层能够为细胞的生命活动提供微环境。在某些优选的实施方案 中，微囊提供了适合细胞粘附和伸展的空间结构和微环境，从而细胞在该结构内能够正常进行增殖、分化、迁移、分泌或新陈代谢，或维持干性。所述微环境指细胞所生长的环境，其包含的要素包括物理因素，比如空间结构、力学强度、温度、湿度、渗透压等；化学因素，比如酸碱度、离子浓度等；生物因素，包括细胞、细胞因子等。这些要素共同构成细胞生命活动的环境，并对在这个环境中生长的细胞的增殖、分化、迁移、分泌和新陈代谢进行动态调控，或维持细胞的干性。在某些优选的实施方案中，所述核层能够为细胞的生命活动提供营养物质。
在某些优选的实施方案中，所述核层地由生物相容性材料制成。
在某些优选的实施方案中，所述微囊还包含封装所述核层的壳层。
在某些优选的实施方案中，微囊的壳层为包裹的细胞提供了力学保护。在某些优选的实施方案中，所述微囊或微囊的壳层具有一定的力学强度，从而能够实现立体堆积。在本发明中，特别优选地，微囊及其壳层具有适当的力学保护性能(例如，具有合适的硬度和/或弹性模量)。一方面，微囊内的细胞在操作过程中(例如，在3D打印过程中)易于因外界压力或剪切力的伤害而受损或死亡。因此，如果微囊及其壳层的硬度和/或弹性模量太低，那么将导致微囊内的细胞存活率在人工操作后显著下降，进而导致微囊的应用受到限制，或者需要使用大量的细胞。另一方面，如果微囊及其壳层的硬度和/或弹性模量太高，那么将导致微囊内的细胞的伸展、迁移受到限制，并且阻碍不同微囊的细胞之间建立细胞连接，不利于构建有机的整体(例如，人工组织)。因此，适当的力学保护性能不仅使得能够对本发明的微囊进行各种操作(例如进行3D生物打印，进行微囊的精确排布等)，而且有利于微囊内的细胞伸展、迁移、建立细胞连接，并形成有机的构建体(例如人工组织)，因此，是特别优选的。
在某些优选的实施方案中，本发明微囊的核层和/或壳层各自任选地经过处理(例如使用核层固定液或壳层固定液进行处理，例如，以改善核层或壳层的力学性能)。
在某些优选的实施方案中，所述微囊、微囊的核层或微囊的壳层各自独立地具有约0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09、0.1、0.15、0.2、0.3、或0.4GPa的硬度。在某些优选的实施方案中，所述微囊、微囊的核层或微囊的壳层各自独立地具有0.01-0.02、0.02-0.03、0.03-0.04、0.04-0.05、0.05-0.06、0.06-0.07、0.07-0.08、0.08-0.09、0.09-0.1、0.1-0.15、0.15-0.2、0.2-0.3、0.3-0.4、0.01-0.4、0.01-0.05、0.05-0.1、0.1-0.2、0.2-0.4、0.05-0.15、或0.06-0.1GPa的硬度。在某些优选的实施方案中，所述微囊、微囊的核层或微囊的壳层各自 独立地具有0.01-0.1GPa或0.01-0.4GPa的硬度。在某些优选的实施方案中，所述微囊、微囊的核层或微囊的壳层具有约0.083GPa的硬度。在某些优选的实施方案中，所述微囊、微囊的核层或微囊的壳层各自独立地具有约0.01、0.05、0.1、0.5、0.8、1、1.2、1.4、1.6、1.8、2、2.4、2.8、3.2、4、10、20、30、40、50、80、或100MPa的弹性模量。在某些优选的实施方案中，所述微囊、微囊的核层或微囊的壳层各自独立地具有0.01-0.05、0.05-0.1、0.1-0.5、0.5-0.8、0.8-1、1-1.2、1.2-1.4、1.4-1.6、1.6-1.8、1.8-2、2-2.4、2.4-2.8、2.8-3.2、3.2-4、4-10、10-20、20-30、30-40、40-50、50-80、80-100、0.5-4、0.5-1、1-1.5、1.5-2、2-3、0.8-1.6、1.4-2.4、0.8-3.2、0.01-100、1-100、10-100、或0.5-50MPa的弹性模量。在某些优选的实施方案中，所述微囊、微囊的核层或微囊的壳层各自独立地具有0.01-1、0.01-10或0.01-100MPa的弹性模量。核层或壳层的力学保护作用(例如，硬度和弹性模量)可通过对核层或壳层的组分和/或含量的配置来控制。
在某些优选的实施方案中，所述壳层也能够为细胞的生命活动提供微环境，例如营养物质。在某些优选的实施方案中，所述壳层由生物相容性材料制成。
在某些优选的实施方案中，用于制备核层和壳层的生物相容性材料可以是相同的或不同的。然而，特别优选地，根据其预期的目的，核层和壳层具有不同的组成。不拘于理论限制，通常认为，壳层提供了主要的力学保护作用，而核层则提供了细胞生命活动所需的主要的营养成分和微环境。因此，在某些优选的实施方案中，与壳层相比，核层具有更多的营养物质。在某些优选的实施方案中，与核层相比，壳层具有较低的降解速率，但具有更高的硬度和/或弹性模量。在某些优选的实施方案中，壳层中不包含细胞。
在某些优选的实施方案中，核层和壳层分别以不同的重量比包含相同的生物相容性材料。换言之，核层和壳层可以由相同的生物相容性材料制成，但以不同的重量比包含生物可降解材料。
在某些优选的实施方案中，所述壳层是通透性的。例如，所述壳层对于水，氧气，和营养物质(糖类例如葡萄糖，脂肪，蛋白质，氨基酸，短肽，矿物质，维生素、细胞因子、核苷酸等)是通透性的。
一般认为，半通透的(即，选择通透的)壳层的使用可能是有利的，因为其能够使得水，氧气，葡萄糖，矿物质，和氨基酸等营养物质透过壳层，进入核层，并提供给细胞，并且能够阻止对细胞有害的物质(例如来自宿主免疫系统的抗体蛋白)进入核层。然而，在本发明的微囊中，通透性壳层的使用是优选的和有利的。特别地，通透性的壳层使得各 种营养物质(包括大分子和小分子营养物质，例如葡萄糖，脂肪，蛋白质，氨基酸，短肽，矿物质，维生素、细胞因子、核苷酸等)能够更加容易、顺畅地进行交换，避免局部区域的细胞无法获得充足的营养物质。例如，当使用本发明的微囊构建大尺寸的人工组织时，通透性的壳层将能够促进各种营养物质的交换，促进人工组织内部/核心区域的微囊内的细胞获得充足的营养物质。此外，通透性的壳层有利于不同微囊之间的细胞进行信号传递和建立细胞连接。特别地，细胞在生长过程中会分泌多种物质(包括细胞外基质的某些组分和多种信号分子)，与邻近的、甚至远端的细胞进行信号传递和/或物质交流，并由此对细胞自身的生命活动以及邻近的、甚至远端的细胞的生命活动产生影响或进行调控。因此，如果使用选择通透性的壳层的话，那么细胞之间的信号传递和/或物质交流将有可能受到影响/阻碍，例如细胞分泌的某些大分子信号物质(例如细胞因子蛋白)可能无法透过壳层，从而可能阻碍不同微囊之间的细胞信号的传递和细胞连接的建立，不利于构建有机的整体(例如，人工组织)。因此，通透性壳层的使用对于本发明的微囊而言是优选的。在本发明中，表述“通透性壳层”意指，各种小分子和大分子物质(例如蛋白质)能够自由通过壳层。例如，在某些优选的实施方案中，所述壳层对于分子量在5000kDa以下的分子是通透的。例如，在某些实施方案中，所述壳层对于分子量在200kDa以下或分子量在200kDa-300kDa、300kDa-400kDa、400kDa-500kDa、500kDa-800kDa、800kDa-1000kDa、1000kDa-1500kDa、1500kDa-2000kDa、2000kDa-3000kDa、3000kDa-4000kDa或4000kDa-5000kDa范围内的分子是通透的。在某些实施方案中，所述壳层对于免疫球蛋白(例如IgG、IgM、IgA、IgD、IgE)是通透的。
在某些优选的实施方案中，所述壳层具有用于微囊内外物质交换的通道或孔。在某些优选的实施方案中，营养物质(糖类例如葡萄糖，脂肪，蛋白质，氨基酸，短肽，矿物质，维生素、细胞因子、核苷酸等)通过所述通道或孔扩散进入所述微囊内。在某些优选的实施方案中，所述通道的直径为至少10、20、50、100、150、200、250、300、350、400、或500nm。在某些优选的实施方案中，所述通道的直径为例如1nm-5μm；10nm-2μm；100nm-1μm；200-800nm等。在某些优选的实施方案中，所述孔的直径为至少100、200、400、600、800、1000、1500、2000、4000、或5000nm。
本发明的微囊的壳层的厚度可以根据实际需要进行选择，而不受特别限制。例如，本发明微囊的壳层的厚度可以为1-20μm，例如5-15μm，例如8-12μm。在某些优选的实施方案中，本发明的微囊的壳层的厚度可以为约0.1、0.5、1、2、5、10、15、20、25、30、或 50μm。在某些优选的实施方案中，本发明的微囊的壳层的厚度可以为0.1-0.5、0.5-1、1-2、2-5、5-10、10-15、15-20、20-25、25-30、30-50、50-100、100-200、200-300、300-400、400-500、0.1-1、1-5、1-10、5-10、10-20、10-30、5-20、或1-20μm。
在某些优选的实施方案中，本发明的微囊的壳层不包含细胞。
在某些优选的实施方案中，本发明所述的生物相容性材料包含生物可降解材料。
在本发明中，使用生物可降解材料来制备微囊是特别优选的。特别地，对于微囊在制备人工组织前体中的用途而言，无法降解的材料的使用是不利的。这是因为，一方面，这些无法降解的材料将被保留在所获得的人工组织中，从而限制人工组织的应用；另一方面，这些无法降解的材料将阻碍不同微囊的细胞之间建立细胞连接，不利于构建有机的整体(例如，人工组织)。因此，生物可降解材料在壳层中的使用对于利用微囊来制备人工组织前体是特别有利的和优选的。
在某些优选的实施方案中，所述生物可降解材料为具有可降解性的生物材料。
在本发明的实施方案中，用于制备微囊的生物可降解材料可以是天然存在的(例如来源于动植物的天然存在的生物可降解材料，例如胶原蛋白，纤维蛋白，壳聚糖，海藻酸盐，淀粉，透明质酸，层粘连蛋白，琼脂糖，明胶，葡聚糖，以及其任意组合)，人工合成的，重组产生的，经过改性的，或者其任何组合。
在某些优选的实施方案中，用于制备微囊的所述生物可降解材料是天然存在的生物可降解材料。在某些优选的实施方案中，所述天然存在的生物可降解材料，选自胶原蛋白，纤维蛋白，壳聚糖，海藻酸盐(例如海藻酸钠或海藻酸钙)，淀粉，透明质酸，层粘连蛋白，琼脂糖，明胶，葡聚糖，甲壳素，纤维素(例如细菌纤维素)，蚕丝蛋白，硫酸软骨素，肝素，纤维蛋白原，纤连蛋白，粘多糖，粘液素，以及其任意组合。在某些优选的实施方案中，用于制备微囊的所述生物可降解材料是经过改性的生物可降解材料，例如经过改性的海藻酸盐，例如氧化海藻酸盐(例如氧化海藻酸钠)，改性明胶(如双醛淀粉DAS交联改性明胶)，改性纤维素(例如羧甲基纤维素，氧化再生纤维素)，以及其任意组合。
在某些优选的实施方案中，用于制备微囊的所述生物可降解材料是人工合成的生物可降解材料，例如聚磷腈，聚丙烯酸及其衍生物(例如聚甲基丙烯酸，丙烯酸和甲基丙烯酸的共聚物)，聚乳酸(PLA)，聚羟基乙酸(PGA)，聚乳酸-乙醇酸共聚物(PLGA)，聚原酸酯(POE)，聚己内酯(PCL)，聚羟基丁酸酯(PHB)，聚氨基酸(例如聚赖氨酸)，可降解性聚氨酯(如 淀粉改性聚氨酯)，聚羟基烷酸酯(PHAs)，聚羟基戊酸酯(PHV)，聚丁二酸丁二醇酯(PBS)，聚乙烯醇，聚对二氧环己酮，聚对二氧杂环己酮，聚二氧杂环己烷酮，聚碳酸丁二醇酯，以及其任何组合。
在某些优选的实施方案中，用于制备微囊的所述生物可降解材料能够被酶(例如细胞分泌的酶)所降解。不同的生物可降解材料的降解速率差异很大，其范围可以为一个月到数年。然而在本发明中，特别优选地，用于制备壳层的生物可降解材料在不超过1个月的时间内降解，例如在不超过30天、不超过25天、不超过20天、不超过15天、不超过10天、不超过5天、不超过4天、不超过3天、不超过2天、或不超过1天的时间内降解。例如，用于制备微囊的生物可降解材料可以在1-2天，2-3天，3-4天，4-5天，5-10天，10-15天，15-20天，20-25天，或25-30天的时间内降解。特别优选地，用于制备微囊的生物可降解材料在不超过10天的时间内降解。降解速率与生物可降解材料的分子组成、分子量大小和分子排列(例如，直链或支链)密切相关。一般情况下，分子量越高、分子排列越紧密，降解时间越长。因此，微囊的降解速率可通过对壳层的组分和/或含量的配置来控制。例如，为了获得更快的降解速率，可使用低含量(例如低于0.5％、1％、2％、3％、4％、或5％)的生物可降解材料、低分子量(例如低于500Da、1kDa、2kDa、3kDa、5kDa、或10kDa)的生物可降解材料，和/或具有疏松分子排布的生物可降解材料。为了获得更慢的降解速率，可使用高含量(例如高于0.5％、1％、2％、3％、4％、或5％)的生物可降解材料、高分子量(例如高于500Da、1kDa、2kDa、3kDa、5kDa、或10kDa)的生物可降解材料，和/或具有紧密分子排布的生物可降解材料。另外，还可通过改变微囊的结构(如：多层包裹、表面多孔、孔隙率大小、比表面积等)来调节生物可降解材料的降解速率。此外，生物可降解材料的降解速率还可以通过改变合成该材料的聚合方式和共聚物比例来进行调节；或者，可通过对该材料的交联来进行调节。此外，用于制备微囊的生物可降解材料的降解速率还可受细胞生命活动的影响。
在本发明中，特别优选的是，微囊内的细胞能够生长、伸展、增殖、迁移，并与其他微囊内的细胞建立细胞连接，形成有机的构建体(例如人工组织)。因此，在某些优选的实施方案中，所述微囊在相对短的时间(例如不超过30天的时间内，例如不超过10天的时间内)降解，以促进不同微囊之间的细胞连接的建立，避免阻碍或影响不同微囊之间的细胞建立相互的细胞连接。在某些优选的实施方案中，所述微囊在不超过30天、不超过25天、不超过20天、不超过15天、不超过10天、不超过5天、不超过4天、不超过3天、 不超过2天、或不超过1天的时间内降解。例如，所述微囊可以在1-2天，2-3天，3-4天，4-5天，5-10天，10-15天，15-20天，20-25天，或25-30天的时间内降解。
各种生物可降解材料是本领域技术人员已知的，并且其降解性能已被进行了广泛研究。参见例如，Alexander D.Augst,Hyun Joon Kong,David J.Mooney,Alginate Hydrogels as Biomaterials,Macromol.Biosci.2006,6,623-633，其通过引用并入本文。
在某些优选的实施方案中，所述微囊的降解能够提供维持或促进所述细胞的生命活动的微环境，例如营养物质。在某些优选的实施方案中，壳层的降解产物为小分子化合物，例如有机酸、单糖(例如葡萄糖)、寡糖、氨基酸、脂质等。此类降解产物可参与到细胞的新陈代谢活动中，用于合成细胞外基质或转化为活动所需的能量。
在某些优选的实施方案中，用于制备微囊的生物可降解材料及其降解产物对于细胞是无毒的，和/或对于宿主是非免疫原性的。
在某些优选的实施方案中，用于制备微囊的生物可降解材料含有细胞外基质或其类似物(例如弹性蛋白)。细胞外基质或其类似物(例如弹性蛋白)的使用能够为微囊内的细胞的生命活动(特别是细胞的生长、粘附、伸展，以及细胞间连接的建立)提供类似于体内的有利的微环境，从而是优选的。
在某些优选的实施方案中，用于制备微囊的生物可降解材料选自胶原蛋白(例如I型，II型，III型胶原蛋白)、纤维蛋白、壳聚糖、海藻酸盐(例如海藻酸钠或海藻酸钙)、氧化海藻酸盐(例如氧化海藻酸钠)、淀粉、透明质酸，层粘连蛋白，弹性蛋白，明胶、葡聚糖、聚氨基酸(例如聚赖氨酸)、琼脂糖，或其任何组合。在某些优选的实施方案中，所述微囊包含海藻酸盐(例如海藻酸钠或海藻酸钙)，例如包含海藻酸钙和明胶，任选地还包含弹性蛋白。
在某些优选的实施方案中，所述微囊包含海藻酸盐(例如海藻酸钠或海藻酸钙)和明胶。
在某些优选的实施方案中，所述微囊包含海藻酸盐(例如海藻酸钠或海藻酸钙)，例如包含海藻酸钙和明胶，任选地还包含弹性蛋白。在某些优选的实施方案中，所述微囊包含氧化海藻酸盐(例如氧化海藻酸钠)。在某些优选的实施方案中，所述微囊包含海藻酸盐(例如海藻酸钠或海藻酸钙)和琼脂糖。
在某些优选的实施方案中，可使用氧化的海藻酸盐(例如氧化的海藻酸钠和氧化的海藻酸钙)来制备微囊，并且可通过控制海藻酸盐的氧化度来调节其降解速度，从而使微囊的降解速度与包裹在其中的细胞生长速度相匹配。
在某些优选的实施方案中，所述微囊包含核层和一个或多个壳层(例如2个、3个、4个或5个)。
在某些优选的实施方案中，用于制备微囊核层的所述生物可降解材料选自：胶原蛋白(例如I型，II型，III型胶原蛋白)，纤维蛋白，壳聚糖，海藻酸盐(例如海藻酸钠或海藻酸钙)，透明质酸，琼脂糖，明胶，淀粉，葡聚糖，聚磷腈，聚丙烯酸及其衍生物，聚乳酸(PLA)，聚氨基酸(例如聚赖氨酸)，可降解性聚氨酯，以及其任意组合。
在某些优选的实施方案中，用于制备微囊壳层的所述生物可降解材料选自：海藻酸盐(例如海藻酸钠或海藻酸钙)，弹性蛋白，聚氨基酸(例如聚赖氨酸)，氧化海藻酸盐，明胶，壳聚糖，以及其任意组合。
在某些优选的实施方案中，用于制备微囊核层的所述生物可降解材料包括胶原蛋白(例如I型，II型，III型胶原蛋白)。
在某些优选的实施方案中，用于制备微囊壳层的所述生物可降解材料选自聚氨基酸(例如聚赖氨酸)和海藻酸盐(例如海藻酸钠或海藻酸钙)。
在某些优选的实施方案中，所述微囊还包含额外的试剂，例如，营养物质、细胞外基质、细胞因子和/或药物活性成分。
在某些优选的实施方案中，所述额外的试剂能够调控(例如促进)细胞的增殖、分化、迁移、分泌和/或新陈代谢，或者，所述额外的试剂能够维持细胞的干性。在某些优选的实施方案中，所述微囊包含至少一种(例如1、2、3、4、5或更多种)能够调控(例如促进)细胞的增殖、分化、迁移、分泌和/或新陈代谢的额外的试剂，或者能够维持细胞干性的额外的试剂。在某些优选的实施方案中，所述微囊能够以受控的方式释放所述额外的试剂。
在某些优选的实施方案中，所述营养物质包括但不限于，核苷酸，氨基酸，多肽，碳水化合物(例如单糖，寡糖，多糖)，脂质，维生素等。
在某些优选的实施方案中，细胞外基质选自多糖，例如糖胺聚糖、蛋白聚糖；结构蛋白，例如胶原和弹性蛋白；粘着蛋白，例如纤粘连蛋白和层粘连蛋白。
在某些优选的实施方案中，所述细胞因子可以是用于调控细胞的增殖、分化、迁移、分泌和/或新陈代谢的细胞因子，包括但不限于：
-与细胞生长相关的细胞因子，例如胰岛素、类胰岛素生长因子(如IGF-Ⅰ、IGF-Ⅱ)、转化生长因子(如TGFα和TGFβ)、血管内皮生长因子、表皮生长因子、成纤细胞生长因子、血小板来源生长因子、骨肉瘤来源生长因子、生长激素释放抑制因子、神经生长因子、白 细胞介素(如IL-1、IL-11、IL-3)、红细胞生长素、集落刺激因子、皮质醇、甲状腺素，或其任何组合；
-与细胞分化相关的细胞因子，例如Oct3/4，Sox2，Klf4，c-Myc，GATA4，TSP1，β-甘油磷酸钠，地塞米松，维生素C，胰岛素，IBMX，吲哚美锌，血小板衍生生长因子BB(PDGF-BB)，5-氮杂胞苷，或其任何组合；
-与细胞迁移相关的细胞因子，例如环磷酸腺苷，三磷酸磷脂酰肌醇，基质细胞衍生因子-1、N-钙粘蛋白，核因子κB，骨连接素，血栓素A2，Ras，或其任何组合；和/或
-与细胞新陈代谢相关的细胞因子，例如胰岛素生长因子1、TRIP-Br2、DKK-1、sRANKL、OPG、TRACP-5b、ALP、SIRT1(2-7)、PGC-1α，PGC-1β、OPG、IL-3、IL-4、IL-6、TGF-β、PGE2、G-CSF、TNF-α，或其任何组合。
在某些优选的实施方案中，所述药物活性成分为能够调控(例如促进)细胞的增殖、分化、迁移、分泌和/或新陈代谢的试剂，或者为能够维持细胞干性的试剂。在某些优选的实施方案中，所述药物活性成分选自rhIL-2、rhIL-11、rhEPO、IFN-α、IFN-β、IFN-γ、G-CSF、GM-CSF、rHuEPO、sTNF-R1、和rhTNF-α。
在某些优选的实施方案中，所述微囊包含能够诱导未分化的细胞向平滑肌细胞或内皮细胞分化的细胞因子，例如TGF-α1、PDGF-BB、VEGF或b-FGF。
在某些优选的实施方案中，所述微囊包括：脂肪间充质干细胞和包裹所述脂肪间充质干细胞的核层，优选地，所述核层由生物可降解材料制成；优选地，所述核层提供维持脂肪间充质干细胞干性的微环境(例如，所述核层包含维持脂肪间充质干细胞干性的额外试剂)；优选地，所述核层提供诱导脂肪间充质干细胞向内皮细胞细胞或平滑肌细胞分化的微环境(例如，所述核层包含诱导脂肪间充质干细胞向内皮细胞或平滑肌细胞分化的诱导因子)。在某些优选实施方案中，所述诱导脂肪间充质干细胞向平滑肌细胞分化的诱导因子选自TGF-α1和PDGF-BB。在某些优选实施方案中，所述诱导脂肪间充质干细胞向内皮细胞分化的诱导因子选自VEGF和b-FGF。
在某些优选的实施方案中，所述微囊包括：脂肪间充质干细胞，包裹所述脂肪间充质干细胞细胞的核层，和，封装所述核层的壳层；优选地，所述核层和壳层各自独立地由生物可降解材料制成；优选地，所述核层提供维持脂肪间充质干细胞干性的微环境(例如，所述核层包含维持脂肪间充质干细胞干性的额外试剂)；优选地，所述核层提供诱 导脂肪间充质干细胞向内皮细胞细胞或平滑肌细胞分化的微环境(例如，所述核层包含诱导脂肪间充质干细胞向内皮细胞或平滑肌细胞分化的诱导因子)。在某些优选实施方案中，此类微囊的壳层也提供诱导脂肪间充质干细胞向内皮细胞或平滑肌细胞分化的微环境(例如，所述壳层包含诱导脂肪间充质干细胞向内皮细胞或平滑肌分化的诱导因子)。在某些优选实施方案中，所述诱导脂肪间充质干细胞向平滑肌细胞分化的诱导因子选自TGF-α1和PDGF-BB。在某些优选实施方案中，所述诱导脂肪间充质干细胞向内皮细胞分化的诱导因子选自VEGF和b-FGF。
在本发明的人工组织前体中，优选地，固体支持物由生物相容性材料制得。
在某些优选的实施方案中，所述生物相容性材料包含生物可降解材料。本发明中，使用生物可降解材料制备固体支持物，可以使得人工组织前体在植入受试者体内后的不断生长过程中，固体支持物逐步降解，最终使得人工组织与被植入者的自体组织完全融合成一体。
在某些优选的实施方案中，所述生物可降解材料为具有可降解性的生物材料。
在某些优选的实施方案中，所述生物可降解材料选自天然存在的生物可降解材料(例如胶原，明胶，壳聚糖，聚羟基丁酸酯(PHB)，甲壳素，海藻酸盐(例如海藻酸钠)，淀粉基生物材料(例如多聚糖淀粉)，纤维素(例如细菌纤维素)，蚕丝蛋白，以及其任意组合)。
在某些优选的实施方案中，所述天然存在的生物可降解材料为淀粉。
在某些优选的实施方案中，所述生物可降解材料选自经过改性的生物可降解材料(例如经过改性的海藻酸盐，例如氧化海藻酸盐(例如氧化海藻酸钠)，改性明胶(例如双醛淀粉DAS交联改性明胶)，改性纤维素(例如羧甲基纤维素，氧化再生纤维素)，以及其任意组合。
