WO2022083037A1 - 仿生人工肝组织及其制备方法与应用 - Google Patents

仿生人工肝组织及其制备方法与应用 Download PDF

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WO2022083037A1
WO2022083037A1 PCT/CN2021/076789 CN2021076789W WO2022083037A1 WO 2022083037 A1 WO2022083037 A1 WO 2022083037A1 CN 2021076789 W CN2021076789 W CN 2021076789W WO 2022083037 A1 WO2022083037 A1 WO 2022083037A1
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cells
liver tissue
liver
tissue
culture
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PCT/CN2021/076789
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English (en)
French (fr)
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姚睿
徐铭恩
冯璐
梁少君
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清华大学
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Priority claimed from CN202011163991.7A external-priority patent/CN114377209B/zh
Priority claimed from CN202011163981.3A external-priority patent/CN114381419B/zh
Priority claimed from CN202011163982.8A external-priority patent/CN114381420B/zh
Application filed by 清华大学 filed Critical 清华大学
Publication of WO2022083037A1 publication Critical patent/WO2022083037A1/zh

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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
    • 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
    • A61L27/14—Macromolecular materials
    • 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
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells

Definitions

  • 202011163982.8 entitled “Liver-like Tissue Structure and Its Preparation Method and Application", with the patent title for "Containing Bile Duct" Priority of Chinese Patent Application No. 202011163991.7 of Chinese Patent Application No. 202011163991.7, the entire disclosure of which is incorporated herein by reference in its entirety.
  • the invention relates to the fields of biological materials and biomedical engineering, in particular to a bionic artificial liver tissue and a preparation method and application thereof.
  • the liver is the largest solid organ in the human body, accounting for about 2% of the total body weight. As the largest digestive gland and metabolic center of the human body, the liver plays an important role in maintaining the metabolic homeostasis of the human body. The liver is responsible for the synthesis of nutrients and is responsible for the synthesis and secretion of serum proteins, amino acids and glycogen in the human body. The liver also undertakes the function of detoxification, metabolizing metabolic wastes, drugs and other compounds from endogenous and exogenous sources. In addition, the liver is also responsible for the metabolism and storage of substances including amino acids, vitamins, lipids and carbohydrates.
  • the commonly used research models at this stage are animal models and flat-cultured human liver parenchyma cells. Due to the species specificity of various pathogens (such as hepatitis C, etc.) and the huge differences in liver functional proteins between animals and humans, the pathological models of animal livers are essentially different from those of humans in many aspects.
  • the source of human hepatocytes cultured in plane is not only extremely limited, but also limited by the plane clustering state of cell surface receptors, resulting in the rapid loss of phenotypic and functional characteristics of mature hepatocytes cultured in vitro.
  • Hepatocytes derived from stem cells or progenitor cells have the characteristics of a wide range of sources, easy access, good function, long-term culture and maintenance in vitro, etc., and have become a hot spot in the field of liver tissue research in recent years.
  • stem cells induced pluripotent stem cells, embryonic stem cells, liver stem cells, liver progenitor cells, endoderm cells, liver endoderm cells, hepatoblasts, mesenchymal stem cells and adult stem cells
  • stem cells induced pluripotent stem cells, embryonic stem cells, liver stem cells, liver progenitor cells, endoderm cells, liver endoderm cells, hepatoblasts, mesenchymal stem cells and adult stem cells
  • the cell density of human natural liver tissue reaches the order of 10 8 to 10 9 cells/mL, dense connections are formed between cells, and there are widely distributed blood vessels as nutrient transport media. It is currently known that there is a close relationship between the structure and physiological function of human tissue, and the cell density close to the physiological conditions in vivo is of great significance for the realization of the physiological function of liver tissue. In vitro, due to the limitations of construction technology and culture conditions, no technical report of such a high-density artificial liver tissue has been seen.
  • an in vitro liver tissue model close to the cell density of human liver tissue (on the order of 10 7 to 10 8 cells/mL) is urgently needed to serve preclinical drug testing, regenerative medicine and in vivo transplantation, bioartificial liver and liver function generation. Compensation, liver disease research and treatment, new drug development and other fields.
  • Natural liver tissue is an organ with complex spatial structures such as bile duct network and blood vessel network composed of various cellular components including hepatocytes and bile duct epithelial cells.
  • the intrahepatic bile duct is composed of bile duct epithelial cells, which secrete and absorb bile from hepatocytes, and then flow into the extrahepatic bile duct, where the bile from the liver is transported to the duodenum or stored in the gallbladder.
  • Blockage of the intrahepatic bile ducts can cause cholestasis, which can lead to acute obstructive suppurative cholangitis, which can be life-threatening.
  • the technical field of constructing artificial liver structures containing bile duct tissue and liver tissue is still blank, and no relevant reports have been seen.
  • the purpose of the present invention is to provide a bionic artificial liver tissue and its preparation method and application.
  • the present invention provides a bionic artificial liver tissue
  • the size of the bionic artificial liver tissue is 0.1-50 cm
  • its macroscopic structure can be columnar, block, flake, cystic, Tubular, mesh, braided or any combination of shapes.
  • the bionic artificial liver tissue includes microfilaments with a diameter of 50-2000 ⁇ m and a hollow channel with an inner diameter of 0.01-300 mm; wherein the microfilaments are formed from biocompatible materials and cells through a casting method or a 3D printing process
  • the hollow channel is formed by surrounding several adjacent microfilaments; the size, shape and distribution density of the hollow channel can be designed according to requirements.
  • the Young's modulus of the bionic artificial liver tissue is 0.1-150KPa.
  • the bionic artificial liver tissue has a very high cell density close to that of human natural liver tissue, and the cell density reaches or even exceeds 10 7 to 10 8 cells/mL. It positively expresses the landmark genes and proteins of mature liver tissue, and has bionic properties It has one or more physiological functions of liver tissue of albumin secretion, nitrogen metabolism, urea synthesis, detoxification and drug metabolism;
  • the bionic artificial liver tissue is an artificial liver structure containing bile duct-like and liver-like tissue structures, has a bile duct-like network structure formed by the connection of bile duct epithelial cells, expresses bile duct epithelial cell markers, and positively expresses the landmark genes of mature liver tissue and protein, with one or more physiological functions of liver tissue of albumin secretion, nitrogen metabolism, urea synthesis, detoxification and drug metabolism;
  • the cells in the biomimetic artificial liver tissue are uniformly dispersed in the whole structure in the form of small-sized (10-50 ⁇ m) clusters, the cell density is in the order of 10 5 to 10 7 cells/mL, has biomimetic physiological functions, and is positively expressed Marker genes and proteins of mature liver tissue.
  • the cells are derived from embryonic stem cells, induced pluripotent stem cells, liver stem cells, liver progenitor cells, endoderm cells, liver endoderm cells, hepatoblasts, mesenchymal stem cells or adult stem cells, and cells differentiated from these cells.
  • Hepatocytes hepatocytes derived from various human tissues and their cell lines; and related cells obtained by gene editing, virus packaging or transformation of all the above cells; preferably liver stem cells and their cell lines, and liver cells differentiated from induced pluripotent stem cells .
  • the cells can also include one or more of bile duct epithelial cells, hepatic stellate cells, hepatic sinusoidal endothelial cells, endothelial cells, fibroblasts, Kupffer cells, etc., including the above-mentioned cells and cell lines thereof, and related cells obtained by gene editing, virus packaging or transformation of the above cells; cells derived from induced pluripotent stem cells, embryonic stem cells, liver stem cells, liver progenitor cells, endoderm cells, liver endoderm cells, mesenchymal stem cells or adult stem cells , obtained from a variety of cell differentiation, or obtained from various human tissues; preferably fibroblasts and/or endothelial cells.
  • the biocompatible material can be selected from natural hydrogel materials and/or synthetic hydrogel materials.
  • the natural hydrogel material can be selected from chitosan, chitosan derivatives, gelatin, gelatin derivatives, alginate, alginate derivatives, agar, matrigel, collagen, collagen derivatives, hyaluronic acid , hyaluronic acid derivatives, cellulose, cellulose-derived materials, proteoglycans, proteoglycan derivatives, glycoproteins, glycoprotein-derived materials, laminin, fibronectin, fibrin (fibrinogen), silk fibroin , at least one of silk fibroin derivatives, vitronectin, osteopontin, peptide hydrogels, DNA hydrogels, etc.; preferably collagen, chitosan, chitosan derivatives, alginate, algae salt derivatives, fibrin, gelatin and/or gelatin derivatives.
  • the synthetic hydrogel material can be selected from polyglycolic acid, polylactic acid, polylactic acid-glycolic acid copolymer, polyglutamic acid-polyethylene glycol, polycaprolactone, polytrimethylene carbonate, poly Glycolic acid, polyethylene glycol-polydioxanone, polyethylene glycol, polytetrafluoroethylene, polyethylene oxide, polyethylene vinyl acetate, polytrimethylene carbonate, polydioxanone, Polyetheretherketone, and at least one of their derivatives or polymers; preferably polyglycolic acid, polylactic acid-glycolic acid copolymer or polylactic acid.
  • the present invention provides a method for preparing bionic artificial liver tissue, comprising:
  • the precursor solution can be prepared into a three-dimensional hydrogel structure according to a pre-designed structure by the following methods: casting method, lost foam method, biological 3D printing method, inkjet printing method, fused deposition modeling method, Electrospinning method, electrostatic drive printing method, stereolithography method or laser sintering method, etc.
  • the method may be to form the three-dimensional structure by controlling the temperature, and the temperature control range is 0°C to 37°C, preferably 4°C to 36°C.
  • the method may also be to shape the three-dimensional structure by light treatment, preferably white light or ultraviolet light.
  • the post-processing method of step (3) includes stabilization treatment and/or sacrificial material treatment.
  • the cross-linking reagent used for the stabilization treatment of the three-dimensional hydrogel structure is selected from divalent cations, genipin, glutaraldehyde, adipic acid dihydrazide, epichlorohydrin, carbodiimide, coagulation At least one of enzymes and derivatives thereof; preferably divalent cations and/or thrombin.
  • the concentration of the cross-linking reagent is 0.1 mM to 10 M, preferably 10 mM to 500 mM.
  • thermosensitive materials such as gelatin, collagen, N-isopropylacrylamide, and polyethylene in the three-dimensional hydrogel structures pyrrolidone, etc.
  • cross-linking reagents etc.
  • step (4) the three-dimensional hydrogel structure is cultured in vitro, including static culture and/or dynamic culture.
  • static culture is performed in a petri dish or multi-well plate; dynamic culture is performed in a bioreactor, a pulsating culture device, a microgravity culture device, a stirring culture device, a wave culture device, a chip or a perfusion culture system.
  • the differentiation of hepatocytes can be regulated by a variety of cytokines to varying degrees, and the cell culture medium used in in vitro culture is based on the basal medium added with cytokines that promote hepatocyte differentiation; wherein, the cytokines that promote hepatocyte differentiation At least one selected from bone morphogenetic protein, fibroblast growth factor, hepatocyte growth factor, tumor suppressor protein M and the like.
  • the cytokines that promote hepatocyte differentiation At least one selected from bone morphogenetic protein, fibroblast growth factor, hepatocyte growth factor, tumor suppressor protein M and the like.
  • bone morphogenetic protein is one of the important members of the transforming growth factor (TGF- ⁇ ) family that regulates cell development, and has the function of regulating the development and differentiation of various stem cells. Studies have shown that bone morphogenetic protein is a variety of stem cells.
  • fibroblast growth factor plays a key role in the regulation of cell development and can stimulate the proliferation of mesoderm-derived cells, neuroectoderm cells, ectodermal cells and endoderm-derived cells, They have chemotactic and mitotic effects on endothelial cells and induce the release of substances that disrupt the basement membrane; hepatocyte growth factor can stimulate hepatocyte proliferation and regulate a variety of cell growth, motility and morphogenesis multifunctional factors.
  • tumor suppressor protein M is an interleukin Cytokines of the -6 family, studies have shown that they are expressed in fetal liver and can promote the maturation of fetal hepatocytes during the embryonic period, and play an important regulatory role in the differentiation and maturation of hepatocytes.
  • the cell culture medium used in the in vitro culture is a basal medium added with factors that induce hepatocyte differentiation and maintain hepatocyte function; wherein, the factors that induce hepatocyte differentiation and maintain hepatocyte function are selected from bone morphogenetic protein, At least one of fibroblast growth factor, hepatocyte growth factor, dimethyl sulfoxide, tumor suppressor protein M and the like.
  • the inducing cholangiocytic differentiation factor is selected from at least one of keratinocyte growth factor, sodium butyrate, sodium taurocholate, epidermal cell growth factor and the like.
  • the cell culture medium comprises 50-120ng/ml activin A, 10-50ng/ml bone morphogenetic protein 2, 10-50ng/ml bone morphogenetic protein 4, 10-50ng/ml fibroblast growth factor 4, 0.1%-2% v/v dimethyl sulfoxide, 10-50 ng/ml hepatocyte growth factor, 1 x 10 -5 -1 x 10 -4 M tumor suppressor protein M, 1 mM ascorbic acid, 0.2 mM N- Acetylcysteine amide and 1 ⁇ 10 -7 M dexamethasone.
  • the synergistic effect of each factor promotes the differentiation and maturation of hepatocytes.
  • the cell culture medium comprises 100-200ng/ml activin A, 100-300ng/ml bone morphogenetic protein 2, 100-300ng/ml bone morphogenetic protein 4, 100-500ng/ml fibroblast growth factor 4, 0.1%-5% v/v dimethyl sulfoxide, 100-300 ng/ml hepatocyte growth factor, 1 ⁇ 10 -5 -1 ⁇ 10 -4 M tumor suppressor protein M and 1 mM ascorbic acid.
  • the synergistic effect of each factor promotes the differentiation and maturation of hepatocytes.
  • the cell culture medium comprises 50-120ng/ml activin A, 10-50ng/ml bone morphogenetic protein 2, 10-50ng/ml bone morphogenetic protein 4, 10-50ng/ml fibroblast growth factor 4, 0.1%-2% v/v dimethyl sulfoxide, 10-50ng/ml hepatocyte growth factor, 1 ⁇ 10-5-1 ⁇ 10-4 M tumor suppressor protein M, 10-50ng/ml keratinocytes Growth factors, 1 x 10 -6 -5 x 10 -6 M sodium butyrate, 1 x 10 -6 -5 x 10 -6 M sodium taurocholate and 1 x 10 -6 -5 x 10 -6 M epidermal cells growth factor.
  • the in vitro culture conditions are: 35°C ⁇ 38°C, 5% CO 2 .
  • the present invention provides a biomimetic artificial liver tissue prepared according to the method.
  • the macroscopic structure of the bionic artificial liver tissue is a column shape, a block shape, a sheet shape, a capsule shape, a tubular shape, a grid shape, a braided shape or any combination of shapes.
  • the size of the bionic artificial liver tissue is 0.1-50 cm.
  • the biomimetic artificial liver tissue comprises microfilaments with a diameter of 50-2000 ⁇ m.
  • the bionic artificial liver tissue has a hollow channel with an inner diameter of 0.01-300 mm.
  • the Young's modulus of the bionic artificial liver tissue is 0.1-150KPa.
  • the biomimetic artificial liver tissue has a very high cell density close to that of human natural liver tissue, and the cell density reaches or even exceeds the order of 10 7 to 10 8 cells/mL.
  • the bionic artificial liver tissue is an artificial liver structure containing bile duct and liver tissue, has a bile duct network structure formed by the connection of bile duct epithelial cells, expresses bile duct epithelial cell markers, has biomimetic physiological functions, and positively expresses markers of mature liver tissue Sex genes and proteins with one or more physiological functions of liver tissue in albumin secretion, nitrogen metabolism, urea synthesis, detoxification and drug metabolism;
  • the cells in the biomimetic artificial liver tissue are uniformly dispersed in the whole structure in the form of small-sized (10-50 ⁇ m) clusters, the cell density is in the order of 10 5 to 10 7 cells/mL, has biomimetic physiological functions, and is positively expressed Marker genes and proteins of mature liver tissue.
  • the present invention provides any of the following applications of the bionic artificial liver tissue:
  • the present invention at least has the following advantages and beneficial effects:
  • the bionic artificial liver tissue provided by the present invention conforms to the physiological tissue characteristics of the human liver with extremely high cell density.
  • the cell density of normal human tissue is about 10 8 to 10 9 cells per milliliter.
  • the present invention can achieve a cell density of about 10 7 to 10 8 cells per milliliter through precise control of culture factors, combined with three-dimensional structure and biomimetic biomaterials. , and has good liver function, filling the current technological gap in constructing high-density, highly bionic, and well-functioning artificial liver structures in vitro.
  • the biomimetic artificial liver tissue provided by the present invention has good liver function, the artificial liver tissue highly expresses the functional proteins and genes related to albumin secretion and drug metabolism that are unique to mature liver tissue/cells, and has good secretion protein and genes.
  • the function of protein and nitrogen metabolism, and the expression level of related genes reaches the level of human liver cells, which is more than 5 times that of flat cultured cells of the same species.
  • the artificial liver tissue of the present invention has highly biomimetic detoxification and drug metabolism functions, which is close to or even exceeds the level of human liver cells, and the expression level of related genes is more than 10 times that of cells of the same species cultured on a plane. Also, the liver function of the tissue can be maintained for a long time.
  • the artificial liver tissue provided by the present invention can be used for drug screening models for preclinical drug detection, as well as regenerative medicine and in vivo transplantation, or bioartificial liver and liver function compensation, and can metabolize and remove exogenous and exogenous compounds inside and outside the body. Metabolized protein and other essential substances. Because of its highly bionic properties, it can be used in the research and treatment of liver disease pathology, new drug development and other fields.
  • the present invention provides an artificial liver tissue with excellent physiological function, which is distributed in a unique uniform dispersion form, which is convenient for observation and various identification and characterization.
  • the cellular phenotypes in artificial liver tissues are highly consistent, which provides a unique liver tissue model for studies on liver development, liver regeneration, and liver disease treatment.
  • the present invention provides that the macroscopic and microscopic morphology of the artificial liver structure can be regulated, and can be customized for production as required. It can be prepared by one or more of the following techniques: casting method, lost foam method, biological 3D printing method, inkjet printing method, fused deposition modeling method, electrospinning method, electrostatic drive printing method, stereolithography method, Laser sintering technology method. Through the above method, a three-dimensional structure with complex shape and controllable size can be manufactured according to specific requirements. The macroscopic shape and microstructure of the three-dimensional structure can also be regulated to meet the needs of different cells for nutrients, oxygen concentration and living microenvironment.
  • the overall structure can form micro-scale artificial liver structures on the scale of micrometers and millimeters, as well as large-scale artificial liver structures on the scale of centimeters or even decimeters.
  • the biomimetic artificial liver tissue of the present invention provides a stable microenvironment for promoting the proliferation, differentiation and function maintenance of hepatocytes.
  • the method of flat cell culture is simple and feasible, it cannot simulate the three-dimensional environment of cell growth: under flat culture conditions, with the increase of cell density, the glycoprotein on the cell membrane conducts signal transduction, which will inhibit cell proliferation.
  • the biological material provided by the invention provides attachment points for cells, and the cells can migrate and aggregate within the structure. The porosity of the biological material ensures the exchange of nutrients and gases between the cells and the culture environment, which is beneficial to the proliferation and differentiation of cells.