在某些优选的实施方案中，所述生物可降解材料选自人工合成的可降解材料(例如脂肪族聚酯(例如聚乳酸(PLA)，聚己内酯(PCL)，聚羟基烷酸酯(PHAs)，聚羟基戊酸酯(PHV)，聚羟基丁酸酯(PHB)，聚丁二酸丁二醇酯(PBS))，聚羟基乙酸(PGA)，聚乳酸-乙醇酸共聚物(PLGA)，聚原酸酯(POE)，可降解性聚氨酯(例如淀粉改性聚氨酯)，聚乙烯醇，聚对二氧环己酮，聚对二氧杂环己酮，聚二氧杂环己烷酮，聚碳酸丁二醇酯，聚磷腈，以及其任何组合)。
在某些优选的实施方案中，所述人工合成的可降解材料选自聚己内酯(PCL)，聚乳酸(PLA)，聚乳酸-羟基乙酸共聚物(PLGA)，聚乙醇酸(PGA)，可降解聚氨酯。
在某些优选的实施方案中，所述生物可降解材料能够被酶(例如细胞分泌的酶)所降解；
在某些优选的实施方案中，所述生物可降解材料在体内的降解时间为1-12个月。
在某些优选的实施方案中，所述生物相容性材料还包含生物不可降解材料(例如尼龙，涤纶，聚丙烯，聚乙烯，聚四氟乙烯，硅橡胶，氟硅橡胶，天然橡胶，聚丙烯酸酯，芳香族聚酯(例如聚对苯二甲酸乙二醇酯(PET))，非降解性聚氨酯，聚醚醚酮，聚丙烯腈，聚硅氧烷，聚甲醛，聚氯乙烯，以及其任何组合)。
在某些优选的实施方案中，所述生物相容性材料包含生物不可降解材料(例如尼龙，涤纶，聚丙烯，聚乙烯，聚四氟乙烯，硅橡胶，氟硅橡胶，天然橡胶，聚丙烯酸酯，芳香族聚酯(例如聚对苯二甲酸乙二醇酯(PET))，非降解性聚氨酯，聚醚醚酮，聚丙烯腈，聚硅氧烷，聚甲醛，聚氯乙烯，以及其任何组合)。
在某些优选的实施方案中，所述生物不可降解材料是生物惰性的。
在某些优选的实施方案中，所述固体支持物为管状固体支持物或片状固体支持物。
在某些优选的实施方案中，所述固体支持物通过模具浸渍、静电纺丝、挤压锻造、3D打印或喷涂制得。
在某些优选的实施方案中，所述固体支持物通过模具浸渍的方法得到。在某些优选的实施方案中，所述模具浸渍法包含以下步骤：
(1)将用于制备固体支持物的材料(例如生物可降解材料)溶于合适的溶剂(例如有机溶剂，例如氯仿，四氢呋喃或N,N-二甲基乙酰胺)中，配制成制备溶液；
(2)将模具浸入所述制备液中，取出模具，使模具上的溶剂挥发；
(3)重复步骤(2)多次，得到固体支持物；
任选地，所述方法还包括以下步骤：
对固体支持物进行干燥、剪切和/或灭菌。
在某些优选的实施方案中，所述制备溶液中，用于制备固体支持物的材料的浓度为0.5wt％-5wt％，例如0.5wt％-1wt％、1wt％-1.5wt％、1.5wt％-2wt％、2wt％-2.5wt％、2.5wt％-3wt％、3wt％-3.5wt％、3.5wt％-4wt％、4wt％-4.5wt％或4.5wt％-5wt％，例如0.5wt％、1wt％、1.5wt％、2wt％、2.5wt％、3wt％、3.5wt％、4wt％、4.5wt％ 或5wt％。
在某些优选的实施方案中，所述固体支持物通过静电纺丝的方法制得。
在某些优选的实施方案中，所述静电纺丝的方法包含以下步骤：
(1)将用于制备固体支持物的材料(例如生物可降解材料)溶于合适的溶剂(例如有机溶剂，例如氯仿)中，配制成制备溶液；
(2)用制备溶液在静电纺丝设备上纺丝，形成固体支持物；
(3)待溶剂挥发后从静电纺丝设备上分离固体支持物。
在某些优选的实施方案中，所述固体支持物在生物构建体的表面制得。
在某些优选的实施方案中，所述固体支持物通过在生物构建体的表面进行3D打印或喷涂制得。
本发明中，可以根据需要将人工组织前体制成任意的形状。在某些优选的实施方案中，使用微囊制备任意形状的生物构建体，与任意形状的固体支持物相结合。在某些优选的实施方案中，在任意形状的固体支持物上，使用微囊制备任意形状的生物构建体。在某些优选的实施方案中，使用微囊制备任意形状的生物构建体，在生物构建体上制备任意形状的固体支持物。
在某些优选的实施方案中，所述人工组织前体包含一个或多个任意形状的生物构建体。
在某些优选的实施方案中，所述人工组织前体为管状(例如圆形管状，例如侧壁开口或不开口的管状)，所述固体支持物为管状(例如圆形管状，例如侧壁开口或不开口的管状)固体支持物，多个所述微囊构成一个或多个管状(例如圆形管状，例如侧壁开口或不开口的管状)生物构建体，并且至少一个管状生物构建体的外壁与所述固体支持物的内壁贴合。
在某些优选的实施方案中，所述人工组织前体包含管状固体支持物和一个侧壁不开口的管状生物构建体，所述管状生物构建体的外壁与所述管状固体支持物的内壁贴合。
在某些优选的实施方案中，所述人工组织前体包含多个管状生物构建体。
图3A-E示例性描述了包含多个管状生物构建体的管状人工组织前体的结构。
图3A为一种管状人工组织前体的侧视图，所述人工组织前体包含管状固体支持物和多个侧壁不开口的管状生物构建体，所述多个侧壁不开口的管状生物构建体在所述管状固 体支持物的内部，并沿管状固体支持物的轴向排列，并且所述多个侧壁不开口的管状生物构建体的外壁均与所述管状固体支持物的内壁贴合。
图3B为一种管状人工组织前体的俯视图，所述人工组织前体包含管状固体支持物和多个侧壁不开口的管状生物构建体，所述多个侧壁不开口的管状生物构建体在所述管状固体支持物的内部，与管状固体支持物同轴设置，并且仅最外侧的侧壁不开口的管状生物构建体的外壁与所述管状固体支持物的内壁贴合。
图3C为一种管状人工组织前体的侧视图，所述人工组织前体包含管状固体支持物和多个侧壁开口的管状生物构建体，所述多个侧壁开口的管状生物构建体在所述管状固体支持物的内部，并沿管状固体支持物的轴向排列，并且所述多个侧壁开口的管状生物构建体的外壁均与所述管状固体支持物的内壁贴合；
图3D为一种管状人工组织前体的俯视图，所述人工组织前体包含管状固体支持物和多个侧壁开口的管状生物构建体，所述多个侧壁开口的管状生物构建体在所述管状固体支持物内部，与管状固体支持物同轴设置并沿径向排列，并且位于仅最外侧的侧壁开口的管状生物构建体的外壁与所述管状支持物的内壁贴合；
图3E为一种管状人工组织前体的俯视图，所述人工组织前体包含管状固体支持物和多个侧壁开口的管状生物构建体，所述多个侧壁开口的管状生物构建体在所述管状固体支持物内部，并与管状固体支持物同轴设置，并且所述多个侧壁开口的管状生物构建体的外壁均与所述管状固体支持物的内壁贴合。
在某些优选的实施方案中，所述人工组织前体包含管状固体支持物、侧壁开口的管状生物构建体和侧壁不开口的管状生物构建体。
本发明中，根据需要对管状人工组织前体及其包含的管状生物构建体和管状固体支持物的尺寸进行设置。
在某些优选的实施方案中，所述人工组织前体的长度为1cm-40cm。
在某些优选的实施方案中，所述人工组织前体的内径为1mm-3cm(例如1-6mm、6-8mm、8-10mm、10-12mm、12mm-3cm)。
在某些优选的实施方案中，所述人工组织前体具有均一或不均一的厚度。例如，所述管状固体支持物内壁的某一部分贴合有管状生物构建体，而其他部分没有贴合管状生物构建体。例如，所述管状固体支持物内壁的不同部分贴合有不同的管状生物构建体。
在某些优选的实施方案中，所述管状固体支持物的长度为1cm-40cm(例如1-10cm、10-20cm、20-30cm或30-40cm)。
在某些优选的实施方案中，所述管状固体支持物的内径为1mm-3cm(例如1-6mm、6-8mm、8-10mm、10-12mm、12mm-3cm)。
在某些优选的实施方案中，所述管状固体支持物的厚度为200μm-1mm(例如200μm-400μm、400μm-600μm、600μm-800μm或800μm-1mm)。
在某些优选的实施方案中，所述管状固体支持物为侧壁开口的圆形管状，所述开口沿轴线方向贯通管状固体支持物的两端，所述管状固体支持物沿径向的切面为扇环；在某些优选的实施方案中，所述扇环的圆心角大于0并且小于360°，例如大于0并且小于30°、30°-60°、60°-90°、90°-120°、120°-150°、150°-180°、180°-210°、210°-240°、240°-270°、270°-300°、300°-330°，或大于330°并且小于360°。
在某些优选的实施方案中，所述管状生物构建体的长度为1cm-40cm(例如1-10cm、10-20cm、20-30cm或30-40cm)。
在某些优选的实施方案中，所述管状生物构建体的内径为1mm-3cm(例如1-6mm、6-8mm、8-10mm、10-12mm、12mm-3cm)。
在某些优选的实施方案中，所述管状生物构建体的厚度为200μm-1mm(例如200μm-400μm、400μm-600μm、600μm-800μm或800μm-1mm)。
在某些优选的实施方案中，所述管状生物构建体为侧壁开口的圆形管状，所述开口沿轴线方向贯通管状生物构建体的两端，所述管状生物构建体沿径向的切面为扇环；在某些优选的实施方案中，所述扇环的圆心角大于0并且小于360°。
在某些优选的实施方案中，所述人工组织前体为片状，所述固体支持物为片状固体支持物，多个所述微囊构成一个或多个片状生物构建体，并且至少一个片状生物构建体与片状固体支持物贴合。
在某些优选的实施方案中，所述片状固体支持物为平面的片状或者弯曲的片状。
在某些优选的实施方案中，所述片状生物构建体为平面的片状或者弯曲的片状。
在某些优选的实施方案中，所述人工组织前体包含片状固体支持物和一个片状生物构建体，所述片状生物构建体的一面与片状固体支持物的一面贴合。
在某些优选的实施方案中，所述人工组织前体包含片状固体支持物和多个片状生物构 建体，多个所述片状生物构建体位于片状固体支持物的一侧，并且各个片状生物构建体的一面均与片状固体支持物的一面贴合。
在某些优选的实施方案中，所述人工组织前体包含片状固体支持物和多个片状生物构建体，多个所述片状生物构建体在片状固体支持物的一侧层叠排布，并且至少一个片状生物构建体的一面与片状固体支持物的一面贴合。
本发明中，根据需要对片状人工组织前体及其包含的片状生物构建体和片状固体支持物的尺寸进行设置。
在某些优选的实施方案中，所述人工组织前体为圆形片状、椭圆形片状、平行四边形(例如矩形)片状、扇形片状或不规则片状。
在某些优选的实施方案中，所述人工组织前体的厚度为0.5mm-3mm(例如0.5mm-1mm、1mm-2mm或2mm-3mm)。
在某些优选的实施方案中，所述人工组织前体的面积为0.5cm2-5cm2(例如0.5cm2-1cm2、1cm2-1.5cm2、1.5cm2-2.5cm2、2.5cm2-2.5cm2或3.5cm2-5cm2)。
在某些优选的实施方案中，所述人工组织前体具有均一或不均一的厚度。例如，所述片状固体支持物的某一部分贴合有片状生物构建体，而其他部分没有贴合片状生物构建体。例如，所述片状固体支持物的不同部分贴合有不同的片状生物构建体。
在某些优选的实施方案中，所述片状固体支持物为圆形片状、椭圆形片状、平行四边形(例如矩形)片状、扇形片状或不规则片状，或近似于圆形、椭圆形、平行四边形(例如矩形)或扇形的片状。
在某些优选的实施方案中，所述片状固体支持物的厚度为0.5mm-3mm(例如0.5mm-1mm、1mm-2mm或2mm-3mm)。
在某些优选的实施方案中，所述片状固体支持物的面积为0.5cm2-5cm2(例如0.5cm2-1cm2、1cm2-1.5cm2、1.5cm2-2.5cm2、2.5cm2-2.5cm2或3.5cm2-5cm2)。
所述片状生物构建体为圆形片状、椭圆形片状、平行四边形(例如矩形)片状、扇形片状或不规则片状，或近似于圆形、椭圆形、平行四边形(例如矩形)或扇形的片状。
在某些优选的实施方案中，所述片状生物构建体的厚度为20μm-3mm(例如20μm-100μm、100μm-500μm、500μm-1mm、1mm-2mm或2mm-3mm)。
在某些优选的实施方案中，所述片状生物构建体的面积为0.5cm2-5cm2(例如0.5 cm2-1cm2、1cm2-1.5cm2、1.5cm2-2.5cm2、2.5cm2-2.5cm2或3.5cm2-5cm2)。
在某些优选的实施方案中，本发明的人工组织前体中，至少一个微囊或者至少一个生物构建体与所述固体支持物固定在一起。
在某些优选的实施方案中，至少一个微囊或者至少一个生物构建体与所述固体支持物通过化学方式贴合。
在某些优选的实施方案中，至少一个生物构建体与所述固体支持物通过粘合剂粘合；
更优选地，所述粘合剂为医用胶。
可用于本发明的医用胶包括但不限于：
软组织类医用粘合剂，例如：以2-氰基丙烯酸辛酯为主体的组织粘合剂；纤维蛋白粘合剂(FS，主要包含纤维蛋白原+凝血酶、Ca2+、VIII因子)；
硬组织类医用粘合剂，例如：用于牙科的合成树脂胶黏剂，例如(1)甲基丙烯酸酯系:4-EMTA(4-甲基丙烯酰氧乙基偏苯三酸酐酯)、phenyp(甲基丙烯酰氧乙基苯基磷酸酯)、配合磷酸酯的Bis-GMA(双甲基丙烯酸缩水甘油酯)等，多用于龋齿填充和牙质粘接，(2)聚羧酸系，例如聚丙烯酸酯+氧化锌或特殊玻璃填料，多用于填充齿洞及粘接有机玻璃补修；骨粘固剂(俗称骨水泥，成分主要为丙烯酸粘固剂(acrylic cement)、聚甲基丙烯酸甲酯(polymethyl methacrylic，PMMA)等。
优选地，所述医用胶包含α-氰基丙烯酸酯(例如α-氰基丙烯酸甲酯，α-氰基丙烯酸乙酯，α-氰基丙烯酸异丁酯，α-氰基丙烯酸异己酯，α-氰基丙烯酸辛酯，例如α-氰基丙烯酸正辛酯)。
在某些优选的实施方案中，所述粘合剂存在于市售的医用胶中，例如白云医用胶EC型(主要成分：α-氰基丙烯酸正辛酯(508)为主体胶，添加附加剂(医用级聚甲基丙烯酸甲酯))；或者福爱乐医用胶(成分：99％的α-氰基丙烯酸正丁酯(NBCA/504)和1％的α-氰基丙烯酸正辛酯(NOCA/508))。
在某些优选的实施方案中，可以调整医用胶的浓度以调整医用胶的凝固时间，以达到良好的粘合效果。可以用适当的溶剂稀释医用胶，例如将医用胶用乙酸乙酯稀释。溶剂可以选择医用级酯类溶剂，如医用级乙酸乙酯，医用级聚甲基丙烯酸甲酯。
另一方面，本申请提供了以下多种制备如上所述的人工组织前体的方法。
方法1：人工组织前体为管状，制备所述人工组织前体的方法包含以下步骤：
(I)制备管状(例如圆形管状；例如侧壁开口或不开口的管状)生物构建体；
(II)将管状生物构建体与管状固体支持物的内壁进行贴合；
在某些优选的实施方案中，制备管状生物构建体通过包含以下步骤的方法进行：
(1)提供一种或多种微囊，其全部或者部分表面附着有第一组分；优选地，所述第一组分包含于第一试剂中；
(2)在临时支持物表面的预设区域上涂布含有第二组分的第二试剂，其中，当所述第一组分与第二组分接触时，能够产生粘连效果，实现粘合作用；所述临时支持物为管状或柱状(例如侧壁不开口的圆形管状、侧壁开口的圆形管状、圆柱状或沿部分圆周设置的柱状)物，所述预设区域位于临时支持物的曲面；任选地，在涂布第二试剂之前，将衬底材料涂布在临时支持物表面的预设区域上；
(3)将步骤(1)中的全部或者部分表面附着有第一组分的微囊放置于涂布了第二试剂的预设区域，使所述微囊表面上的第一组分与预设区域上的第二组分接触，产生粘连效果，从而将所述微囊组装(粘合)成第一层结构，所述第一层结构为管状结构；
任选地，所述方法还包括以下步骤：
(4)在前一步骤产生的结构上涂布第二试剂；
(5)将步骤(1)中的全部或者部分表面附着有第一组分的微囊放置于前一步骤产生的结构上，使所述微囊表面上的第一组分与前一步骤产生的结构上的第二组分接触，产生粘连效果，从而在前一步骤产生的结构上，将所述微囊组装(粘合)成另一层结构；
(6)任选地，重复步骤(4)和(5)一次或多次；例如至少1次，至少2次，至少3次，至少4次，至少5次，至少10次，至少15次，至少20次，至少30次，至少40次，至少50次，至少100次，至少200次，至少500次，或更多次；
从而得到管状生物构建体。
任选地，所述方法还包括：将侧壁开口的圆形管状生物构建体进行粘合，得到侧壁不开口的圆形管状生物构建体。
任选地，所述方法还包括：将管状生物构建体从临时支持物上分离。
在某些优选的实施方案中，所述临时支持物为具有曲面的打印平台，例如3D打印机的旋转杆。
在某些优选的实施方案中，所述衬底材料是由温敏性材料，例如明胶、聚N-异丙基丙 烯酰胺、聚N-异丙基丙烯酰胺—聚乙二醇嵌段共聚物、聚乙二醇共聚物(例如聚乙烯醇-聚乙二醇共聚物)，聚羟乙基丙烯酸酯、琼脂糖、Matrigel、壳聚糖/甘油磷酸钠体系或Pluronic F127。
在某些优选的实施方案中，所述临时支持物为温敏性材料(例如明胶、聚N-异丙基丙烯酰胺、聚N-异丙基丙烯酰胺—聚乙二醇嵌段共聚物、聚乙二醇共聚物，聚羟乙基丙烯酸酯、琼脂糖、Matrigel、壳聚糖/甘油磷酸钠体系或Pluronic F127)制成的圆柱状物或圆管状物。
在某些优选的实施方案中，所述临时支持物为圆柱状物。图4A示例性描述了作为临时支持物的圆柱状物。
在某些优选的实施方案中，所述临时支持物为圆柱状物，所述预设区域为圆柱状物的整个侧面,如图4B所示，从而，步骤(3)得到的第一层结构为侧壁不开口的圆形管状结构。
在某些优选的实施方案中，所述临时支持物为圆柱状物，所述预设区域在展开的圆柱状物的侧面上为一矩形，并且所述预设区域沿圆柱状物的轴向贯通圆柱状物的侧面，如图4C所示，从而，步骤(3)得到的第一层结构为侧壁不开口的圆形管状结构。
在某些优选的实施方案中，所述临时支持物为圆柱状物，所述预设区域在展开的圆柱状物的侧面上为一矩形，并且所述预设区域沿圆柱状物的圆周方向贯通柱状物侧面，如图4D所示，从而，步骤(3)得到的第一层结构为侧壁不开口的圆形管状结构。
在某些优选的实施方案中，所述临时支持物为圆柱状物，所述预设区域在展开的圆柱状物的侧面上为一矩形，并且未沿圆柱状物的圆周方向和轴向贯通圆柱状物侧面，如图4E所示，从而，步骤(3)得到的第一层结构为侧壁开口的圆形管状结构。
在某些优选的实施方案中，步骤(3)中，在将全部或部分表面附着有第一组分的微囊放置于步骤(2)的涂布了第二试剂的预设区域之后，静置0.1-60s；(例如0.1-1s，1-5s，5-10s，10-15s，15-20s，20-25s，25-30s，30-35s，35-40s，40-45s，45-50s，50-55s，或55-60s)。该静置步骤有利于所述微囊表面上的第一组分与预设区域上的第二组分充分接触，并发生相互作用，从而将所述微囊组装(粘合)成第一层结构。
在某些优选的实施方案中，通过生物打印法来实施制备管状生物构建体的方法。
在某些优选的实施方案中，使用打印机(例如3D生物打印机)来进行生物打印法；或者，使用自动化或非自动化机械过程来进行生物打印法；或者，通过使用手工放置或手工沉积法(例如使用移液器)来进行生物打印法。
在某些优选的实施方案中，通过挤出式打印法或模块化打印法来对微囊进行打印。
在某些优选的实施方案中，使用模块化打印法，挤出式打印法，或喷墨式打印法来对所述第二试剂进行打印。
在某些优选的实施方案中，使用模块化打印法，挤出式打印法，或喷墨式打印法来对所述辅助材料进行打印。
在某些优选的实施方案中，使用3D生物打印机来制备生物构建体。
在某些优选的实施方案中，所述3D生物打印机包含：第一墨盒，其用于提供微囊；第二墨盒，其用于提供第二试剂；第一打印喷头；以及，连接至第二墨盒的第二打印喷头。
在某些优选的实施方案中，所述3D生物打印机还包含：第三墨盒，其用于提供衬底材料；以及，第三打印喷头。
在某些优选的实施方案中，所述3D生物打印机还包含：用于提供第一试剂的第四墨盒。
在某些优选的实施方案中，所述方法包括下述步骤：
(1)在3D生物打印机的第一墨盒中提供微囊，其全部或者部分表面附着有第一组分，并且在3D生物打印机的第二墨盒中提供含有第二组分的第二试剂；其中，当所述第一组分与第二组分接触时，能够产生粘连效果，实现粘合作用；
(2)通过3D生物打印机的连接至第二墨盒的第二打印喷头，在旋转杆曲面的预设区域上打印第二试剂；任选地，在打印第二试剂之前，将衬底材料打印在预设区域上；
(3)通过3D生物打印机的第一打印喷头，将步骤(1)中的微囊打印至步骤(2)的打印了第二试剂的预设区域上，使所述微囊表面上的第一组分与所述预设区域上的第二组分接触，产生粘连效果，从而将所述微囊组装(粘合)成第一层结构；
任选地，所述方法还包括下述步骤：
(4)通过所述第二打印喷头，在前一步骤获得的结构上打印第二试剂；
(5)通过所述第一打印喷头，将步骤(1)中的微囊打印至前一步骤产生的结构上，使所述微囊表面上的第一组分与所述结构上的第二组分接触，产生粘连效果，从而在前一步骤产生的结构上，将所述微囊组装(粘合)成另一层结构；
(6)任选地，重复步骤(4)和(5)一次或多次；例如至少1次，至少2次，至少3次，至少4次，至少5次，至少10次，至少15次，至少20次，至少30次，至少40次，至少50次，至少100次，至少200次，至少500次，或更多次；
从而，制备得到管状生物构建体。
方法2：人工组织前体为管状，制备所述人工组织前体的方法包含以下步骤：
(I)制备管状(例如圆形管状；例如侧壁开口或不开口的管状)生物构建体；
(II)将管状生物构建体与管状固体支持物的内壁进行贴合。
在某些优选的实施方案中，制备管状生物构建体通过包含以下步骤的方法进行：
(1)提供一种或多种微囊，其全部或者部分表面附着有第一组分；优选地，所述第一组分包含于第一试剂中；
(2)在临时支持物的表面上，用含有第二组分的第二试剂绘制预设的环形(例如圆环或扇环)图案，其中，当所述第一组分与第二组分接触时，能够产生粘连效果，实现粘合作用；所述临时支持物具有至少一个平面，所述环形图案位于临时支持物的平面；
(3)将步骤(1)中的全部或者部分表面附着有第一组分的微囊放置于第二试剂绘制的预设的环形图案上，使所述微囊表面上的第一组分与所述环形图案上的第二组分接触，产生粘连效果，从而将所述微囊组装(粘合)成第一层结构，所述第一层结构为环形结构；
(4)在环形结构上涂布第二试剂；
(5)将步骤(1)中的全部或者部分表面附着有第一组分的微囊放置于前一步骤产生的结构上，使所述微囊表面上的第一组分与前一步骤产生的结构上的第二组分接触，产生粘连效果，从而在前一步骤产生的结构上，将所述微囊组装(粘合)成另一层结构；
(6)任选地，重复步骤(4)和(5)一次或多次；例如至少1次，至少2次，至少3次，至少4次，至少5次，至少10次，至少15次，至少20次，至少30次，至少40次，至少50次，至少100次，至少200次，至少500次，或更多次；
从而得到管状生物构建体；
任选地，所述方法还包括：将侧壁开口的圆形管状生物构建体进行粘合，得到侧壁不开口的圆形管状生物构建体；
在某些优选的实施方案中，所述临时支持物为3D打印机的打印平台；
在某些优选的实施方案中，步骤(3)中，在将全部或部分表面附着有第一组分的微囊放置于步骤(2)的绘制的预设的环形图案上之后，静置0.1-60s；(例如0.1-1s，1-5s，5-10s，10-15s，15-20s，20-25s，25-30s，30-35s，35-40s，40-45s，45-50s，50-55s，或55-60s)。该静置步骤有利于所述微囊表面上的第一组分与预设区域上的第二组分充 分接触，并发生相互作用，从而将所述微囊组装(粘合)成第一层结构。