  • By adjusting the components of the medium for inducing differentiation cells can be induced to differentiate and mature, and the physiological functions of mature liver cells can be maintained.
  • the construction method provided by the present invention has a stable process, which can not only rapidly proliferate cells, obtain extremely high cell density similar to natural tissue, but also obtain liver tissue with good function. It can be mass-produced, which is convenient for large-scale research and application.
  • the obtained artificial liver tissue has high yield, low cost, stable method, controllable process, small intra-batch/inter-batch variation, and can realize mass production, so as to carry out downstream large-scale research and development. It can be used in the fields of liver tissue development research, liver tissue regeneration research, liver disease occurrence and development research, preclinical drug testing, and new drug testing and development.
  • the elastic modulus of the artificial liver tissue constructed by the present invention is 0.1-150KPa, which is close to the elastic modulus of healthy adult liver tissue.
  • the artificial liver tissue constructed by the invention has good structural stability, can maintain a three-dimensional structure to realize long-term culture and dynamic culture, is conducive to uniform and sufficient nutrient and gas exchange, and promotes cell proliferation and function maintenance.
  • the present invention has successfully constructed an artificial liver tissue that simultaneously realizes the coexistence of hepatocytes and bile duct structures.
  • an artificial liver tissue that simultaneously realizes the coexistence of hepatocytes and bile duct structures.
  • the human liver tissue provided by the invention contains various cell types such as hepatocytes and bile duct epithelial cells, and has a multi-branched bile duct network structure of micron level, which fills the technical gap in the field of artificial liver structures at present.
  • the biomimetic liver tissue provided by the present invention has a bile duct-like network structure composed of bile duct epithelial cells and dense hepatocyte clusters, which are close to the natural tissue structure in which the interlobular intrahepatic bile ducts and the liver plate are tightly connected in a physiological liver. Because the function maintenance of mature hepatocytes is demanding on the culture environment, it is difficult to culture in vitro and the function maintenance time is short.
  • the biomimetic liver tissue containing single-tube tissue provided by the present invention has a high biomimetic physiological structure, has a good secretion function of mature liver tissue, highly expresses functional proteins and genes related to albumin secretion and drug metabolism, and the liver tissue of the tissue is highly biomimetic. The function can be maintained for a long time.
  • the present invention provides that the macroscopic and microscopic morphology of the artificial liver structure can be controlled, and can be customized for production as required.
  • the artificial liver structure can be prepared by one or more of the following techniques: casting technology, lost foam technology, biological 3D printing technology, inkjet printing, fused deposition modeling, electrospinning, electrostatic drive printing, stereolithography, laser Sintering technology.
  • three-dimensional structures with complex shapes and various sizes can be manufactured according to specific needs, and then the macroscopic shape and microscopic structure of the three-dimensional structure can be regulated to meet the needs of different cells for nutrients, oxygen concentration and living microenvironment.
  • the overall structure can form micro-scale artificial liver structures on the scale of micrometers and millimeters, as well as large-scale artificial liver structures on the scale of centimeters or even decimeters.
  • FIG. 1 is a schematic diagram of a three-dimensional structure of a bionic artificial liver tissue in a preferred embodiment of the present invention.
  • A is a schematic diagram of a three-dimensional three-dimensional structure prepared by a casting method
  • B is a schematic diagram of a three-dimensional three-dimensional structure prepared by a 3D printing method
  • C is a schematic diagram of a biomimetic artificial liver tissue with an extremely high density of hepatocyte distribution prepared by the casting method. Shaped as clusters of hepatocytes within the tissue.
  • FIG. 2 is a schematic diagram of an extrusion bio-3D printing process and a multi-layer grid structure in a preferred embodiment of the present invention.
  • FIG. 3 is a microscopic topography diagram of a bionic artificial liver tissue in a preferred embodiment of the present invention. Densely distributed hepatocytes can be seen under the light microscope, and hepatocytes exist in the form of tightly packed cell clusters.
  • FIG. 4 is the cell proliferation of the bionic artificial liver tissue in the preferred embodiment of the present invention.
  • Figure 5 shows the expression of key landmark proteins in mature hepatocytes of biomimetic artificial liver tissue in a preferred embodiment of the present invention.
  • FIG. 6 shows the function of the bionic artificial liver tissue in the preferred embodiment of the present invention.
  • A represents the secretion level of albumin and urea in the bionic artificial liver tissue of the present invention, which is compared with the same conditions of two-dimensional culture
  • B represents the expression of key landmark genes in the liver tissue normalized to the level of the same type of cells in plane culture, which is compared with the same conditions of two-dimensional culture. Comparison of the same conditions for 2D culture. *, ** and *** indicate statistically significant differences between different treatment groups, * indicates P ⁇ 0.05, ** indicates P ⁇ 0.01, and *** indicates P ⁇ 0.001.
  • FIG. 7 is a schematic diagram of different design structures for constructing biomimetic artificial liver tissue according to the present invention.
  • FIG. 8 is a schematic diagram of a three-dimensional structure of an artificial liver tissue with excellent physiological function in a preferred embodiment of the present invention.
  • A is a schematic diagram of a three-dimensional structure prepared by a casting method
  • B is a schematic diagram of a three-dimensional three-dimensional structure prepared by a 3D printing method
  • C is a schematic diagram of an artificial liver structure with excellent physiological functions prepared by the casting method, wherein the cell phenotype Highly consistent, distributed within the structure in a uniformly dispersed form.
  • FIG. 9 is a schematic diagram of the extrusion bio-3D printing process and the multi-layer grid structure in the preferred embodiment of the present invention.
  • FIG. 10 is a cell topography diagram of an artificial liver tissue with excellent physiological function in a preferred embodiment of the present invention. A large number of evenly distributed hepatocytes were seen under the light microscope.
  • Figure 11 shows the expression of key marker proteins of mature hepatocytes in the artificial liver tissue with excellent physiological function in the preferred embodiment of the present invention, and the phenotypes of all cells are highly consistent.
  • Fig. 12 shows the function of the artificial liver tissue in the preferred embodiment of the present invention.
  • A represents the albumin synthesis and urea secretion levels of the artificial liver tissue of the present invention, the same kind of cells cultured on a plane, and human hepatocytes
  • B the detoxification and urea secretion levels of the artificial liver tissue of the present invention, the same kind of cells cultured on a plane, and human hepatocytes
  • Expression levels of key genes in drug metabolism ** and *** indicate that the difference between different treatment groups is statistically significant, ** indicates P ⁇ 0.01, *** indicates P ⁇ 0.001.
  • FIG. 13 is a schematic diagram of an artificial liver structure containing bile duct tissue and liver tissue in a preferred embodiment of the present invention.
  • A is the schematic diagram of the three-dimensional structure prepared by the casting method
  • B is the schematic diagram of the three-dimensional three-dimensional structure prepared by the 3D printing method
  • C is the schematic diagram of the artificial liver structure containing bile duct tissue and liver tissue prepared by the casting method, wherein the clusters
  • the shape is the hepatocyte cluster in the tissue
  • the line is the bile duct network structure in the tissue.
  • Fig. 14 is the topography of the artificial liver structure containing bile duct tissue and liver tissue in a preferred embodiment of the present invention.
  • the spherical shape is a cluster of hepatocytes, as shown by the star mark; the spindle shape is the network structure formed by bile duct epithelial cells, as shown by the black arrow.
  • Figure 15 shows the expression of key marker proteins of bile duct epithelial cells and mature hepatocytes in the artificial liver structure containing bile duct tissue and liver tissue in the preferred embodiment of the present invention.
  • A staining results of CYP3A4 and CK19 proteins
  • B staining results of ALB and CK19 proteins
  • C staining results of CK19 proteins and DAPI proteins
  • D 3D layer scan of CK19 proteins in the structure.
  • Figure 16 shows the functional characterization and gene expression of the artificial liver tissue in the preferred embodiment of the present invention.
  • A represents the albumin and urea secretion levels of the artificial liver structure, compared with the same conditions of the two-dimensional culture
  • B represents the expression of the key landmark genes of the artificial liver structure normalized to the level of the flat cultured hepatocytes, compared with the two-dimensional culture. comparison of the same conditions.
  • This embodiment provides a bionic artificial liver tissue constructed by using 3D bioprinting technology, as shown in FIG. 1 .
  • iPSCs induced pluripotent stem cells
  • the specific implementation steps are as follows: iPS cells (Stem Cell) are dissociated using Cell Dispase (Dispase, Roche Diagnostics) and seeded on a Matrigel substrate (Becton Dickinson). Subsequently, iPSCs were cultured in L-WNT3A (CRL2647; ATCC)-expressing cell-conditioned RPMI 1640 medium (Sigma) for 3 to 4 days to differentiate into the restricted endoderm stage.
  • L-WNT3A CTL2647; ATCC
  • RPMI 1640 medium Sigma
  • Human fibroblasts (HFL1, CCL-153) were purchased from ATCC Company. Human fibroblasts were cultured in flat adherent culture medium, the medium was changed every 2 days, and the cells were passaged at a ratio of 1:3 when the cells reached 85% confluence.
  • the composition of the human fibroblast medium is: DMEM medium (Gibco, 11960044) supplemented with 10% FBS serum (Gibco, 16000), 1% fibroblast growth additive (RayBiotech, 230-00791-100), 1% Penicillin streptomycin (Gibco, 15140122).
  • a 21% solution of polylactic acid (Sigma-Aldrich, 765112, molecular weight 10000 Da) and a 21% solution of fibrinogen (Sigma-Aldrich, F3879) were prepared.
  • liver progenitor cells obtained in step 1 PBS was first added to rinse the surface, and then trypsin (Gibco, 25200072) was added to cover the cell surface, digested at 37°C for 3 min, collected, and centrifuged to obtain a single cell suspension.
  • PBS was added to the human fibroblasts in step 2 to rinse the surface, then trypsin (Gibco, 25200072) was added to cover the cell surface, digested at 37°C for 3 min, collected, and centrifuged to obtain the pellet of human fibroblasts. Resuspend to obtain a single cell suspension.
  • printing solution A single cell suspension A: the concentration of liver progenitor cells is 2.5 ⁇ 10 6 cells/mL, 7% Polylactic acid solution and 7% fibrinogen solution
  • printing solution B single cell suspension B: human fibroblasts at a concentration of 2.5 x 10 cells/mL, 7% polylactic acid solution and 7% fibrinogen solution .
  • Three-dimensional structures were constructed using extrusion printing equipment (Regenvo, Bio-architect X) of Genofei Biotechnology Co., Ltd.
  • the obtained printable single-cell suspensions A and B were loaded onto a dual-nozzle 3D bioprinter respectively, and the temperature in the printer cavity, the temperature of the printing plate and the nozzle were controlled to be 20 °C.
  • the designed CAD file and computer path it was possible to Build a variety of three-dimensional structures with complex structures.
  • a double-jet printer is used to construct a three-dimensional hexagonal mesh with 6 microfilaments in each layer (the composition of the microfilaments is the same as that of the printing solutions A and B), a total of 6 layers, and each microfilament is 2 cm long and 0.5 cm high.
  • the lattice structure is shown in Figure 2. After construction, the structure was soaked in 500 mM thrombin solution for 10 min to complete post-stabilization treatment, and a three-dimensional artificial liver structure was obtained.
  • the artificial liver structure was constructed, it was cultured in programmed induction medium for 10 days at 37°C and 5% CO 2 to obtain a biomimetic artificial liver tissue with extremely high cell density.
  • the specific components are: RPMI 1640 medium, 20ng/ml bone morphogenetic protein 2 (BMP2, Gibco, PHC7146), 20ng/ml bone morphogenetic protein 4 (BMP4, Gibco, PHC9533), 30ng/ml fibroblast growth factor 4 (FGF4, R&D SYSTEMS, 233-FB-025), 20ng/ml hepatocyte growth factor (HGF, R&D systems, 294-HGN-005), 0.1% DMSO (Sigma) and 1 ⁇ B-27 Supplement (Gibco, 17504044).
  • hepatocyte medium (HCM; Lonza), containing 1% epidermal growth factor (R&D systems, 236-EG-200), 5 ⁇ 10 -5 M tumor suppressor Protein M (INVITROGEN, PHC5015), 2% GlutaMAX TM Supplement (Gibco, 35050061), 5 ⁇ 10 -5 M hydrocortisone sodium succinate (Changzhou Siyao), 0.2% dimethyl sulfoxide (Sigma, D2650) , 0.05% insulin (Sigma, I9278), 0.05% transferrin (Transferrin, Sigma, T8158), 0.05% epinephrine (Sigma, E4642), 1 mM ascorbic acid (Sigma, 1043003), 0.2 mM N-acetylcysteine Amide (Sigma, A0737), 1 ⁇ 10 ⁇ 7 M dexamethasone (D4902).
  • HCM hepatocyte medium
  • R&D systems 236-EG-200
  • the cell culture medium of the present invention is improved on the basis of existing research, and the factors that maintain the function of liver cells are added: bone morphogenetic protein, fibroblast growth factor, hepatocyte growth factor and tumor suppressor protein M and other factors .
  • the dynamic culture mode was used to continue the culture for 10 days.
  • pulsed culture is used, and for the pulsed bioreactor, see ZL200910079726.8.
  • the peristaltic pump JD-200 produced by Chongyang Zhongcheng Stainless Steel Parts Service Department of Chongzhou City is used to provide the corresponding circulating power.
  • the working voltage is set to 12V and the flow rate is 60ml/min;
  • the DC motor is produced by Beijing Aikes Motor Co., Ltd.
  • the motor is ZGB37RH52i, set its working voltage to 12V and rotation speed to 100r/min; use a 100ml syringe; make the self-made guide rod and slide rail, and various components, such as DC motor, guide rod, slide rail, syringe with self-made
  • the bracket is fixed on the base plate and connects the various components.
  • the cell culture solution circulation part consists of a culture solution bottle, a peristaltic pump and a culture box. Each part is connected by a silicone tube.
  • the culture solution is pumped from the culture solution bottle into the culture box (built-in engineered tissue) through the silicone tube by a peristaltic pump.
  • the tube flows back to the culture solution bottle; the guide rail slider, the syringe and the DC motor form a pulsating part, the DC motor is connected with the guide rail slider to push the syringe piston to reciprocate, and the syringe is connected to the liquid outlet of the peristaltic pump and then connected to the culture box, thus forming pulsating flow; a pressure gauge is set on the incubator to detect the pressure of the culture fluid in the tissue placed in the incubator.
  • the connecting tube and syringe of the pulsatile bioreactor were disassembled and sterilized by high temperature and high pressure. Then turn on the pulsating bioreactor, connect the peristaltic pump and the DC motor, first add a small amount of 75% alcohol to the culture solution bottle, and use the alcohol to flow sterilize in the pulsating circulation system; pour out the alcohol and then add it to the culture solution bottle A certain amount of sterilized PBS solution is used to rinse residual alcohol.
  • the DC motor After the DC motor runs smoothly, it drives the slider to push the syringe piston to reciprocate on the guide rail.
  • the culture medium When the piston is pulled out, the culture medium is sucked from the culture medium bottle.
  • the sucked culture medium When extruding, the sucked culture medium is injected into the circulation system formed by the peristaltic pump and flows through the culture box.
  • the engineered tissue in the fluid flow back into the culture medium bottle.
  • the pressure at the cultured engineered tissue can be adjusted by adjusting the amount of the culture fluid drawn and extruded from the syringe each time.
  • the peristaltic pump and the DC motor continue to move, and the pulsatile bioreactor provides a pulsatile circulating culture fluid flow to achieve pulsatile culture of the engineered tissue.
  • FIG. 3 is the microscopic morphology of the biomimetic artificial liver tissue with extremely high cell density prepared in Example 1.
  • a proliferation detection kit (Cell counting kit-8, Dojindo) was used, and the operation was performed according to the instructions of the kit. Specific operation steps: rinse the sample once with PBS, add 800 ⁇ l of cell culture medium and 80 ⁇ l of CCK-8 solution to immerse the structure, and incubate at 37° C. for 2 h. Then pipette 110 ⁇ l of the incubation reaction solution into a 96-well plate, and use a microplate reader (BIO-RAD, Model 680) to detect the OD value of the sample at 450 nm. Each sample was replicated 3 times as blank control. The assays were selected on days 1, 10 and 20 of culture.
  • the cell density of the finally obtained artificial liver tissue was 1.2 ⁇ 10 8 cells/mL.
  • the obtained bionic liver tissue is a three-dimensional hexagonal grid structure with a height of 1 mm, a total of 6 layers, each microfilament is 2 cm long, and the Young's modulus is 1 KPa.
  • the artificial liver tissue includes microfilaments with a diameter of about 500 ⁇ m and a hollow channel with an inner diameter of about 800 ⁇ m.
  • Immunofluorescence staining was used to detect the expression of key proteins of hepatocyte function (such as CYP3A4 and ALB), enzyme-linked immunosorbent assay (Elisa) was used to detect the liver function level of the constructed three-dimensional tissue, and qPCR technology was used to detect the transcription level of marker genes of mature hepatocytes. .
  • the flat culture method is generally carried out in a conventional petri dish, such as a 6-well plate.
  • qPCR detection technology extraction of cellular RNA Operation steps: wash the structures once with PBS, add 1 ml of Trizol (Gibco, 15596026) to each structure, mix by repeated pipetting, let stand for 10 minutes at room temperature, and then transfer to 1.5 ml of EP Into the tube, add 200 ul of chloroform, shake rapidly for 30 seconds, leave at room temperature for 5 minutes, and then centrifuge at 12000g for 10 minutes at 4°C. Remove the supernatant, add an equal volume of isopropanol, and centrifuge at 12,000 g for 10 minutes at 4°C.
  • Trizol Gibco, 15596026
  • RNA reverse transcription operation steps PrimeScript TM II 1st strand cDNA Synthesis Kit (TaKaRa, 6210) was used, and the operation was carried out in accordance with the kit instructions. The RNA content was adjusted to 5ng. Primers are: Oligo dT Primer. The reverse transcription PCR program was: 42°C for 50 min, 95°C, 5 min, and 4°C incubation, using a PCR machine (ABI, SimpliAmpTM thermal cycler).
  • Fluorescence quantitative PCR operation steps Use the Maxima SYBR Green qPCR Master Mix (Thermo Scientific, K0251) kit, and operate according to the kit instructions. After adding the reaction solution as required, place the reaction plate on the qPCR instrument for detection.
  • the reaction program is: 95°C, 10min, 95°C for 15s, 60°C for 30s, 40 cycles, 72°C for 30s, and 72°C for 10min.
  • the primer sequences used for qPCR are as follows (5'-3'):
  • ALB and CYP3A4 are the marker proteins of the secretion function and drug metabolism of mature hepatocytes
  • MRP2 is the marker protein of polarity and bile canalicular structure after hepatocytes form organized arrangement.
  • the detection results show that the expression levels of various genes in the artificial liver tissue provided by the present invention are higher than those of the same kind of cells cultured on a plane, wherein, the gene transcription level of CYP3A4 is under the same conditions (the same culture medium is used, only the cell culture methods are different, Cell culture was 3.5 times that of two-dimensional cultured cells under flat conditions, such as in a 6-well plate, and the ALB gene transcription level in the three-dimensional printed structure was 12.7 times that of two-dimensional cultured cells under the same conditions. MRP2 in the three-dimensional printed structure The gene transcription level was 2.1 times that of two-dimensional cultured cells under the same conditions, and the data were all significantly different.