在某些优选的实施方案中，通过生物打印法来实施制备管状生物构建体的方法。
在某些优选的实施方案中，使用打印机(例如3D生物打印机)来进行生物打印法；或者，使用自动化或非自动化机械过程来进行生物打印法；或者，通过使用手工放置或手工沉积法(例如使用移液器)来进行生物打印法。
在某些优选的实施方案中，通过挤出式打印法或模块化打印法来对微囊进行打印。
在某些优选的实施方案中，使用模块化打印法，挤出式打印法，或喷墨式打印法来对所述第二试剂进行打印。
在某些优选的实施方案中，使用模块化打印法，挤出式打印法，或喷墨式打印法来对所述辅助材料进行打印。
其中，使用3D生物打印机来制备生物构建体；
在某些优选的实施方案中，所述3D生物打印机包含：第一墨盒，其用于提供微囊；第二墨盒，其用于提供第二试剂；第一打印喷头；以及，连接至第二墨盒的第二打印喷头。
在某些优选的实施方案中，所述3D生物打印机还包含：第三墨盒，其用于提供辅助材料；以及，第三打印喷头。
在某些优选的实施方案中，所述3D生物打印机还包含：用于提供第一试剂的第四墨盒。
在某些优选的实施方案中，所述方法包括下述步骤：
(1)在3D生物打印机的第一墨盒中提供微囊，其全部或者部分表面附着有第一组分，并且在3D生物打印机的第二墨盒中提供含有第二组分的第二试剂；其中，当所述第一组分与第二组分接触时，能够产生粘连效果，实现粘合作用；
(2)通过3D生物打印机的连接至第二墨盒的第二打印喷头，在打印平台上用所述第二试剂绘制环形(例如圆环或扇环)图案；
(3)通过3D生物打印机的第一打印喷头，将步骤(1)中的微囊打印至步骤(2)绘制的环形图案上，使所述微囊表面上的第一组分与所述环形图案上的第二组分接触，产生粘连效果，从而将所述微囊组装(粘合)成第一层结构；
任选地，所述方法还包括下述步骤：
(4)通过所述第二打印喷头，在前一步骤获得的结构上打印第二试剂；
(5)通过所述第一打印喷头，将步骤(1)中的微囊打印至前一步骤产生的结构上，使所述微囊表面上的第一组分与所述结构上的第二组分接触，产生粘连效果，从而在前一步骤产生的结构上，将所述微囊组装(粘合)成另一层结构；
(6)任选地，重复步骤(4)和(5)一次或多次；例如至少1次，至少2次，至少3次，至少4次，至少5次，至少10次，至少15次，至少20次，至少30次，至少40次，至少50次，至少100次，至少200次，至少500次，或更多次；
从而，制备得到管状生物构建体。
示例性的圆环和扇环图案如图5A所示。
方法3：人工组织前体为片状，制备所述人工组织前体的方法包含以下步骤：
(I)制备片状(例如平面的片状或弯曲的片状)生物构建体；
(II)将片状生物构建体与片状固体支持物进行贴合。
在某些优选的实施方案中，制备片状生物构建体通过包含以下步骤的方法进行：
(1)提供一种或多种微囊，其全部或者部分表面附着有第一组分；优选地，所述第一组分包含于第一试剂中；
(2)在临时支持物表面的预设区域上涂布含有第二组分的第二试剂，其中，当所述第一组分与第二组分接触时，能够产生粘连效果，实现粘合作用；所述临时支持物具有至少一个平面，所述预设的图案位于临时支持物的平面；
(3)将步骤(1)中的全部或者部分表面附着有第一组分的微囊放置于涂布了第二试剂的预设区域，使所述微囊表面上的第一组分与预设区域上的第二组分接触，产生粘连效果，从而将所述微囊组装(粘合)成第一层结构，所述第一层结构为平面的片状结构；
任选地，所述方法还包括以下步骤：
(4)在前一步骤产生的结构上涂布第二试剂；
(5)将步骤(1)中的全部或者部分表面附着有第一组分的微囊放置于前一步骤产生的结构上，使所述微囊表面上的第一组分与前一步骤产生的结构上的第二组分接触，产生粘连效果，从而在前一步骤产生的结构上，将所述微囊组装(粘合)成另一层结构；
(6)任选地，重复步骤(4)和(5)一次或多次；例如至少1次，至少2次，至少3次，至少4次，至少5次，至少10次，至少15次，至少20次，至少30次，至少40 次，至少50次，至少100次，至少200次，至少500次，或更多次；
从而得到平面的片状生物构建体。
任选地，所述方法还包括，对平面的片状生物构建体进行弯折，得到弯曲的片状生物构建体。
在某些优选的实施方案中，所述预设区域为平行四边形(例如矩形)区域、圆形区域、椭圆形区域、扇形区域或不规则形区域。
在某些优选的实施方案中，所述临时支持物为3D打印机的打印平台。
在某些优选的实施方案中，步骤(3)中，在将全部或部分表面附着有第一组分的微囊放置于步骤(2)的涂布了第二试剂的预设区域之后，静置0.1-60s。
在某些优选的实施方案中，步骤(3)中，在将全部或部分表面附着有第一组分的微囊放置于步骤(2)的涂布了第二试剂的预设区域之后，静置0.1-60s；(例如0.1-1s，1-5s，5-10s，10-15s，15-20s，20-25s，25-30s，30-35s，35-40s，40-45s，45-50s，50-55s，或55-60s)。该静置步骤有利于所述微囊表面上的第一组分与预设区域上的第二组分充分接触，并发生相互作用，从而将所述微囊组装(粘合)成第一层结构。
在某些优选的实施方案中，通过生物打印法来实施制备管状生物构建体的方法。
在某些优选的实施方案中，使用打印机(例如3D生物打印机)来进行生物打印法；或者，使用自动化或非自动化机械过程来进行生物打印法；或者，通过使用手工放置或手工沉积法(例如使用移液器)来进行生物打印法。
在某些优选的实施方案中，通过挤出式打印法或模块化打印法来对微囊进行打印。
在某些优选的实施方案中，使用模块化打印法，挤出式打印法，或喷墨式打印法来对所述第二试剂进行打印。
在某些优选的实施方案中，使用模块化打印法，挤出式打印法，或喷墨式打印法来对所述辅助材料进行打印。
在某些优选的实施方案中，使用3D生物打印机来制备生物构建体；
在某些优选的实施方案中，所述3D生物打印机包含：第一墨盒，其用于提供微囊；第二墨盒，其用于提供第二试剂；第一打印喷头；以及，连接至第二墨盒的第二打印喷头。
在某些优选的实施方案中，所述3D生物打印机还包含：用于提供第一试剂的第三墨盒。
在某些优选的实施方案中，所述方法包括下述步骤：
(1)在3D生物打印机的第一墨盒中提供微囊，其全部或者部分表面附着有第一组分， 并且在3D生物打印机的第二墨盒中提供含有第二组分的第二试剂；其中，当所述第一组分与第二组分接触时，能够产生粘连效果，实现粘合作用；
(2)通过3D生物打印机的连接至第二墨盒的第二打印喷头，在打印平台的预设区域上打印第二试剂；
(3)通过3D生物打印机的第一打印喷头，将步骤(1)中的微囊打印至步骤(2)的打印了第二试剂的预设区域上，使所述微囊表面上的第一组分与所述预设区域上的第二组分接触，产生粘连效果，从而将所述微囊组装(粘合)成第一层结构；
任选地，所述方法还包括下述步骤：
(4)通过所述第二打印喷头，在前一步骤获得的结构上打印第二试剂；
(5)通过所述第一打印喷头，将步骤(1)中的微囊打印至前一步骤产生的结构上，使所述微囊表面上的第一组分与所述结构上的第二组分接触，产生粘连效果，从而在前一步骤产生的结构上，将所述微囊组装(粘合)成另一层结构；
(6)任选地，重复步骤(4)和(5)一次或多次；例如至少1次，至少2次，至少3次，至少4次，至少5次，至少10次，至少15次，至少20次，至少30次，至少40次，至少50次，至少100次，至少200次，至少500次，或更多次；
从而，制备得到片状生物构建体。
任选地，所述方法还包括，对平面的片状生物构建体进行弯折，得到弯曲的片状生物构建体；
在某些优选的实施方案中，所述预设区域为平行四边形(例如矩形)区域、圆形区域或椭圆形区域。
在某些优选的实施方案中，所述临时支持物为3D打印机的打印平台。
在某些优选的实施方案中，步骤(3)中，在将全部或部分表面附着有第一组分的微囊放置于步骤(2)的涂布了第二试剂的预设区域之后，静置0.1-60s。
在某些优选的实施方案中，使用3D生物打印机来制备片状生物构建体。
在某些优选的实施方案中，所述3D生物打印机包含：第一墨盒，其用于提供微囊；第二墨盒，其用于提供第二试剂；第一打印喷头；以及，连接至第二墨盒的第二打印喷头。
在某些优选的实施方案中，所述3D生物打印机还包含：用于提供第一试剂的第三墨盒。
在某些优选的实施方案中，所述方法包括下述步骤：
(1)在3D生物打印机的第一墨盒中提供微囊，其全部或者部分表面附着有第一组分， 并且在3D生物打印机的第二墨盒中提供含有第二组分的第二试剂；其中，当所述第一组分与第二组分接触时，能够产生粘连效果，实现粘合作用；
(2)通过3D生物打印机的连接至第二墨盒的第二打印喷头，在打印平台的预设区域上打印第二试剂；
(3)通过3D生物打印机的第一打印喷头，将步骤(1)中的微囊打印至步骤(2)的打印了第二试剂的预设区域上，使所述微囊表面上的第一组分与所述预设区域上的第二组分接触，产生粘连效果，从而将所述微囊组装(粘合)成第一层结构；
任选地，所述方法还包括下述步骤：
(4)通过所述第二打印喷头，在前一步骤获得的结构上打印第二试剂；
(5)通过所述第一打印喷头，将步骤(1)中的微囊打印至前一步骤产生的结构上，使所述微囊表面上的第一组分与所述结构上的第二组分接触，产生粘连效果，从而在前一步骤产生的结构上，将所述微囊组装(粘合)成另一层结构；
(6)任选地，重复步骤(4)和(5)一次或多次；例如至少1次，至少2次，至少3次，至少4次，至少5次，至少10次，至少15次，至少20次，至少30次，至少40次，至少50次，至少100次，至少200次，至少500次，或更多次；
从而，制备得到片状生物构建体。
方法4：人工组织前体为片状，制备所述人工组织前体的方法包含以下步骤：
(I)由方法3中制备片状生物构建体的方法，制备片状生物构建体；
(II)提供制备固体支持物的材料(例如生物相容性材料)，在片状生物构建体上，制备片状固体支持物。
在某些优选的实施方案中，通过3D打印或喷涂的方法制备片状固体支持物。
方法5：人工组织前体为管状，制备所述人工组织前体的方法包含以下步骤：
(I)通过方法3中制备片状生物构建体方法，制备片状生物构建体；
(II)将步骤(I)制得的片状生物构建体进行弯折，和/或将片状生物构建体的边缘进行粘合，得到管状生物构建体；
(III)将管状生物构建体与管状固体支持物的内壁进行贴合。
方法6：人工组织前体为管状，制备所述人工组织前体的方法包含以下步骤：
(I)由方法1或方法2任一项中的制备管状生物构建体的方法，制备管状生物构建体；
或者，由方法3中的制备片状生物构建体的方法，制备片状生物构建体；之后，对制得的片状生物构建体进行弯折，和/或将片状生物构建体的边缘进行粘合，得到管状生物构建体；
(II)提供制备固体支持物的材料(例如生物相容性材料)，在管状生物构建体的外壁上，制备管状固体支持物；
在某些优选的实施方案中，通过3D打印或喷涂的方法制备管状固体支持物。
方法1-6任一项定义的制备人工组织前体的方法，还包括对生物构建体进行定型。
在某些优选的实施方案中，可以根据生物构建体的结构稳定性及厚度的需要，在生物构建体上喷涂定型剂(例如市售的含α-氰基丙烯酸酯的医用胶)。喷涂定型剂的层数越多，则生物构建体的结构越稳定，和/或具有更大的厚度。
在某些优选的实施方案中，对生物构建体进行定型的方法包括以下步骤：
1)在生物构建体的表面喷涂一层医用胶，待医用胶凝固；
2)在喷涂了医用胶的生物构建体的表面滴加并均匀涂抹细胞培养基；
3)再次喷涂医用胶，在培养基中阴离子的作用下，医用胶快速凝固；
4)任选地，重复步骤2)和3)。
可通过上述方法在生物构建体的表面形成医用胶层，使生物构建体稳定坚固。该方法还可用于调节生物构建体的厚度，方便与固体支持物相匹配。
方法7：人工组织前体为管状或片状，制备所述人工组织前体的方法包含以下步骤：
(1)提供一种或多种微囊，其全部或者部分表面附着有第一组分；在某些优选的实施方案中，所述第一组分包含于第一试剂中；
(2)提供固体支持物，在所述固体支持物表面的预设区域上涂布含有第二组分的第二试剂，其中，当所述第一组分与第二组分接触时，能够产生粘连效果，实现粘合作用；
(3)将步骤(1)中的全部或者部分表面附着有第一组分的微囊放置于涂布了第二试剂的预设区域，使所述微囊表面上的第一组分与预设区域上的第二组分接触，产生粘连效果，从而在固体支持物表面，将所述微囊组装(粘合)成第一层结构；
任选地，所述方法还包括以下步骤：
(4)在前一步骤产生的结构上涂布第二试剂；
(5)将步骤(1)中的全部或者部分表面附着有第一组分的微囊放置于前一步骤产生的结构上，使所述微囊表面上的第一组分与前一步骤产生的结构上的第二组分接触，产生粘连效果，从而在前一步骤产生的结构上，将所述微囊组装(粘合)成另一层结构；
(6)任选地，重复步骤(4)和(5)一次或多次；例如至少1次，至少2次，至少3次，至少4次，至少5次，至少10次，至少15次，至少20次，至少30次，至少40次，至少50次，至少100次，至少200次，至少500次，或更多次；
从而得到人工组织前体；
在某些优选的实施方案中，所述固体支持物为管状或片状；
在某些优选的实施方案中，所述固体支持物为管状固体支持物，所述预设区域位于固体支持物的内壁；
在某些优选的实施方案中，步骤(3)中，在将全部或部分表面附着有第一组分的微囊放置于步骤(2)的涂布了第二试剂的预设区域之后，静置0.1-60s；(例如0.1-1s，1-5s，5-10s，10-15s，15-20s，20-25s，25-30s，30-35s，35-40s，40-45s，45-50s，50-55s，或55-60s)。该静置步骤有利于所述微囊表面上的第一组分与预设区域上的第二组分充分接触，并发生相互作用，从而将所述微囊组装(粘合)成第一层结构。
在某些优选的实施方案中，使用3D生物打印机来制备人工组织前体。
在某些优选的实施方案中，所述3D生物打印机包含：第一墨盒，其用于提供微囊；第二墨盒，其用于提供第二试剂；第一打印喷头；以及，连接至第二墨盒的第二打印喷头。
在某些优选的实施方案中，所述3D生物打印机还包含：用于提供第一试剂的第三墨盒。
在某些优选的实施方案中，所述方法包括下述步骤：
(1)在3D生物打印机的第一墨盒中提供微囊，其全部或者部分表面附着有第一组分，并且在3D生物打印机的第二墨盒中提供含有第二组分的第二试剂；其中，当所述第一组分与第二组分接触时，能够产生粘连效果，实现粘合作用；
(2)通过3D生物打印机的连接至第二墨盒的第二打印喷头，在固体支持物的预设区域上打印第二试剂；
(3)通过3D生物打印机的第一打印喷头，将步骤(1)中的微囊打印至步骤(2)的打印了第二试剂的预设区域上，使所述微囊表面上的第一组分与所述预设区域上的第二组分接触，产生粘连效果，从而将所述微囊组装(粘合)成第一层结构；
任选地，所述方法还包括下述步骤：
(4)通过所述第二打印喷头，在前一步骤获得的结构上打印第二试剂；
(5)通过所述第一打印喷头，将步骤(1)中的微囊打印至前一步骤产生的结构上，使所述微囊表面上的第一组分与所述结构上的第二组分接触，产生粘连效果，从而在前一步骤产生的结构上，将所述微囊组装(粘合)成另一层结构；
(6)任选地，重复步骤(4)和(5)一次或多次；例如至少1次，至少2次，至少3次，至少4次，至少5次，至少10次，至少15次，至少20次，至少30次，至少40次，至少50次，至少100次，至少200次，至少500次，或更多次；
从而，制备得到人工组织前体。
在本发明的制备的人工组织前体的方法中，优选地，所述第一组分和/或第二组分为生物相容性材料。在某些优选的实施方案中，所述第一组分和/或第二组分为来源于生物的材料。在某些优选的实施方案中，所述第一组分和/或第二组分为生物可降解材料。
在某些优选的实施方案中，所述第一组分与第二组分在接触时能够发生强烈相互作用(例如，能够发生化学反应)，并产生粘连效果，实现粘合作用。此类粘合作用不仅能够实现细胞与细胞之间、细胞与组织之间、组织与组织之间的粘合，而且能够实现细胞/组织与外部物质之间的粘合。特别优选地，此类粘合作用具备至少一种选自以下的性质：(1)其是安全、可靠、无毒性、不致癌、不致畸、不致突变的；(2)其具有良好的生物相容性，不妨碍机体组织的自身愈合；(3)其能够在有血液和组织液的条件下使用；(4)其能够在常温、常压下实现快速粘合；(5)其具有良好的粘合强度及持久性，粘合部分具有一定的弹性和韧性；(6)其在使用过程中对机体组织无刺激性；(7)在达到粘合效果后，相关的组分能够逐渐被降解和吸收；和，(8)粘合部位能够被细胞迁移通过。
在某些优选实施方案中，所述第一组分与第二组分接触而产生的粘连效果可用于将两个微囊粘合在一起，形成生物构建体；并且由此所获得的生物构建体的拉伸模量不低于10Pa，例如不低于20Pa，不低于30Pa，不低于40Pa，不低于50Pa，不低于60Pa，不低于70Pa，不低于80Pa，不低于90Pa，不低于100Pa，不低于200Pa，不低于300 Pa，不低于400Pa，不低于500Pa，不低于600Pa，不低于700Pa，不低于800Pa，不低于900Pa或不低于1000Pa。在某些优选实施方案中，所获得的构建体的拉伸模量可达到1KPa-10Mpa，例如1-5KPa，5-10KPa，10-50KPa，50-100KPa，100-500KPa，500-1000KPa，1-5MPa，或5-10MPa。在某些优选实施方案中，微囊中的细胞能够迁移通过粘合部位，进入邻近的微囊中或者更远端的微囊中。由此，微囊中的细胞能够在整个构建体内的生长、迁移、分化和增殖。
在某些优选的实施方案中，所述第一组分和第二组分是选自下列的组合：
(1)纤维蛋白原和凝血酶；
(2)海藻酸盐(例如海藻酸钠)或氧化的海藻酸盐(例如氧化的海藻酸钠)和含有Ca2+、Mg2+、Ba2+、Sr2+、或Fe3+的物质(例如含有Ca2+、Mg2+、Ba2+、Sr2+、或Fe3+的溶液或半固体(例如凝胶))；
(3)含马来酰亚胺基团的分子(例如含有马来酰亚胺基团的聚乙二醇(MAL-PEG))和含自由巯基的分子(例如含有自由巯基的聚乙二醇(PEG-SH))；
(4)含有阴离子的物质(例如含有阴离子的溶液或半固体(例如凝胶))和α-氰基丙烯酸酯(例如α-氰基丙烯酸甲酯，α-氰基丙烯酸乙酯，α-氰基丙烯酸异丁酯，α-氰基丙烯酸异己酯，α-氰基丙烯酸正辛酯)；
(5)纤维蛋白原和α-氰基丙烯酸酯(例如α-氰基丙烯酸甲酯，α-氰基丙烯酸乙酯，α-氰基丙烯酸异丁酯，α-氰基丙烯酸异己酯，α-氰基丙烯酸正辛酯)；
(6)血清白蛋白(例如，牛血清白蛋白)和戊二醛；
(7)含氨基甲酸酯基团(-NHCOO-)或异氰酸酯基团(-NCO)的分子(例如含氨基甲酸酯基团的聚乙二醇或含异氰酸酯基团的聚乙二醇)和含活泼氢的分子(例如含羧基的聚乙二醇)；
(8)明胶-间苯二酚和戊二醛；
(9)碳化二亚胺交联明胶和聚L-谷氨酸(PLGA)；和
(10)胺基化明胶和醛基化多糖。
应当特别指出的是，只要所述第一组分与第二组分接触时，能够产生粘连效果，实现粘合作用，即可用于实施本发明的实施方案。本发明的第一组分与第二组分并不局限于上述特定的组合。此外，当将某一组合用作第一组分和第二组分时，第一组分可以是该组合的任一成员，且第二组分是该组合的另一成员。例如，当使用纤维蛋白原和凝血 酶这个组合时，第一组分可以是纤维蛋白原(此时第二组分为凝血酶)，或者可以是凝血酶(此时第二组分为纤维蛋白原)。
在某些优选的实施方案中，所述第一组分为纤维蛋白原，且第二组分为凝血酶。在某些优选的实施方案中，所述第一组分为海藻酸盐(例如海藻酸钠)或氧化的海藻酸盐(例如氧化的海藻酸钠)，且第二组分为含有Ca2+、Mg2+、Ba2+、Sr2+、或Fe3+的物质，例如含有Ca2+、Mg2+、Ba2+、Sr2+、或Fe3+的溶液或半固体(例如凝胶)。在某些优选的实施方案中，所述第一组分为含马来酰亚胺基团的分子(例如含有马来酰亚胺基团的聚乙二醇(MAL-PEG))，且第二组分为含自由巯基的分子(例如含有自由巯基的聚乙二醇(PEG-SH))。在某些优选的实施方案中，所述第一组分为含有阴离子的物质，例如含有阴离子的溶液或半固体(例如凝胶)，且第二组分为α-氰基丙烯酸酯(例如α-氰基丙烯酸甲酯，α-氰基丙烯酸乙酯，α-氰基丙烯酸异丁酯，α-氰基丙烯酸异己酯，α-氰基丙烯酸正辛酯)。在某些优选的实施方案中，所述第一组分为纤维蛋白原，且第二组分为α-氰基丙烯酸酯(例如α-氰基丙烯酸甲酯，α-氰基丙烯酸乙酯，α-氰基丙烯酸异丁酯，α-氰基丙烯酸异己酯，α-氰基丙烯酸正辛酯)。在某些优选的实施方案中，所述第一组分为血清白蛋白(例如，牛血清白蛋白)，且第二组分为戊二醛。在某些优选的实施方案中，所述第一组分为含氨基甲酸酯基团(-NHCOO-)或异氰酸酯基团(-NCO)的分子(例如含氨基甲酸酯基团的聚乙二醇或含异氰酸酯基团的聚乙二醇)，且第二组分为含活泼氢的分子(例如含羧基的聚乙二醇)。在某些优选的实施方案中，所述第一组分为明胶-间苯二酚，且第二组分为戊二醛。在某些优选的实施方案中，所述第一组分为碳化二亚胺交联明胶，且第二组分为聚L-谷氨酸(PLGA)。在某些优选的实施方案中，所述第一组分为胺基化明胶，且第二组分为醛基化多糖。
在某些优选的实施方案中，在所述第一试剂中，第一组分的浓度为0.01-50wt％。例如，在某些优选的实施方案中，所述第一组分的浓度为0.01-0.05wt％，0.05-0.1wt％，0.1-0.5wt％，0.5-1wt％，1-5wt％，5-10wt％，10-15wt％，15-20wt％，20-25wt％，25-30wt％，30-35wt％，35-40wt％，40-45wt％，或者45-50wt％。
在某些优选的实施方案中，在所述第二试剂中，第二组分的浓度为0.01-50wt％。例如，在某些优选的实施方案中，所述第二组分的浓度为0.01-0.05wt％，0.05-0.1wt％，0.1-0.5wt％，0.5-1wt％，1-5wt％，5-10wt％，10-15wt％，15-20wt％，20-25wt％，25-30wt％，30-35wt％，35-40wt％，40-45wt％，或者45-50wt％。
在某些优选的实施方案中，可通过选择第一组分和第二组分的类别和/或浓度来控制粘合作用的强度和/或持续时间。例如，当纤维蛋白原和凝血酶接触时，二者能够发生相互作用并产生力学强度较弱的纤维蛋白。因此，在某些优选的实施方案中，可将纤维蛋白原和凝血酶用作第一组分和第二组分，并且，此类试剂特别适合用于构建力学强度较小的组织，如弹性模量小于10MPa的组织。例如，α-氰基丙烯酸酯能够与含有阴离子的溶液发生强烈的聚合反应，生成力学强度较大的聚合物。因此，在某些优选的实施方案中，可将含有阴离子的物质和α-氰基丙烯酸酯用作第一组分和第二组分，并且，此类试剂盒特别适合用于构建力学强度较大的组织，如弹性模量大于10MPa的组织。