  • the present embodiment provides a kind of bionic artificial liver tissue with extremely high cell density prepared by using the casting method, including the following steps:
  • HepaRG is a liver stem cell obtained from human hepatoma tissue, which has the ability of bidirectional differentiation into liver cells and bile duct epithelial cells.
  • HepaRG cells (Sigma, HPRGC1) were cultured in cell expansion medium, the composition of cell expansion medium was Williams'Medium E medium (Sigma, W4125) supplemented with 10% FBS serum (Gibco, 16000), 0.05 % Insulin (Sigma, I9278), 5 x 10-7 M Hydrocortisone Succinate (Sigma, H4881), 1% Penicillin Streptomycin (Gibco, 15140122) and 1% GlutaMAX TM Supplement (Gibco, 35050061). When the cells are 90% confluent, passage at a ratio of 1:5 and change the culture medium every 2-3 days.
  • the schematic diagram of the mold used in this example is shown in Figure 1, forming a hollow cylinder-like three-dimensional structure with an outer cylinder diameter of 3cm, a central cylinder diameter of 1cm, and a cylinder wall thickness of 300um.
  • the mold was cross-linked at 20° C. for 15 min. After cross-linking, the formed structure was taken out, immersed in 5% (w/v) glutaraldehyde solution for cross-linking, and then immersed in 500 mM calcium chloride solution for cross-linking. Then, the cells were cultured in a differentiation medium for 15 days to obtain a biomimetic artificial liver tissue (three-dimensional structure).
  • the components of the differentiation medium are: hepatocyte medium (HCM; Lonza), 30ng/ml fibroblast growth factor 4 (FGF4, R&D SYSTEMS, 233-FB-025), 20ng/ml hepatocyte growth factor (HGF, R&D systems , 294-HGN-005), 0.05% epinephrine (Sigma, E4642), 0.2% dimethyl sulfoxide (Sigma, D2650), 1 mM ascorbic acid (Sigma, 1043003), 0.2 mM N-acetylcysteine amide ( Sigma, A0737) and 1 ⁇ 10 ⁇ 7 M dexamethasone (D4902).
  • the cell culture medium of the invention is improved on the basis of the existing research, and the factors that promote the differentiation of hepatocytes and maintain the function of hepatocytes are added: fibroblast growth factor, hepatocyte growth factor and epinephrine and other substances.
  • the three-dimensional structures were cultured at 37°C and 5% CO 2 , and the medium was replaced with fresh medium every 2-3 days. During the period, the morphological changes of the cells can be observed with an optical microscope.
  • the present invention uses the mixed solution of 2uM Calcein-AM (Dojindo, C326) and 4.5uM PI (Dojindo, P346) to dye live (green)/dead (red) cells respectively, and the dyeing is performed in the dark for 15 minutes. Recordings were observed using a laser scanning confocal microscope (LSCM, Nikon, Z2). Statistics of the photos of live and dead staining showed that the cell survival rate in the artificial tissue was about 90% or more at each time point.
  • a proliferation detection kit (Cell counting kit-8, Dojindo) was used, and the procedures were followed in accordance with the instructions for use of the kit. Specific operation steps: on the 20th day of culture, the samples were washed once with PBS, 800 ⁇ l of cell culture medium and 80 ⁇ l of CCK-8 solution were added to immerse the structures, and incubated at 37° C. for 2 h. Then pipette 110 ⁇ l of the incubation reaction solution into a 96-well plate, and use a microplate reader (BIO-RAD, Model 680) to detect the OD value of the sample at 450 nm. Each sample was replicated 3 times as blank control.
  • Cell counting kit-8 Cell counting kit-8, Dojindo
  • the obtained bionic liver tissue is a hollow cylinder-like three-dimensional structure with a cylindrical wall thickness of 300um, the diameter of the outer cylinder is 3cm, the diameter of the central cylinder is 1cm, and the Young's modulus is 1KPa.
  • immunofluorescence staining was used to detect the expression of key proteins (such as CYP3A4 and ALB) that mark the function of hepatocytes.
  • key proteins such as CYP3A4 and ALB
  • qPCR technology was used to detect the transcription level of marker genes in mature hepatocytes ( Figure 6).
  • CYP3A4 is a marker protein of drug metabolism function of mature hepatocytes
  • ALB is a marker protein of secretion function of mature hepatocytes. Recordings were observed with a laser confocal microscope (LSCM, Nikon, Z2).
  • Figure 5(A) is the staining result of CYP3A4 protein and DAPI
  • Figure 5(B) is the staining result of ALB protein and DAPI. It can be seen from the figure that in the artificial liver tissue, both CYP3A4 and ALB proteins are highly expressed.
  • RNA reverse transcription operation steps PrimeScript TM II 1st strand cDNA Synthesis Kit (TaKaRa, 6210) was used, and the operation was carried out in accordance with the kit instructions. The RNA content was adjusted to 5ng. The primer was Oligo dT Primer. The reverse transcription PCR program was: 42°C for 50 min, 95°C, 5 min, and 4°C incubation, using a PCR instrument (ABI, SimpliAmpTM thermal cycler).
  • Fluorescence quantitative PCR operation steps Use the Maxima SYBR Green qPCR Master Mix (Thermo Scientific, K0251) kit, and operate according to the kit instructions. After adding the reaction solution as required, place the reaction plate in the qPCR instrument for detection.
  • the reaction program is: 95°C, 10min, 95°C for 15s, 60°C for 30s, 40 cycles, 72°C for 30s, and 72°C for 10min. Gene expression at different time points was obtained.
  • the primer sequences used for qPCR are as follows (5'-3'):
  • ALB and CYP3A4 are the marker proteins of mature hepatocyte secretory function and drug metabolism function, and MRP2 is the marker protein of polarity and bile canalicular structure after hepatocytes form organized arrangement.
  • the test results are shown in Figure 6(B), the expression levels of various genes in the artificial liver tissue are higher than those in the same cells cultured in the plane, and the gene transcription level of CYP3A4 is the same as that in the two-dimensional culture under the same conditions (the culture used in 2.9 times that of cells, the ALB gene transcription level in the 3D printed structure is 19.2 times that of the 2D cultured cells under the same conditions, and the MRP2 gene transcription level in the 3D printed structure is 1.56 times that of the 2D cultured cells under the same conditions. , the data are significantly different.
  • Example 3 Using 3D bioprinting to prepare artificial liver tissue with excellent physiological functions
  • This embodiment provides an artificial liver tissue with excellent physiological function constructed by a 3D bioprinting device, as shown in FIG. 8 .
  • induced pluripotent stem cells use induced pluripotent stem cells to induce differentiation to obtain liver progenitor cells.
  • the specific implementation steps are as follows: the induced pluripotent stem cells are seeded on Matrigel substrate (Becton Dickinson) and cultured for 3-4 days.
  • the induced pluripotent stem cells were differentiated to the restricted endoderm stage, and the medium composition was: L-WNT3A (CRL2647; ATCC)-expressing cell-conditioned RPMI 1640 medium (Life, C11875500BT), 100ng/ ml Activin A (ActivinA, Gibco, PHG9014), 1% GlutaMAX TM Supplement (Gibco, 35050061), 1% Penicillin Streptomycin (Gibco, 15140122), 0.2% FBS (Bioind, 04-001-1A), and 1 xB-27 Supplement (Gibco, 17504044). Following 5 days of culture, cells at the defined endoderm stage were induced to differentiate into liver progenitor cells.
  • the components of the culture medium are: hepatocyte medium (HCM; Lonza), 30ng/ml bone morphogenetic protein 4 (BMP4, Gibco, PHC9533), 30ng/ml fibroblast growth factor 4 (FGF4, R&D SYSTEMS, 233-FB- 025), 1% GlutaMAX TM Supplement (Gibco, 35050061) and 1 ⁇ B-27 Supplement (Gibco, 17504044).
  • Human hepatocytes were purchased from Corning Corporation (Gentest Human Frozen Hepatocytes, 454550). The cells were seeded in 6-well plates (Thermo Scientific, 150239) coated with rat tail collagen type I (Corning, BioCoat 354236) according to the manufacturer's instructions. Human primary hepatocyte culture medium (LONZA, CC-3198) was used for culturing, and the culture medium was replaced every 2-3 days for a total of 5 days of culture. Human hepatocytes were used as controls.
  • GelMA is a derivative of gelatin, which is prepared by the reaction of gelatin and methacrylic anhydride (MA). A large number of amino groups present on the side chain of gelatin are replaced by methacryloyl groups in methacrylic anhydride to form modified gelatin, which has the The characteristics of covalent cross-linking and long-term stable structure.
  • the preparation method is as follows: 1) The gelatin is dissolved in phosphate buffered saline (PBS, pH 7.4). After complete dissolution, add methacrylic anhydride to the mixture and stir well; 2) Dilute the mixed solution with PBS to stop the reaction, then put the diluted solution into a dialysis membrane and dialyze it in ultrapure water to remove toxic and undesired substances.
  • PBS phosphate buffered saline
  • GelMA hydrogels can be formed from an aqueous solution of GelMA containing a photoinitiator (Irgacure 2959) by irradiation with ultraviolet (UV).
  • a photoinitiator Irgacure 2959
  • Trypsin (Gibco, 25200072) was added to digest the liver progenitor cells obtained in step 1 to obtain a single cell suspension. After counting and counting, it was mixed with the pre-heated GelMA solution/fibrinogen mixed solution to obtain the final cell concentration in the printing solution. A solution of 1 x 107 cells/mL, 7 % GelMA and 7% fibrinogen.
  • the three-dimensional structures were constructed using the bio-3D printing equipment (Regenvo, Bio-architect X) of Genofei Biotechnology Co., Ltd. Load the printing solution obtained in step 2 onto the printer, control the temperature in the printer cavity, the temperature of the printing base plate and the nozzle to be 10 °C and 20 °C respectively. According to the designed CAD file and computer path, a variety of complex structures can be constructed.
  • three-dimensional structure In this example, a three-dimensional grid structure with 6 microfilaments per layer (the composition of the microfilaments is the same as that of the printing solution) is constructed, with a total of 4 layers, a length and a width of 2 cm and a height of 1 mm.
  • the schematic diagram is shown in Figure 9. After construction, the structure was soaked in 200 mM thrombin solution for 20 min to complete post-stabilization treatment, and an artificial three-dimensional structure was obtained.
  • the artificial liver tissue with excellent physiological function was obtained by culturing in an induction medium for 20 days under the condition of 37° C. and 5% CO 2 .
  • the components of induction medium are: RPMI 1640 medium, 200ng/ml bone morphogenetic protein 2 (BMP2, Gibco, PHC7146), 300ng/ml fibroblast growth factor 4 (FGF4, R&D SYSTEMS, 233-FB-025), 200ng /ml hepatocyte growth factor (HGF, R&D systems, 294-HGN-005), 5 x 10-5 M tumor suppressor protein M (OSM, INVITROGEN, PHC5015), 5% dimethyl sulfoxide (Sigma) and 5% Epinephrine (Sigma, E4642).
  • the induction medium of the present invention contains high concentrations of various cytokines that promote the differentiation of hepatocytes, such as bone morphogenetic protein, fibroblast growth factor, hepatocyte growth factor and tumor suppressor protein M and the like.
  • This differentiation scheme enables liver progenitor cells to go beyond the expansion stage and directly enter the high-efficiency differentiation stage.
  • an artificial liver tissue with uniform cell phenotype, uniform distribution and excellent physiological functions is finally obtained.
  • the size of the artificial liver tissue structure prepared in this example is: length 2 cm x width 2 cm x height 1 mm, has a 4-layer grid structure, and a Young's modulus of 0.5KPa.
  • the cellular phenotypes in the artificial liver tissue structures were highly consistent, and were uniformly dispersed throughout the structure in the form of small-sized (10-50 ⁇ m) cell clusters, each of which contained less than 50 cells.
  • the bionic artificial liver tissue includes microfilaments with a diameter of about 300 ⁇ m and a hollow channel with an inner diameter of about 400 mm.
  • the present invention uses the mixed solution of 2uM Calcein-AM (Dojindo, C326) and 4.5uM PI (Dojindo, P346) to dye live (green)/dead (red) cells respectively, and the dyeing is performed in the dark for 15 minutes. Recordings were observed using a laser scanning confocal microscope (LSCM, Nikon, Z2). Statistics of the photos of live and dead staining showed that the cell survival rate of the artificial liver tissue at each time point was about 85% or more.
  • immunofluorescence staining was used to detect the expression of key proteins that mark the function of hepatocytes (such as ALB and CYP3A4) (Fig. 11), and enzyme-linked immunosorbent assay (Elisa) was used to detect the expression of the three-dimensional tissue constructed. Liver function level, using qPCR technology to detect the transcription level of marker genes in mature hepatocytes.
  • Immunofluorescence staining three-dimensional structures were washed with phosphate buffered saline (PBS) (BI, 02-024-1AC); 4% paraformaldehyde was fixed at room temperature for 30 minutes, washed three times with PBS for 5 minutes each; containing 0.3 The mixture of % Triton-X (Sigma, X100) and 5% bovine serum albumin (BSA) (Multicell, 800-096-EG) was blocked for 1 hour; the blocking buffer was aspirated, and the diluted primary antibody was added (with 0.3% Triton-X and 1% BSA), ALB (abcam, ab83465) and CYP3A4 (abcam, ab3572), incubated at 4°C overnight.
  • PBS phosphate buffered saline
  • BSA bovine serum albumin
  • ALB is a marker protein for the secretion function of mature hepatocytes
  • CYP3A4 is a marker protein for the drug metabolism function of mature hepatocytes. It can be seen that the hepatocytes with excellent functions are discretely and uniformly distributed in the biomaterial, and have mature albumin secretion and drug metabolism functions.
  • Albumin secretion detection kit (Bethyl, E80-129, E101, E115) and urea secretion detection kit (BIO ASSAY SYSTEMS, DIUR-500) were used to detect the albumin secretion and urea secretion function of the obtained liver tissue according to the kit instructions.
  • the results show that, compared with the cells of the conventional plane culture (the same medium used), the three-dimensional structure of the liver tissue prepared in this example, the albumin secretion level of the artificial liver tissue is 7.5 times that of the cells of the same kind in the plane culture, and the urea secretion level is 11.3 times of the same type of cells were cultured on the plane, and the data were all significantly different.
  • Extraction of cellular RNA Wash the three-dimensional structures once with PBS, add 1 ml of Trizol (Gibco, 15596026) to each structure, mix by repeated pipetting, let stand for 10 minutes at room temperature, then transfer to a 1.5 ml EP tube, add 200 ul of chloroform, shake quickly for 30 seconds, stand at room temperature for 5 minutes, and then centrifuge at 12000g for 10 minutes at 4°C. Remove the supernatant, add an equal volume of isopropanol, and centrifuge at 12,000g for 10 minutes at 4°C. Discard the supernatant, wash the precipitate with 75% absolute ethanol, air-dry to obtain RNA, and dissolve in DEPC water.
  • Trizol Gibco, 15596026
  • RNA concentration and purity were checked with a spectrophotometer (Thermo Scientific).
  • RNA reverse transcription operation steps use PrimeScript TM II 1st strand cDNA Synthesis Kit (TaKaRa, 6210), and operate according to the kit instructions. The RNA content was adjusted to 5ng.
  • the primer was Oligo dT Primer.
  • the reverse transcription PCR program was: 42°C for 50 min, 95°C, 5 min, and 4°C incubation, using a PCR instrument (ABI, SimpliAmpTM thermal cycler).
  • Fluorescence quantitative PCR operation steps Use Applied Biosystems 60x gene detection kit, and operate according to the kit instructions. After adding the reaction solution as required, place the reaction plate on the qPCR instrument for detection.
  • the reaction program is: 95°C, 10 min, 95°C for 15s, 60°C for 30s, 40 cycles, 72°C for 30s, and 72°C for 10min. Gene expression at different time points was obtained.
  • the primers used for qPCR are shown in Table 1:
  • liver tissue Human hepatocytes are the current "gold standard" in the field of liver tissue research, and are carried out in a conventional flat culture method (the flat culture method is generally carried out in a conventional culture dish, such as a 6-well plate); artificial liver tissue is the
  • the liver tissue prepared in this example has a three-dimensional structure and contains certain material components and specific mechanical characteristics.
  • the proteins encoded by the above genes are marker proteins for the detoxification and drug metabolism functions of mature hepatocytes. It can be seen from the experimental results that the expression level of key liver genes in the artificial liver tissue of the present invention reaches or even exceeds the level of human hepatocytes, and is significantly higher than the gene expression level of the same cells in plane culture (there are significant differences in data).
  • the present embodiment provides artificial liver tissue with excellent physiological functions of various structures prepared by a casting method, including the following steps:
  • HepaRG is a liver stem cell obtained from human hepatoma tissue, which has the ability of bidirectional differentiation into liver cells and bile duct epithelial cells.
  • HepaRG cells (Sigma, HPRGC1) were cultured in cell expansion medium.
  • the composition of cell expansion medium was: Williams'Medium E medium (Sigma, W4125) supplemented with 10% FBS serum (Gibco, 16000), 0.05% insulin (Sigma, I9278), 5 x 10-5 M hydrocortisone succinate (Sigma, H4881), 1% penicillin streptomycin (Gibco, 15140122) and 1% GlutaMAX TM (Gibco, 35050061).
  • the cells were 90% confluent, the cells were passaged at a ratio of 1:5, the culture medium was changed every 2-3 days, and the cells were co-cultured for 5 days. The cells were collected and used when they reached 90% confluence.
  • Trypsin (Gibco, 25200072) was added to the HepaRG cells cultured in step 1, digested at 37°C for 3 min, collected, centrifuged to obtain the precipitate of liver stem cells, resuspended in basal medium to obtain a single cell suspension, and the cells were subjected to After counting, it was diluted in proportion, and then mixed with the pre-heated polylactic acid-glycolic acid copolymer/collagen mixed solution, and the cell concentration in the final printing solution (precursor solution) was 1 ⁇ 10 7 cells/mL, 4% Polylactic-glycolic acid copolymer solution and 15% collagen solution.
  • the schematic diagram of the mold used in this example is shown in Figure 1, forming a cylindrical three-dimensional structure with a volume of 3 cm in outer diameter, a hollow cylinder with a diameter of 500 ⁇ m evenly distributed in the center, and a height of 3 cm.
  • the mold was cross-linked at 37° C. for 30 min. After cross-linking, the formed structure was taken out and immersed in a 5% (w/v) glutaraldehyde solution for cross-linking. Then, the induced differentiation medium is used for culturing for 20-25 days to obtain bionic liver tissue.
  • the components of differentiation medium are: hepatocyte medium (HCM; Lonza), 100ng/ml activin A (ActivinA, Gibco, PHG9014), 300ng/ml fibroblast growth factor 4 (FGF4, R&D SYSTEMS, 233-FB-025 ), 500ng/ml hepatocyte growth factor (HGF, R&D systems, 294-HGN-005), 1 ⁇ B-27 (Gibco, 17504044), 2% GlutaMAX TM Supplement (Gibco, 35050061), 5 ⁇ 10 -3 M Hydrocortisone sodium succinate (Changzhou Siyao), 10% DMSO (Sigma) and 5% insulin (Sigma, I9278).
  • the induction medium of the present invention contains high concentrations of various cytokines that promote the differentiation of hepatocytes, such as activin A, fibroblast growth factor, and hepatocyte growth factor, so as to promote the efficient differentiation of liver stem cells. With the assistance, an artificial liver tissue with uniform cell phenotype, uniform distribution and excellent physiological function was finally obtained.
  • various cytokines that promote the differentiation of hepatocytes, such as activin A, fibroblast growth factor, and hepatocyte growth factor, so as to promote the efficient differentiation of liver stem cells.
  • the structures were cultured at 37°C with 5% CO 2 , and the medium was replaced with fresh medium every 2 to 3 days. During the period, the morphological changes of the cells can be observed with an optical microscope.
  • pulsed culture is used, and for the pulsed bioreactor, see ZL200910079726.8.
  • the peristaltic pump JD-200 produced by Chongyang Zhongcheng Stainless Steel Parts Service Department of Chongzhou City is used to provide the corresponding circulating power.
  • the working voltage is set to 12V and the flow rate is 60ml/min;
  • the DC motor is produced by Beijing Aikes Motor Co., Ltd.
  • the motor is ZGB37RH52i, set its working voltage to 12V and rotation speed to 100r/min; use a 100ml syringe; make the self-made guide rod and slide rail, and various components, such as DC motor, guide rod, slide rail, syringe with self-made
  • the bracket is fixed on the base plate and connects the various components.
  • the cell culture solution circulation part consists of a culture solution bottle, a peristaltic pump and a culture box. Each part is connected by a silicone tube.
  • the culture solution is pumped from the culture solution bottle into the culture box (built-in engineered tissue) through the silicone tube by a peristaltic pump.
  • the tube flows back to the culture solution bottle;
  • the guide rail slider, the syringe and the DC motor form a pulsating part, the DC motor is connected with the guide rail slider to push the syringe piston to reciprocate, and the syringe is connected to the liquid outlet of the peristaltic pump and then connected to the culture box.
  • pulsating flow a pressure gauge is set on the incubator to detect the pressure of the culture fluid in the tissue placed in the incubator.
  • the connecting tube and syringe of the pulsatile bioreactor were disassembled and sterilized by high temperature and high pressure. Then turn on the pulsation bioreactor, the peristaltic pump and the DC motor are turned on, first add a small amount of 75% alcohol to the culture solution bottle, and use alcohol to flow sterilize in the pulsation circulation system; pour out the alcohol and then add it to the culture solution bottle A certain amount of sterilized PBS solution is used to rinse residual alcohol.
  • the DC motor After the DC motor runs smoothly, it drives the slider to push the syringe piston to reciprocate on the guide rail. When the piston is pulled out, the culture medium is sucked from the culture medium bottle. The engineered tissue in the cassette flows back into the medium bottle. The pressure at the cultured engineered tissue can be adjusted by adjusting the amount of the culture fluid drawn and extruded from the syringe each time. As a result, the peristaltic pump and the DC motor continue to move, and the pulsatile bioreactor provides a pulsatile circulating culture fluid flow to achieve pulsatile culture of the engineered tissue.
  • the present invention uses the mixed solution of 2uM Calcein-AM (Dojindo, C326) and 4.5uM PI (Dojindo, P346) to dye live (green)/dead (red) cells respectively, and the dyeing is performed in the dark for 15 minutes. Recordings were observed using a laser scanning confocal microscope (LSCM, Nikon, Z2). Statistics of the photos of live and dead staining showed that the cell survival rate in the artificial liver tissue at each detection time point was higher than 85%.
  • immunofluorescence staining was used to detect the expression of key proteins (such as CYP3A4 and ALB) that mark the function of hepatocytes, and enzyme-linked immunosorbent assay (Elisa) was used to detect the level of liver function in the constructed three-dimensional tissue. Transcription levels of marker genes in mature hepatocytes were detected by qPCR.
  • CYP3A4 is a marker protein of drug metabolism function of mature hepatocytes
  • ALB is a marker protein of secretion function of mature hepatocytes. It can be seen that hepatocytes are discretely and uniformly distributed and structured within microfilaments, with mature secretory and metabolic functions.
  • the artificial liver tissue structure prepared in this example is a cylinder with a diameter of 3 cm ⁇ a height of 3 cm, and 19 hollow cylinders with a diameter of 500 ⁇ m are evenly distributed in the center of the cylinder ( FIG. 1 ).
  • the Young's modulus of the structure was 1 KPa.
  • the cell phenotypes in the artificial liver tissue structure were highly consistent, and were uniformly dispersed throughout the structure in the form of small-sized cell clusters, and the number of cells in each small-sized cell cluster (10-50 ⁇ m) was less than 50.
  • the bionic artificial liver tissue includes microfilaments with a diameter of about 400 ⁇ m and a hollow channel with an inner diameter of about 1000 mm.
  • Procedure for extracting cellular RNA Wash the three-dimensional structures once with PBS, add 1 ml of Trizol (Gibco, 15596026) to each structure, mix by repeated pipetting, let stand for 10 minutes at room temperature, and then transfer to a 1.5 ml EP tube. 200ul of chloroform was added, shaken rapidly for 30 seconds, placed at room temperature for 5 minutes, and centrifuged at 12000g at 4°C for 10 minutes. Discard the supernatant, add an equal volume of isopropanol, and centrifuge at 12,000g at 4°C for 10 minutes. Discard the supernatant, wash the precipitate with 75% absolute ethanol, air-dry to obtain RNA, and dissolve in DEPC water.
  • Trizol Gibco, 15596026
  • RNA concentration and purity were checked with a spectrophotometer (Thermo Scientific).
  • RNA reverse transcription operation steps use PrimeScript TM II 1st strand cDNA Synthesis Kit (TaKaRa, 6210), and operate according to the kit instructions. The RNA content was adjusted to 5ng.
  • the primer was Oligo dT Primer.
  • the reverse transcription PCR program was: 42°C for 50 min, 95°C, 5 min, and 4°C incubation, using a PCR instrument (ABI, SimpliAmpTM thermal cycler). Fluorescence quantitative PCR operation steps: use Applied Biosystems 60x gene detection kit, and operate according to the kit instructions. After adding the reaction solution as required, place the reaction plate in the qPCR instrument for detection.
  • the reaction program is: 95°C, 10min, 95°C 15s, 60°C 30s, 40 cycles, 72°C 30s, 72°C 10min. Obtain gene expression at different time points.
  • the primer sequences used for qPCR are shown in
  • the proteins encoded by the above genes are marker proteins for the detoxification and drug metabolism functions of mature hepatocytes.
  • the test results are shown in Figure 12B and Table 2.
  • the expression levels of various genes in the artificial liver tissue are at or higher than those in human hepatocytes, and are significantly higher than the gene expression levels in the same cells cultured in planes. The data are significant. difference.
  • the artificial bionic artificial liver tissue provided by the present invention has a certain structure, material composition, material arrangement and mechanical properties.
  • the growth and development of cells in tissues is completely different from the conventional flat culture, which is the key to successfully inducing specific three-dimensional structures of liver tissue and physiological functions, providing downstream applications closer to human tissue.
  • the research model fills the gap in this field.
  • This embodiment provides an artificial liver structure that can be constructed by a biological 3D printing device, as shown in FIG. 2 .
  • Human normal hepatocytes (L-02) were purchased from Procell Company, the product number is HL-7702. Human normal hepatocytes were cultured in plane adherent culture using expansion medium, the medium was changed every 2 days, and the cells were passaged at a ratio of 1:3 when the cells reached 85% confluence.
  • the composition of the medium of human normal hepatocytes is: Williams'Medium E medium (Sigma, W4125) supplemented with 10% FBS serum (Gibco, 16000), 1% penicillin streptomycin (Gibco, 15140122), 20ng/mL liver Cell Growth Factor (HGF, R&D Systems) and 50 ⁇ M ⁇ -mercaptoethanol.
  • Human intrahepatic bile duct epithelial cells were purchased from Procell (CP-H042). Human intrahepatic bile duct epithelial cells were cultured in plane adherent culture using expansion medium, the culture medium was changed every 2 days, and the cells were passaged at a ratio of 1:3 when the cells reached 85% confluence.
  • composition of the medium of human intrahepatic bile duct epithelial cells is: Vivo 15 medium (Lonza 04-418Q) supplemented with 10% FBS serum (Gibco, 16000), 1% epithelial cell growth additive (Procell, CP-H042), 5 ⁇ 10 -5 mol/L hydrocortisone sodium succinate (Changzhou Siyao), 0.05% insulin (Sigma, I9278), 0.05% transferrin (Transferrin, Sigma, T8158), 0.05% epinephrine hydrochloride (Epinephrine hydrochloride) , Sigma, E4642), 1% penicillin-streptomycin (Gibco, 15140122).
  • polyglycolic acid Shanghai Yuanye Biotechnology Co., Ltd., S26878, molecular weight 10,000-20,000 Da
  • fibrinogen solution Sigma-Aldrich, F3879
  • printing solution A single cell suspension A: the concentration of normal human hepatocytes is 0.5 ⁇ 10 5 cells/mL, 7% polyglycolic acid solution and 7% fibrinogen solution; and printing solution B (single cell suspension B): human intrahepatic bile duct epithelial cells at a concentration of 0.5 x 105 cells/mL, 7% polyglycolic acid solution and 7% fibrinogen solution.
  • Three-dimensional structures were constructed using extrusion printing equipment (Regenvo, Bio-architect X) of Genofei Biotechnology Co., Ltd.
  • the obtained printable single-cell suspensions A and B were loaded onto a dual-nozzle 3D bioprinter respectively, and the temperature in the printer cavity, the temperature of the printing plate and the nozzle were controlled to be 20 °C.
  • the designed CAD file and computer path it was possible to Build a variety of three-dimensional structures with complex structures.
  • a double-jet printer is used to construct a three-dimensional hexagonal mesh with 6 microfilaments in each layer (the composition of the microfilaments is the same as that of the printing solutions A and B), a total of 6 layers, and each microfilament is 2 cm long and 0.5 cm high.
  • the lattice structure is shown in Figure 2. After construction, a 200 mM thrombin solution was used for soaking and cross-linking to complete post-stabilization treatment, and a three-dimensional hydrogel structure was obtained.
  • the co-culture medium was cultured for 10 days at 37°C and 5% CO 2 to obtain a biomimetic artificial structure with bile duct tissue and liver tissue.
  • the components of the co-culture medium are Williams'Medium E medium (Sigma, W4125), 10% FBS serum (Gibco, 16000), 100ng/ml Activin A (Activin A, Gibco, PHG9014), 20ng/ml bone morphogenesis Protein 2 (Gibco, PHC7146), 20ng/ml bone morphogenetic protein 4 (Gibco, PHC9533), 1% dimethyl sulfoxide (Sigma, D2650), 20ng/ml hepatocyte growth factor (R&D SYSTEMS, 294-HGN- 005), 5 ⁇ 10 -5 M tumor suppressor protein M (INVITROGEN, PHC5015), 20ng/ml keratinocyte growth factor (R&D SYSTEMS, 251-KG-010), 1.8 ⁇ 10
  • the cell culture medium of the invention is improved on the basis of the existing research, and the factors that maintain the function of hepatocytes are added: activin A, bone morphogenetic protein, dimethyl sulfoxide, hepatocyte growth factor and tumor suppressor protein M , and factors that induce differentiation into bile duct epithelial cells and maintain cholangiocyte function: keratinocyte growth factor, sodium butyrate, sodium taurocholate, and epidermal cell growth factor.
  • pulsed culture is used, and for the pulsed bioreactor, see ZL200910079726.8.
  • the peristaltic pump JD-200 produced by Chongyang Zhongcheng Stainless Steel Parts Service Department of Chongzhou City is used to provide the corresponding circulating power.
  • the working voltage is set to 12V and the flow rate is 60ml/min;
  • the DC motor is produced by Beijing Aikes Motor Co., Ltd.
  • the motor ZGB37RH52i set its working voltage to 12V and the rotation speed to 100r/min; use a 100ml syringe; make the self-made guide rod and slide rail, as well as various components such as DC motor, guide rod, slide rail, and syringe with self-made
  • the liver tissue is fixed on the base plate and the components are connected.
  • the cell culture solution circulation part consists of a culture solution bottle, a peristaltic pump and a culture box. Each part is connected by a silicone tube.
  • the culture solution is pumped from the culture solution bottle into the culture box (built-in engineered tissue) through the silicone tube by a peristaltic pump.
  • the tube flows back to the culture solution bottle; the guide rail slider, the syringe and the DC motor form a pulsating part, the DC motor is connected with the guide rail slider to push the syringe piston to reciprocate, and the syringe is connected to the liquid outlet of the peristaltic pump and then connected to the culture box, thus forming pulsating flow; a pressure gauge is set on the incubator to detect the pressure of the culture fluid in the tissue placed in the incubator.
  • the connecting tube and syringe of the pulsatile bioreactor were disassembled and sterilized by high temperature and high pressure. Then turn on the pulsating bioreactor, connect the peristaltic pump and the DC motor, first add a small amount of 75% alcohol to the culture solution bottle, and use the alcohol to flow sterilize in the pulsating circulation system; pour out the alcohol and then add it to the culture solution bottle A certain amount of sterilized PBS solution is used to rinse residual alcohol.
  • the DC motor After the DC motor runs smoothly, it drives the slider to push the syringe piston to reciprocate on the guide rail.
  • the culture medium When the piston is pulled out, the culture medium is sucked from the culture medium bottle.
  • the sucked culture medium When extruding, the sucked culture medium is injected into the circulation system formed by the peristaltic pump and flows through the culture box.
  • the engineered tissue in the fluid flow back into the culture medium bottle.
  • the pressure at the cultured engineered tissue can be adjusted by adjusting the amount of the culture fluid drawn and extruded from the syringe each time.
  • the peristaltic pump and the DC motor continue to move, and the pulsatile bioreactor provides a pulsatile circulating culture fluid flow to achieve pulsatile culture of the engineered tissue.
  • the microscopic morphology of the in vitro artificial liver structure with both hepatocyte clusters (indicated by asterisks) and bile duct networks (indicated by black arrows) was obtained.
  • the through-channels (ie hollow channels) inside the structure can be seen from the figure.
  • Live and dead staining detection Use a mixed solution of 2uM Calcein-AM (Dojindo, C326) and 4.5uM PI (Dojindo, P346) to stain live (green)/dead (red) cells, respectively, in the dark for 15 minutes . Recordings were observed using a laser scanning confocal microscope (LSCM, Nikon, Z2). Statistics of the photos of live and dead staining were performed, and the cell survival rate of the Day0 structure after printing was about 95%.
  • Biological detection In order to detect the formation of the bile duct network in the three-dimensional structure and the maintenance of hepatocyte function, immunofluorescence staining was used to detect the expression of specific marker proteins (such as CYP3A4, ALB and CK19) in hepatocytes and intrahepatic bile duct epithelial cells ( Figure 15), the enzyme-linked immunosorbent assay (Elisa) was used to detect the liver function level of the constructed three-dimensional tissue, and the qPCR technology was used to detect the transcription level of genes related to hepatocytes and bile duct epithelial cells.
  • specific marker proteins such as CYP3A4, ALB and CK19
  • Immunofluorescence staining structures were washed with phosphate buffered saline (PBS) (BI, 02-024-1AC); fixed in 4% paraformaldehyde for 30 min at room temperature and washed 3 times with PBS for 5 min each; with 0.3% Triton A mixture of -X (Sigma, X100) and 5% bovine serum albumin (BSA) (Multicell, 800-096-EG) was blocked for 1 hour; the blocking buffer was aspirated, and the diluted primary antibody (containing 0.3% Triton-X and 1% BSA), CYP3A4 (abcam, ab3572), ALB (abcam, ab83465) and CK19 (RD, MAB3506) were incubated overnight at 4°C.
  • PBS phosphate buffered saline
  • BSA bovine serum albumin
  • Figure 15 shows the results of immunofluorescence staining of key proteins in three-dimensional biomimetic liver tissue, wherein ALB and CYP3A4 are marker proteins of mature hepatocytes, and CK19 is a marker protein of bile duct epithelial cells.
  • A is the staining result of CYP3A4 and CK19 protein
  • B is the staining result of ALB and CK19 protein
  • C is the staining result of CK19 protein and DAPI
  • D is the three-dimensional layer scan of CK19 protein in the structure.
  • all the above-mentioned proteins are highly expressed.
  • CYP3A4 is a marker protein of the drug metabolism function of mature hepatocytes
  • CK19 is a marker of bile duct epithelial cells, and it can be seen that bile duct epithelial cells form a network structure within the structural microfilaments
  • ALB is a marker protein of the secretion function of mature hepatocytes . It can be seen that the hepatocyte mass has mature secretory and metabolic functions, and the bile duct epithelial cells form a network structure around the hepatocyte mass.
  • the bile duct cells self-assemble to form a three-dimensional hollow tubular structure (the inner diameter of the hollow tubular structure is about It is 800 ⁇ m, and the average interval distance between the pipes is about 500 ⁇ m, and the distribution density of the pipes is about 50 pieces/cm 2 ), which is in line with the physiological structure of the human liver.
  • Albumin secretion detection kit (Bethyl, E80-129, E101, E115) and urea secretion detection kit (BIO ASSAY SYSTEMS, DIUR-500) were used to detect the albumin secretion and urea secretion function of the obtained liver tissue according to the kit instructions.
  • the results show that the hepatic tissue containing bile duct structure prepared by the present invention is 7.9 times of the albumin secretion level of the three-dimensional biomimetic liver tissue compared with the conventional plane cultured cells, and the urea secretion level is 7.9 times that of the plane cultured cells (the medium used is the same composition). Culture cells 2.7 times.
  • the obtained three-dimensional bionic liver tissue is a three-dimensional hexagonal grid structure with a height of 2 mm, a total of 10 layers, and each microfilament is 2 cm long.
  • the Young's modulus of the bionic liver tissue was 1.5KPa.
  • the bionic liver tissue contains microfilaments with a diameter of about 500 ⁇ m and a hollow channel with an inner diameter of about 800 ⁇ m.
  • the flat culture method is generally carried out in a conventional petri dish, such as a 6-well plate.
  • qPCR gene detection extraction of cellular RNA Operation steps: Wash the structures once with PBS, add 1 ml of Trizol (Gibco, 15596026) to each structure, mix by repeated pipetting, let stand for 10 minutes at room temperature, and then transfer to a 1.5 ml EP tube , add 200ul of chloroform, shake quickly for 30 seconds, place at room temperature for 5 minutes, and then centrifuge at 12000g for 10 minutes at 4°C. Remove the supernatant, add an equal volume of isopropanol, and centrifuge at 12,000 g for 10 minutes at 4°C.
  • Trizol Gibco, 15596026
  • RNA reverse transcription operation steps PrimeScript TM II 1st strand cDNA Synthesis Kit (TaKaRa, 6210) was used, and the operation was carried out in accordance with the kit instructions. The RNA content was adjusted to 5ng. Primers are: Oligo dT Primer.
  • the reverse transcription PCR program was: 42°C for 50 min, 95°C, 5 min, and 4°C incubation, using a PCR instrument (ABI, SimpliAmpTM thermal cycler).
  • Fluorescence quantitative PCR operation steps use Maxima SYBR Green qPCR Master Mix (Thermo Scientific, K0251) kit, and operate according to the kit instructions. After adding the reaction solution as required, place the reaction plate on the qPCR instrument for detection.
  • the reaction program is: 95°C, 10 min, 95°C for 15s, 60°C for 30s, 40 cycles, 72°C for 30s, and 72°C for 10min. Obtain gene expression at different time points.