在某些优选的实施方案中，所述第二试剂为液体或半固体(例如凝胶)。在某些优选的实施方案中，第二试剂用于绘制预设的图案或涂布在预设的区域。因此，特别优选地，所述第二试剂具有适当的粘度，以便其在用于绘制图案时，能够稳定维持图案或区域的形状/模式/轮廓，而不任意流动。因此，在某些优选的实施方案中，所述第二试剂的粘度为1-1000Pa·s，例如30-160Pa·s。在某些优选的实施方案中，所述第二试剂的粘度为约1、2、3、4、5、6、7、8、9、10、12、14、16、18、20、25、30、50、80、100、200、300、400、500、800、或1000Pa·s。在某些优选的实施方案中，所述第二试剂的粘度为1-2、2-3、3-4、4-5、5-6、6-7、7-8、8-9、9-10、10-12、12-14、14-16、16-18、18-20、20-25、25-30、30-50、50-80、80-100、100-200、200-300、300-400、400-500、500-800、或800-1000、1-3、3-8、8-16、3-10、10-20、20-50、50-160Pa·s、或30-160Pa·s。
在某些优选的实施方案中，所述第二试剂还含有第三组分，所述第三组分为粘性剂。可通过调整第三组分(粘性剂)的含量来方便地调整第二试剂的粘度，以便第二试剂能够维持特定的形状，从而适合用于绘制图案或进行涂布。在某些优选的实施方案中，所述第三组分为生物相容性材料。在某些优选的实施方案中，所述第三组分为来源于生物的材料。在某些优选的实施方案中，所述第三组分为生物可降解材料。在某些优选的实施方案中，所述第三组分为温敏性材料。在某些优选的实施方案中，所述温敏性材料在不同的温度下具有不同的形态。例如，所述温敏性材料(例如明胶)在较低的温度下呈固态或半固态，而在较高的温度下呈液态。在某些优选的实施方案中，所述温敏性材料的相变温度在5-40℃之间，例如5-10℃，10-15℃，15-20℃，20-25℃，25-30℃，30-35℃，35-40℃。在某些优选的实施方案中，所述温敏性材料选自明胶，聚N-异丙基丙烯酰胺-聚乙二醇嵌段共聚物，聚乙二醇共聚物(例如聚乙烯醇-聚乙二醇共聚物)，琼脂糖， Matrigel，壳聚糖/甘油磷酸钠体系，Pluronic F127，和聚N-异丙基丙烯酰胺(PNIPAAm)水凝胶。在某些优选的实施方案中，所述第三组分(粘性剂)选自明胶、嵌段聚合物F-127、琼脂糖、聚乙二醇、瓜尔胶、聚乙烯醇、壳聚糖、胶原、透明质酸、甲壳素、纤维素及其衍生物(例如羟丙基纤维素)、聚氨基酸、聚N-异丙基丙烯酰胺-聚乙二醇嵌段共聚物、聚乙二醇共聚物(例如聚乙烯醇-聚乙二醇共聚物)、海藻酸盐(例如海藻酸钠)、改性的海藻酸盐(例如氧化的海藻酸盐，例如氧化的海藻酸钠)、Matrigel、壳聚糖/甘油磷酸钠体系、和聚N-异丙基丙烯酰胺(PNIPAAm)水凝胶。
在某些优选的实施方案中，所述第三组分(粘性剂)为明胶。
在某些优选的实施方案中，在所述第二试剂中，第三组分的浓度为0.01-50wt％。例如，在某些优选的实施方案中，所述第三组分的浓度为0.01-0.05wt％，0.05-0.1wt％，0.1-0.5wt％，0.5-1wt％，1-5wt％，5-10wt％，10-15wt％，15-20wt％，20-25wt％，25-30wt％，30-35wt％，35-40wt％，40-45wt％，或者45-50wt％。
在某些优选实施方案中，在步骤(1)中，全部或部分表面附着有第一组分的微囊是通过将包含所述第一组分的第一试剂涂布于所述微囊的表面而获得的。因此，在某些优选实施方案中，步骤(1)包括，将所述第一组分涂布于所述微囊的全部或部分表面，从而提供全部或部分表面附着有所述第一试剂的微囊。
在某些优选实施方案中，在步骤(1)中，全部或者部分表面附着有第一组分的微囊是通过将所述微囊在包含所述第一组分的第一试剂中浸渍而获得的。
在某些优选实施方案中，将所述微囊在第一试剂中浸渍1-30min，例如1-5min，5-10min，10-15min，15-20min，20-25min，或25-30min。在某些优选实施方案中，在步骤(1)中，在摇晃或振荡的条件下，将所述微囊在第一试剂中浸渍。摇晃或振荡条件可用于促进第一试剂附着至微囊表面。在某些优选实施方案中，步骤(1)是在室温条件(例如15-37℃)下进行的。在某些优选实施方案中，步骤(1)是在低温条件(例如4-15℃)下进行的。
在某些优选实施方案中，步骤(1)还包括，在浸渍于第一试剂中后，洗涤所述微囊。在某些优选实施方案中，使用缓冲液(例如生理缓冲溶液)或培养基溶液来洗涤所述微囊。在某些优选实施方案中，在浸渍于第一试剂中后，通过将所述微囊浸于缓冲液(例如生理缓冲溶液)或培养基溶液中来洗涤所述微囊。洗涤步骤可以用于去除微囊表面所 附着的过量第一试剂。在某些优选实施方案中，洗涤步骤可进行1-5min或5-10min。在某些优选实施方案中，洗涤步骤可在室温条件(例如15-37℃)下或低温条件(例如4-15℃)下进行。
在某些优选实施方案中，步骤(3)是在室温条件(例如15-37℃)下或者在低温条件(例如4-15℃)下进行的。
在某些优选实施方案中，在步骤(5)中，在将全部或部分表面附着有第一组分的微囊放置于前一步骤产生的结构上后，静置0.1-60s(例如0.1-1s，1-5s，5-10s，10-15s，15-20s，20-25s，25-30s，30-35s，35-40s，40-45s，45-50s，50-55s，或55-60s)。该静置步骤有利于所述微囊表面上的第一试剂与所述结构上的第二试剂充分接触，并发生相互作用，从而在前一步骤产生的结构上，将所述微囊组装(粘合)成新的结构层。在某些优选实施方案中，步骤(6)是在室温条件(例如15-37℃)下或者在低温条件(例如4-15℃)下进行的。
在某些优选实施方案中，在进行步骤(2)-(6)期间，还在所产生的结构内部或外周添加辅助材料(例如用于形成支架的辅助材料)。(例如用于支撑的辅助材料)。在某些优选实施方案中，所述辅助材料不包含细胞。优选地，此类辅助材料的添加/使用能够帮助限定所产生的人工组织前体的形状，和/或帮助维持或增强所产生的人工组织前体的稳定性。在某些优选实施方案中，所述辅助材料包含于本发明方法所制备的人工组织前体中。在某些优选实施方案中，所述辅助材料包含于本发明方法所制备的人工组织前体中，并且其随后能够被降解。在此类情况下，所述辅助材料仅仅是暂时性地构成了人工组织前体的一部分。在某些优选实施方案中，所述辅助材料包含于本发明方法所制备的人工组织前体中，并且其是不可降解的。在此类情况下，所述辅助材料直接(稳定地)构成了人工组织前体的一部分。在某些优选实施方案中，此类辅助材料是生物相容性和/或生物可降解的。在某些优选实施方案中，所述辅助材料是为温敏性材料。在某些优选的实施方案中，所述温敏性材料在不同的温度下具有不同的形态。例如，所述温敏性材料(例如明胶)在较低的温度下呈固态或半固态，而在较高的温度下呈液态。在某些优选的实施方案中，所述温敏性材料的相变温度在5-40℃之间，例如5-10℃，10-15℃，15-20℃，20-25℃，25-30℃，30-35℃，35-40℃。在某些优选实施方案中，所述温敏性材料选自明胶，聚N-异丙基丙烯酰胺-聚乙二醇嵌段共聚物，聚乙二醇共聚物(例如聚乙烯醇-聚乙二醇共聚物)，聚羟乙基丙烯酸酯，琼脂糖，Matrigel，壳聚糖/甘油磷酸钠 体系，Pluronic F127，和聚N-异丙基丙烯酰胺(PNIPAAm)水凝胶。
在某些优选的实施方案中，所述辅助材料可以具有任何想要的尺寸。在某些优选的实施方案中，所述辅助材料具有微米至厘米级别的尺寸，例如1μm-10cm，例如，1μm-2μm，2μm-3μm，3μm-4μm，4μm-5μm，5μm-6μm，6μm-7μm，7μm-8μm，8μm-9μm，9μm-10μm，10μm-20μm，20μm-30μm，30μm-40μm，40μm-50μm，50μm-60μm，60μm-70μm，70μm-80μm，80μm-90μm，90μm-100μm，100μm-200μm，200μm-300μm，300μm-400μm，400μm-500μm，500μm-600μm，600μm-700μm，700μm-800μm，800μm-900μm，900μm-1mm，1mm-2mm，2mm-3mm，3mm-4mm，4mm-5mm，5mm-6mm，6mm-7mm，7mm-8mm，8mm-9mm，9mm-10mm，10mm-20mm，20mm-30mm，30mm-40mm，40mm-50mm，50mm-60mm，60mm-70mm，70mm-80mm，80mm-90mm，90mm-100mm，100μm-5mm，500μm-1mm，100μm-800μm，300μm-600μm。
在某些优选的实施方案中，所述辅助材料可以具有任何想要的形状。例如，所述辅助材料可以是片状结构(例如长方形，正方形，圆形，椭圆形，六角形或不规则形状的片状结构)，或中空管状结构，或中空三维结构(例如中空立方体，中空球体，中空的矩形棱柱体，中空圆柱体，或中空的不规则形状的三维结构)，或实心三维结构(例如实心立方体，实心球体，实心矩形棱柱体，实心圆柱体，或实心不规则形状的三维结构)，或其任何组合。在某些优选的实施方案中，所述辅助材料的形状模拟天然组织或器官的形状。
在某些优选的实施方案中，用于制备生物构建体的微囊存在于生物墨汁中。在某些优选的实施方案中，所述生物墨汁还包含载体。
在某些优选的实施方案中，所述载体及其降解产物对于细胞是无毒的，和/或对于宿主是非免疫原性的。在某些优选的实施方案中，所述载体包含生物可降解材料。在某些优选的实施方案中，所述载体中的生物可降解材料是生物相容性的。
在某些优选的实施方案中，所述载体中的生物可降解材料的降解能够提供维持或促进微囊(例如生物砖)内的细胞的生命活动的微环境，例如营养物质。在某些优选的实施方案中，降解产物为小分子化合物，例如有机酸、单糖(例如葡萄糖)、寡糖、氨基酸、脂质等。此类降解产物可参与到细胞的新陈代谢活动中(例如用于合成细胞外基质)，用于合成细胞外基质或转化为活动所需的能量。
在某些优选的实施方案中，所述载体中的生物可降解材料是天然存在的(例如来源于 动植物的天然存在的生物可降解材料，例如胶原蛋白，纤维蛋白，壳聚糖，海藻酸盐，淀粉，透明质酸，层粘连蛋白，琼脂糖，明胶，葡聚糖，以及其任意组合)，人工合成的，重组产生的，经过改性的，或者其任何组合。
在某些优选的实施方案中，所述载体中的生物可降解材料是天然存在的可降解聚合物。优选地，所述可降解聚合物选自胶原蛋白，纤维蛋白，壳聚糖，海藻酸盐，淀粉，透明质酸，层粘连蛋白，明胶，葡聚糖，弹性蛋白，以及其任意组合。
在某些优选的实施方案中，所述载体中的生物可降解材料是经过改性的可降解聚合物，例如经过改性的海藻酸盐，例如氧化海藻酸盐(例如氧化海藻酸钠)。
在某些优选的实施方案中，所述载体中的生物可降解材料是合成的可降解聚合物。此类可降解聚合物包括但不限于，聚磷腈，聚丙烯酸及其衍生物(例如聚甲基丙烯酸，丙烯酸和甲基丙烯酸的共聚物)，聚乳酸(PLA)，聚羟基乙酸(PGA)，聚乳酸-乙醇酸共聚物(PLGA)，聚原酸酯(POE)，聚己内酯(PCL)，聚羟基丁酸酯(PHB)，聚氨基酸(例如聚赖氨酸)，可降解性聚氨酯，以及其任何组合。
在某些优选的实施方案中，所述载体还包含水，无机盐，pH缓冲剂，稳定剂，防腐剂，或其任何组合。
在某些优选的实施方案中，所述载体促进微囊(例如生物砖)在构建体上的安置，和/或将生物砖固定在构建体上。
在某些优选的实施方案中，载体为液体或半液体(例如凝胶)。在某些优选的实施方案中，所述载体的粘度为1-1000Pas，例如30-160Pas。在某些优选的实施方案中，所述载体的粘度为约1、2、3、4、5、6、7、8、9、10、12、14、16、18、20、25、30、50、80、100、200、300、400、500、800、或1000Pas。在某些优选的实施方案中，所述载体的粘度为1-2、2-3、3-4、4-5、5-6、6-7、7-8、8-9、9-10、10-12、12-14、14-16、16-18、18-20、20-25、25-30、30-50、50-80、80-100、100-200、200-300、300-400、400-500、500-800、或800-1000、1-3、3-8、8-16、3-10、10-20、20-50、50-160Pas、或30-160Pas。
在某些优选的实施方案中，步骤(3)中使用的全部或部分表面附着有第一组分的微囊与步骤(5)中使用的全部或部分表面附着有第一组分的微囊可以是相同或不同的。在步骤(5)使用与步骤(3)不同的全部或部分表面附着有第一组分微囊的情况下，可在进行步骤(5)之前，提供所述不同的微囊并使微囊的全部或部分表面附着有第一组分， 从而提供与步骤(3)不同的全部或部分表面附着有第一组分的微囊。
在通常情况下，根据想要制备的人工组织前体的细胞分布模式来选择步骤(3)和步骤(5)中使用的微囊(特别是，微囊中包含的细胞)。例如，当想要制备的管状生物构建体仅包含一种细胞时，可在步骤(3)和步骤(5)中使用包含相同细胞的微囊。当想要制备的管状生物构建体仅包含两种或更多种细胞时，可在步骤(3)和步骤(5)中使用两种或更多种的微囊，其各自包含不同的细胞或细胞组合；或者可在步骤(3)和步骤(5)中使用两种或更多种的微囊，其包含相同的未分化的细胞，但包含可将未分化的细胞诱导为不同成体细胞的诱导因子。
如上文中所详细描述的，本发明的第一组分和第二组分不局限于特定的组合。相应地，本发明的方法也不限于特定的第一组分和第二组分的组合。此外，对于每一轮的绘制/涂布，和微囊组装(例如，步骤(2)和(3)构成一轮的绘制/涂布，和微囊组装，且步骤(4)和(5)构成另一轮的绘制/涂布，和微囊组装)，可使用相同或不同的第一组分和第二组分的组合。例如，本发明方法的步骤(2)和(3)可以使用第一组分和第二组分的第一种组合，而步骤(4)和(5)可以使用相同的组合或者不同的组合(即，第一组分和第二组分的另一种组合)。
在某些实施方案中，例如方法7的某些实施方案中，步骤(2)和(3)使用的是第一组分和第二组分的第一种组合(例如含有阴离子的物质和α-氰基丙烯酸酯的组合)；而步骤(4)和(5)使用与步骤(2)和(3)不同的组合(例如与含有阴离子的物质和α-氰基丙烯酸酯的组合不同的组合，例如纤维蛋白原和凝血酶的组合)。
如上文所描述的，每一次的步骤(4)和(5)的重复各自构成了一轮的微囊组装。对于每一轮的微囊单元组装，可使用相同或不同的微囊；和/或，相同或不同的第一组分和第二组分的组合。在某些优选的实施方案中，对于每一轮的微囊组装，可通过更换相应墨盒中的墨汁，或者，可通过提供额外的墨盒，来实现微囊、包含第一组分的第一试剂和包含第二组分的第二试剂的更替。
在某些优选的实施方案中，本发明方法中的生物打印步骤(例如步骤(2)-(6))是连续的和/或基本连续的。在某些优选的实施方案中，在本发明方法的步骤(2)-(6)中，连续地生物打印多层结构，以获得具有预定模式的、包含多层结构的生物构建体或人工组织前体。在某些优选的实施方案中，在本发明方法的步骤(2)-(6)中，对于每一层结构，可使用相同或不同的微囊来进行打印。在某些优选的实施方案中，根据预定 的模式，使用一种或多种微囊来打印多层结构。在某些优选的实施方案中，在本发明方法的步骤(2)-(6)中，连续地生物打印多个区段，以获得具有预定模式的、包含多个区段的生物构建体或人工组织前体。在某些优选的实施方案中，在本发明方法的步骤(2)-(6)中，对于每一个区段，可使用相同或不同的微囊来进行打印。在某些优选的实施方案中，根据预定的模式，使用一种或多种微囊来打印多个区段。
在某些优选的实施方案中，本发明的人工组织前体，用于组织移植(例如管腔移植，例如血管移植)。在某些优选的实施方案中，在实施本发明的方法之前，获得组织或组织损伤位点的细胞分布信息。在某些优选的实施方案中，本发明的方法还包括，获得组织或组织损伤位点的细胞分布信息，然后根据所述细胞分布信息制备人工组织前体。在某些优选的实施方案中，本发明方法所使用的微囊中的细胞来源于受试者。在某些优选的实施方案中，本发明方法所使用的微囊中的细胞来源于与所述受试者具有相似或相同特征(例如，物种，年龄，性别，遗传信息等)的其他受试者。在某些优选的实施方案中，本发明方法所使用的微囊中的细胞来源于同种异体。在某些优选的实施方案中，本发明方法所使用的微囊中的细胞来源于细胞系。在某些优选的实施方案中，本发明的制备人工组织前体的方法在体外进行。
在本发明的某些实施方案中(例如方法1-3或方法5的某些实施方案中)，包括将生物构建体与固体支持物固定在一起。
在某些优选的实施方案中，将生物构建体与固体支持物通过化学方式贴合。
在某些优选的实施方案中，将生物构建体与固体支持物通过粘合剂粘合。
在某些优选的实施方案中，所述粘合剂为α-氰基丙烯酸酯(例如α-氰基丙烯酸甲酯，α-氰基丙烯酸乙酯，α-氰基丙烯酸异丁酯，α-氰基丙烯酸异己酯，α-氰基丙烯酸辛酯)。
在另一个方面，本申请还涉及一种生物构建体，其通过方法1、2或3中任一项定义的制备的生物构建体的方法制得。
在另一个方面，本申请还涉及一种用于制备人工组织前体的试剂盒，所述试剂盒包含微囊，以及彼此分离的第一试剂和第二试剂，其中，所述微囊包含细胞和包裹细胞的生物 相容性材料，所述第一试剂含有第一组分，所述第二试剂含有第二组分，并且当所述第一组分与第二组分接触时，能够产生粘连效果，实现粘合作用。
在某些优选的实施方案中，所述第一组分与第二组分在接触时能够发生强烈相互作用(例如，能够发生化学反应)，并产生粘连效果，实现粘合作用。此类粘合作用不仅能够实现细胞与细胞之间、细胞与组织之间、组织与组织之间的粘合，而且能够实现细胞/组织与外部物质之间的粘合。特别优选地，此类粘合作用具备至少一种选自以下的性质：(1)其是安全、可靠、无毒性、不致癌、不致畸、不致突变的；(2)其具有良好的生物相容性，不妨碍机体组织的自身愈合；(3)其能够在有血液和组织液的条件下使用；(4)其能够在常温、常压下实现快速粘合；(5)其具有良好的粘合强度及持久性，粘合部分具有一定的弹性和韧性；(6)其在使用过程中对机体组织无刺激性；(7)在达到粘合效果后，相关的组分能够逐渐被降解和吸收；和，(8)粘合部位能够被细胞迁移通过。
在某些优选实施方案中，所述第一组分与第二组分接触而产生的粘连效果可用于将两个微囊粘合在一起，形成构建体；并且由此所获得的构建体的拉伸模量不低于10Pa，例如不低于20Pa，不低于30Pa，不低于40Pa，不低于50Pa，不低于60Pa，不低于70Pa，不低于80Pa，不低于90Pa，不低于100Pa，不低于200Pa，不低于300Pa，不低于400Pa，不低于500Pa，不低于600Pa，不低于700Pa，不低于800Pa，不低于900Pa，不低于1000Pa。在某些优选实施方案中，所获得的构建体的拉伸模量可达到1KPa-10Mpa，例如1-5KPa，5-10KPa，10-50KPa，50-100KPa，100-500KPa，500-1000KPa，1-5MPa，或5-10MPa。在某些优选实施方案中，微囊中的细胞能够迁移通过粘合部位，进入邻近的微囊中或者更远端的微囊中。由此，微囊中的细胞能够在整个构建体内的生长、迁移、分化和增殖。
在某些优选的实施方案中，所述第一组分和第二组分是选自下列的组合：
(1)纤维蛋白原和凝血酶；
(2)海藻酸盐(例如海藻酸钠)或氧化的海藻酸盐(例如氧化的海藻酸钠)和含有Ca2+、Mg2+、Ba2+、Sr2+、或Fe3+的物质(例如含有Ca2+、Mg2+、Ba2+、Sr2+、或Fe3+的溶液或半固体(例如凝胶))；
(3)含马来酰亚胺基团的分子(例如含有马来酰亚胺基团的聚乙二醇(MAL-PEG))和含自由巯基的分子(例如含有自由巯基的聚乙二醇(PEG-SH))；
(4)含有阴离子的物质(例如含有阴离子的溶液或半固体(例如凝胶))和α-氰基 丙烯酸酯(例如α-氰基丙烯酸甲酯，α-氰基丙烯酸乙酯，α-氰基丙烯酸异丁酯，α-氰基丙烯酸异己酯，α-氰基丙烯酸正辛酯)；
(5)纤维蛋白原和α-氰基丙烯酸酯(例如α-氰基丙烯酸甲酯，α-氰基丙烯酸乙酯，α-氰基丙烯酸异丁酯，α-氰基丙烯酸异己酯，α-氰基丙烯酸正辛酯)；
(6)血清白蛋白(例如，牛血清白蛋白)和戊二醛；
(7)含氨基甲酸酯基团(-NHCOO-)或异氰酸酯基团(-NCO)的分子(例如含氨基甲酸酯基团的聚乙二醇或含异氰酸酯基团的聚乙二醇)和含活泼氢的分子(例如含羧基的聚乙二醇)；
(8)明胶-间苯二酚和戊二醛；
(9)碳化二亚胺交联明胶和聚L-谷氨酸(PLGA)；和
(10)胺基化明胶和醛基化多糖。
应当特别指出的是，只要所述第一组分与第二组分接触时，能够产生粘连效果，实现粘合作用，即可用于实施本发明的实施方案。本发明的第一组分与第二组分并不局限于上述特定的组合。此外，当将某一组合用作第一组分和第二组分时，第一组分可以是该组合的任一成员，且第二组分是该组合的另一成员。例如，当使用纤维蛋白原和凝血酶这个组合时，第一组分可以是纤维蛋白原(此时第二组分为凝血酶)，或者可以是凝血酶(此时第二组分为纤维蛋白原)。
在某些优选的实施方案中，所述第一组分为纤维蛋白原，且第二组分为凝血酶。在某些优选的实施方案中，所述第一组分为海藻酸盐(例如海藻酸钠)或氧化的海藻酸盐(例如氧化的海藻酸钠)，且第二组分为含有Ca2+、Mg2+、Ba2+、Sr2+、或Fe3+的物质，例如含有Ca2+、Mg2+、Ba2+、Sr2+、或Fe3+的溶液或半固体(例如凝胶)。在某些优选的实施方案中，所述第一组分为含马来酰亚胺基团的分子(例如含有马来酰亚胺基团的聚乙二醇(MAL-PEG))，且第二组分为含自由巯基的分子(例如含有自由巯基的聚乙二醇(PEG-SH))。在某些优选的实施方案中，所述第一组分为含有阴离子的物质，例如含有阴离子的溶液或半固体(例如凝胶)，且第二组分为α-氰基丙烯酸酯(例如α-氰基丙烯酸甲酯，α-氰基丙烯酸乙酯，α-氰基丙烯酸异丁酯，α-氰基丙烯酸异己酯，α-氰基丙烯酸正辛酯)。在某些优选的实施方案中，所述第一组分为纤维蛋白原，且第二组分为α-氰基丙烯酸酯(例如α-氰基丙烯酸甲酯，α-氰基丙烯酸乙酯，α-氰基丙烯酸异丁酯，α-氰基丙烯酸异己酯，α-氰基丙烯酸正辛酯)。在某些优选的实施方案中，所述 第一组分为血清白蛋白(例如，牛血清白蛋白)，且第二组分为戊二醛。在某些优选的实施方案中，所述第一组分为含氨基甲酸酯基团(-NHCOO-)或异氰酸酯基团(-NCO)的分子(例如含氨基甲酸酯基团的聚乙二醇或含异氰酸酯基团的聚乙二醇)，且第二组分为含活泼氢的分子(例如含羧基的聚乙二醇)。在某些优选的实施方案中，所述第一组分为明胶-间苯二酚，且第二组分为戊二醛。在某些优选的实施方案中，所述第一组分为碳化二亚胺交联明胶，且第二组分为聚L-谷氨酸(PLGA)。在某些优选的实施方案中，所述第一组分为胺基化明胶，且第二组分为醛基化多糖。
在某些优选的实施方案中，在所述第一试剂中，第一组分的浓度为0.01-50wt％。例如，在某些优选的实施方案中，所述第一组分的浓度为0.01-0.05wt％，0.05-0.1wt％，0.1-0.5wt％，0.5-1wt％，1-5wt％，5-10wt％，10-15wt％，15-20wt％，20-25wt％，25-30wt％，30-35wt％，35-40wt％，40-45wt％，或者45-50wt％。
在某些优选的实施方案中，在所述第二试剂中，第二组分的浓度为0.01-50wt％。例如，在某些优选的实施方案中，所述第二组分的浓度为0.01-0.05wt％，0.05-0.1wt％，0.1-0.5wt％，0.5-1wt％，1-5wt％，5-10wt％，10-15wt％，15-20wt％，20-25wt％，25-30wt％，30-35wt％，35-40wt％，40-45wt％，或者45-50wt％。
在某些优选的实施方案中，所述第二试剂还含有第三组分，所述第三组分为粘性剂。可通过调整第三组分(粘性剂)的含量来方便地调整第二试剂的粘度，以便第二试剂能够维持特定的形状，从而适合用于绘制图案或进行涂布。在某些优选的实施方案中，所述第三组分为生物相容性材料。在某些优选的实施方案中，所述第三组分为来源于生物的材料。在某些优选的实施方案中，所述第三组分为生物可降解材料。在某些优选的实施方案中，所述第三组分为温敏性材料。在某些优选的实施方案中，所述温敏性材料在不同的温度下具有不同的形态。例如，所述温敏性材料(例如明胶)在较低的温度下呈固态或半固态，而在较高的温度下呈液态。在某些优选的实施方案中，所述温敏性材料的相变温度在5-40℃之间，例如5-10℃，10-15℃，15-20℃，20-25℃，25-30℃，30-35℃，35-40℃。在某些优选的实施方案中，所述温敏性材料选自明胶，聚N-异丙基丙烯酰胺-聚乙二醇嵌段共聚物，聚乙二醇共聚物(例如聚乙烯醇-聚乙二醇共聚物)，琼脂糖，Matrigel，壳聚糖/甘油磷酸钠体系，Pluronic F127，和聚N-异丙基丙烯酰胺(PNIPAAm)水凝胶。在某些优选的实施方案中，所述第三组分(粘性剂)选自明胶、嵌段聚合物F-127、琼脂糖、聚乙二醇、瓜尔胶、聚乙烯醇、壳聚糖、胶原、透明质酸、甲壳素、纤维素及 其衍生物(例如羟丙基纤维素)、聚氨基酸、聚N-异丙基丙烯酰胺-聚乙二醇嵌段共聚物、聚乙二醇共聚物(例如聚乙烯醇-聚乙二醇共聚物)、海藻酸盐(例如海藻酸钠)、改性的海藻酸盐(例如氧化的海藻酸盐，例如氧化的海藻酸钠)、Matrigel、壳聚糖/甘油磷酸钠体系、和聚N-异丙基丙烯酰胺(PNIPAAm)水凝胶。在某些优选的实施方案中，所述第三组分(粘性剂)为明胶。
在某些优选的实施方案中，在所述第二试剂中，第三组分的浓度为0.01-50wt％。例如，在某些优选的实施方案中，所述第三组分的浓度为0.01-0.05wt％，0.05-0.1wt％，0.1-0.5wt％，0.5-1wt％，1-5wt％，5-10wt％，10-15wt％，15-20wt％，20-25wt％，25-30wt％，30-35wt％，35-40wt％，40-45wt％，或者45-50wt％。
在某些优选的实施方案中，所述试剂盒中包含的微囊为上述任一项定义的微囊。
在另一个方面，本发明提供了一种用于制备管状生物构建体的套盒(package)，其包含一个或多个如上所定义的试剂盒。在某些优选的实施方案中，不同的试剂盒使用相同的第一试剂和第二试剂的组合。在某些优选的实施方案中，不同的试剂盒使用不同的第一试剂和第二试剂的组合。
在某些优选的实施方案中，本发明的人工组织前体可进行进一步的培养。因此，本申请还涉及一种人工组织，所述人工组织通过对本发明的人工组织前体进行培养(例如体外培养或体内培养)得到。
在某些优选的实施方案中，所述人工组织为人工管腔。
在某些优选的实施方案中，所述管腔为包含上皮细胞的管腔(例如血管，食管，气管，胃，胆管，肠道(包括小肠和大肠，例如十二指肠、空肠、回肠、盲肠(包括阑尾)、升结肠、结肠右曲、横结肠、结肠左曲、降结肠、乙状结肠、直肠)，输卵管，输精管，输尿管，膀胱或淋巴管)。
在某些优选的实施方案中，所述人工管腔为管状人工管腔或片状人工管腔。
在某些优选的实施方案中，所述人工管腔为人工血管或血管补片。
在某些优选的实施方案中，在允许微囊内的细胞增殖、分化、迁移、分泌和/或新陈代 谢的条件下，培养所述人工组织前体。培养条件取决于微囊内的细胞类型，所使用的微囊的类型，人工组织前体的结构和形状，培养的目的等等。本领域技术人员能够选择合适的培养条件，例如培养基，pH，温度，CO2水平和持续时间。一般的组织和细胞培养条件可参见例如，Doyle,Alan,and J.Bryan Griffiths,eds.Cell and tissue culture:laboratory procedures in biotechnology.New York:Wiley,1998。在某些优选的实施方案中，培养人工组织前体至少0、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、25、或30天。在某些优选的实施方案中，培养人工组织前体1-3、3-5、5-7、7-10、10-14、14-21、21-28、1-7、7-14、1-14、或14-28天。在某些优选的实施方案中，在3D培养箱中培养所获得的人工组织前体。在某些优选的实施方案中，在生物反应器中培养所获得的人工组织前体。在某些优选的实施方案中，在37℃，5％CO2的条件下培养所获得的人工组织前体。在某些优选的实施方案中，在培养过程中对人工组织前体施加物理刺激(例如压力，剪切力，光照，加热等)。在某些优选的实施方案中，在培养过程中对人工组织前体施加化学刺激(例如激素，细胞因子，化学试剂等)。
在某些优选的实施方案中，在培养过程中，微囊中的生物可降解材料至少一部分被降解。在某些优选的实施方案中，此类生物可降解材料的降解产物为微囊中的细胞提供了营养物质和/或细胞外基质。在某些优选的实施方案中，微囊中的生物可降解材料被降解至少10％、20％、30％、40％、50％、60％、70％、80％、或90％。
在某些优选的实施方案中，人工组织前体中的细胞在培养过程中分泌分泌物，并且这些分泌物整合入人工组织前体中。在某些优选的实施方案中，微囊内的细胞在培养过程中彼此连接。在某些优选的实施方案中，微囊之间的细胞在培养过程中彼此连接。在某些优选的实施方案中，所述生物构建体在培养后具有高细胞密度(例如至少100、200、500、1000、2000、5000、10000、20000、50000、或100000细胞/mm3)。在某些优选的实施方案中，微囊内的细胞在培养后增殖至少2、5、10、20、50、100、200、500、1000、2000、5000、10000、20000、50000、或100000倍。
在某些优选的实施方案中，将所述人工组织前体植入非人受试者体内，并在所述非人受试者体内进行培养。
在某些优选的实施方案中，所述非人受试者为哺乳动物，例如牛科动物、马科动物、羊科动物、猪科动物、犬科动物、猫科动物、啮齿类动物、灵长类动物。
在又一方面，本发明提供了一种管腔植入体，所述管腔植入体包含本发明的人工组织前体(例如管状人工组织前体或片状人工组织前体)或人工管腔。
在某些优选的实施方案中，所述管腔植入体包含一个或多个(例如2个、3个、4个或5个)本发明的人工组织前体(例如管状人工组织前体或片状人工组织前体)，或一个或多个(例如2个、3个、4个或5个)本发明的人工管腔(例如管状人工管腔或片状人工管腔)。
在某些优选的实施方案中，所述管腔植入体包含多个(例如2个、3个、4个或5个)本发明的管状人工组织前体，多个所述管状人工组织前体之间流体连通。
在某些优选的实施方案中，所述管腔植入体包含多个(例如2个、3个、4个或5个)本发明的管状人工管腔，多个所述管状人工管腔之间流体连通。
在某些优选的实施方案中，所述管腔植入体为线性的管状结构，或者为枝化的管状结构。
在某些优选的实施方案中，所述管腔植入体为X形管状、Y形管状或T形管状。
在某些优选的实施方案中，所述管腔为包含上皮细胞的管腔(例如血管，食管，气管，胃，胆管，肠道(包括小肠和大肠，例如十二指肠、空肠、回肠、盲肠(包括阑尾)、升结肠、结肠右曲、横结肠、结肠左曲、降结肠、乙状结肠、直肠)，输卵管，输精管，输尿管，膀胱或淋巴管)。
在某些优选的实施方案中，所述包含上皮细胞的管腔为血管。
在某些优选的实施方案中，所述管腔植入体为血管植入体，其包含本发明的人工血管或血管补片。
在某些优选的实施方案中，所述管腔植入体还包含药物活性成分，例如用于防止血栓、钙化、感染和/或排异反应的药物活性成分。
在某些优选的实施方案中，所述管腔植入体还包含感应装置，所述感应装置用于检测管腔内的流体参数。
在某些优选的实施方案中，所述管腔植入体还包含调节装置，所述调节装置用于调节管腔内的流体参数。
在某些优选的实施方案中，所述管腔植入体被植入受试者的体内。
在某些优选的实施方案中，所述受试者患有以下疾病中的一种或多种：心血管疾病、脑血管疾病、周围血管疾病、骨科疾病、泌尿系统疾病或肿瘤疾病；
在某些优选的实施方案中，所述受试者具有以下疾病中的一种或多种：冠心病、脑缺血性卒中、血管瘤、恶性肿瘤侵犯血管、血栓闭塞性脉管炎、血液运输受阻引起的骨科疾病、慢性肾功能衰竭；
在某些优选的实施方案中，所述受试者为哺乳动物，例如牛科动物、马科动物、羊科动物、猪科动物、犬科动物、猫科动物、啮齿类动物、灵长类动物；其中，特别优选的受试者为人。
本发明的管腔植入体，例如血管植入体，可用于替换受试者的狭窄、闭塞、扩张、损伤或畸形的管腔(例如血管)，或用于构建管腔旁路，从功能上取代狭窄、闭塞、扩张、损伤或畸形的管腔。在受试者自体的管腔供给不足的情况下，本发明的管腔植入体可被用做管腔替代品。例如本发明的血管植入体可用于冠状动脉旁路移植术(CABG)，施用于患有冠心病的受试者，或用于动静脉造瘘术，施用于患有慢性肾功能衰竭的受试者。
在又一方面，本申请提供了一种替换或修补受试者的管腔(例如血管)的方法，包括将本发明的管腔植入体植入受试者体内。
在某些优选的实施方案中，所述方法被用于治疗目的。例如，用本发明的管腔植入体替代受试者的狭窄、闭塞、扩张、损伤或畸形的管腔。
在某些优选的实施方案中，所述方法被用于非治疗目的。例如，用本发明的管腔植入体替换非人受试者的正常管腔，以用于医学研究。
在某些优选的实施方案中，所述受试者为哺乳动物，例如牛科动物、马科动物、羊科动物、猪科动物、犬科动物、猫科动物、啮齿类动物、灵长类动物；例如，所述受试者为人。
在又一方面，本发明提供了一种管腔模型，所述管腔模型包含本发明的人工管腔。
在某些优选的实施方案中，所述管腔模型包含一个或多个(例如2个、3个、4个或5个)本发明的人工管腔(例如管状人工管腔，例如人工血管)。
在某些优选的实施方案中，，所述管腔模型包含多个(例如2个、3个、4个或5个)本发明的管状人工管腔，多个所述管状人工管腔之间流体连通。
在某些优选的实施方案中，所述管腔模型为线性的管状结构，或者为枝化的管状结构。
在某些优选的实施方案中，所述管腔模型为X形管状、Y形管状或T形管状。
在某些优选的实施方案中，所述管腔模型还包含感应装置，所述感应装置用于检测管腔内的流体参数。
在某些优选的实施方案中，所述管腔模型还包含调节装置，所述调节装置用于调节管腔内的流体参数。
在某些优选的实施方案中，所述管腔模型用于医学教学演示、药物(例如用于预防和/或治疗血管疾病的药物，例如药物的有效成分)的筛选、生物研究或医学研究(例如血管流体力学的研究)。
图5B示例性地显示了本发明的管腔植入体或管腔模型的形状。可已通过对线性的管状人工组织前体(或管状人工管腔)进行连接(例如粘接或缝合)，得到枝化的管状人工组织前体(或管状人工管腔)，进而得到枝化的管腔植入体或管腔模型。
在又一方面，本发明提供了本发明的人工组织前体用于制备人工组织、管腔植入体或管腔模型的用途。
在某些优选的实施方案中，所述人工组织为如上任一项所述的人工组织(例如人工管腔)。
在某些优选的实施方案中，所述管腔植入体为如上任一项所述的管腔植入体。
在某些优选的实施方案中，所述管腔模型为如上任一项所述的管腔模型。
在又一方面，本发明提供了本发明的人工组织用于制备管腔植入体或管腔模型的用途。
在某些优选的实施方案中，所述管腔植入体为如上任一项所述的管腔植入体。
在某些优选的实施方案中，所述管腔模型为如上任一项所述的管腔模型。
发明的有益效果
与现有技术相比，本发明的技术方案具有以下有益效果中的一个或多个：
1、本发明的人工组织前体中，微囊中的细胞数量总体上是一致的，微囊为细胞的分化和/或生长提供了合适的微环境，有利于细胞保持干性，维持其分化能力，构建出的组织内细胞分布比较均匀，有利于形成结构和功能完整的组织。
2、本发明的人工组织前体，能够形成稳固的结构，使包裹在其中的细胞能够停留在特定的位置。微囊可以为细胞提供力学保护，使其在制备人工组织前体过程中，以及在植入体内后，能够经受管腔内体液的冲击过程，而不易受到损伤或造成脱落。
3、本发明的人工组织中，细胞分布均匀，人工组织容易形成完整的结构和功能。
4、在某些优选的实施方案中，使用脂肪间充质干细胞制备人工组织前体。脂肪间充质干细胞容易获取，细胞安全性高，自从脂肪间充质干细胞用于体内外研究后，还未见关于脂肪间充质干细胞成瘤性的报道。
5、本发明的人工组织前体可以根据病人的需求进行个性化定制，实现个性化制备。
6、本发明的人工组织前体中，固体支持物与微囊或由微囊构成的生物构建体紧密贴合，不发生相对移动。
具体实施方式
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。
实施例中未指明其来源的试剂、试剂盒或仪器均为市场上商购可得的常规产品。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。
实施例1包含恒河猴脂肪间充质干细胞的生物砖的制备
1、脂肪间充质干细胞的收集和培养
(1)收集脂肪间充质干细胞：以恒河猴为动物模型，从腹股沟处剪取脂肪组织置于50mL离心管中；使用胰酶对脂肪组织进行消化，离心收集脂肪间充质干细胞。
(2)使用无血清Lonza培养基对脂肪间充质干细胞进行扩增培养。原代培养第四代的细胞的显微照片如图6所示。从图中可见，细胞形态均一，生长状态良好。离心收集细胞。
2、胶原溶液的配制
使用牛I型胶原进行配制。
(1)高温灭菌处理100mL烧杯和搅拌磁子；
(2)将溶液容器外表面消毒后，放入生物安全柜内；
(3)取Co60辐照灭菌处理的胶原固体0.5g置于烧杯中，加入25mL无菌去离子水(经0.22μm滤器过滤处理)；
(4)使用磁力搅拌器搅拌，使胶原固体浸入水中；
(5)滴加醋酸溶液(经0.22μm滤器过滤处理)至pH＝3；
(6)搅拌溶液直至胶原固体完全溶解；4℃保存。
注：如原料为胶原固体，可以按照如上操作；如原料为胶原溶液，可以直接使用或稀释后使用。
根据实际使用的浓度，可以使用超纯水(经0.22μm滤器过滤处理)对胶原溶液进行稀释。
3、含有脂肪间充质干细胞的生物砖的制备
(1)制备U型底超疏水孔板：在超净室内将U型底孔板用酒精清洗干净后，将U型底孔板置入过氧化氢/浓硫酸溶液(30％(v/v)，H2O2:H2SO4＝1:3)中，80℃反应1小时，以进行羟基化处理。将羟基化的U型底孔板放入浓度为1％的1H,1H,2H,2H-全氟癸基三乙氧基硅烷(购自Sigma)溶液中12小时，再在100℃烘箱中加热4h，以进行硅化处理。最后，清洗U型底孔板并风干。
(2)制备含种子细胞的胶原溶液：将45μL NaOH溶液(4mol/L)与1mL I型胶原(4mg/mL)混合，配制成pH＝7的胶原溶液。使用该溶液与恒河猴脂肪间充质干细胞混合形成细胞悬液(细胞总浓度为2×107/mL)。
(3)制备聚赖氨酸溶液：将聚赖氨酸(购自Sigma，数均分子量(Mn)为150,000-300,000)溶于pH7.2的DMEM高糖培养基，得到浓度为1wt％的聚赖氨酸溶液。
(4)胶原滴加(形成核层结构)：采用能够吸取和排出纳升级液体的电子式吸取装置精密吸取0.1μL含种子细胞的胶原溶液，滴入步骤1制备的U型底超疏水孔板中，形成液滴，在37℃恒温保持30min，使其成型。
可选的电子式吸取装置为Eppendorf Xplorer 0.5-10uL或TransferpetteElectronic0.5-10uL，借助他们的分液功能，每分液量最低可达到0.1μL；或者采用SGE自动进样器1μL或0.5μL，可分别实现10次和5次的0.1μL液体滴定；特殊地，可选用锥形特制针头进行滴定，提高精确度。
(5)滴加聚赖氨酸溶液(形成壳层结构)：更换吸头后，精密吸取0.5μL步骤(3)制备的聚赖氨酸溶液，在超疏水孔板中央位置将其滴入步骤(4)中成型的核层表面，反应10min，以形成包含恒河猴脂肪间充质干细胞的生物砖，其形态如图7所示。图8为激光共聚焦显微镜拍摄的生物砖照片，其中绿色荧光为生物砖的壳，红色荧光为脂肪间 充质干细胞。
实施例2使用3D生物打印机制备生物砖-膨体聚四氟乙烯人工血管前体，以及人工血管前体的体内应用和评价
1、制备过程：
(1)配制纤维蛋白原溶液(5wt％)，用作第一试剂：称取纤维蛋白原0.1g，将其溶解于2mL生理盐水中(如果需要，可以在37℃水浴中充分溶解)；随后，将纤维蛋白原溶液通过0.22μm滤器，进行过滤灭菌；将经过滤的纤维蛋白原溶液保存，备用。
(2)配制凝血酶溶液(2000U/mL)，用作第二试剂：称取0.0011g CaCl2，加入2000U凝血酶中(Ca2+浓度为10mmol/mL)，然后加入1mL生理盐水充分溶解；随后，将凝血酶溶液通过0.22μm滤器，进行过滤灭菌；保存，备用。
(3)将实施例1制得的含有恒河猴脂肪间充质干细胞的生物砖浸泡于5％的纤维蛋白原溶液(用作第一试剂)中5分钟，以在其表面附着/组装纤维蛋白原分子(如果需要，可以进行温和摇晃，以便于组装)。加入H-DMEM培养基继续浸泡5分钟，以洗去其表面未组装的纤维蛋白原分子，从而得到经浸渍的生物砖。
(4)使用蓝光英诺血管生物打印机，在4℃条件下，在旋转杆上打印一层明胶，明胶的厚度为1mm，待明胶凝固后，将凝血酶溶液(用作第二试剂)喷涂于明胶表面。
(5)将生物砖打印在明胶表面，在凝血酶的作用下，生物砖表面的纤维蛋白原相互交联，使生物砖连接成一体，形成侧壁无开口的圆形管状生物构建体，其长度为20mm，直径为6mm，壁厚为1mm。
(6)加热旋转杆至37℃，然后从旋转杆上取下管状构建体。
(7)管状生物构建体的外壁喷涂一层医用胶(白云医用胶医用EC型)。将膨体聚四氟乙烯管状固体支持物套在管状生物构建体的外侧，通过医用胶将生物构建体的外壁与膨体聚四氟乙烯管状固体支持物的内壁进行粘合，得到人工血管前体，其形态如图9所示。
2、体内植入
步骤(1)对恒河猴进行开腹，暴露腹主动脉。
步骤(2)切断腹主动脉，将两个断端分别与得到的人工血管前体进行缝合。
步骤(3)对动物腹部的伤口进行缝合。
3、取材及病理检测
术后5天将人工血管取出。图10A显示了人工血管的形态，图10B为去除管状支持物得到的血管组织，图10C为将组织纵向剖开后的形态。对组织进行HE染色及免疫组化染色，并与正常血管进行对比，结果如图11-13所示。
图11为HE染色结果，图11A为正常血管，图11B为人工血管。如图所示，人工血管与正常血管有相似的细胞排列、内皮细胞层(细箭头所指)和平滑肌细胞层(粗箭头所指)。
图12为α-SMA染色结果，α-SMA阳性细胞为平滑肌细胞。图12A为正常血管，图12B为人工血管。如图所示，构成人工血管的脂肪间充质干细胞向平滑肌细胞分化，并出现与正常血管相似的细胞形态、排列及方向性。
图13为CD31染色结果，CD31阳性细胞为内皮细胞。图13A为正常血管，图13B为人工血管。如图所示，在与血液接触面，构成人工血管的脂肪间充质干细胞向内皮细胞分化，并出现与正常血管相似的细胞形态及排列。
实施例3使用3D生物打印机制备生物砖-聚己内酯人工血管前体，以及人工血管前体的体内应用和评价
1、聚己内酯管状固体支持物的制备
步骤(1)称取一定量聚己内酯溶于四氢呋喃中，配制成浓度为2wt％的制备溶液。
步骤(2)将人工血管模具浸入制备溶液中，缓慢取出，待溶剂挥发后重复操作，直至得到管壁厚度为0.5mm的聚己内酯管状固体支持物。
步骤(3)将聚己内酯管状固体支持物从模具上取下，放入超纯水清洗。
步骤(4)将聚己内酯管状固体支持物干燥并剪切成所需长度，进行环氧乙烷灭菌后备用。
2、使用3D打印机制备生物砖-聚己内酯人工血管前体
使用实施例1制得的含有恒河猴脂肪间充质干细胞的生物砖和聚己内酯管状固体支持物，按照实施例2的步骤，使用3D打印机制备侧壁无开口的圆形管状生物构建体，并使用医用胶(白云医用胶医用EC型)，将聚己内酯管状固体支持物套在管状生物构建体的外侧)将生物构建体的外壁与聚己内酯管状固体支持物的内壁进行粘合，得到人工血管前体。
3、人工血管前体的体内应用和评价
将实施例3制得的人工血管前体植入恒河猴体内，术后6天对所形成的人工血管的形态和血流方向进行检测，结果如图14所示。
图14A为超声检测结果，从图中可以看出，人工血管的管腔通畅。
图14B为彩色多普勒检测结果，结果显示，人工血管两侧血流方向一致，证明血管通畅。
术后20天将人工血管取出，使用免疫组化法对人工血管进行检测，结果如图15A和15B所示(图中的标尺均为200μm)。
图15A显示了α-SMA染色结果，如图中粗箭头所指，人工血管中，脂肪间充质干细胞向平滑肌细胞进行分化。图15B显示了CD31染色结果，如图中细箭头所指，人工血管血管中，脂肪间充质干细胞向内皮细胞进行分化。
图15C为天狼星红染色结果，如图所示，人工血管形成与正常血管相似的胶原结构。
实施例4手工构建生物砖-膨体聚四氟乙烯人工血管前体，以及人工血管前体的体内应用和评价
1、制备过程
(1)将实施例1的生物砖浸泡于5％的纤维蛋白原溶液中5分钟，随后去除纤维蛋白原溶液，加入H-DMEM培养基继续浸泡5分钟。
(2)截取长度为1cm的膨体聚四氟乙烯人工血管(戈尔人工血管，型号：S0604，流水号：3425)，作为管状固体支持物，吸取8μl医用胶(白云医用胶医用EC型)，均匀涂抹于膨体聚四氟乙烯人工血管的内壁。
(3)将生物砖逐一贴附于膨体聚四氟乙烯人工血管内壁，在医用胶的作用下，生物砖与膨体聚四氟乙烯人工血管牢固粘合在一起，形人工血管前体。
2、体内应用和评价
将人工血管前体植入恒河猴体内14天后取材，使用免疫组化染色进行检测，结果如图16A和16B所示(图中的标尺均为50μm)。
图16A为α-SMA染色结果，如图中粗箭头所指，人工血管中有脂肪间充质干细胞向平滑肌细胞进行分化。
图16B为CD31染色结果，如图中细箭头所指，人工血管中有脂肪间充质干细胞向 内皮细胞进行分化。
实施例5包含恒河猴脂肪间充质干细胞的微囊的制备
(1)制备U型底超疏水孔板：在超净室内将U型底孔板用酒精清洗干净后，将U型底孔板置入过氧化氢/浓硫酸溶液(30％(v/v)，H2O2:H2SO4＝1:3)中，80℃反应1小时，以进行羟基化处理。将羟基化的U型底孔板放入浓度为1％的1H,1H,2H,2H-全氟癸基三乙氧基硅烷(购自Sigma)溶液中12小时，再在100℃烘箱中加热4h，以进行硅化处理。最后，清洗U型底孔板并风干。
(2)制备含种子细胞的胶原溶液：将45μl NaOH溶液(4mol/L)与1mL I型胶原(4mg/mL)混合，配制成pH＝7的胶原溶液。使用该溶液与实施例1中收集的恒河猴脂肪间充质干细胞混合形成细胞悬液(细胞总浓度为2×107/mL)。