  • the primer sequences used for qPCR are as follows (5'-3'):
  • CYP3A4 is a marker protein of drug metabolism in mature hepatocytes, and its gene transcription level is the same as that of the two-dimensional cultured cells under the same conditions.
  • ALB is a marker protein of the secretory function of mature hepatocytes, and the ALB gene transcription level in the three-dimensional printing structure is the same condition (the same medium is used, only the cell culture method is different, and the cells are cultured under flat conditions, such as 6 wells).
  • CK19 is a marker protein specifically expressed in bile duct epithelial cells, and the transcription level of CK19 gene in the three-dimensional printed structure is 3.4 times that of two-dimensional cultured cells under the same conditions. The data are all significant difference.
  • This embodiment provides a method for preparing an artificial liver structure by a casting method, as shown in FIG. 16 and FIG. 7 . It includes the following steps:
  • iPSCs Use induced pluripotent stem cells (iPSCs) to induce differentiation to obtain liver stem cells.
  • the specific implementation steps are as follows: iPS cells (Anhui Zhongsheng Suyuan Biotechnology Co., Ltd.) are dissociated using Cell Dispase (Dispase, Roche Diagnostics) and inoculated on BD Matrigel Growth Factor Reduced Basement Membrane Matrix (Becton Dickinson). Subsequently, iPSCs were placed in L-WNT3A (CRL2647; ATCC)-expressing cell-conditioned RPMI 1640 medium (Sigma) for 4 days to differentiate into the restricted endoderm stage.
  • L-WNT3A CTL2647; ATCC
  • RPMI 1640 medium Sigma
  • the medium composition included: 100ng/mL Activin A ( R&D Systems), 1% GlutaMAX (Thermo Fisher Scientific), 1% Penicillin Streptomycin (Gibco), 0.2% FBS and 1 x B-27 (Supplement, Thermo Fisher Scientific), cells at the defined endoderm stage were obtained. Subsequently, cells at the defined endoderm stage were cultured in RPMI 1640 basal medium for 8 days to obtain liver stem cells, and the medium contained: 30ng/mL bone morphogenetic protein (BMP4, R&D Systems) and 20ng/mL fibroblasts Cell Growth Factor (FGF4, R&D Systems), 1% GlutaMAX and 1 ⁇ B-27 (Thermo Fisher Scientific).
  • BMP4 bone morphogenetic protein
  • FGF4 Cell Growth Factor
  • liver stem cells For the liver stem cells during the proliferation period, first add PBS to rinse the surface, then add trypsin (Gibco, 25200072) to cover the cell surface, digest at 37°C for 3 min, collect, centrifuge to obtain the precipitate of liver stem cells, and resuspend in basal medium. The single-cell suspension was obtained, the cells were counted and then diluted in proportion, and then mixed with the pre-heated sodium alginate/gelatin mixed solution, and the cell concentration in the final precursor solution was 1 ⁇ 10 5 cells/mL, 4 % Sodium Alginate and 15% Gelatin.
  • trypsin Gibco, 25200072
  • the schematic diagram of the structure of the mold used in this example is shown in Figure 13, forming a hollow hexahedron-like three-dimensional structure with a cross-sectional hexagonal diameter of 6 cm, a central cylinder diameter of 2 cm, and a height of 6 cm.
  • the mold was cross-linked at 15°C for 15 min.
  • the formed structure was taken out and immersed in a 5% (w/v) glutaraldehyde solution for cross-linking. Then, it was immersed in 300mM calcium chloride solution for cross-linking and then cultured in the bidirectional differentiation medium of liver stem cells.
  • the specific components were: RPMI 1640 medium containing 20ng/mL hepatocyte growth factor (HGF, R&D Systems) 1% GlutaMAX (Thermo Fisher Scientific), 1% Penicillin Streptomycin (Gibco), 1 ⁇ B27 Supplement Minus Vitamin A, 1 ⁇ 10 -5 M Interleukin 6 (Miltenyi), 1 ⁇ M Sodium Taurocholate (Sigma), 1 ⁇ M Acetone sodium (Sigma), and cultured for 15 days to obtain an artificial liver structure.
  • HGF hepatocyte growth factor
  • GlutaMAX Thermo Fisher Scientific
  • Penicillin Streptomycin Gabco
  • 1 ⁇ B27 Supplement Minus Vitamin A 1 ⁇ 10 -5 M Interleukin 6 (Miltenyi)
  • 1 ⁇ M Sodium Taurocholate Sigma
  • 1 ⁇ M Acetone sodium Sigma
  • hepatocyte medium HCM; Lonza
  • 25% human ES/iPS stem cell serum-free medium mTeSR TM 1 medium (StemCell, 05850 )
  • 20ng/ml bone morphogenetic protein 2 BMP2, Gibco, PHC7146
  • 20ng/ml bone morphogenetic protein 4 BMP4, Gibco, PHC9533
  • 30ng/ml fibroblast growth factor 4 FGF4, R&D SYSTEMS, 233 -FB-025)
  • 20ng/ml hepatocyte growth factor HGF, R&D systems, 294-HGN-005
  • 5 ⁇ 10-5 M tumor suppressor protein M OSM, INVITROGEN, PHC5015)
  • 20ng/ml keratinocyte growth Factor KGF, R&D, 251-KG-010
  • the cell culture medium of the present invention is improved on the basis of existing research, and the factors that induce hepatocyte differentiation and maintain hepatocyte function are added: bone morphogenetic protein, fibroblast growth factor, hepatocyte growth factor, dimethyl Sulfoxide and tumor suppressor protein M, and factors that induce cholangiocytic differentiation: keratinocyte growth factor, sodium butyrate, sodium taurocholate, and epidermal cell growth factor and other factors.
  • Live and dead staining detection Use a mixed solution of 2uM Calcein-AM (Dojindo, C326) and 4.5uM PI (Dojindo, P346) to stain live (green)/dead (red) cells, respectively, in the dark for 15 minutes . Recordings were observed using a laser scanning confocal microscope (LSCM, Nikon, Z2). Statistics of the photos of live and dead staining were performed, and the cell survival rate of the Day0 structure after printing was about 95%.
  • Biological detection In order to detect the formation of bile duct network and the maintenance of hepatocyte function in the three-dimensional structure, immunofluorescence staining was used to detect the expression of specific marker proteins (such as CYP3A4, ALB and CK19) in hepatocytes and intrahepatic bile duct epithelial cells, Enzyme-linked immunosorbent assay (Elisa) was used to detect the liver function level of the constructed three-dimensional tissues ( Figure 16A), and qPCR technology was used to detect the transcription levels of genes related to hepatocytes and bile duct epithelial cells ( Figure 16B). The immunofluorescence staining method was the same as that in Example 5.
  • specific marker proteins such as CYP3A4, ALB and CK19
  • qPCR gene detection extraction of cellular RNA Operation steps: Wash the structures once with PBS, add 1 ml of Trizol (Gibco, 15596026) to each structure, mix by repeated pipetting, let stand for 10 minutes at room temperature, and then transfer to a 1.5 ml EP tube , add 200ul of chloroform, shake quickly for 30 seconds, place at room temperature for 5 minutes, and then centrifuge at 12000g for 10 minutes at 4°C. Remove the supernatant, add an equal volume of isopropanol, and centrifuge at 12,000 g for 10 minutes at 4°C.
  • Trizol Gibco, 15596026
  • RNA reverse transcription operation steps PrimeScript TM II 1st strand cDNA Synthesis Kit (TaKaRa, 6210) was used, and the operation was carried out in accordance with the kit instructions. The RNA content was adjusted to 5ng.
  • the primer was Oligo dT Primer.
  • the reverse transcription PCR program was: 42°C for 50 min, 95°C, 5 min, and 4°C incubation, using a PCR instrument (ABI, SimpliAmpTM thermal cycler).
  • Fluorescence quantitative PCR operation steps use Maxima SYBR Green qPCR Master Mix (Thermo Scientific, K0251) kit, and operate according to the kit instructions. After adding the reaction solution as required, place the reaction plate on the qPCR instrument for detection.
  • the reaction program is: 95°C, 10 min, 95°C for 15s, 60°C for 30s, 40 cycles, 72°C for 30s, and 72°C for 10min.
  • the gene expression profiles at different time points were obtained (Fig. 16B).
  • the primer sequences used for qPCR are as follows (5'-3'):
  • CYP3A4 is a marker protein of the drug metabolism function of mature hepatocytes.
  • its gene transcription level is 2.3 times that of two-dimensional cultured cells under the same conditions
  • ALB is a marker protein for the secretory function of mature hepatocytes
  • the ALB gene transcription level in the three-dimensional printed structure is 36.2 times that of two-dimensional cultured cells under the same conditions
  • CK19 is a marker protein specifically expressed in bile duct epithelial cells.
  • the transcription level of CK19 gene in the three-dimensional printed structure is 18.7 times that of two-dimensional cultured cells under the same conditions. ** and *** indicate significant differences in data.
  • the cross-section of the obtained three-dimensional artificial liver tissue is a hexagon with a circumscribed circle diameter of 6 cm and a height of 6 cm; the center contains a cylindrical structure with a diameter of 2 cm and 6 spoke support structures (Fig. 13).
  • the cell density was about 8 ⁇ 10 6 cells/cm 3 , and the Young's modulus was 1.5 KPa.
  • the bionic artificial liver tissue provided by the invention can be used in the fields of drug preclinical detection, regenerative medicine and in vivo transplantation, bioartificial liver and liver function compensation, liver disease research and treatment, drug testing and new drug development.