(3)胶原滴加(形成核层结构)：采用能够吸取和排出纳升级液体的电子式吸取装置精密吸取0.1μL含种子细胞的胶原溶液，滴入步骤1制备的U型底超疏水孔板中，形成液滴，在37℃恒温保持30min，使其成型，得到含有恒河猴脂肪间充质干细胞的微囊。
实施例6手工构建微囊-膨体聚四氟乙烯人工组织前体，以及人工血管前体的体内应用和评价
1、制备过程
(1)将实施例5的微囊浸泡于5％的纤维蛋白原溶液中5分钟，随后去除纤维蛋白原溶液，加入H-DMEM培养基继续浸泡5分钟。
(2)截取长度1cm的膨体聚四氟乙烯人工血管(戈尔人工血管，型号：S0604，流水号：3425)，吸取8μl医用胶(白云医用胶医用EC型)，均匀涂抹于膨体聚四氟乙烯人工血管的内壁。
(3)将微囊逐一贴附于膨体聚四氟乙烯人工血管内壁，在医用胶的作用下，微囊与膨体聚四氟乙烯人工血管牢固粘合在一起，形人工血管前体。
2、体内应用和评价
将人工血管前体植入恒河猴体内14天后取材，图17A为得到的人工血管的剖面图。使用免疫组化法进行检测，结果如图17B和17C所示(图中的标尺均为50μm)。
图17B为α-SMA染色结果，如图中粗箭头所指，人工血管中有脂肪间充质干细胞 向平滑肌细胞进行分化。
图17C为CD31染色结果，如图中细箭头所指，人工血管中有脂肪间充质干细胞向内皮细胞进行分化。
实施例7使用3D生物打印机制备加强型生物砖-膨体聚四氟乙烯人工血管前体
(1)将实施例1的生物砖浸泡于5％的纤维蛋白原溶液中5分钟，随后去除纤维蛋白原溶液，加入H-DMEM培养基继续浸泡5分钟。
(2)制备外径为4mm的旋转杆，将步骤(1)中制备好的生物砖逐一打印在旋转杆上，形成管状生物构建体。
(3)吸取8μL医用胶均匀涂抹在管状生物构建体的外壁；
(4)准备长度为1cm、内径为6mm的膨体聚四氟乙烯人工血管，吸取8μL医用胶均匀涂抹在人工血管的内壁。由左至右，将人工血管套在管状生物构建体上。涂抹于人工血管内壁的医用胶在阴离子作用下固化，使得人工血管与管状生物构建体粘合在一起，形成人工血管前体。
图18显示了上述制备过程。图158：将生物砖打印在旋转杆上，形成管状生物构建体；图18B和18C：将人工血管由左至右，套在管状生物构建体上；图18D：将人工血管与管状生物构建体粘合在一起，形成人工血管前体。
实施例8手工构建生物砖-聚乳酸人工血管前体
使用可降解的聚乳酸管状支持物，以及实施例1制得的生物砖，制备人工血管前体。图19描述了制备的过程。
图19A和19B显示了以聚乳酸为基本材料，采用电纺丝的方法制造出的管状固体支持物。
图19C显示了以下操作：将聚乳酸管状支持物剪开，然后在管状支持物的一面滴加医用胶，另一面对应位置放置生物砖。
图19D显示了，医用胶可以渗透通过管壁，使得生物砖与内壁相粘合在一起，得到人工血管前体。
上述操作仅为了方便观察和拍照。在实际制备中，是在聚乳酸管状固体支持物的外壁滴加医用胶，使医用胶渗透至内壁。由于医用胶可以渗透过电纺聚乳酸管壁，从而将生物 砖固定。上述结果一方面说明，聚乳酸可以作为固体支持物使用，另一方面说明，电纺得到的固体支持物，由于其孔结构具有渗透性，医用胶可以渗透通过管壁，可以在固体支持物的一面滴加医用胶，在另一面放置生物砖，从而固定生物砖，得到人工组织前体。
实施例9.利用生物砖、纤维蛋白原和凝血酶来制备管状生物构建体
1、含有鼠骨髓间充质干细胞的生物砖的制备
制备生物砖，其包含鼠骨髓间充质干细胞，且其核层包含胶原，壳层包含海藻酸钠；并且其制备方法如中国申请201610211570.4中所述。
实验材料：生物砖(根据中国专利申请201610211570.4中描述的方法制备)、纤维蛋白原(牛源)、凝血酶(牛源)、生理盐水(医用级)、CaCl2、无菌水、和明胶(猪源)；
2、管状生物构建体的制备
(1)配制纤维蛋白原溶液(5wt％)：称取纤维蛋白原0.1g，将其溶解于2mL生理盐水中(如果需要，可以在37℃水浴中充分溶解)；随后，将纤维蛋白原溶液通过0.22μm滤器，进行过滤灭菌；将经过滤的纤维蛋白原溶液(用作第一试剂)保存，备用。
(2)配制凝血酶溶液(2000U/mL)：称取0.0011g CaCl2，加入2000U凝血酶中(Ca2+浓度为10mmol/mL)，然后加入1mL生理盐水充分溶解；随后，将凝血酶溶液通过0.22μm滤器，进行过滤灭菌；保存，备用。
(3)配制明胶溶液(10wt％)：称取1g明胶，加入10mL无菌水，于37℃水浴中充分溶解；随后，将明胶溶液通过0.22μm滤器，进行过滤灭菌；保存，备用。
(4)将1mL凝血酶溶液和1mL明胶溶液均匀混合，然后置于37℃水浴中备用，即第二试剂。
(5)将生物砖浸入第一试剂10min，以在其表面附着/组装纤维蛋白原分子(如果需要，可以进行温和摇晃，以便于组装)。然后将生物砖浸于细胞培养基5min，以洗去其表面未组装的纤维蛋白原分子，从而得到经浸渍的生物砖。
(6)利用纤维蛋白原和凝血酶的凝血反应来连接和组装生物砖，以形成预设的三维结构。构建步骤如下：
a.在0℃冰浴条件下，在玻璃平皿中用第二试剂绘制圆环图案(如果需要，可以在圆环外填充明胶溶液，作为构建管状结构的辅助材料)；
b.将生物砖沿圆环图案放置并静置3s，形成由生物砖构成的圆环状结构(第一层)；
c.在圆环状结构的上表面滴加第二试剂，绘制圆环图案；
d.将生物砖沿圆环图案放置并静置3s，形成由生物砖构成的圆环状结构(第二层)；
e.根据需要，重复步骤c-d，形成不同层数的由生物砖构成的圆环状结构，即侧壁无开口的圆形管状结构(如果需要，可以将含有辅助材料的管状结构置于37℃环境中，并洗去辅助材料)。
图20展示了利用生物砖、纤维蛋白原和凝血酶来制备管状三维构建体的实验步骤和实验结果；其中，图20A显示的是，在生物砖表面附着/组装纤维蛋白原；图20B显示的是，用辅助材料构建环状辅助结构(任选的步骤)；图20C显示的是，沿环状辅助结构滴加第二试剂，绘制圆环图案；图20D显示的是，将组装单元沿圆环图案放置形成环状结构；图20E显示的是，在环状结构的上表面用第二试剂绘制圆环图案，然后将组装单元沿圆环图案放置(任选地，可以重复该步骤一次或多次，以构建含有多层结构的构建体)；图20F显示的是，构建得到的管状结构；图20G显示的是，去除辅助结构(任选的步骤)。
此外，参考GB/T228.1-2010，在电子拉力测试仪(Model 5967,Instron)上测试所获得的生物构建体的拉伸模量，其中，载荷为10N，拉伸速度为20mm/min，温度为25℃，并且在测试过程中，保持样品润湿状态。测量结果显示，所获得的管状结构的拉伸模量为1.25KPa。
还使用OLYMPUS IX83显微镜来观察刚刚制备获得的管状结构。观察结果示于图21A(Bar，200μm)。结果显示，在刚刚制备获得的管状结构中，生物砖尚未发生相互融合，细胞在各自的生物砖中均匀分布。将该管状结构在DMEM高糖培养基中培养3天，随后再次使用OLYMPUS IX83显微镜进行观察。观察结果示于图21B(Bar，200μm)。结果显示，在经培养的管状结构中，生物砖已完全相互融合，紧密连接在一起，形成了完整的生物构建体。这些结果表明，管状结构中的细胞能够正常生长，并且可以迁移通过生物砖之间的粘合部位，并最终实现生物砖的融合。
人工血管前体的体内应用和评价
实施例10-14中，以恒河猴为动物模型，将实施例2制得的包含生物砖和膨体聚四氟乙烯的人工血管前体植入恒河猴体内(即，用作血管植入体)，并对植入后的情况进行评价。
实施例10-14中，将人工血管前体与恒河猴自体腹主动脉进行血管吻合的步骤均参照实 施例2的步骤进行。
实施例10
将11只恒河猴分别编号为NO.1-NO.11，其中NO.11为对照组。将人工血管前体与NO.1-NO.10恒河猴的自体腹主动脉进行血管吻合。
按照下表所示的植入后的时间，对各恒河猴体内的血管植入体进行取材和检测。
编号 时间
NO.1 4h，7d
NO.2 8h，7d
NO.3 12h，7d
NO.4 24h，7d
NO.5 3d，7d
NO.6 5d，7d
NO.7 7d
NO.8 14d
NO.9 21d
NO.10 28d
(1)使用HE染色法对血管植入体的组织结构进行观察，图22显示了检测结果，图中的标尺均为200μm。结果显示：植入4小时后，生物砖之间仍存在缝隙，彼此独立不相连；植入8小时-24小时后，生物砖逐渐相互融合成一体；随着植入时间的增加，生物砖融合成的人工血管逐渐形成与正常血管相类似的组织学结构。
(2)使用免疫组化染色方法对CD31的表达进行检测，图23和图24显示了检测结果。图23为放大100倍的结果，图中的标尺均为200μm。图24为放大400倍的结果，图中的标尺均为50μm。结果显示：植入5天后，血管植入体与血液接触的腔面出现内皮细胞；随着植入时间的增加，内皮细胞不断增加，在28天时，形成与正常血管相似的较完整的内皮细胞层。
(3)使用免疫组化染色方法对α-SMA的表达进行检测，图25显示了检测结果，图中的标尺均为200μm。结果显示：植入8小时后，生物砖内包裹的脂肪间充质干细胞开始向 平滑肌细胞进行分化并表达α-SMA；植入3天后，脂肪间充质干细胞的形态逐渐向平滑肌细胞转变且α-SMA的表达进一步增加；随着植入时间的增加，平滑肌细胞量逐渐增加并形成与正常血管相似的平滑肌细胞层。
实施例11
将人工血管前体与恒河猴自体腹主动脉进行血管吻合。将恒河猴分为4组，分别在植入后的7天、14天、21天和28天，对血管植入体与恒河猴自体血管连接处进行取材。使用HE染色法对连接处的组织结构进行观察，使用免疫组织化学染色法分别对CD31和α-SMA的表达进行检测。另以不实施植入的恒河猴作为对照组，取正常血管进行检测。
图26显示了检测结果。第一行图片为HE染色法的检测结果，图中的标尺为200μm；第二行图片为CD31的检测结果，图中的标尺为50μm；第三行图片为α-SMA的检测结果，图中的标尺为200μm。图中的粗箭头表示自体血管，细箭头表示血管植入体。
结果显示：在植入7天时，血管植入体与恒河猴自体血管相互连接，但彼此组织结构有显著差别，内皮细胞层连续但不完整，平滑肌细胞层不连续；随着植入时间的增加，血管植入体与恒河猴自体血管不断相互融合；在植入28天时，血管植入体与恒河猴自体血管融合成一体，内皮细胞层及平滑肌细胞层连续完整，形成与正常血管相似的组织结构。
实施例12
将人工血管前体与恒河猴自体腹主动脉进行血管吻合。将恒河猴分为4组，分别在植入后的5天、7天、21天和28天，对血管植入体进行取材。使用天狼猩红染色法对血管胶原进行染色，结果如图27所示，图中的标尺为100μm。结果显示：血管植入体植入5天后，开始出现胶原的表达；随着植入时间的增加，表达的胶原逐渐增多并开始分层，形成与正常血管相似的胶原结构。
实施例13
将人工血管前体与恒河猴自体腹主动脉进行血管吻合。使用超声分别于术后的5天、18天和61天对血管植入体进行检测，结果如图28所示，第一行图片为超声检测结果，第二行图片为彩色多普勒检测结果。结果显示，血管植入体内血管通畅，血流连续，管腔内表面光滑无血栓形成，无异常增生，与正常血管连接处无狭窄。
实施例14
将人工血管前体与恒河猴自体腹主动脉进行血管吻合。使用增强CT分别于植入后的19天和62天进行检测，结果如图29所示。结果显示，血管植入体血流通畅，无堵塞。
实施例15使用3D生物打印机制备生物砖-膨体聚四氟乙烯血管补片前体
(1)将市售的膨体聚四氟乙烯人工血管(管壁厚度：0.56mm，内径：8mm)裁剪成长4cm、宽1cm、具有一定弧度的近似矩形片状，得到片状固体支持物，如图30A所示；
(2)在片状固体支持物上喷涂一层医用胶(白云医用胶医用EC型)；
(3)将实施例1制得的包含恒河猴脂肪间充质干细胞的生物砖浸泡于5％的纤维蛋白原溶液中5分钟，随后去除纤维蛋白原溶液，加入H-DMEM培养基继续浸泡5分钟。
(4)使用3D生物打印机，将浸泡后的生物砖逐一打印在医用胶表面，铺满片状固体支持物，形成血管补片前体，如图30B所示。
实施例16使用3D生物打印机制备微囊-聚己内酯血管补片前体
(1)参照实施例3的方法，制备厚度为0.5mm的平整的聚己内酯片状固体支持物，将其裁剪为长3.5cm、宽1cm的近似矩形片状，如图30C所示；
(2)在片状固体支持物上喷涂一层医用胶(白云医用胶医用EC型)；
(3)使用3D生物打印机，将实施例5制得的包含恒河猴脂肪间充质干细胞的微囊逐一打印在医用胶表面，铺满片状固体支持物，形成血管补片前体，如图30D所示。
实施例17血管补片前体的体内应用和评价
以恒河猴为动物模型，将实施例15和16制得的血管补片前体进行体内植入。在恒河猴腹主动脉上制造血管缺损后，根据具体的血管缺损情况，将血管补片前体修剪成合适的长椭圆形，并将血管补片前体与缺损部位进行缝合。
图31A显示了在恒河猴腹主动脉上制造血管缺损，图31B显示了将血管补片前体与缺损部位进行缝合。图31B图中，粗箭头所指为实施例15制得的包含生物砖的血管补片前体，细箭头所指为实施例16制得的包含微囊的血管补片前体。
植入7天后，将血管补片取出。图32A和32B分别显示了由包含生物砖的血管补片前体和包含微囊的血管补片前体形成的血管组织。如图所示，补片内生物砖或微囊融合成一体，形成完整的内膜。
对血管组织进行CD31和α-SMA免疫组化染色，结果如图33所示。
图33A和33B显示了由包含生物砖的血管补片前体形成的血管组织的检测结果。结果显示，体内植入7天后，生物砖内脂肪间充质干细胞向内皮细胞(图33A)和平滑肌细胞(图33B)分化。
图33C和33D显示了由包含微囊的血管补片前体形成的血管组织的检测结果。结果显示，体内植入7天后，微囊内脂肪间充质干细胞向内皮细胞(图33C)和平滑肌细胞(图33D)分化。
实施例18双壳层生物砖的制备和力学性能测定
1、生物砖的制备
(1)制备U型底超疏水孔板：在超净室内将U型底孔板用酒精清洗干净后，将U型底孔板置入过氧化氢/浓硫酸溶液(30％(v/v)，H2O2:H2SO4＝1:3)中，80℃反应1h，以进行羟基化处理。将羟基化的U型底孔板放入浓度为1％的1H,1H,2H,2H-全氟癸基三乙氧基硅烷(Sigma)溶液中12h，再在100℃烘箱中加热4h，以进行硅化处理。最后，清洗U型底孔板并风干。
(2)制备含种子细胞的胶原溶液：将45μL NaOH溶液(4mol/L)与1mL I型胶原(4mg/mL)混合，配制成pH＝7的胶原溶液。使用该溶液与离心获得的恒河猴脂肪干细胞混合形成细胞悬液(细胞总浓度为2x107/mL)。
(3)制备聚赖氨酸溶液：将聚赖氨酸(Sigma，数均分子量Mn为150,000-300,000)溶于pH7.2的H-DMEM培养基，得到浓度为1wt％的聚赖氨酸溶液。
(4)制备海藻酸钠溶液：将海藻酸钠(Sigma)溶于pH7.2的H-DMEM培养基中，得到浓度为1wt％的海藻酸钠溶液
(5)胶原滴加(形成核层结构)：采用能够吸取和排出纳升级液体的电子式吸取装置精密吸取0.1μL步骤2制备的I型胶原溶液，滴入步骤1制备的U型底超疏水孔板中，形成液滴，在37℃恒温保持30min，使其成型。
可选的电子式吸取装置为Eppendorf Xplorer 0.5-10uL或TransferpetteElectronic0.5-10uL，借助他们的分液功能，每分液量最低可达到0.1μL；或者采用SGE自动进样 器1μL或0.5μL，可分别实现10次和5次的0.1μL液体滴定；特殊地，可选用锥形特制针头进行滴定，提高精确度。
(6)滴加聚赖氨酸溶液：更换吸头后，精密吸取0.5μL步骤3制备的聚赖氨酸溶液，在超疏水孔板中央位置将其滴入步骤5中成型的核层表面，反应10min，以形成生物砖壳的第1层。
(7)使用H-DMEM培养基对步骤6的产物冲洗2次。
(8)滴加海藻酸钠溶液：更换吸头后，精密吸取0.5μL步骤4制备的聚赖氨酸溶液，在超疏水孔板中央位置将其滴入步骤6中成型的壳层表面，反应10min，以形成生物砖壳的第2层，获得壳为2层的生物砖；制备的生物砖直径约300μm。
任选地，将制备好的生物砖置于PBS中，形成生物砖悬液。
3、弹性模量检测：
使用Piuma Nanoindenter纳米压痕仪对生物砖的弹性模量进行检测。
3.1受检样品制备：
(1)吸管取1mL生物砖悬液，滴加在塑料培养皿中间；
(2)静置10min后，吸干液体，可见生物砖吸附于培养皿底部；
(3)使用纳米压痕仪移动平台使探头逐步逼近生物砖表面，在接近生物砖时，使用吸管取一滴水滴于探头与生物砖之间，保证生物砖处于正常生理状态。
(4)此时生物砖吸附于底部，不会移动，可以进行检测(参见图34)。
3.2样品测试：
测试条件：
Figure PCTCN2017101738-appb-000001
测试结果：
图35为本实施例的生物砖的应力应变曲线。本实施例的生物砖的有效杨氏模量数值为24.77kPa。
实施例19使用3D生物打印机制备包含双壳层生物砖的人工血管前体，以及人工血管前体的体内应用和评价
1、胶原溶液(溶液A)的配制
使用牛I型胶原配制胶原溶液，作为生物墨汁的载体。
(1)高温灭菌处理100mL烧杯和搅拌磁子；
(2)将溶液容器外表面消毒后，放入生物安全柜内；
(3)取Co60辐照灭菌处理的胶原固体0.5g置于烧杯中，加入25mL无菌去离子水(经0.22μm滤器过滤处理)；
(4)使用磁力搅拌器搅拌，使胶原固体浸入水中；
(5)滴加醋酸溶液(经0.22μm滤器过滤处理)至pH＝3；
(6)搅拌溶液直至胶原固体完全溶解；
(7)根据打印环节需要，调控胶原溶液浓度。如无特殊要求，胶原溶液浓度为约2wt％。标记，命名为溶液A，4℃保存。
2、人工血管前体的制备
2.1准备
(1)将溶液A与生物砖按1:1的体积比共混，制备成墨汁A(生物墨汁)，并填装于打印墨盒A中，保持墨盒温度为4℃；
(2)将市售的白云医用胶作为墨汁B填装于打印墨盒B中，保持墨盒温度为室温；
(3)将墨盒A、B分别与相对应的打印喷头相连接；
(4)根据患者影像学检测结果，确定所需血管内径，选择相对应的旋生仪并安装；
(5)启动3D生物打印机，运行自检程序，测量喷头高度，确定3D生物打印机各部分正常；
(6)在电脑工作站中进行参数设置，包括：直径，长度，打印顺序，打印厚度，旋生仪温度，喷头温度。
图36示意性地展示了本实施例所使用的3D生物打印机。
2.2打印生物构建体并与管状固体支持物组装
(1)启动3D生物打印机制备程序；
(2)运行喷头A，打印墨汁A，使其在旋生仪的旋转杆上形成长度为20mm的管状生物构建体，厚度约1mm(如图37所示)；
(3)运行B喷头，在墨汁A形成的生物构建体上均匀喷涂墨汁B；
(4)将尺寸相匹配的Gore膨体聚四氟乙烯人工血管作为管状固体支持物，套在管状生物构建体的外表面，进行组装，通过墨汁B(医用胶)将两者粘合成一体，形成人工血管前体(如图38所示)，并从旋转杆上取出。
3、将人工血管前体植入恒河猴体内，进行体内应用和评价
3.1手术流程
(1)沿腹部中线做5-7cm的纵向皮肤切口，推荐使用高频电刀，因为它既可切割，又可止血。分离皮下组织及肌肉层直至腹膜，进入腹腔，打开腹腔后，将小肠轻柔的外翻出来，使用生理盐水浸湿的纱布包裹避免脱水，暴露腹主动脉；
(2)经静脉注射0.5mg kg-1肝素钠进行抗凝；
(3)在动脉下方穿过两根0-0缝合丝线，一根靠近肠系膜动脉下部，另一根靠近髂总动脉分支处，在需要时用于结扎阻断血流；
(4)使用动脉夹阻断肾下腹主动脉血流，两个动脉夹之间的距离约为3cm，从中间切除约2cm的腹主动脉；
(5)使用7-0聚乙烯缝合线，采用端端吻合法将2cm长的人工血管前体与恒河猴自体腹主动脉相缝合；
(6)松开动脉远心端动脉夹，使人工血管前体内的空气排空；
(7)松开动脉近心端动脉夹，使用消毒纱布按压血管缝合处数分钟，防止渗血直至缝合口无血液渗出；
(8)检测动脉远心端搏动情况，如搏动正常，则植入成功。图39显示了植入后的人工血管前体。
3.2组织学检测
体内植入170天后，对血管植入体进行取材。使用免疫荧光法检测植入体的内皮细胞及平滑肌细胞形成情况。
使用绿色荧光对血管内皮细胞进行荧光标记，结果显示，血管植入体形成了完整的内皮细胞层，荧光显微照片如图40所示，图中标尺为200μm。
使用红色荧光对血管平滑肌细胞进行荧光标记，结果显示，血管植入体形成了完整的平滑肌细胞层，荧光显微照片如图41所示，图中标尺为200μm。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公开的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (37)

  1. 一种人工组织前体，其包含固体支持物和多个微囊，其中，至少一个微囊与固体支持物贴合，所述微囊包含细胞和包裹细胞的生物相容性材料；
    优选地，所述人工组织前体为管腔(例如循环管腔、消化管腔、呼吸管腔、泌尿管腔或生殖管腔)前体；
    优选地，所述管腔为包含上皮细胞的管腔(例如血管，食管，气管，胃，胆管，肠道(包括小肠和大肠，例如十二指肠、空肠、回肠、盲肠(包括阑尾)、升结肠、结肠右曲、横结肠、结肠左曲、降结肠、乙状结肠、直肠)，输卵管，输精管，输尿管，膀胱或淋巴管)；
    优选地，所述人工组织前体为管状或片状；
    优选地，多个所述微囊构成一个或多个生物构建体；
    优选地，一个或多个生物构建体与固体支持物贴合。
  2. 权利要求1的人工组织前体，所述微囊在生理环境下(例如4-37℃，例如pH在6-8之间，例如在生理环境的流体剪切力下)具有稳定的结构；
    优选地，所述微囊具有在吸取或挤压中不会造成微囊破碎的力学强度；
    优选地，所述微囊为包裹的细胞提供了力学保护，并且能够为细胞的生命活动提供微环境(例如，营养物质)；
    优选地，所述微囊为生物砖；
    优选地，所述微囊的尺寸各自独立地为20-2000μm，例如30-1900μm，40-1800μm，50-1700μm，60-1600μm，70-1500μm，80-1400μm，90-1300μm，100-1200μm，200-1000μm，300-800μm，400-600μm，100-500μm；
    优选地，所述微囊各自独立地为球形，或者任何期望的形状(例如立方体，矩形棱柱，六棱柱，圆柱，或不规则的形状)；
    优选地，所述微囊各自独立地为固体或半固体，例如凝胶态；
    优选地，所述微囊以混合物的形式存在；
    优选地，所述微囊是分离的微囊；
    优选地，所述微囊提供于容器中。
  3. 权利要求1或2的人工组织前体，所述微囊中包含上皮细胞，例如内皮细胞(例如血管内皮细胞)、平滑肌细胞(例如血管平滑肌细胞)和/或未分化的细胞；
    优选地，所述微囊中的细胞为未分化的细胞，例如干细胞(例如脂肪间充质干细胞、骨髓间充质干细胞、诱导多能干细胞和胚胎干细胞)；
    优选地，所述未分化的细胞能够分化为上皮细胞(例如内皮细胞)和/或平滑肌细胞；
    优选地，所述未分化的细胞选自干细胞(例如脂肪间充质干细胞、骨髓间充质干细胞、诱导多能干细胞和胚胎干细胞)和祖细胞(例如内皮祖细胞)中的一种或多种；
    优选地，所述细胞获自动物，例如哺乳动物，例如人、猿、猴、大猩猩、牛、猪、犬、绵羊和山羊；
    优选地，所述细胞来源于选自下述的组织：结缔组织(例如，疏松结缔组织、致密结缔组织、弹性组织、网状结缔组织和脂肪组织)、肌肉组织(例如，骨骼肌、平滑肌和心肌)、泌尿生殖组织、胃肠组织、肺组织、骨组织、神经组织和上皮组织(例如，单层上皮和复层上皮)、内胚层来源的组织、中胚层来源的组织和外胚层来源的组织；
    优选地，所述微囊各自独立地包含一个或多个细胞，例如1-106个细胞，例如10-900、20-800、30-700、40-600、50-500、60-400、70-300、80-200、10-100个、10-103个、10-104个、10-105个、10-106个细胞。
  4. 权利要求1-3任一项的人工组织前体，所述微囊包含细胞和包裹所述细胞的核层；
    优选地，所述核层能够为细胞的生命活动提供微环境(例如，营养物质)；
    优选地，所述核层由生物可降解材料制成；
    优选地，所述微囊还包含封装所述核层的壳层；
    优选地，所述壳层能够为微囊提供力学保护；
    优选地，所述壳层能够为细胞的生命活动提供微环境，例如营养物质；
    优选地，所述壳层和/或核层是经过处理的(例如，使用壳层固定液或核层固定液进行了处理，例如以改善壳层和/或核层的力学性能)；
    优选地，所述壳层由生物可降解材料制成；
    优选地，所述壳层为通透性的；例如，所述壳层对于水，氧气，和营养物质(糖类例如葡萄糖，脂肪，蛋白质，氨基酸，短肽，矿物质，维生素、细胞因子、核苷酸)是通透性的；
    优选地，所述壳层具有用于微囊内外物质交换的通道或孔；
    优选地，所述壳层的厚度为0.1-50μm，例如0.1-0.5、0.5-1、1-2、2-5、5-10、10-15、15-20、20-25、25-30、30-50、50-100、100-200、200-300、300-400、400-500、0.1-1、1-5、1-10、5-10、10-20、10-30、5-20、或1-20μm；
    优选地，所述微囊、微囊的核层或微囊的壳层各自独立地具有0.01-0.02、0.02-0.03、0.03-0.04、0.04-0.05、0.05-0.06、0.06-0.07、0.07-0.08、0.08-0.09、0.09-0.1、0.1-0.15、0.15-0.2、0.2-0.3、0.3-0.4、0.01-0.4、0.01-0.05、0.05-0.1、0.1-0.2、0.2-0.4、0.05-0.15、或0.06-0.1GPa的硬度；
    优选地，所述微囊、微囊的核层或微囊的壳层各自独立地具有0.01-0.1GPa或0.01-0.4GPa的硬度；
    优选地，所述微囊、微囊的核层或微囊的壳层各自独立地具有0.01-0.05、0.05-0.1、0.1-0.5、0.5-0.8、0.8-1、1-1.2、1.2-1.4、1.4-1.6、1.6-1.8、1.8-2、2-2.4、2.4-2.8、2.8-3.2、3.2-4、4-10、10-20、20-30、30-40、40-50、50-80、80-100、0.5-4、0.5-1、1-1.5、1.5-2、2-3、0.8-1.6、1.4-2.4、0.8-3.2、0.01-100、1-100、10-100、或0.5-50MPa的弹性模量；
    优选地，所述微囊、微囊的核层或微囊的壳层各自独立地具有0.01-1、0.01-10或0.01-100MPa的弹性模量。
  5. 权利要求1-4任一项的人工组织前体，所述生物相容性材料包含生物可降解材料；
    优选地，所述生物可降解材料为具有可降解性的生物材料；
    优选地，所述生物可降解材料是天然存在的(例如来源于动植物的天然存在的生物可降解材料)，人工合成的，重组产生的，经过改性的，或者其任何组合；
    优选地，所述生物可降解材料包含天然存在的生物可降解材料，例如胶原蛋白，纤维蛋白，壳聚糖，海藻酸盐(例如海藻酸钠或海藻酸钙)，淀粉，透明质酸，层粘连蛋白，琼脂糖，明胶，葡聚糖，甲壳素，纤维素(例如细菌纤维素)，蚕丝蛋白，硫酸软骨素，肝素，纤维蛋白原，纤连蛋白，粘多糖，粘液素，以及其任意组合；经过改性的生物可降解材料，例如经过改性的海藻酸盐，例如氧化海藻酸盐(例如氧化海藻酸钠)，改性明胶(例如双醛淀粉DAS交联改性明胶)，改性纤维素(例如羧甲基纤维素，氧化再生纤维素)，以及其任意组合；和/或人工合成的生物可降解材料，例如聚磷腈，聚丙烯酸及其衍生物(例如聚甲基丙烯酸，丙烯酸和甲基丙烯酸的共聚物)，聚乳酸(PLA)，聚羟基乙酸(PGA)，聚乳 酸-乙醇酸共聚物(PLGA)，聚原酸酯(POE)，聚己内酯(PCL)，聚羟基丁酸酯(PHB)，聚氨基酸(例如聚赖氨酸)，可降解性聚氨酯(如淀粉改性聚氨酯)，聚羟基烷酸酯(PHAs)，聚羟基戊酸酯(PHV)，聚丁二酸丁二醇酯(PBS)，聚乙烯醇，聚对二氧环己酮，聚对二氧杂环己酮，聚二氧杂环己烷酮，聚碳酸丁二醇酯，以及其任何组合；
    优选地，所述生物可降解材料能够被酶(例如细胞分泌的酶)所降解；
    优选地，所述生物可降解材料的降解能够提供维持或促进所述细胞的生命活动的营养物质。
  6. 权利要求1-5任一项的人工组织前体，其中，所述微囊还包含额外的试剂，例如，营养物质、细胞外基质、细胞因子和/或药物活性成分；
    优选地，所述额外的试剂能够调控(例如促进)细胞的增殖、分化、迁移、分泌和/或新陈代谢，或维持细胞的干性；
    优选地，所述营养物质包括但不限于，微量元素，核苷酸，氨基酸，多肽，碳水化合物(例如单糖，寡糖，多糖)，脂质，维生素；
    优选地，细胞外基质选自多糖，例如糖胺聚糖、蛋白聚糖；结构蛋白，例如胶原和弹性蛋白；粘着蛋白，例如纤粘连蛋白和层粘连蛋白；
    优选地，所述细胞因子是用于调控细胞的增殖、分化、迁移、分泌和/或新陈代谢的细胞因子，包括但不限于：诱导未分化的细胞向平滑肌细胞或内皮细胞分化的细胞因子，例如TGF-α1、PDGF-BB、VEGF或b-FGF；
    优选地，所述药物活性成分为能够调控(例如促进)细胞的增殖、分化、迁移、分泌和/或新陈代谢的试剂，或者为能够维持细胞干性的试剂；优选地，所述药物活性成分选自rhIL-2、rhIL-11、rhEPO、IFN-α、IFN-β、IFN-γ、G-CSF、GM-CSF、rHuEPO、sTNF-R1、和rhTNF-α。
  7. 权利要求1-6任一项的人工组织前体，所述固体支持物由生物相容性材料制得；
    优选地，所述生物相容性材料包含生物可降解材料；
    优选地，所述生物可降解材料为具有可降解性的生物材料；
    优选地，所述生物可降解材料选自天然存在的生物可降解材料(例如胶原，明胶，壳聚糖，聚羟基丁酸酯(PHB)，甲壳素，海藻酸盐(例如海藻酸钠)，淀粉基生物材料(例如 多聚糖淀粉)，纤维素(例如细菌纤维素)，蚕丝蛋白，以及其任意组合)；
    优选地，所述天然存在的生物可降解材料为淀粉；
    优选地，所述生物可降解材料选自经过改性的生物可降解材料(例如经过改性的海藻酸盐，例如氧化海藻酸盐(例如氧化海藻酸钠)，改性明胶(例如双醛淀粉DAS交联改性明胶)，改性纤维素(例如羧甲基纤维素，氧化再生纤维素)，以及其任意组合；)
    优选地，所述生物可降解材料选自人工合成的生物可降解材料(例如脂肪族聚酯(例如聚乳酸(PLA)，聚己内酯(PCL)，聚羟基烷酸酯(PHAs)，聚羟基戊酸酯(PHV)，聚羟基丁酸酯(PHB)，聚丁二酸丁二醇酯(PBS))，聚羟基乙酸(PGA)，聚乳酸-乙醇酸共聚物(PLGA)，聚原酸酯(POE)，可降解性聚氨酯(例如淀粉改性聚氨酯)，聚乙烯醇，聚对二氧环己酮，聚对二氧杂环己酮，聚二氧杂环己烷酮，聚碳酸丁二醇酯，聚磷腈，以及其任何组合)；
    优选地，所述人工合成的生物可降解材料选自聚己内酯(PCL)，聚乳酸(PLA)，聚乳酸-羟基乙酸共聚物(PLGA)，聚乙醇酸(PGA)，可降解聚氨酯；
    优选地，所述生物可降解材料能够被酶(例如细胞分泌的酶)所降解；
    优选地，所述生物可降解材料在体内的降解时间为1-12个月；
    优选地，所述生物相容性材料还包含生物不可降解材料(例如尼龙，涤纶，聚丙烯，聚乙烯，聚四氟乙烯，硅橡胶，氟硅橡胶，天然橡胶，聚丙烯酸酯，芳香族聚酯(例如聚对苯二甲酸乙二醇酯(PET))，非降解性聚氨酯，聚醚醚酮，聚丙烯腈，聚硅氧烷，聚甲醛，聚氯乙烯，以及其任何组合)；
    优选地，所述生物不可降解材料是生物惰性的；
    优选地，所述生物相容性材料包含生物不可降解材料(例如尼龙，涤纶，聚丙烯，聚乙烯，聚四氟乙烯，硅橡胶，氟硅橡胶，天然橡胶，聚丙烯酸酯，芳香族聚酯(例如聚对苯二甲酸乙二醇酯(PET))，非降解性聚氨酯，聚醚醚酮，聚丙烯腈，聚硅氧烷，聚甲醛，聚氯乙烯，以及其任何组合)；
    优选地，所述生物不可降解材料是生物惰性的；
    优选地，所述固体支持物为管状固体支持物或片状固体支持物；
    优选地，所述固体支持物在生物构建体的表面制得；
    优选地，所述固体支持物通过静电纺丝、挤压锻造、3D打印或喷涂制得。
  8. 权利要求1-7任一项的人工组织前体，所述人工组织前体为管状(例如圆形管状， 例如侧壁开口或不开口的管状)，所述固体支持物为管状(例如圆形管状，例如侧壁开口或不开口的管状)固体支持物，多个所述微囊构成一个或多个管状(例如圆形管状，例如侧壁开口或不开口的管状)生物构建体，并且至少一个管状生物构建体的外壁与所述固体支持物的内壁贴合；
    优选地，所述人工组织前体包含管状固体支持物和一个侧壁不开口的管状生物构建体，所述管状生物构建体的外壁与所述管状固体支持物的内壁贴合；
    优选地，所述人工组织前体包含多个管状生物构建体；
    优选地，所述人工组织前体包含管状固体支持物和多个侧壁不开口的管状生物构建体，所述多个侧壁不开口的管状生物构建体在所述管状固体支持物的内部，并沿管状固体支持物的轴向排列，并且所述多个侧壁不开口的管状生物构建体的外壁均与所述管状固体支持物的内壁贴合；
    优选地，所述人工组织前体包含管状固体支持物和多个侧壁不开口的管状生物构建体，所述多个侧壁不开口的管状生物构建体在所述管状固体支持物的内部，与管状固体支持物同轴设置，并且仅最外侧的侧壁不开口的管状生物构建体的外壁与所述管状固体支持物的内壁贴合；
    优选地，所述人工组织前体包含管状固体支持物和多个侧壁开口的管状生物构建体，所述多个侧壁开口的管状生物构建体在所述管状固体支持物的内部，并沿管状固体支持物的轴向排列，并且所述多个侧壁开口的管状生物构建体的外壁均与所述管状固体支持物的内壁贴合；
    优选地，所述人工组织前体包含管状固体支持物和多个侧壁开口的管状生物构建体，所述多个侧壁开口的管状生物构建体在所述管状固体支持物内部，与管状固体支持物同轴设置并沿径向排列，并且仅最外侧的侧壁开口的管状生物构建体的外壁与所述管状固体支持物的内壁贴合；
    优选地，所述人工组织前体包含管状固体支持物和多个侧壁开口的管状生物构建体，所述多个侧壁开口的管状生物构建体在所述管状固体支持物内部，并与管状固体支持物同轴设置，并且所述多个侧壁开口的管状生物构建体的外壁均与所述管状固体支持物的内壁贴合；
    优选地，所述人工组织前体包含管状固体支持物、侧壁开口的管状生物构建体和侧壁不开口的管状生物构建体。
  9. 权利要求8的人工组织前体，所述人工组织前体的长度为1cm-40cm；
    优选地，所述人工组织前体的内径为1mm-3cm(例如1-6mm、6-8mm、8-10mm、10-12mm、12mm-3cm)；
    优选地，所述人工组织前体具有均一或不均一的厚度；
    优选地，所述管状固体支持物的长度为1cm-40cm；
    优选地，所述管状固体支持物的内径为1mm-3cm(例如1-6mm、6-8mm、8-10mm、10-12mm、12mm-3cm)；
    优选地，所述管状固体支持物的厚度为200μm-1mm；
    优选地，所述管状固体支持物为侧壁开口的圆形管状，所述开口沿轴线方向贯通管状固体支持物的两端，所述管状固体支持物沿径向的切面为扇环；优选地，所述扇环的圆心角大于0并且小于360°；
    优选地，所述管状生物构建体的长度为1cm-40cm；
    优选地，所述管状生物构建体的内径为1mm-3cm(例如1-6mm、6-8mm、8-10mm、10-12mm、12mm-3cm)；
    优选地，所述管状生物构建体的厚度为200μm-1mm；
    优选地，所述管状生物构建体为侧壁开口的圆形管状，所述开口沿轴线方向贯通管状生物构建体的两端，所述管状生物构建体沿径向的切面为扇环；优选地，所述扇环的圆心角大于0并且小于360°。
  10. 权利要求1-7任一项的人工组织前体，所述人工组织前体为片状，所述固体支持物为片状固体支持物，多个所述微囊构成一个或多个片状生物构建体，并且至少一个片状生物构建体与片状固体支持物贴合；
    优选地，所述片状固体支持物为平面的片状或者弯曲的片状；
    优选地，所述片状生物构建体为平面的片状或者弯曲的片状；
    优选地，所述人工组织前体包含片状固体支持物和一个片状生物构建体，所述片状生物构建体的一面与片状固体支持物的一面贴合；
    优选地，所述人工组织前体包含片状固体支持物和多个片状生物构建体，多个所述片状生物构建体位于片状固体支持物的一侧，并且各个片状生物构建体的一面均与片状固体 支持物的一面贴合；
    优选地，所述人工组织前体包含片状固体支持物和多个片状生物构建体，多个所述片状生物构建体在片状固体支持物的一侧层叠排布，并且至少一个片状生物构建体的一面与片状固体支持物的一面贴合。
  11. 权利要求10的人工组织前体，所述人工组织前体为圆形片状、椭圆形片状、平行四边形(例如矩形)片状、扇形片状或不规则片状；
    优选地，所述人工组织前体的厚度为0.5mm-3mm；
    优选地，所述人工组织前体的面积为0.5cm2-5cm2；
    优选地，所述人工组织前体具有均一或不均一的厚度；
    优选地，所述片状固体支持物为圆形片状、椭圆形片状、平行四边形(例如矩形)片状、扇形片状或不规则片状；
    优选地，所述片状固体支持物的厚度为0.5mm-3mm；
    优选地，所述片状固体支持物的面积为0.5cm2-5cm2；
    优选地，所述片状生物构建体为圆形片状、椭圆形片状、平行四边形(例如矩形)片状、扇形片状或不规则片状；
    优选地，所述片状生物构建体的厚度为20μm-3mm；
    优选地，所述片状生物构建体的面积为0.5cm2-5cm2。
  12. 权利要求1-11任一项的人工组织前体，其中，至少一个微囊或者至少一个生物构建体与所述固体支持物固定在一起；
    优选地，至少一个微囊或者至少一个生物构建体与所述固体支持物通过化学方式贴合；
    优选地，至少一个生物构建体与所述固体支持物通过粘合剂粘合；
    更优选地，所述粘合剂为医用胶；
    优选地，所述医用胶选自：软组织类医用粘合剂和硬组织类医用粘合剂(例如，用于牙科或骨科的医用粘合剂)；
    优选地，所述医用胶选自：以2-氰基丙烯酸辛酯为主体的组织粘合剂、纤维蛋白粘合剂、合成树脂粘合剂(例如甲基丙烯酸酯系粘合剂或聚羧酸系粘合剂)、骨粘固剂或以聚甲基丙烯酸甲酯为主体的粘合剂)；
    优选地，所述医用胶包含α-氰基丙烯酸酯(例如α-氰基丙烯酸甲酯，α-氰基丙烯酸乙酯，α-氰基丙烯酸异丁酯，α-氰基丙烯酸异己酯，α-氰基丙烯酸辛酯，例如α-氰基丙烯酸正辛酯)。
  13. 制备权利要求1-12任一项的人工组织前体的方法，所述人工组织前体为管状，其包含以下步骤：
    (I)制备管状(例如圆形管状；例如侧壁开口或不开口的管状)生物构建体；
    (II)将管状生物构建体与管状固体支持物的内壁进行贴合；
    优选地，制备管状生物构建体通过包含以下步骤的方法进行：
    (1)提供一种或多种微囊，其全部或者部分表面附着有第一组分；优选地，所述第一组分包含于第一试剂中；
    (2)在临时支持物表面的预设区域上涂布含有第二组分的第二试剂，其中，当所述第一组分与第二组分接触时，能够产生粘连效果，实现粘合作用；所述临时支持物为管状或柱状(例如侧壁不开口的圆形管状、侧壁开口的圆形管状、圆柱状或沿部分圆周设置的柱状)物，所述预设区域位于临时支持物的曲面；任选地，在涂布第二试剂之前，将衬底材料涂布在临时支持物表面的预设区域上；
    (3)将步骤(1)中的全部或者部分表面附着有第一组分的微囊放置于涂布了第二试剂的预设区域，使所述微囊表面上的第一组分与预设区域上的第二组分接触，产生粘连效果，从而将所述微囊组装(粘合)成第一层结构，所述第一层结构为管状结构；
    任选地，所述方法还包括以下步骤：
    (4)在前一步骤产生的结构上涂布第二试剂；
    (5)将步骤(1)中的全部或者部分表面附着有第一组分的微囊放置于前一步骤产生的结构上，使所述微囊表面上的第一组分与前一步骤产生的结构上的第二组分接触，产生粘连效果，从而在前一步骤产生的结构上，将所述微囊组装(粘合)成另一层结构；
    (6)任选地，重复步骤(4)和(5)一次或多次；
    从而得到管状生物构建体；
    任选地，所述方法还包括：将侧壁开口的圆形管状生物构建体进行粘合，得到侧壁不开口的圆形管状生物构建体；
    任选地，所述方法还包括：将管状生物构建体从临时支持物上分离；
    优选地，所述临时支持物为具有曲面的打印平台，例如3D打印机的旋转杆；
    优选地，所述衬底材料是温敏性材料，例如明胶、聚N-异丙基丙烯酰胺、聚N-异丙基丙烯酰胺-聚乙二醇嵌段共聚物、聚乙二醇共聚物(例如聚乙烯醇-聚乙二醇共聚物)，聚羟乙基丙烯酸酯、琼脂糖、Matrigel、壳聚糖/甘油磷酸钠体系或Pluronic F127；
    优选地，所述临时支持物为由温敏性材料(例如明胶、聚N-异丙基丙烯酰胺、聚N-异丙基丙烯酰胺—聚乙二醇嵌段共聚物、聚乙二醇共聚物，聚羟乙基丙烯酸酯、琼脂糖、Matrigel、壳聚糖/甘油磷酸钠体系或Pluronic F127)制成的圆柱状物或圆管状物；
    优选地，所述临时支持物为圆柱状物，所述预设区域为圆柱状物的整个侧面；
    优选地，所述临时支持物为圆柱状物，所述预设区域在展开的圆柱状物的侧面上为一矩形，并且所述预设区域沿圆柱状物的轴向贯通圆柱状物的侧面；
    优选地，所述临时支持物为圆柱状物，所述预设区域在展开的圆柱状物的侧面上为一矩形，并且所述预设区域沿圆柱状物的圆周方向贯通柱状物侧面；
    优选地，所述临时支持物为圆柱状物，所述预设区域在展开的圆柱状物的侧面上为一矩形，并且未沿圆柱状物的圆周方向和轴向贯通圆柱状物侧面；
    优选地，步骤(3)中，在将全部或部分表面附着有第一组分的微囊放置于步骤(2)的涂布了第二试剂的预设区域之后，静置0.1-60s；
    优选地，使用3D生物打印机来制备管状生物构建体。
  14. 制备权利要求1-12任一项的人工组织前体的方法，所述人工组织前体为管状，其包含以下步骤：
    (I)制备管状(例如圆形管状；例如侧壁开口或不开口的管状)生物构建体；
    (II)将管状生物构建体与管状固体支持物的内壁进行贴合；
    制备管状生物构建体通过包含以下步骤的方法进行：
    (1)提供一种或多种微囊，其全部或者部分表面附着有第一组分；优选地，所述第一组分包含于第一试剂中；
    (2)在临时支持物的表面上，用含有第二组分的第二试剂绘制预设的环形(例如圆环或扇环)图案，其中，当所述第一组分与第二组分接触时，能够产生粘连效果，实现粘合作用；所述临时支持物具有至少一个平面，所述环形图案位于临时支持物的平面；
    (3)将步骤(1)中的全部或者部分表面附着有第一组分的微囊放置于第二试剂绘 制的预设的环形图案上，使所述微囊表面上的第一组分与所述环形图案上的第二组分接触，产生粘连效果，从而将所述微囊组装(粘合)成第一层结构，所述第一层结构为环形结构；
    (4)在环形结构上涂布第二试剂；
    (5)将步骤(1)中的全部或者部分表面附着有第一组分的微囊放置于前一步骤产生的结构上，使所述微囊表面上的第一组分与前一步骤产生的结构上的第二组分接触，产生粘连效果，从而在前一步骤产生的结构上，将所述微囊组装(粘合)成另一层结构；
    (6)任选地，重复步骤(4)和(5)一次或多次；
    从而得到管状生物构建体；
    任选地，所述方法还包括：将侧壁开口的圆形管状生物构建体进行粘合，得到侧壁不开口的圆形管状生物构建体；
    优选地，所述临时支持物为3D打印机的打印平台；
    优选地，步骤(3)中，在将全部或部分表面附着有第一组分的微囊放置于步骤(2)的第二试剂绘制的预设的环形图案上之后，静置0.1-60s；
    优选地，使用3D生物打印机来制备管状生物构建体。
  15. 制备权利要求1-12任一项的人工组织前体的方法，所述人工组织前体为片状，其包含以下步骤：
    (I)制备片状(例如平面的片状或弯曲的片状)生物构建体；
    (II)将片状生物构建体与片状固体支持物进行贴合；
    优选地，制备片状生物构建体通过包含以下步骤的方法进行：
    (1)提供一种或多种微囊，其全部或者部分表面附着有第一组分；优选地，所述第一组分包含于第一试剂中；
    (2)在临时支持物表面的预设区域上涂布含有第二组分的第二试剂，其中，当所述第一组分与第二组分接触时，能够产生粘连效果，实现粘合作用；所述临时支持物具有至少一个平面，所述预设的图案位于临时支持物的平面；
    (3)将步骤(1)中的全部或者部分表面附着有第一组分的微囊放置于涂布了第二试剂的预设区域，使所述微囊表面上的第一组分与预设区域上的第二组分接触，产生粘连效果，从而将所述微囊组装(粘合)成第一层结构，所述第一层结构为平面的片状结 构；
    任选地，所述方法还包括以下步骤：
    (4)在前一步骤产生的结构上涂布第二试剂；
    (5)将步骤(1)中的全部或者部分表面附着有第一组分的微囊放置于前一步骤产生的结构上，使所述微囊表面上的第一组分与前一步骤产生的结构上的第二组分接触，产生粘连效果，从而在前一步骤产生的结构上，将所述微囊组装(粘合)成另一层结构；
    (6)任选地，重复步骤(4)和(5)一次或多次，得到平面的片状生物构建体；
    任选地，所述方法还包括，对平面的片状生物构建体进行弯折，得到弯曲的片状生物构建体；
    优选地，所述预设区域为平行四边形(例如矩形)区域、圆形区域或椭圆形区域、扇形区域或不规则区域；
    优选地，所述临时支持物为3D打印机的打印平台；
    优选地，步骤(3)中，在将全部或部分表面附着有第一组分的微囊放置于步骤(2)的涂布了第二试剂的预设区域之后，静置0.1-60 s；
    优选地，使用3D生物打印机来制备片状生物构建体。