  • the present invention provides an artificial liver structure containing bile ducts and liver tissue, which is a widely used liver model and is used for drug preclinical detection, environmental monitoring, toxicology detection, tissue engineering, regenerative medicine, new drug research and development, and research on liver tissue development. , study the occurrence and development of diseases and other fields.

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Abstract

一种仿生人工肝组织及其制备方法与应用。该仿生人工肝组织具有以下三种特征之一：（1）含有接近人体天然组织的极高的细胞密度，具有仿生的生理功能；（2）含有类胆管和类肝脏组织结构，肝细胞呈肝板样或团簇形态，胆管上皮细胞呈胆管样三维脉管网络形态，组织功能良好；（3）所述仿生人工肝组织中细胞以小尺寸团簇的形式均匀分散在整个结构中，具有仿生的生理功能。仿生肝组织的制备方法以及在药物临床前检测、再生医学和体内移植、生物人工肝和肝功能代偿、肝脏疾病学、新药研发、组织发育、肝组织再生、药物毒理学检测、环境、大气、水体污染物和毒理学检测等领域被提及。

Description

仿生人工肝组织及其制备方法与应用
交叉引用
本申请要求2020年10月19日提交的专利名称为“仿生人工肝组织及其制备方法与应用”的第202011120076.X号中国专利申请、专利名称为“类肝组织结构体及其制备方法与应用”的第202011120070.2号中国专利申请、专利名称为“含有胆管和肝脏组织的人工肝结构体及其制备方法与应用”的第202011120082.5号中国专利申请以及2020年10月27日提交的专利名称为“仿生人工肝组织及其制备方法与应用”的第202011163981.3号中国专利申请、专利名称为“类肝组织结构体及其制备方法与应用”的第202011163982.8号中国专利申请、专利名称为“含有胆管和肝脏组织的人工肝结构体及其制备方法与应用”的第202011163991.7号中国专利申请的优先权，其全部公开内容通过引用整体并入本文。
技术领域
本发明涉及生物材料和生物医学工程领域，具体地说，涉及一种仿生人工肝组织及其制备方法与应用。
背景技术
肝脏是人体最大的实体器官，约占人体总重量的2％。作为人体最大的消化腺和新陈代谢中心，肝脏在维持人体的代谢稳态承担着重要角色。肝脏承担着营养物质的合成功能，负责人体内的血清蛋白、氨基酸和糖原的合成和分泌。肝脏还承担着解毒功能，代谢来自内源和外源的代谢废物、药物等化合物。此外，肝脏还承担着包括氨基酸、维生素、脂质和碳水化合物等物质的代谢和储存。
由于人体试验的伦理和安全限制，现阶段常用的研究模型是动物模型和平面培养的人体肝实质细胞。由于多种病原体(如丙型肝炎等)的种属特异性以及动物与人类的肝脏功能性蛋白的巨大差异，导致动物的肝脏的病理模型在许多方面都与人体有着本质的差别。而平面培养的人体肝细胞，不仅来源极其有限，而且受限于细胞表面受体的平面群聚状态，导致体外平面培养的成熟肝细胞很快丧失其表型与功能特征。这些问题导致目前常用的研究模型无法在临床前药物检测中准确的预测药物的导致的人类肝脏毒性和肝损伤，难以进行新药的临床前筛选与开发。
人体肝实质细胞来源极其有限，体外增殖能力有限且易分化，在一般体外培养条件下极易丧失其表型和功能特征。从干细胞或祖细胞衍生获得的肝细胞，具有来源广泛、易获取、功能良好、体外可长期培养和维持功能等特点，是近年来肝组织研究领域的热点。多种干细胞(诱导多能干细胞、胚胎干细胞、肝脏干细胞、肝脏祖细胞、内胚层细胞、肝脏内胚层细胞、肝母细胞、间充质干细胞和成体干细胞)都具有向肝脏细胞分化的潜力，已经有大量的研究致力于优化和规范不同干细胞分化至肝细胞的过程。但是，现阶段干细胞分化研究面临一个普遍问题：分化不可控(分化不均一、效率较低而且可重复性差)、得到的细胞不成熟而且生理功能较差、分化过程耗时长且产率低等问题，无法获得 大量的、无批次内差异、功能稳定的干细胞衍生的肝细胞，无法满足组织发育、肝脏再生和肝病发生与发展方面的基础与应用研究的需求。
人体天然肝组织的细胞密度达到10 8～10 9个/mL量级，细胞和细胞之间形成致密连接，并有广泛分布的血管作为营养传输媒介。目前公知，人体组织的结构与生理功能之间存在密切联系，接近体内生理条件的细胞密度，对于肝组织的生理功能实现具有重要意义。而在体外，由于构建技术和培养条件的局限性，尚未见到如此高密度的人工肝组织的技术报道。因此，目前亟需接近人体肝组织细胞密度(达到10 7～10 8个/mL量级)的体外肝组织模型，服务于药物临床前检测、再生医学和体内移植、生物人工肝和肝功能代偿、肝脏疾病研究与治疗、新药开发等领域。
天然肝脏组织是由包括肝细胞、胆管上皮细胞等多种细胞成分组成的含有胆管网络、血管网络等复杂空间结构的器官，其中肝内胆管系统是一套由肝内胆管上皮细胞形成的负责肝内胆汁的产生与转运的复杂管道系统。肝内胆管由胆管上皮细胞组成，分泌并吸收来自于肝细胞的胆汁，进而汇入肝外胆管，将来自于肝脏的胆汁输送到十二指肠或存储到胆囊中。肝内胆管堵塞会引起胆汁淤积，进而引起急性梗阻性化脓性胆管炎，可危及生命。目前，构建含有胆管组织和肝组织的人工肝结构体技术领域仍是空白，尚未见到相关报道。
发明内容
本发明的目的是提供一种仿生人工肝组织及其制备方法与应用。
为了实现本发明目的，第一方面，本发明提供一种仿生人工肝组织，所述仿生人工肝组织的尺寸大小为0.1～50cm，其宏观结构可以是柱状、块状、片状、囊状、管状、网格状、编织状或任意形状组合。
所述仿生人工肝组织包含直径大小为50～2000μm的微丝和内径大小为0.01～300mm的中空通道；其中，所述微丝是由生物相容性材料和细胞通过铸模法或3D打印工艺形成的，呈丝状或圆柱状结构；所述中空通道是由相邻的数根微丝围绕形成的；中空通道的大小、形状和分布密度可以根据需求设计。
所述仿生人工肝组织的杨氏模量为0.1-150KPa。
本发明的仿生人工肝组织还具有以下任一特征：
①所述仿生人工肝组织具有接近人体天然肝组织的极高的细胞密度，细胞密度达到甚至超过10 7～10 8个/mL量级，阳性表达成熟肝组织的标志性基因和蛋白，具有仿生的生理功能，具有白蛋白分泌、氮代谢、尿素合成、解毒和药物代谢的一种或多种肝组织生理功能；
②所述仿生人工肝组织为含有类胆管和类肝脏组织结构的人工肝结构体，具有胆管上皮细胞连接形成的类胆管网络结构，表达胆管上皮细胞标志物，阳性表达成熟肝组织的标志性基因和蛋白，具有白蛋白分泌、氮代谢、尿素合成、解毒和药物代谢的一种或多种肝组织生理功能；
③所述仿生人工肝组织中细胞以小尺寸(10-50μm)团簇的形式均匀分散在整个结构中，细胞密度为10 5～10 7个/mL量级，具有仿生的生理功能，阳性表达成熟肝组织的标志性基因和蛋白。
优选地，所述细胞来源于胚胎干细胞、诱导多能干细胞、肝脏干细胞、肝脏祖细胞、内胚层细胞、 肝脏内胚层细胞、肝母细胞、间充质干细胞或成体干细胞，以及这些细胞分化得到的肝细胞；人体各种组织来源的肝细胞及其细胞系；以及上述所有细胞经过基因编辑、病毒包装或改造获得的相关细胞；优选肝脏干细胞及其细胞系、诱导多能干细胞分化得到的肝脏细胞。
进一步地，所述细胞还可以包括胆管上皮细胞、肝星状细胞、肝窦内皮细胞、内皮细胞、成纤维细胞、枯否细胞等中的一种或多种，包括上述细胞及其细胞系，以及上述细胞经过基因编辑、病毒包装或改造获得的相关细胞；细胞来源于诱导多能干细胞、胚胎干细胞、肝脏干细胞、肝脏祖细胞、内胚层细胞、肝脏内胚层细胞、间充质干细胞或成体干细胞，由多种细胞分化得到，或人体各种组织获得；优选成纤维细胞和/或内皮细胞。
本发明中，所述生物相容性材料可选自天然水凝胶材料和/或人工合成的水凝胶材料。
所述天然水凝胶材料可选自壳聚糖、壳聚糖衍生物、明胶、明胶衍生物、藻酸盐、藻酸盐衍生物、琼脂、基质胶、胶原、胶原衍生物、透明质酸、透明质酸衍生物、纤维素、纤维素衍生材料、蛋白多糖、蛋白多糖衍生物、糖蛋白、糖蛋白衍生材料、层连接蛋白、纤连接蛋白、纤维蛋白(纤维蛋白原)、丝素蛋白、丝素蛋白衍生物、玻连蛋白、骨桥蛋白、肽段水凝胶、DNA水凝胶等中的至少一种；优选胶原、壳聚糖、壳聚糖衍生物、藻酸盐、藻酸盐衍生物、纤维蛋白、明胶和/或明胶衍生物。
所述人工合成的水凝胶材料可选自聚乙醇酸、聚乳酸、聚乳酸-羟基乙酸共聚物、聚谷氨酸-聚乙二醇、聚己内酯、聚三亚甲基碳酸酯、聚乙醇酸、聚乙二醇-聚二氧六环酮、聚乙二醇、聚四氟乙烯、聚氧化乙烯、聚乙烯醋酸乙烯酯、聚三亚甲基碳酸酯、聚对二氧环己酮、聚醚醚酮，以及它们的衍生物或聚合物等中的至少一种；优选聚乙醇酸、聚乳酸-羟基乙酸共聚物或聚乳酸。
第二方面，本发明提供仿生人工肝组织的制备方法，包括：
(1)将生物相容性材料与细胞均匀混合得到含有细胞的前体溶液；
(2)将所述前体溶液按照预先设计的结构(图7)制备成三维水凝胶结构体；
(3)对三维水凝胶结构体进行后处理；
(4)三维水凝胶结构体的体外培养和/或细胞诱导分化获得仿生人工肝组织。
步骤(2)可采用如下方法将所述前体溶液按照预先设计的结构制备成三维水凝胶结构体：铸模法、消失模法、生物3D打印法、喷墨打印法、熔融沉积成型法、静电纺丝法、静电驱动打印法、立体光刻法或激光烧结法等。
所述方法可以是通过控制温度使三维结构成型，温度控制范围在0℃～37℃，优选4℃～36℃。
所述方法还可以是通过光处理使三维结构成型，优选白光或紫外光。
步骤(3)所述后处理方法包括稳定化处理和/或牺牲材料处理。
其中，对三维水凝胶结构体进行稳定化处理所用的交联试剂选自二价阳离子、京尼平、戊二醛、已二酸二酰肼、环氧氯丙烷、碳化二亚胺、凝血酶及其衍生物等中的至少一种；优选二价阳离子和/或凝血酶。
所述交联试剂的浓度为0.1mM～10M，优选10mM～500mM。
对三维水凝胶结构体进行牺牲材料处理，包括去除多余材料，所述多余材料包括三维水凝胶结构体中的温敏材料(如明胶、胶原蛋白、N-异丙基丙烯酰胺和聚乙烯吡咯烷酮等)、交联试剂等。
步骤(4)对三维水凝胶结构体进行体外培养，包括静置培养和/或动态培养。
优选地，静置培养在培养皿、多孔板中进行；动态培养在生物反应器、脉动培养装置、微重力培养装置、搅拌培养装置、波浪式培养装置、芯片或灌注等培养系统中进行。
肝细胞的分化可以受到多种细胞因子不同程度的调控，体外培养所用细胞培养液是在基础培养液的基础上添加了促进肝细胞分化的细胞因子；其中，所述促进肝细胞分化的细胞因子选自骨形态发生蛋白、成纤维细胞生长因子、肝细胞生长因子、抑癌蛋白M等中的至少一种。其中，骨形态发生蛋白是调控细胞发育的转化生长因子(TGF-β)家族的重要成员之一，具有调控多种干细胞发育分化的功能，研究表明，骨形态发生蛋白是多种干细胞向肝细胞分化过程中必不可少的关键细胞因子；成纤维细胞生长因子对细胞的发育调控起着关键作用，可促进刺激中胚层来源细胞、神经外胚层细胞、外胚层细胞和内胚层来源细胞的增殖，它们对内皮细胞具有趋化和有丝分裂作用，并诱导破坏基底膜的物质的释放；肝细胞生长因子可以刺激肝细胞的增殖，并调节多种细胞生长、运动和形态发生的多功能因子。通过旁分泌或自分泌机制，借助上皮间质的相互作用，在胚胎发生、创伤愈合、血管发生、组织器官再生、形态发生和致癌作用等方面发挥重要作用；抑癌蛋白M是一种属于白介素-6家族的细胞因子，研究表明在胎肝中表达，并可以在胚胎时期促进胎肝细胞的成熟，在肝细胞的分化成熟阶段起着重要的调控作用。
体外培养所用细胞培养液是在基础培养液的基础上添加了诱导肝细胞分化和维持肝细胞功能的因子；其中，所述诱导肝细胞分化和维持肝细胞功能的因子选自骨形态发生蛋白、成纤维细胞生长因子、肝细胞生长因子、二甲基亚砜、抑癌蛋白M等中的至少一种。所述诱导胆管细胞分化因子选自角质细胞生长因子、丁酸钠、牛黄胆酸钠、表皮细胞生长因子等中的至少一种。
优选地，所述细胞培养液包含50-120ng/ml激活素A，10-50ng/ml骨形态发生蛋白2，10-50ng/ml骨形态发生蛋白4，10-50ng/ml成纤维细胞生长因子4，0.1％-2％v/v二甲基亚砜，10-50ng/ml肝细胞生长因子，1×10 -5-1×10 -4M抑癌蛋白M，1mM抗坏血酸，0.2mM N-乙酰半胱氨酸酰胺和1×10 -7M地塞米松。各因子之间协同作用，共同促进肝细胞的分化和成熟。
优选地，所述细胞培养液包含100-200ng/ml激活素A，100-300ng/ml骨形态发生蛋白2，100-300ng/ml骨形态发生蛋白4，100-500ng/ml成纤维细胞生长因子4，0.1％-5％v/v二甲基亚砜，100-300ng/ml肝细胞生长因子，1×10 -5-1×10 -4M抑癌蛋白M和1mM抗坏血酸。各因子之间协同作用，共同促进肝细胞的分化和成熟。
优选地，所述细胞培养液包含50-120ng/ml激活素A，10-50ng/ml骨形态发生蛋白2，10-50ng/ml骨形态发生蛋白4，10-50ng/ml成纤维细胞生长因子4，0.1％-2％v/v二甲基亚砜，10-50ng/ml肝细胞生长因子，1×10 -5-1×10 -4M抑癌蛋白M，10-50ng/ml角质细胞生长因子，1×10 -6-5×10 -6M丁酸钠，1×10 -6-5×10 -6M牛黄胆酸钠和1×10 -6-5×10 -6M表皮细胞生长因子。
体外培养条件为：35℃～38℃，5％CO 2。
第三方面，本发明提供按照所述方法制备的仿生人工肝组织。
所述仿生人工肝组织的宏观结构为柱状、块状、片状、囊状、管状、网格状、编织状或任意形状组合。
优选地，所述仿生人工肝组织的尺寸大小为0.1～50cm。
优选地，所述仿生人工肝组织包含直径大小为50～2000μm的微丝。
优选地，所述仿生人工肝组织具有内径大小为0.01～300mm的中空通道。
优选地，所述仿生人工肝组织的杨氏模量为0.1-150KPa。
本发明的仿生人工肝组织还具有以下任一特征：
①所述仿生人工肝组织具有接近人体天然肝组织的极高的细胞密度，细胞密度达到甚至超过10 7～10 8个/mL量级，具有仿生的生理功能，阳性表达成熟肝组织的标志性基因和蛋白，具有白蛋白分泌、氮代谢、尿素合成、解毒和药物代谢的一种或多种肝组织生理功能；
②所述仿生人工肝组织为含有胆管和肝脏组织的人工肝结构体，具有胆管上皮细胞连接形成的胆管网络结构，表达胆管上皮细胞标志物，具有仿生的生理功能，阳性表达成熟肝组织的标志性基因和蛋白，具有白蛋白分泌、氮代谢、尿素合成、解毒和药物代谢的一种或多种肝组织生理功能；
③所述仿生人工肝组织中细胞以小尺寸(10-50μm)团簇的形式均匀分散在整个结构中，细胞密度为10 5～10 7个/mL量级，具有仿生的生理功能，阳性表达成熟肝组织的标志性基因和蛋白。
第四方面，本发明提供所述仿生人工肝组织的以下任一应用：
1)药物临床前检测；
2)用作再生医学和体内移植的材料；
3)用作生物人工肝和肝功能代偿的研究；
4)肝脏疾病病理学研究；
5)新药研发；
6)肝组织发育研究；
7)肝组织再生研究；
8)药物毒理学检测和研究；
9)环境、大气、水体污染物和毒理学检测和研究。
借由上述技术方案，本发明至少具有下列优点及有益效果：
(一)本发明提供的仿生人工肝组织符合人类肝脏极高细胞密度的生理组织特性。正常人体组织细胞密度在每毫升10 8～10 9个细胞左右。针对特定需求，本发明可通过培养因子的精准化控制，结合三维结构和仿生的生物材料，所构建的仿生人工肝组织中的细胞密度可达到每毫升10 7～10 8个细胞左右的量级，并且具有良好的肝脏功能，填补了目前体外构建高密度、高度仿生、功能良好的人工肝结构体的技术空白。
(二)本发明提供的仿生人工肝组织具有良好的肝脏功能，该人工肝组织高表达成熟肝组织/细胞所特有的白蛋白分泌相关和药物代谢相关的功能蛋白和基因，具有良好的分泌白蛋白和氮代谢的功能，相关基因的表达水平达到人体肝细胞的水平，是平面培养同种细胞的5倍以上。本发明的人工肝组织具有高度仿生的解毒和药物代谢功能，接近或达到甚至超过人体肝细胞水平，相关基因表达水平是平面培养同种细胞的10倍以上。并且，组织的肝功能可维持较长时间。本发明提供的人工肝组织可用于药物临床前检测的药筛模型，以及再生医学和体内移植，或生物人工肝和肝功能代偿，可代谢和清除体内外内外源化合物，补充需肝脏合成或代谢的蛋白质等必须物质。因其高度仿生的特性，可用于肝脏疾病病理学研究与治疗、新药研发等领域。
(三)本发明提供了一种生理功能优异的人工肝组织，以独特的均匀分散形式分布，方便观察以及多种鉴定和表征。此外，人工肝组织中的细胞表型高度一致，这为肝脏发育、肝脏再生和肝脏疾病治疗等方面的研究提供了独特的肝组织模型。
(四)本发明提供人工肝结构体的宏观和微观形态可调控，可根据需要定制化生产。可采用以下的一种或多种技术制备：铸模法、消失模法、生物3D打印法、喷墨打印法、熔融沉积成型法、静电纺丝法、静电驱动打印法、立体光刻技术法、激光烧结技术法。通过上述方法，可以根据具体需求，制造出形状复杂、尺寸可控的三维结构。还可对三维结构的宏观形状与微观结构进行调控，满足不同细胞对营养物质、氧气浓度和生存微环境的需求。整体结构上既可形成微米和毫米级尺度的微型人工肝结构体，也可形成厘米、甚至分米级尺度的大型人工肝结构体。
(五)本发明的仿生人工肝组织提供了促进肝细胞增殖、分化和功能维持的稳定微环境。平面培养细胞的方法虽简单易行，却不能模拟细胞生长的三维环境：平面培养条件下，随着细胞密度的增加，细胞膜上糖蛋白进行信号转导，会抑制细胞的增殖。本发明提供的生物材料为细胞提供了附着点，细胞可以在结构内迁移和聚集，生物材料的多孔性保证了细胞与培养环境中的营养物质和气体交换，利于细胞的增殖和分化。通过调节诱导分化培养液的成分，可以诱导细胞在分化成熟，并维持成熟肝脏细胞的生理功能。
(六)本发明提供的构建方法工艺稳定，不仅可以使细胞快速增殖，获得与天然组织类似的极高的细胞密度，且获得的肝组织功能良好。可批量生产，便于开展大规模研究和应用。采用本发明的人工肝组织制备方法，得到的人工肝组织产率高、成本低、方法稳定、过程可控、批次内/间差异小，可以实现大批量生产，以开展下游大规模研究和应用，可用于肝组织发育研究、肝组织再生研究、肝脏疾病发生与发展研究、临床前药物检测和新药测试与开发等领域。本发明构建的人工肝组织的弹性模量在0.1-150KPa，接近健康成人肝组织的弹性模量。本发明构建的人工肝组织结构稳定性好，可以维持三维立体结构实现长期培养和动态培养，有利于均匀、充足的养分和气体交换，促进细胞增殖和功能维持。
(七)本发明首次成功构建出同时实现肝细胞与胆管结构共存的人工肝组织，通过精准控制的程序性生长因子，结合生物材料和3D结构，实现干细胞向肝脏细胞和胆管上皮细胞的双向分化，或 肝细胞与胆管上皮细胞的共培养和功能维持。本发明提供的人体肝组织含有肝细胞和胆管上皮细胞等多种细胞类型，并且具有微米级的多分支胆管网络结构，填补了目前人工肝结构体领域技术的空白。
(八)本发明提供的仿生肝组织具有胆管上皮细胞组成的胆管样网络结构与致密肝细胞团簇，接近生理肝脏中小叶间肝内胆管与肝板紧密连接的天然组织结构。由于成熟肝细胞的功能维持对培养环境要求苛刻，故体外培养难度大，功能维持时间短。本发明提供的含有单管组织的仿生肝组织，因其生理结构的高度仿生性，具有良好的成熟肝组织分泌功能，高表达白蛋白分泌、药物代谢相关的功能蛋白和基因，并且组织的肝功能可维持较长时间。
(九)本发明提供人工肝结构体的宏观和微观形态可调控，可根据需要定制化生产。可采用以下的一种或多种技术制备人工肝结构体：铸模技术、消失模技术、生物3D打印技术、喷墨打印、熔融沉积成型、静电纺丝、静电驱动打印、立体光刻技术、激光烧结技术。通过上述方法，可以根据具体需求，制造出复杂形状、各种尺寸的三维结构，进而对三维结构的宏观形状与微观结构进行调控，满足不同细胞对营养物质、氧气浓度和生存微环境的需求，在此基础上，整体结构上既可形成微米和毫米级尺度的微型人工肝结构体，也可形成厘米、甚至分米级尺度的大型人工肝结构体。
附图说明
图1为本发明较佳实施例中仿生人工肝组织的三维结构示意图。其中，A为用铸模法制备的三维立体结构示意图，B为用3D打印法制备三维立体结构的示意图，C为铸模法制备的具有极高密度肝细胞分布的仿生人工肝组织的示意图，其中团簇状为组织内的肝细胞团簇。
图2为本发明较佳实施例中挤出式生物3D打印工艺与多层网格结构示意图。
图3为本发明较佳实施例中仿生人工肝组织的显微形貌图。光学显微镜下可见密集分布的肝细胞，肝细胞以紧密排列的细胞团簇形式存在。
图4为本发明较佳实施例中仿生人工肝组织的细胞增殖情况。