  16. 制备权利要求1-12任一项的人工组织前体的方法，所述人工组织前体为片状，其包含以下步骤：
    (I)由权利要求15定义的制备片状生物构建体的方法，制备片状生物构建体；
    (II)提供制备固体支持物的材料(例如生物相容性材料)，在片状生物构建体上，制备片状固体支持物；
    优选地，通过3D打印或喷涂的方法制备片状固体支持物。
  17. 制备权利要求1-12任一项的人工组织前体的方法，所述人工组织前体为管状，其包含以下步骤：
    (I)通过权利要求15定义的制备片状生物构建体的方法，制备片状生物构建体；
    (II)将步骤(I)制得的片状生物构建体进行弯折，和/或将片状生物构建体的边缘进行粘合，得到管状生物构建体；
    (III)将管状生物构建体与管状固体支持物的内壁进行贴合。
  18. 制备权利要求1-12任一项的人工组织前体的方法，所述人工组织前体为管状，其包含以下步骤：
    (I)由权利要求13或14任一项定义的制备管状生物构建体的方法，制备管状生物构建体；
    或者，由权利要求15定义的制备片状生物构建体的方法，制备片状生物构建体；之后，对制得的片状生物构建体进行弯折，和/或将片状生物构建体的边缘进行粘合，得到管状生物构建体；
    (II)提供制备固体支持物的材料(例如生物相容性材料)，在管状生物构建体的外壁上，制备管状固体支持物；
    优选地，通过3D打印或喷涂的方法制备管状固体支持物。
  19. 制备权利要求1-12任一项的人工组织前体的方法，其包含以下步骤：
    (1)提供一种或多种微囊，其全部或者部分表面附着有第一组分；优选地，所述第一组分包含于第一试剂中；
    (2)提供固体支持物，在所述固体支持物表面的预设区域上涂布含有第二组分的第二试剂，其中，当所述第一组分与第二组分接触时，能够产生粘连效果，实现粘合作用；
    (3)将步骤(1)中的全部或者部分表面附着有第一组分的微囊放置于涂布了第二试剂的预设区域，使所述微囊表面上的第一组分与预设区域上的第二组分接触，产生粘连效果，从而在固体支持物表面，将所述微囊组装(粘合)成第一层结构；
    任选地，所述方法还包括以下步骤：
    (4)在前一步骤产生的结构上涂布第二试剂；
    (5)将步骤(1)中的全部或者部分表面附着有第一组分的微囊放置于前一步骤产生的结构上，使所述微囊表面上的第一组分与前一步骤产生的结构上的第二组分接触，产生粘连效果，从而在前一步骤产生的结构上，将所述微囊组装(粘合)成另一层结构；
    (6)任选地，重复步骤(4)和(5)一次或多次；
    从而得到人工组织前体；
    优选地，所述固体支持物为管状或片状；
    优选地，所述固体支持物为管状固体支持物，所述预设区域位于固体支持物的内壁；
    优选地，步骤(3)中，在将全部或部分表面附着有第一组分的微囊放置于步骤(2)的涂布了第二试剂的预设区域之后，静置0.1-60 s；
    优选地，使用3D生物打印机来制备人工组织前体。
  20. 权利要求13-19任一项的方法，其中，所述第一组分和/或第二组分为生物相容性材料，为来源于生物的材料，和/或为生物可降解材料；
    优选地，所述第一组分与第二组分接触而产生的粘连效果可用于将两个微囊粘合在一起，形成生物构建体；并且由此所获得的生物构建体的拉伸模量不低于10Pa，例如不低于20Pa，不低于30Pa，不低于40Pa，不低于50Pa，不低于60Pa，不低于70Pa，不低于80Pa，不低于90Pa，不低于100Pa，不低于200Pa，不低于300Pa，不低于400Pa，不低于500Pa，不低于600Pa，不低于700Pa，不低于800Pa，不低于900Pa，或不低于1000Pa；
    优选地，所述第一组分和第二组分是选自下列的组合：
    (1)纤维蛋白原和凝血酶；
    (2)海藻酸盐(例如海藻酸钠)或氧化的海藻酸盐(例如氧化的海藻酸钠)和含有Ca2+、Mg2+、Ba2+、Sr2+、或Fe3+的物质(例如含有Ca2+、Mg2+、Ba2+、Sr2+、或Fe3+的溶液或半固体(例如凝胶))；
    (3)含马来酰亚胺基团的分子(例如含有马来酰亚胺基团的聚乙二醇(MAL-PEG))和含自由巯基的分子(例如含有自由巯基的聚乙二醇(PEG-SH))；
    (4)含有阴离子的物质(例如含有阴离子的溶液或半固体(例如凝胶))和α-氰基丙烯酸酯(例如α-氰基丙烯酸甲酯，α-氰基丙烯酸乙酯，α-氰基丙烯酸异丁酯，α-氰基丙烯酸异己酯，α-氰基丙烯酸正辛酯)；
    (5)纤维蛋白原和α-氰基丙烯酸酯(例如α-氰基丙烯酸甲酯，α-氰基丙烯酸乙酯，α-氰基丙烯酸异丁酯，α-氰基丙烯酸异己酯，α-氰基丙烯酸正辛酯)；
    (6)血清白蛋白(例如，牛血清白蛋白)和戊二醛；
    (7)含氨基甲酸酯基团(-NHCOO-)或异氰酸酯基团(-NCO)的分子(例如含氨基甲酸酯基团的聚乙二醇或含异氰酸酯基团的聚乙二醇)和含活泼氢的分子(例如含羧基的聚乙二醇)；
    (8)明胶-间苯二酚和戊二醛；
    (9)碳化二亚胺交联明胶和聚L-谷氨酸(PLGA)；和
    (10)胺基化明胶和醛基化多糖；
    优选地，所述第一组分为纤维蛋白原，且第二组分为凝血酶；或者，
    所述第一组分为海藻酸盐(例如海藻酸钠)或氧化的海藻酸盐(例如氧化的海藻酸钠)，且第二组分为含有Ca2+、Mg2+、Ba2+、Sr2+、或Fe3+的物质，例如含有Ca2+、Mg2+、Ba2+、Sr2+、或Fe3+的溶液或半固体(例如凝胶)；或者，
    所述第一组分为含马来酰亚胺基团的分子(例如含有马来酰亚胺基团的聚乙二醇(MAL-PEG))，且第二组分为含自由巯基的分子(例如含有自由巯基的聚乙二醇(PEG-SH))；或者，
    所述第一组分为含有阴离子的物质，例如含有阴离子的溶液或半固体(例如凝胶)，且第二组分为α-氰基丙烯酸酯(例如α-氰基丙烯酸甲酯，α-氰基丙烯酸乙酯，α-氰基丙烯酸异丁酯，α-氰基丙烯酸异己酯，α-氰基丙烯酸正辛酯)；或者
    所述第一组分为纤维蛋白原，且第二组分为α-氰基丙烯酸酯(例如α-氰基丙烯酸甲酯，α-氰基丙烯酸乙酯，α-氰基丙烯酸异丁酯，α-氰基丙烯酸异己酯，α-氰基丙烯酸正辛酯)；或者
    所述第一组分为血清白蛋白(例如，牛血清白蛋白)，且第二组分为戊二醛；或者
    所述第一组分为含氨基甲酸酯基团(-NHCOO-)或异氰酸酯基团(-NCO)的分子(例如含氨基甲酸酯基团的聚乙二醇或含异氰酸酯基团的聚乙二醇)，且第二组分为含活泼氢的分子(例如含羧基的聚乙二醇)；或者
    所述第一组分为明胶-间苯二酚，且第二组分为戊二醛；或者
    述第一组分为碳化二亚胺交联明胶，且第二组分为聚L-谷氨酸(PLGA)；或者
    所述第一组分为胺基化明胶，且第二组分为醛基化多糖；
    优选地，在所述第一试剂中，第一组分的浓度为0.01-50wt％；
    优选地，在所述第二试剂中，第二组分的浓度为0.01-50wt％；
    优选地，所述第二试剂为液体或半固体(例如凝胶)；
    优选地，所述第二试剂的粘度为1-1000Pa·s，例如30-160Pa·s；
    优选地，所述第二试剂还含有第三组分，所述第三组分为粘性剂；
    优选地，所述粘性剂用于调节第二试剂的粘度；
    优选地，所述粘性剂选自明胶、嵌段聚合物F-127、琼脂糖、聚乙二醇、瓜尔胶、 聚乙烯醇、壳聚糖、胶原、透明质酸、甲壳素、纤维素及其衍生物(例如羟丙基纤维素)、聚氨基酸、聚N-异丙基丙烯酰胺-聚乙二醇嵌段共聚物、聚乙二醇共聚物(例如聚乙烯醇-聚乙二醇共聚物)、海藻酸盐(例如海藻酸钠)、改性的海藻酸盐(例如氧化的海藻酸盐，例如氧化的海藻酸钠)、Matrigel、壳聚糖/甘油磷酸钠体系、和聚N-异丙基丙烯酰胺(PNIPAAm)水凝胶；
    优选地，所述第三组分为生物相容性材料；为来源于生物的材料；为生物可降解材料；和/或，为温敏性材料；
    优选地，所述温敏性材料选自明胶，聚N-异丙基丙烯酰胺-聚乙二醇嵌段共聚物，聚乙二醇共聚物(例如聚乙烯醇-聚乙二醇共聚物)，琼脂糖，Matrigel，壳聚糖/甘油磷酸钠体系，Pluronic F127，和聚N-异丙基丙烯酰胺(PNIPAAm)水凝胶；
    优选地，所述第三组分的浓度为0.01-50wt％。
  21. 权利要求13-19任一项的方法，其中，在步骤(1)中，所述全部或部分表面附着有第一组分的微囊是通过将包含所述第一组分的第一试剂涂布于所述微囊的表面而获得的。
  22. 权利要求13-19任一项的方法，其中，在步骤(1)中，全部或者部分表面附着有第一组分的微囊是通过将所述微囊在包含所述第一组分的第一试剂中浸渍而获得的；
    优选地，将所述微囊在第一试剂中浸渍1-30min，例如1-5min，5-10min，10-15min，15-20min，20-25min，或25-30min；
    优选地，在步骤(1)中，在摇晃或振荡的条件下，将所述微囊在第一试剂中浸渍；
    优选地，步骤(1)是在室温条件(例如15-37℃)下，或是在低温条件(例如4-15℃)下进行的；
    优选地，步骤(1)还包括，在使微囊的全部或部分表面附着有第一试剂后，洗涤所述微囊；
    优选地，使用缓冲液(例如生理缓冲溶液)或培养基溶液来洗涤所述微囊；
    优选地，洗涤步骤进行1-5min或5-10min；
    优选地，洗涤步骤可在室温条件(例如15-37℃)下或低温条件(例如4-15℃)下进行。
  23. 权利要求13-19任一项的方法，其中，步骤(3)是在室温条件(例如15-37℃)下或者在低温条件(例如4-15℃)下进行的；
    优选地，在步骤(5)中，在将全部或部分表面附着有第一组分的微囊放置于前一步骤产生的结构上后，静置0.1-60s(例如0.1-1s，1-5s，5-10s，10-15s，15-20s，20-25s，25-30s，30-35s，35-40s，40-45s，45-50s，50-55s，或55-60s)；
    优选地，步骤(5)是在室温条件(例如15-37℃)下或者在低温条件(例如4-15℃)下进行的。
  24. 权利要求13-19任一项的方法，其中，在进行步骤(2)-(6)期间，还在所产生的结构内部或外周添加辅助材料；
    优选地，所述辅助材料不包含细胞；
    优选地，所述辅助材料是生物相容性和/或生物可降解的；优选地，所述辅助材料是温敏性材料；
    优选地，所述温敏性材料选自明胶、聚N-异丙基丙烯酰胺、聚N-异丙基丙烯酰胺—聚乙二醇嵌段共聚物、聚乙二醇共聚物(例如聚乙烯醇-聚乙二醇共聚物)、琼脂糖、Matrigel、壳聚糖/甘油磷酸钠体系或Pluronic F127。
  25. 权利要求13-19任一项的方法，其中，步骤(5)中使用的全部或部分表面附着有第一组分的微囊与步骤(1)中使用的全部或部分表面附着有第一组分的微囊是相同或不同的；
    优选地，不同的微囊包含不同的细胞和/或附着不同的第一组分。
  26. 权利要求13-25任一项的方法，其中，所述方法通过生物打印法来实施；
    优选地，使用打印机(例如3D生物打印机)来进行生物打印法；或者，使用自动化或非自动化机械过程来进行生物打印法；或者，通过使用手工放置或手工沉积法(例如使用移液器)来进行生物打印法；
    优选地，通过挤出式打印法或模块化打印法来对微囊进行打印；
    优选地，使用模块化打印法，挤出式打印法，或喷墨式打印法来对所述第二试剂进 行打印；
    优选地，使用模块化打印法，挤出式打印法，或喷墨式打印法来对所述辅助材料进行打印。
  27. 权利要求13-15或17任一项的方法，包括将生物构建体与固体支持物固定在一起；
    优选地，将生物构建体与固体支持物通过化学方式贴合；
    优选地，将生物构建体与固体支持物通过粘合剂粘合；
    优选地，所述粘合剂为α-氰基丙烯酸酯(例如α-氰基丙烯酸甲酯，α-氰基丙烯酸乙酯，α-氰基丙烯酸异丁酯，α-氰基丙烯酸异己酯，α-氰基丙烯酸辛酯)。
  28. 一种生物构建体，由权利要求13-15任一项定义的制备生物构建体的方法得到。
  29. 一种用于制备人工组织前体的试剂盒，所述试剂盒包含微囊，以及彼此分离的第一试剂和第二试剂，其中，所述微囊包含细胞和包裹细胞的生物相容性材料，所述第一试剂含有第一组分，所述第二试剂含有第二组分，并且当所述第一组分与第二组分接触时，能够产生粘连效果，实现粘合作用；
    优选地，所述第一组分与第二组分接触而产生的粘连效果可用于将两个微囊粘合在一起，形成生物构建体；并且由此所获得的生物构建体的拉伸模量不低于10Pa(例如不低于100Pa)；
    优选地，所述第一组分和/或第二组分为生物相容性材料；为来源于生物的材料；和/或，为生物可降解材料；
    优选地，所述第一组分和第二组分是选自下列的组合：
    (1)纤维蛋白原和凝血酶；
    (2)海藻酸盐(例如海藻酸钠)或氧化的海藻酸盐(例如氧化的海藻酸钠)和含有Ca2+、Mg2+、Ba2+、Sr2+、或Fe3+的物质(例如含有Ca2+、Mg2+、Ba2+、Sr2+、或Fe3+的溶液或半固体(例如凝胶))；
    (3)含马来酰亚胺基团的分子(例如含有马来酰亚胺基团的聚乙二醇(MAL-PEG))和含自由巯基的分子(例如含有自由巯基的聚乙二醇(PEG-SH))；
    (4)含有阴离子的物质(例如含有阴离子的溶液或半固体(例如凝胶))和α-氰基 丙烯酸酯(例如α-氰基丙烯酸甲酯，α-氰基丙烯酸乙酯，α-氰基丙烯酸异丁酯，α-氰基丙烯酸异己酯，α-氰基丙烯酸正辛酯)；
    (5)纤维蛋白原和α-氰基丙烯酸酯(例如α-氰基丙烯酸甲酯，α-氰基丙烯酸乙酯，α-氰基丙烯酸异丁酯，α-氰基丙烯酸异己酯，α-氰基丙烯酸正辛酯)；
    (6)血清白蛋白(例如，牛血清白蛋白)和戊二醛；
    (7)含氨基甲酸酯基团(-NHCOO-)或异氰酸酯基团(-NCO)的分子(例如含氨基甲酸酯基团的聚乙二醇或含异氰酸酯基团的聚乙二醇)和含活泼氢的分子(例如含羧基的聚乙二醇)；
    (8)明胶-间苯二酚和戊二醛；
    (9)碳化二亚胺交联明胶和聚L-谷氨酸(PLGA)；和
    (10)胺基化明胶和醛基化多糖。
  30. 权利要求29的试剂盒，优选地，所述第一组分为纤维蛋白原，且第二组分为凝血酶；或者，
    所述第一组分为海藻酸盐(例如海藻酸钠)或氧化的海藻酸盐(例如氧化的海藻酸钠)，且第二组分为含有Ca2+、Mg2+、Ba2+、Sr2+、或Fe3+的物质，例如含有Ca2+、Mg2+、Ba2+、Sr2+、或Fe3+的溶液或半固体(例如凝胶)；或者，
    所述第一组分为含马来酰亚胺基团的分子(例如含有马来酰亚胺基团的聚乙二醇(MAL-PEG))，且第二组分为含自由巯基的分子(例如含有自由巯基的聚乙二醇(PEG-SH))；或者，
    所述第一组分为含有阴离子的物质，例如含有阴离子的溶液或半固体(例如凝胶)，且第二组分为α-氰基丙烯酸酯(例如α-氰基丙烯酸甲酯，α-氰基丙烯酸乙酯，α-氰基丙烯酸异丁酯，α-氰基丙烯酸异己酯，α-氰基丙烯酸正辛酯)；或者
    所述第一组分为纤维蛋白原，且第二组分为α-氰基丙烯酸酯(例如α-氰基丙烯酸甲酯，α-氰基丙烯酸乙酯，α-氰基丙烯酸异丁酯，α-氰基丙烯酸异己酯，α-氰基丙烯酸正辛酯)；或者
    所述第一组分为血清白蛋白(例如，牛血清白蛋白)，且第二组分为戊二醛；或者
    所述第一组分为含氨基甲酸酯基团(-NHCOO-)或异氰酸酯基团(-NCO)的分子(例如含氨基甲酸酯基团的聚乙二醇或含异氰酸酯基团的聚乙二醇)，且第二组分为含活泼氢 的分子(例如含羧基的聚乙二醇)；或者
    所述第一组分为明胶-间苯二酚，且第二组分为戊二醛；或者
    述第一组分为碳化二亚胺交联明胶，且第二组分为聚L-谷氨酸(PLGA)；或者
    所述第一组分为胺基化明胶，且第二组分为醛基化多糖；
    优选地，在所述第一试剂中，第一组分的浓度为0.01-50wt％；
    优选地，在所述第二试剂中，第二组分的浓度为0.01-50wt％；
    优选地，所述第二试剂为液体或半固体(例如凝胶)；
    优选地，所述第二试剂的粘度为1-1000Pa·s，例如30-160Pa·s；
    优选地，所述第二试剂还含有第三组分，所述第三组分为粘性剂；
    优选地，所述粘性剂用于调节第二试剂的粘度；
    优选地，所述第三组分(粘性剂)选自明胶、嵌段聚合物F-127、琼脂糖、聚乙二醇、瓜尔胶、聚乙烯醇、壳聚糖、胶原、透明质酸、甲壳素、纤维素及其衍生物(例如羟丙基纤维素)、聚氨基酸、聚N-异丙基丙烯酰胺-聚乙二醇嵌段共聚物、聚乙二醇共聚物(例如聚乙烯醇-聚乙二醇共聚物)、海藻酸盐(例如海藻酸钠)、改性的海藻酸盐(例如氧化的海藻酸盐，例如氧化的海藻酸钠)、Matrigel、壳聚糖/甘油磷酸钠体系、和聚N-异丙基丙烯酰胺(PNIPAAm)水凝胶；
    优选地，所述第三组分为生物相容性材料；为来源于生物的材料；为生物可降解材料；和/或，为温敏性材料；
    优选地，所述温敏性材料选自明胶，聚N-异丙基丙烯酰胺-聚乙二醇嵌段共聚物，聚乙二醇共聚物(例如聚乙烯醇-聚乙二醇共聚物)，琼脂糖，Matrigel，壳聚糖/甘油磷酸钠体系，Pluronic F127，和聚N-异丙基丙烯酰胺(PNIPAAm)水凝胶；
    优选地，所述第三组分的浓度为0.01-50wt％。
  31. 权利要求30的试剂盒，所述微囊为权利要求2-6任一项定义的微囊。
  32. 一种用于制备人工组织前体的套盒，其包含一个或多个权利要求29-31任一项的试剂盒；
    优选地，不同的试剂盒使用相同的第一试剂和第二试剂的组合；
    优选地，不同的试剂盒使用不同的第一试剂和第二试剂的组合。
  33. 一种人工组织，所述人工组织通过对权利要求1-12任一项的人工组织前体进行培养(例如体外培养或体内培养)得到；
    优选地，所述人工组织为人工管腔；
    优选地，所述管腔为包含上皮细胞的管腔(例如血管，食管，气管，胃，胆管，肠道(包括小肠和大肠，例如十二指肠、空肠、回肠、盲肠(包括阑尾)、升结肠、结肠右曲、横结肠、结肠左曲、降结肠、乙状结肠、直肠)，输卵管，输精管，输尿管，膀胱或淋巴管)；
    优选地，所述人工管腔为管状人工管腔或片状人工管腔；
    优选地，所述人工管腔为人工血管或血管补片；
    优选地，在允许微囊内的细胞增殖、分化、迁移、分泌和/或新陈代谢的条件下，培养所述人工组织前体；
    例如，培养人工组织前体1-3、3-5、5-7、7-10、10-14、14-21、21-28、1-7、7-14、1-14、或14-28天；
    例如，在3D培养箱中或在生物反应器中培养人工组织前体；
    优选地，将所述人工组织前体植入非人受试者体内，并在所述非人受试者体内进行培养；
    优选地，所述非人受试者为哺乳动物，例如牛科动物、马科动物、羊科动物、猪科动物、犬科动物、猫科动物、啮齿类动物、灵长类动物。
  34. 一种管腔植入体，所述管腔植入体包含权利要求1-12任一项的人工组织前体(例如管状人工组织前体或片状人工组织前体)或权利要求33的人工管腔；
    优选地，所述管腔植入体包含一个或多个(例如2个、3个、4个或5个)权利要求1-12任一项的人工组织前体(例如管状人工组织前体或片状人工组织前体)，或一个或多个(例如2个、3个、4个或5个)权利要求33的人工管腔(例如管状人工管腔或片状人工管腔)；
    优选地，所述管腔植入体包含多个(例如2个、3个、4个或5个)权利要求1-12任一项的管状人工组织前体，多个所述管状人工组织前体之间流体连通；
    优选地，所述管腔植入体包含多个(例如2个、3个、4个或5个)权利要求33的管状人工管腔，多个所述管状人工管腔之间流体连通；
    优选地，所述管腔植入体为线性的管状结构，或者为枝化的管状结构；
    优选地，所述管腔植入体为X形管状、Y形管状或T形管状；
    优选地，所述管腔为包含上皮细胞的管腔(例如血管，食管，气管，胃，胆管，肠道(包括小肠和大肠，例如十二指肠、空肠、回肠、盲肠(包括阑尾)、升结肠、结肠右曲、横结肠、结肠左曲、降结肠、乙状结肠、直肠)，输卵管，输精管，输尿管，膀胱或淋巴管)；
    优选地，所述包含上皮细胞的管腔为血管；
    优选地，所述管腔植入体为血管植入体，其包含权利要求33的人工血管或血管补片；
    优选地，所述管腔植入体还包含药物活性成分(例如用于防止血栓、钙化、感染和/或排异反应的药物活性成分)；
    优选地，所述管腔植入体还包含感应装置，所述感应装置用于检测管腔内的流体参数；
    优选地，所述管腔植入体还包含调节装置，所述调节装置用于调节管腔内的流体参数；
    优选地，所述管腔植入体被植入受试者的体内；
    优选地，所述受试者患有以下疾病中的一种或多种：心血管疾病、脑血管疾病、周围血管疾病、骨科疾病、泌尿系统疾病或肿瘤疾病；
    优选地，所述受试者具有以下疾病中的一种或多种：冠心病、脑缺血性卒中、血管瘤、恶性肿瘤侵犯血管、血栓闭塞性脉管炎、血液运输受阻引起的骨科疾病、慢性肾功能衰竭；
    优选地，所述受试者为哺乳动物，例如牛科动物、马科动物、羊科动物、猪科动物、犬科动物、猫科动物、啮齿类动物、灵长类动物；其中，特别优选的受试者为人。
  35. 一种管腔(例如血管)模型，所述管腔模型包含权利要求33的人工管腔(例如人工血管)；
    优选地，所述管腔模型包含一个或多个(例如2个、3个、4个或5个)权利要求33的人工管腔(例如管状人工管腔，例如人工血管)；
    优选地，所述管腔模型包含多个(例如2个、3个、4个或5个)权利要求33的管状人工管腔，多个所述管状人工管腔之间流体连通；
    优选地，所述管腔模型为线性的管状结构，或者为枝化的管状结构；
    优选地，所述管腔模型为X形管状、Y形管状或T形管状；
    优选地，所述管腔模型还包含感应装置，所述感应装置用于检测管腔内的流体参数；
    优选地，所述管腔模型还包含调节装置，所述调节装置用于调节管腔内的流体参数；
    优选地，所述管腔模型用于医学教学演示、药物(例如用于预防和/或治疗血管疾病的药物，例如药物的有效成分)的筛选、生物研究或医学研究(例如血管流体力学的研究)。
  36. 权利要求1-12任一项的人工组织前体用于制备人工组织、管腔植入体或管腔模型的用途；
    优选地，所述人工组织为权利要求33的人工组织(例如人工管腔)；
    优选地，所述管腔植入体为权利要求34的管腔植入体；
    优选地，所述管腔模型为权利要求35的管腔模型。
  37. 权利要求33的人工组织用于制备管腔植入体或管腔模型的用途；
    优选地，所述管腔植入体为权利要求34的管腔植入体；
    优选地，所述管腔模型为权利要求35的管腔模型。
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