图5为本发明较佳实施例中仿生人工肝组织的成熟肝细胞的关键标志性蛋白表达情况。
图6为本发明较佳实施例中仿生人工肝组织的功能情况。其中，A表示本发明仿生人工肝组织中白蛋白和尿素分泌水平，与二维培养的同种条件比较；B表示肝组织的关键标志性基因表达标准化到平面培养的同种细胞的水平，与二维培养的同种条件比较。*，**和***表示不同处理组之间的差异具有统计学意义，*表示P<0.05，**表示P<0.01，***表示P<0.001。
图7为本发明用于构建仿生人工肝组织的不同设计结构的示意图。
图8为本发明较佳实施例中生理功能优异的人工肝组织的三维结构示意图。其中，A为用铸模法制备的三维立体结构示意图，B为用3D打印法制备三维立体结构的示意图，C为铸模法制备的一种生理功能优异的人工肝结构体的示意图，其中细胞表型高度一致，以均匀分散形式分布于结构体内。
图9为本发明较佳实施例中挤出式生物3D打印工艺与多层网格结构示意图。
图10为本发明较佳实施例中生理功能优异的人工肝组织的细胞形貌图。光学显微镜下可见大量均匀离散分布的肝细胞。
图11为本发明较佳实施例中生理功能优异的人工肝组织中成熟肝细胞关键标志性蛋白表达情况，所有细胞表型高度一致。
图12为本发明较佳实施例中人工肝组织的功能情况。其中，A表示本发明人工肝组织、平面培养的同种细胞和人体肝细胞的白蛋白合成和尿素分泌水平；B表示本发明人工肝组织、平面培养的同种细胞、人体肝细胞的解毒和药物代谢关键基因表达水平。**和***表示不同处理组之间的差异具有统计学意义，**表示P<0.01，***表示P<0.001。
图13为本发明较佳实施例中含有胆管组织和肝组织的人工肝结构体的示意图。其中，A为用铸模法制备的三维立体结构示意图，B为用3D打印法制备三维立体结构的示意图，C为铸模法制备的含有胆管组织和肝组织的人工肝结构体的示意图，其中团簇状为组织内的肝细胞团簇，线条状为组织内的胆管网络结构。
图14为本发明较佳实施例中含有胆管组织和肝组织的人工肝结构体形貌。含有胆管组织和肝组织的人工肝结构体显微形貌，类球形为肝细胞团簇，如星形标记所示；梭形为胆管上皮细胞形成的网络结构，如黑色箭头所示。
图15为本发明较佳实施例中含有胆管组织和肝组织的人工肝结构体中胆管上皮细胞和成熟肝细胞的关键标志性蛋白表达情况。其中，A：CYP3A4与CK19蛋白染色结果，B：ALB与CK19蛋白染色结果，C：CK19蛋白与DAPI染色结果，D：CK19蛋白在结构内的三维层扫图。
图16为本发明较佳实施例中人工肝组织功能表征与基因表达情况。其中，A表示人工肝结构体白蛋白和尿素分泌水平，与二维培养的同种条件比较；B表示人工肝结构体的关键标志性基因表达标准化到平面培养肝细胞的水平，与二维培养的同种条件比较。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段，所用原料均为市售商品。
实施例1 双喷头三维打印设备制备仿生人工肝组织
本实施例提供一种利用生物3D打印技术构建的一种仿生人工肝组织，如图1所示。
1、肝脏祖细胞的获得与培养
使用诱导性多能干细胞(iPSCs)进行诱导分化，获得肝脏祖细胞。具体实施步骤为：将iPS细胞(Stem Cell)使用细胞分散酶(Dispase,Roche Diagnostics)解离并接种于Matrigel基底上(Becton Dickinson)上。随后，将iPSCs培养于L-WNT3A(CRL2647；ATCC)-expressing cell-conditioned RPMI 1640培养液中(Sigma)培养3～4天向限定性内胚层阶段进行分化，培养液成分包括：50ng/mL激活素A(Activin A，R&D Systems)，1％GlutaMAX TM Supplement(Gibco,35050061)，1％青链霉素(Gibco)，2％FBS和1×B-27(Thermo Fisher Scientific)，获得限定性内胚层阶段的细胞。随后，将限定性内胚层阶段的细胞培养于RPMI 1640基础培养液中培养6天，获得肝脏祖细胞，培养液中包含：30ng/mL骨形态发生蛋白(BMP4,R&D Systems)和30ng/mL成纤维细胞生长因子(FGF4,R&D Systems)，1％ GlutaMAX TM Supplement和1×B-27(Thermo Fisher Scientific)。
2、成纤维细胞的平面培养
人成纤维细胞(HFL1,CCL-153)购自ATCC公司。使用扩增培养基对人成纤维细胞进行平面贴壁培养，每2天更换培养液，当细胞达到85％汇合时按照1:3的比例传代。人成纤维细胞的培养基的成分为：DMEM培养液(Gibco,11960044)中添加10％FBS血清(Gibco,16000)，1％成纤维细胞生长添加剂(RayBiotech，230-00791-100)，1％青链霉素(Gibco,15140122)。
3、打印材料的制备
配制21％的聚乳酸(Sigma-Aldrich，765112，分子量10000Da)溶液和21％的纤维蛋白原溶液(Sigma-Aldrich，F3879)。
向步骤1获得的肝脏祖细胞中，先加入PBS润洗表面，然后加入胰酶(Gibco，25200072)覆盖细胞表面，37℃条件下消化3min后收集，离心得到单细胞悬液。
向步骤2的人成纤维细胞中加入PBS润洗表面，然后加入胰酶(Gibco，25200072)覆盖细胞表面，37℃条件下消化3min后收集，离心得到人成纤维细胞的沉淀，用基础培养基重悬，得到单细胞悬液。
分别将两种细胞悬液与提前加热的聚乳酸溶液/纤维蛋白原溶液混合均匀，获得打印溶液A(单细胞悬液A)：肝脏祖细胞浓度为2.5×10 6个细胞/mL，7％聚乳酸溶液和7％纤维蛋白原溶液，以及打印溶液B(单细胞悬液B)：人成纤维细胞浓度为2.5×10 6个细胞/mL，7％聚乳酸溶液和7％纤维蛋白原溶液。
4、生物3D打印制备仿生人工肝结构体
使用捷诺飞生物科技股份有限公司的挤出式打印设备(Regenovo，Bio-architect X)构建三维结构体。分别将获得的可打印的单细胞悬液A和B装载至双喷头生物3D打印机上，控制打印机腔内温度、打印底板温度和喷头温度为20℃，按照设计好的CAD文件与计算机路径，可以构建多种具有复杂结构的三维立体结构。本实施例中使用双喷头打印机，构建了每层6根微丝(微丝的成分同打印溶液A、B)，一共6层，每根微丝长2cm，高0.5cm的立体六边形网格结构，结构示意图如图2所示。构建后使用500mM凝血酶溶液浸泡结构体10min完成稳定化后处理，得到三维人工肝结构体。
5、人工肝组织培养与功能成熟
人工肝结构体构建完成后，于37℃5％CO 2条件下采用程序性诱导培养基培养10天，获得具有极高细胞密度的仿生人工肝组织。前5～6天，采用第一阶段诱导培养基，具体成分为：RPMI 1640培养液，20ng/ml骨形态发生蛋白2(BMP2,Gibco,PHC7146)，20ng/ml骨形态发生蛋白4(BMP4,Gibco,PHC9533)，30ng/ml成纤维细胞生长因子4(FGF4,R&D SYSTEMS，233-FB-025)，20ng/ml肝细胞生长因子(HGF,R&D systems,294-HGN-005)，0.1％DMSO(Sigma)和1×B-27 Supplement(Gibco,17504044)。之后更换为第二阶段诱导培养基，具体成分为：肝细胞培养基(HCM；Lonza)，含有1％表皮细胞生长因子(R&D systems,236-EG-200)，5×10 -5M抑癌蛋白M(INVITROGEN,PHC5015)，2％GlutaMAX TM Supplement(Gibco,35050061),5×10 -5M氢化可的松琥珀酸钠(常州四药)，0.2％二甲基亚砜(Sigma,D2650)，0.05％胰岛素(Sigma,I9278)，0.05％转铁蛋白(Transferrin，Sigma,T8158)，0.05％ 肾上腺素(Sigma,E4642)，1mM抗坏血酸(Sigma,1043003)，0.2mM N-乙酰半胱氨酸酰胺(Sigma,A0737)，1×10 -7M地塞米松(D4902)。
本发明的细胞培养液在已有研究的基础上进行了改良，增加了维持肝细胞功能的因子：骨形态发生蛋白，成纤维细胞生长因子，肝细胞生长因子和抑癌蛋白M等多种因子。
6、人工肝组织动态培养
静置培养10天后，再采用动态培养模式继续培养10天。本实施例中采用脉动培养，脉动生物反应器参见ZL200910079726.8。
采用崇州市崇阳众诚不锈钢配件服务部生产的蠕动泵JD-200来提供相应的循环动力，设定其工作电压为12V，流速为60ml/min；直流电机为北京艾克斯电机有限公司生产的电机ZGB37RH52i，设定其工作电压为12V、转速为100r/min；采用100ml的注射器；将自制导杆和滑块导轨，以及各部件，如直流电机、导杆、滑块导轨、注射器用自制的支架固定在底板上，连接各部件。
细胞培养液循环部分由培养液瓶、蠕动泵和培养盒构成，各部分由硅胶管连接，培养液经硅胶管由蠕动泵从培养液瓶泵入培养盒(内置工程化组织)，然后经硅胶管流回培养液瓶；导轨滑块、注射器和直流电机构成脉动部分，直流电机与导轨滑块连接推动注射器活塞往复运动，注射器与蠕动泵的出液端连接后和培养盒连接，由此形成脉动流；压力表设置在培养盒上，检测置于培养盒内的组织内的培养液压力。
在进行体外培养以前，先拆卸脉动生物反应器的连接管、注射器，利用高温高压灭菌。然后接通脉动生物反应器，蠕动泵和直流电机接通，先在培养液瓶中加入少量75％的酒精，利用酒精在脉动循环系统中流动灭菌；倒掉酒精然后在培养液瓶中加入一定量已灭菌的PBS溶液，利用该溶液冲洗残余酒精。
关掉电源，然后在培养液瓶中加入待培养需使用的培养液，用灭菌好的镊子夹住步骤1-5制备的工程化肝组织接到培养盒的接头上。为使工程化组织牢固地接在接头上，用已灭菌的细线固定工程化组织的两头。待脉动生物反应器系统完全连接好后，接通电源，调整蠕动泵的电压为12V，调节人造组织处所受压力到0.1MPa，即可可以持续运行脉动生物反应器对工程化组织进行脉动培养。
在培养过程中保持上述电压和组织的压力，使线性控制脉动培养过程中脉动频率在100次/分钟。
直流电机运行平稳后，带动滑块在导轨上推动注射器活塞往复运动，活塞拉出时从培养液瓶中吸取培养液，挤出时把吸入的培养液注入蠕动泵形成的循环系统中流经培养盒中的工程化组织流回培养液瓶。通过调节注射器每次吸入、挤出的培养液的量可调节培养工程化组织处的压力。由此，蠕动泵和直流电机持续运动，脉动生物反应器就提供了一个脉动循环的培养液流实现了对工程化组织的脉动培养。
7、细胞形态观察
形态观察：第0天(打印后24小时以内)、第1天、第10天、第20天分别用光学显微镜(Olympus,CX40)每天观察细胞形态变化，并拍摄记录人工肝组织内细胞生长形态和细胞额形成情况(图3)。从第5～6 天开始观察到细胞形成团簇，且细胞团簇随着时间逐渐变大；在第20天，人工肝组织内的肝细胞持续增殖，形成了致密的团簇，且紧密而均匀地分布于结构内。图3为实施例1制备的具有极高细胞密度的仿生人工肝组织的显微形貌。
8、细胞增殖检测分析
为了检测仿生人工肝组织的细胞增殖水平，使用增殖检测试剂盒(Cell counting kit-8，Dojindo)，按照试剂盒使用说明步骤操作。具体操作步骤：将样品用PBS冲洗1次，加入800μl细胞培养液和80μl CCK-8溶液浸没结构体，37℃孵育2h。然后吸取110μl孵育反应液到96孔板中，使用酶标仪(BIO-RAD，Model 680)在450nm处检测样品OD值。每个样品3个重复，做空白对照。选择在培养的第1天、第10天和第20天进行检测。结果如图4所示，最终得到的人工肝组织的细胞密度为1.2×10 8个/mL。所得仿生肝组织为高1mm的立体六边形网格结构，一共6层，每根微丝长2cm，杨氏模量为1KPa。该人工肝组织包含直径大小约为500μm的微丝和内径大小约为800μm的中空通道。
9、人工肝组织的表型和功能检测
采用免疫荧光染色检测肝细胞功能的关键蛋白表达(如CYP3A4和ALB)，采用酶联免疫吸附试验(Elisa)检测构建三维组织的肝功能水平，采用qPCR技术检测成熟肝细胞标志性基因的转录水平。
1)免疫荧光染色：用磷酸缓冲液(PBS)(BI,02-024-1AC)洗涤结构；4％多聚甲醛在室温下固定30分钟，用PBS洗涤3次，每次5分钟；含0.3％Triton-X(Sigma,X100)和5％牛血清白蛋白(bovine serum albumin,BSA)(Multicell,800-096-EG)的混合液封闭1小时；吸出封闭缓冲液，加入稀释后的一抗(含0.3％Triton-X和1％BSA)，CYP3A4(abcam，ab3572)和ALB(abcam，ab83465)，4℃过夜孵育。用PBS洗涤3次，每次5分钟；加入对应二抗Alexa
Figure PCTCN2021076789-appb-000001
594(abcam，ab150080)和Alexa
Figure PCTCN2021076789-appb-000002
488(abcam，ab150113)，室温避光孵育2小时后，用PBS洗涤3次，每次5分钟；接着加入DAPI染细胞核，室温避光孵育5分钟。
2)采用白蛋白分泌检测试剂盒(Bethyl，E80-129、E101、E115)和尿素分泌检测试剂盒(BIO ASSAY SYSTEMS，DIUR-500)按照试剂盒说明书检测所得肝组织的白蛋白分泌和尿素分泌功能。结果显示，与常规平面培养(所用培养基成分相同)的同种细胞相比，本发明制备的人工肝组织的白蛋白分泌水平是平面培养细胞的29倍，尿素分泌水平是平面培养细胞的2.3倍，数据均有显著性差异。
注：平面培养方式一般在常规培养皿中，如6孔板中进行。
3)qPCR检测技术：提取细胞RNA操作步骤：用PBS洗涤结构1次，每个结构加入1ml Trizol(Gibco,15596026)，反复吹打混匀，在室温静置10分钟，然后转移至1.5ml的EP管中，加入200ul氯仿，快速摇30秒，室温放置5分钟后，在4℃以12000g条件离心10分钟。去除上清液，加入等体积异丙醇，在4℃以12000g条件离心10分钟。弃去上清，用75％无水乙醇洗涤沉淀，风干后可获得RNA，使用DEPC水溶解。用spectrophotometer(Thermo Scientific)来检测RNA浓度及纯度。
RNA反转录操作步骤：采用PrimeScript TM II 1st strand cDNA Synthesis Kit(TaKaRa，6210)，完全按照试剂盒说明书来进行操作。RNA含量均调整为5ng。引物为：Oligo dT Primer。反转录PCR程序为： 42℃ 50min，95℃，5min，4℃保温，所用PCR仪(ABI，SimpliAmpTM热循环仪)。
荧光定量PCR操作步骤：使用Maxima SYBR Green qPCR Master Mix(Thermo Scientific,K0251)试剂盒，完全按照试剂盒说明书进行操作。按要求加入反应液后，将反应板置于qPCR仪进行检测，反应程序为：95℃，10min，95℃ 15s，60℃ 30s，40个循环，72℃ 30s，72℃ 10min。
qPCR所用引物序列如下(5′-3′)：
GAPDH引物序列：
Forward：TGCACCACCAACTGCTTAGC
Reverse：GGCATGGACTGTGGTCATGAG
ALB引物序列：
Forward：GCACAGAATCCTTGGTGAACAG
Reverse：ATGGAAGGTGAATGTTTCAGCA
CYP3A4引物序列：
Forward：TAACAGTCTTTCCATTCCTC
Reverse：GGACTCAGTTTCTTTTGAAT
MRP2引物序列：
Forward：TGAGCAAGTTTGAAACGCACAT
Reverse：AGCTCTTCTCCTGCCGTCTCT
ALB和CYP3A4是成熟肝细胞分泌功能和药物代谢功能的标志性蛋白，MRP2是肝细胞形成组织性排列后出现极性和胆小管结构的标志性蛋白。检测结果显示，本发明提供的人工肝组织各种基因的表达水平均高于平面培养的同种细胞，其中，CYP3A4的基因转录水平是同等条件(采用相同的培养基，仅细胞培养方式不同，细胞培养在平面条件下，如6孔板中进行)下二维培养细胞的3.5倍，三维打印结构体中ALB基因转录水平是同等条件下二维培养细胞的12.7倍，三维打印结构体中MRP2基因转录水平是同等条件下二维培养细胞的2.1倍，数据均有显著性差异。
实施例2 利用铸模法制备极高细胞密度的人工肝组织
本实施例提供一种利用铸模法制备多种结构的具有极高细胞密度的仿生人工肝组织，包括如下步骤：
1、肝脏干细胞培养
HepaRG是一种由人体肝癌组织获得的肝脏干细胞，具有向肝脏细胞和胆管上皮细胞双向分化的能力。将HepaRG细胞(Sigma，HPRGC1)在细胞扩增培养液中进行培养，细胞扩增培养液的成分为Williams'Medium E培养液(Sigma,W4125)中添加10％FBS血清(Gibco,16000)，0.05％胰岛素(Sigma,I9278)，5×10 -7M氢化可的松琥珀酸酯(Sigma,H4881)，1％青链霉素(Gibco,15140122)和1％GlutaMAX TM Supplement(Gibco,35050061)。当细胞90％汇合时，按照1:5的比例传代，每2-3天更换一次培养液。
2、铸模前体溶液的制备
配制16％的海藻酸钠溶液和30％的明胶溶液。
对增殖期间的HepaRG细胞，先加入PBS润洗表面，然后加入胰酶(Gibco，25200072)覆盖细胞表面，37℃条件下消化3min后收集，离心得到单细胞悬液。对细胞进行计数后按比例稀释，然后与提前加热的海藻酸钠/明胶混合溶液混合均匀，最终获得的打印溶液(前体溶液)中细胞浓度为1×10 5个细胞/mL，4％海藻酸钠溶液和15％明胶溶液。
3、铸模法构建含有肝细胞的三维结构体
将步骤2得到的前体溶液倒入预先设计好的模具中。本实施例中所用的模具示意图如图1所示，形成体积为外圆柱直径为3cm，中央圆柱直径1cm，圆柱壁厚300um的中空圆柱体样三维结构体。将模具至于20℃下进行交联15min，交联后将成型的结构取出，浸入5％(w/v)的戊二醛溶液中进行交联，随后浸入500mM的氯化钙溶液进行交联。然后采用分化培养基培养15天，得到仿生人工肝组织(三维结构体)。分化培养基成分为：肝细胞培养基(HCM；Lonza)，30ng/ml成纤维细胞生长因子4(FGF4,R&D SYSTEMS，233-FB-025)，20ng/ml肝细胞生长因子(HGF,R&D systems,294-HGN-005)，0.05％肾上腺素(Sigma,E4642)，0.2％二甲基亚砜(Sigma,D2650)，1mM抗坏血酸(Sigma,1043003)，0.2mM N-乙酰半胱氨酸酰胺(Sigma,A0737)和1×10 -7M地塞米松(D4902)。
本发明的细胞培养液在已有研究的基础上进行了改良，增加了促进肝细胞分化和维持肝细胞功能的因子：成纤维细胞生长因子，肝细胞生长因子和肾上腺素等物质。
加入分化培养液后，将三维结构体于37℃5％CO 2条件下进行培养，每2～3天更换新鲜培养基。期间可用光学显微镜观察细胞形态变化。
4、人工仿生肝组织观察与细胞活死检测
1)第0天、第1天、第7天、第14天、第20天分别用光学显微镜(Olympus,CX40)每天观察细胞形态变化，并拍摄记录三维结构体内细胞生长形态和细胞团簇形成情况。从第7天开始观察到明显的细胞团簇，且细胞团簇随着时间逐渐变大；在第20天，在结构内看到肝细胞增殖后，形成了致密的团簇，且均匀分布于人工肝组织内部。
2)第1天、第7天、第14天、第20天分别对三维结构体内细胞进行活死染色检测。本发明使用2uM Calcein-AM(Dojindo，C326)和4.5uM PI(Dojindo，P346)的混合溶液分别对活(绿色)/死(红色)细胞进行染色，染色避光进行，持续15分钟。使用激光扫描共聚焦显微镜(LSCM，Nikon，Z2)观察记录。对活死染色的照片进行数据统计，各时间点人工组织内细胞存活率约在90％以上。
5、仿生肝组织细胞的增殖检测分析
为了检测三维结构体内的细胞增殖水平，使用增殖检测试剂盒(Cell counting kit-8，Dojindo)，按照试剂盒使用说明步骤操作。具体操作步骤：在培养的第20天，将样品用PBS冲洗1次，加入800μl细胞培养液和80μl CCK-8溶液浸没结构体，37℃孵育2h。然后吸取110μl孵育反应液到96孔板中，使用酶标仪(BIO-RAD，Model 680)在450nm处检测样品OD值。每个样品3个重复，做空白对照。结果 显示，实施例2制备的类肝组织的细胞密度与常规平面培养的细胞相比，三维仿生肝组织的细胞增殖水平是平面培养同种细胞的19.44倍，最终得到的三维仿生肝组织的细胞数可达每毫升8.1×10 7个细胞。所得仿生肝组织为圆柱壁厚300um的中空圆柱体样三维结构体，外圆柱直径为3cm，中央圆柱直径1cm，杨氏模量为1KPa。
6、仿生人工肝组织的功能检测
为了检测三维结构体中肝细胞的功能，采用免疫荧光染色检测了标记肝细胞功能的关键蛋白表达(如CYP3A4和ALB)，如图5所示，采用酶联免疫吸附试验(Elisa)检测构建三维组织的肝功能水平，采用qPCR技术检测成熟肝细胞标志性基因的转录水平(图6)。
1)免疫荧光染色：用磷酸缓冲液(PBS)(BI,02-024-1AC)洗涤结构；4％多聚甲醛在室温下固定30分钟，用PBS洗涤3次，每次5分钟；含0.3％Triton-X(Sigma,X100)和5％牛血清白蛋白(bovine serum albumin,BSA)(Multicell,800-096-EG)的混合液封闭1小时；吸出封闭缓冲液，加入稀释后的一抗(含0.3％Triton-X和1％BSA)，CYP3A4(abcam，ab3572)和ALB(abcam，ab83465)，4℃过夜孵育。用PBS洗涤3次，每次5分钟；加入对应二抗Alexa
Figure PCTCN2021076789-appb-000003
594(abcam，ab150080)和Alexa
Figure PCTCN2021076789-appb-000004
488(abcam，ab150113)，室温避光孵育2小时后，用PBS洗涤3次，每次5分钟；接着加入DAPI染细胞核，室温避光孵育5分钟。其中，CYP3A4是成熟肝细胞药物代谢功能的标志性蛋白；ALB是成熟肝细胞分泌功能的标志性蛋白。用激光共聚焦显微镜(LSCM，Nikon，Z2)观察记录。图5(A)是CYP3A4蛋白与DAPI染色结果，图5(B)是ALB蛋白与DAPI染色结果。由图可知，在人工肝组织中，CYP3A4和ALB蛋白均高表达。
2)采用白蛋白分泌检测试剂盒(Bethyl，E80-129、E101、E115)和尿素分泌检测试剂盒(BIO ASSAY SYSTEMS，DIUR-500)按照试剂盒说明书检测所得肝组织的白蛋白分泌和尿素分泌功能，结果如图6(A)所示，结果显示实施例2制备的具有类组织密度的肝组织三维结构体与常规平面培养(所用培养基成分相同)的细胞相比，三维仿生肝组织的白蛋白分泌水平是平面培养细胞的36倍，尿素分泌水平是平面培养细胞的1.9倍。
3)qPCR检测技术
提取细胞RNA操作步骤：用PBS洗涤三维结构体1次，每个结构体加入1ml Trizol(Gibco,15596026)，反复吹打混匀，在室温静置10分钟，然后转移至1.5ml的EP管中，加入200ul氯仿，快速摇30秒，室温放置5分钟后，在4℃以12000g条件离心10分钟。去除上清液，加入等体积异丙醇，在4℃以12000g条件离心10分钟。弃去上清，用75％无水乙醇洗涤沉淀，风干后可获得RNA，使用DEPC水溶解。用spectrophotometer(Thermo Scientific)来检测RNA浓度及纯度。
RNA反转录操作步骤：采用PrimeScript TM II 1st strand cDNA Synthesis Kit(TaKaRa，6210)，完全按照试剂盒说明书来进行操作。RNA含量均调整为5ng。引物为Oligo dT Primer。反转录PCR程序为：42℃ 50min，95℃，5min，4℃保温，所用PCR仪(ABI，SimpliAmpTM热循环仪)。
荧光定量PCR操作步骤：使用Maxima SYBR Green qPCR Master Mix(Thermo Scientific,K0251)试 剂盒，完全按照试剂盒说明书进行操作。按要求加入反应液后，将反应板置于qPCR仪进行检测，反应程序为：95℃，10min，95℃ 15s，60℃ 30s，40个循环，72℃ 30s，72℃ 10min。获得基因在不同时间点的表达。
qPCR所用引物序列如下(5′-3′)：
GAPDH引物序列：
Forward：TGCACCACCAACTGCTTAGC
Reverse：GGCATGGACTGTGGTCATGAG
ALB引物序列：
Forward：GCACAGAATCCTTGGTGAACAG
Reverse：ATGGAAGGTGAATGTTTCAGCA
CYP3A4引物序列：
Forward：TAACAGTCTTTCCATTCCTC
Reverse：GGACTCAGTTTCTTTTGAAT
MRP2引物序列：
Forward：TGAGCAAGTTTGAAACGCACAT
Reverse：AGCTCTTCTCCTGCCGTCTCT
ALB和CYP3A4是成熟肝细胞分泌功能和药物代谢功能的标志性蛋白，MRP2是肝细胞形成组织性排列后出现极性和胆小管结构的标志性蛋白。检测结果如图6(B)所示，人工肝组织各种基因的表达水平均高于平面培养的同种细胞基因表达水平，其中，CYP3A4的基因转录水平是同等条件下二维培养(所用培养基成分相同)细胞的2.9倍，三维打印结构体中ALB基因转录水平是同等条件下二维培养细胞的19.2倍，三维打印结构体中MRP2基因转录水平是同等条件下二维培养细胞的1.56倍，数据均有显著性差异。
实施例3 利用生物3D打印制备生理功能优异的人工肝组织
本实施例提供一种通过生物3D打印设备构建的一种生理功能优异的人工肝组织，如图8所示。
1、肝脏祖细胞、人体肝细胞的获得与培养
肝脏祖细胞的获得与培养：使用诱导性多能干细胞进行诱导分化，获得肝脏祖细胞。具体实施步骤为：将诱导多能性干细胞接种于Matrigel基底(Becton Dickinson)上培养3～4天。随后培养4天，将诱导性多能干细胞向限定性内胚层阶段进行分化，培养液成分为：L-WNT3A(CRL2647；ATCC)-expressing cell-conditioned RPMI 1640培养液(Life,C11875500BT)，100ng/ml激活素A(ActivinA,Gibco,PHG9014)，1％GlutaMAX TM Supplement(Gibco,35050061)，1％青链霉素(Gibco,15140122)，0.2％FBS(Bioind,04-001-1A)，以及1×B-27 Supplement(Gibco,17504044)。随后培养5天，将限定性内胚层阶段的细胞诱导分化为肝脏祖细胞。培养液成分为：肝细胞培养基(HCM；Lonza)，30ng/ml骨形态发生蛋白4(BMP4,Gibco,PHC9533)，30ng/ml成纤维细胞生长因子4(FGF4, R&D SYSTEMS，233-FB-025)，1％GlutaMAX TM Supplement(Gibco,35050061)和1×B-27 Supplement(Gibco,17504044)。
人体肝细胞的获得与培养：人体肝细胞购自美国康宁公司(Gentest人类冷冻肝细胞，454550)。按照说明书将细胞接种于用大鼠尾巴Ι型胶原蛋白(康宁，BioCoat 354236)包被的6孔板(Thermo Scientific，150239)内培养。使用人原代肝细胞培养基(LONZA，CC-3198)进行培养，每2-3天更换一次培养液，共培养5天。人体肝细胞用作对照。
2、打印溶液的制备
GelMA是明胶的衍生物，是由明胶与甲基丙烯酸酐(MA)反应制得，明胶侧链上存在的大量氨基被甲基丙烯酸酐中的甲基丙烯酰基取代，形成改性明胶，具有可共价交联、结构长期稳定的特点。制备方法如下：1)将明胶溶于磷酸盐缓冲液(PBS，pH7.4)中。完全溶解后，向混合物中加入甲基丙烯酸酐，并搅拌均匀；2)将混合溶液用PBS稀释以终止反应，然后将稀释溶液放入透析膜中在超纯水中透析，以去除有毒和未反应的MA以及其他副产物；3)将最终溶液冷冻干燥储存；4)通过在紫外线(UV)照射，可以将含有光引发剂(Irgacure 2959)的GelMA的水溶液形成GelMA水凝胶。
配制21％的GelMA水凝胶溶液和21％的纤维蛋白原溶液。
加入胰酶(Gibco，25200072)消化步骤1获得的肝脏祖细胞，得到单细胞悬液，计数统计之后，与提前加热的GelMA溶液/纤维蛋白原混合溶液混合均匀，最终获得的打印溶液中细胞浓度为1×10 7个细胞/mL，7％GelMA和7％纤维蛋白原溶液。
3、三维打印构建含有细胞的类肝组织三维结构体
使用捷诺飞生物科技股份有限公司的生物3D打印设备(Regenovo，Bio-architect X)构建三维结构体。将步骤2获得的打印溶液装载至打印机上，控制打印机腔内温度、打印底板温度和喷头温度分别为10℃和20℃，按照设计好的CAD文件与计算机路径，可以构建多种具有复杂结构的三维立体结构。本实施例中构建了每层6根微丝(微丝的成分同打印溶液)，一共4层，长、宽各2cm，高1mm的立体网格结构，结构示意图如图9所示。构建后使用200mM的凝血酶溶液浸泡结构体20min完成稳定化后处理，得到人工三维结构体。
4、人工肝组织培养与功能成熟
人工三维结构体构建完成后，于37℃ 5％CO 2条件下采用诱导培养基培养20天，获得生理功能优异的人工肝组织。诱导培养基成分为：RPMI 1640培养液，200ng/ml骨形态发生蛋白2(BMP2,Gibco,PHC7146)，300ng/ml成纤维细胞生长因子4(FGF4,R&D SYSTEMS，233-FB-025)，200ng/ml肝细胞生长因子(HGF,R&D systems,294-HGN-005)，5×10 -5M抑癌蛋白M(OSM,INVITROGEN,PHC5015)，5％二甲基亚砜(Sigma)和5％肾上腺素(Sigma,E4642)。
本发明的诱导培养基含有高浓度的多种促进肝细胞分化的细胞因子，如骨形态发生蛋白，成纤维细胞生长因子，肝细胞生长因子和抑癌蛋白M等。该分化方案促使肝脏祖细胞跨越了扩增阶段，直接进入高效分化阶段，在生物材料与三维微环境的辅助下，最终获得细胞表型均一、分布均匀、生理功 能优异的人工肝组织。本实施例制备的人工肝组织结构体的尺寸为：长2cm×宽2cm×高1mm，具有4层网格结构，杨氏模量为0.5KPa。人工肝组织结构体中的细胞表型高度一致，以小尺寸(10-50μm)细胞团簇的形式均匀分散在整个结构中，每个小尺寸细胞团簇中的细胞数量小于50个。该仿生人工肝组织包含直径大小约为300μm的微丝和内径大小约为400mm的中空通道。
5、人工肝组织观察与细胞活死比例检测
1)第1天、第7天、第14天、第21天分别用光学显微镜(Olympus,CX40)每天观察细胞形态变化，并拍摄记录三维结构体内细胞生长形态和细胞团簇形成情况。在第21天，可观察到肝细胞离散存在，且均匀地分布于人工肝组织内部，本实施例制备的一种生理功能优异的人工肝组织的显微形貌见图10。
2)第1天、第7天、第14天、第21天分别对人工肝组织进行细胞活死染色检测。本发明使用2uM Calcein-AM(Dojindo，C326)和4.5uM PI(Dojindo，P346)的混合溶液分别对活(绿色)/死(红色)细胞进行染色，染色避光进行，持续15分钟。使用激光扫描共聚焦显微镜(LSCM，Nikon，Z2)观察记录。对活死染色的照片进行数据统计，各时间点人工肝组织的细胞存活率约在85％以上。
6、人工肝组织的功能检测
为了检测三维结构体中肝细胞的功能，采用免疫荧光染色检测了标记肝细胞功能的关键蛋白表达(如ALB和CYP3A4)(图11)，采用酶联免疫吸附试验(Elisa)检测构建三维组织的肝功能水平，采用qPCR技术检测成熟肝细胞标志性基因的转录水平。
免疫荧光染色：用磷酸缓冲液(PBS)(BI,02-024-1AC)洗涤三维结构体；4％多聚甲醛在室温下固定30分钟，用PBS洗涤3次，每次5分钟；含0.3％Triton-X(Sigma,X100)和5％牛血清白蛋白(bovine serum albumin,BSA)(Multicell,800-096-EG)的混合液封闭1小时；吸出封闭缓冲液，加入稀释后的一抗(含0.3％Triton-X和1％BSA)，ALB(abcam，ab83465)和CYP3A4(abcam，ab3572)，4℃过夜孵育。用PBS洗涤3次，每次5分钟；加入对应二抗Alexa
Figure PCTCN2021076789-appb-000005
594(abcam，ab150080)和Alexa
Figure PCTCN2021076789-appb-000006
488(abcam，ab150113)，室温避光孵育2小时后，用PBS洗涤3次，每次5分钟；接着加入DAPI染细胞核，室温避光孵育5分钟。用激光共聚焦显微镜(LSCM，Nikon，Z2)观察记录。图11分别为ALB蛋白与CYP3A4蛋白染色结果。由图可知，ALB蛋白与CYP3A4蛋白均高表达。其中，ALB是成熟肝细胞分泌功能的标志性蛋白，CYP3A4是成熟肝细胞药物代谢功能的标志性蛋白。可以看出功能优异的肝细胞离散均匀的分布在生物材料中，具有成熟的白蛋白分泌和药物代谢功能。
采用白蛋白分泌检测试剂盒(Bethyl，E80-129、E101、E115)和尿素分泌检测试剂盒(BIO ASSAY SYSTEMS，DIUR-500)按照试剂盒说明书检测所得肝组织的白蛋白分泌和尿素分泌功能。结果显示，本实施例制备的肝组织三维结构体与常规平面培养(所用培养基成分相同)的细胞相比，人工肝组织白蛋白分泌水平是平面培养同种细胞的7.5倍，尿素分泌水平是平面培养同种细胞的11.3倍，数据均有显著性差异。
qPCR检测：
提取细胞RNA操作：用PBS洗涤三维结构体1次，每个结构体加入1ml Trizol(Gibco,15596026)，反复吹打混匀，在室温静置10分钟，然后转移至1.5ml的EP管中，加入200ul氯仿，快速摇30秒，室温放置5分钟后，在4℃以12000g条件离心10分钟。去除上清液，加入等体积异丙醇，4℃ 12000g离心10分钟。弃上清，用75％无水乙醇洗涤沉淀，风干后可获得RNA，使用DEPC水溶解。用spectrophotometer(Thermo Scientific)来检测RNA浓度及纯度。RNA反转录操作步骤：采用PrimeScript TM II 1st strand cDNA Synthesis Kit(TaKaRa，6210)，按照试剂盒说明书进行操作。RNA含量均调整为5ng。引物为Oligo dT Primer。反转录PCR程序为：42℃ 50min，95℃，5min，4℃保温，所用PCR仪(ABI，SimpliAmpTM热循环仪)。荧光定量PCR操作步骤：使用Applied Biosystems 60x基因检测试剂盒，按照试剂盒说明书进行操作。按要求加入反应液后，将反应板置于qPCR仪进行检测，反应程序为：95℃，10min，95℃ 15s，60℃ 30s，40个循环，72℃ 30s，72℃ 10min。获得基因在不同时间点的表达。qPCR所用引物见表1：
表1
Figure PCTCN2021076789-appb-000007
注：人体肝细胞是目前肝组织研究领域的“黄金标准”，是采用常规平面培养方式进行的(平面培养方式一般在常规培养皿中，如6孔板中进行)；人工肝组织是本实施例中制备的肝组织，具有三维结构，包含一定的物质成分和特定的力学特征。
以上基因编码的蛋白是成熟肝细胞解毒和药物代谢功能的标志性蛋白。从实验结果可以看出，本 发明人工肝组织的关键肝脏基因表达水平达到甚至超过人体肝细胞的水平，显著性高于平面培养的同种细胞基因表达水平(数据有显著性差异)。
实施例4 利用铸模法制备生理功能优异的人工肝组织
本实施例提供利用铸模法制备多种结构的生理功能优异的人工肝组织，包括如下步骤：
1、肝脏干细胞培养
HepaRG是一种由人体肝癌组织获得的肝脏干细胞，具有向肝脏细胞和胆管上皮细胞双向分化的能力。将HepaRG细胞(Sigma，HPRGC1)在细胞扩增培养液中进行培养，细胞扩增培养液的成分为：Williams'Medium E培养液(Sigma,W4125)中添加10％FBS血清(Gibco,16000)，0.05％胰岛素(Sigma,I9278)，5×10 -5M氢化可的松琥珀酸酯(Sigma,H4881)，1％青链霉素(Gibco,15140122)和1％GlutaMAX TM(Gibco,35050061)。当细胞90％汇合时按照1:5的比例传代，每2-3天更换一次培养液，共培养5天，细胞达到90％汇合时收集使用。
2、铸模前体溶液的制备
配制16％的聚乳酸-羟基乙酸共聚物(Sigma，P2191，分子量30000～60000Da，丙交酯和乙交酯的摩尔比为50:50)溶液和30％的Ι型鼠尾胶原(康宁，BioCoat 354236)溶液。
向步骤1培养的HepaRG细胞中加入胰酶(Gibco，25200072)，在37℃条件下消化3min后收集，离心得到肝脏干细胞的沉淀，用基础培养基重悬，得到单细胞悬液，对细胞进行计数后按比例稀释，然后与提前加热的聚乳酸-羟基乙酸共聚物/胶原混合溶液混合均匀，最终获得的打印溶液(前体溶液)中细胞浓度为1×10 7个细胞/mL，4％聚乳酸-羟基乙酸共聚物溶液和15％胶原溶液。
3、铸模法构建含有细胞的结构体
将步骤2得到的前体溶液倒入预先设计好的模具中。本实施例中所用的模具示意图如图1所示，形成体积为外圆直径3cm，中央均匀分布直径500um的中空圆柱，高3cm的圆柱体样三维结构体。将模具至于37℃条件下进行交联30min，交联后将成型的结构取出，浸入5％(w/v)的戊二醛溶液进行交联。然后采用诱导分化培养基培养20-25天，得到仿生肝组织。分化培养基成分为：肝细胞培养基(HCM；Lonza)，100ng/ml激活素A(ActivinA,Gibco,PHG9014)，300ng/ml成纤维细胞生长因子4(FGF4,R&D SYSTEMS，233-FB-025)，500ng/ml肝细胞生长因子(HGF,R&D systems,294-HGN-005)，1×B-27(Gibco,17504044)，2％GlutaMAX TM Supplement(Gibco,35050061)，5×10 -3M氢化可的松琥珀酸钠(常州四药)，10％DMSO(Sigma)和5％胰岛素(Sigma,I9278)。
本发明的诱导培养基含有高浓度的多种促进肝细胞分化的细胞因子，如激活素A、成纤维细胞生长因子、肝细胞生长因子，促使肝脏干细胞高效分化，在生物材料与三维微环境的辅助下，最终获得细胞表型均一、分布均匀、生理功能优异的人工肝组织。
加入培养液后，结构体于37℃5％CO 2条件下进行培养，每2～3天更换新鲜培养基。期间可用光学显微镜观察细胞形态变化。
4、人工肝组织的动态培养
本实施例中采用脉动培养，脉动生物反应器参见ZL200910079726.8。
采用崇州市崇阳众诚不锈钢配件服务部生产的蠕动泵JD-200来提供相应的循环动力，设定其工作电压为12V，流速为60ml/min；直流电机为北京艾克斯电机有限公司生产的电机ZGB37RH52i，设定其工作电压为12V、转速为100r/min；采用100ml的注射器；将自制导杆和滑块导轨，以及各部件，如直流电机、导杆、滑块导轨、注射器用自制的支架固定在底板上，连接各部件。
细胞培养液循环部分由培养液瓶、蠕动泵和培养盒构成，各部分由硅胶管连接，培养液经硅胶管由蠕动泵从培养液瓶泵入培养盒(内置工程化组织)，然后经硅胶管流回培养液瓶；导轨滑块、注射器和直流电机构成脉动部分，直流电机与导轨滑块连接推动注射器活塞往复运动，注射器与蠕动泵的出液端连接后和培养盒连接，由此形成脉动流；压力表设置在培养盒上，检测置于培养盒内的组织内的培养液压力。
在进行体外培养以前，先拆卸脉动生物反应器的连接管、注射器，利用高温高压灭菌。然后接通脉动生物反应器，蠕动泵和直流电机接通，首先在培养液瓶中加入少量75％的酒精，利用酒精在脉动循环系统中流动灭菌；倒掉酒精然后在培养液瓶中加入一定量已灭菌的PBS溶液，利用该溶液冲洗残余酒精。
关掉电源，然后在培养液瓶中加入待培养需使用的培养液，用灭菌好的镊子夹住步骤1～3制备的工程化肝组织接到培养盒的接头上。为使工程化组织牢固地接在接头上，用已灭菌的细线固定工程化组织的两头。待脉动生物反应器系统完全连接好后，接通电源，调整蠕动泵的电压为12V，调节人造组织处所受压力到0.1MPa，然后就可以持续运行脉动生物反应器对工程化组织进行脉动培养了。
在培养过程中保持上述电压和组织的压力，使线性控制脉动培养过程中脉动频率在100次/分钟。
直流电机运行平稳后，带动滑块在导轨上推动注射器活塞往复运动，活塞拉出的时候从培养液瓶中吸取培养液，挤出时把吸入的培养液注入蠕动泵形成的循环系统中流经培养盒中的工程化组织流回培养液瓶。通过调节注射器每次吸入、挤出的培养液的量可调节培养工程化组织处的压力。由此，蠕动泵和直流电机持续运动，脉动生物反应器就提供了一个脉动循环的培养液流实现了对工程化组织的脉动培养。
5、人工肝组织观察与细胞活死比例检测
1)第1天、第7天、第14天、第21天分别用光学显微镜(Olympus,CX40)每天观察细胞形态变化，并拍摄记录三维结构体内细胞生长形态和细胞团簇形成情况。在第21天，在结构内看到肝细胞离散存在，且均匀地分布于结构内。用光学显微镜(Olympus,CX40)每天观察细胞形态变化，最终获得的具有肝细胞离散均匀分布表征的人体肝组织结构体的显微形貌。
2)第1天、第7天、第14天、第21天分别对人工肝组织内细胞进行活死染色检测。本发明使用2uM Calcein-AM(Dojindo，C326)和4.5uM PI(Dojindo，P346)的混合溶液分别对活(绿色)/死(红色)细胞进行染色，染色避光进行，持续15分钟。使用激光扫描共聚焦显微镜(LSCM，Nikon，Z2)观察记录。对活死染色的照片进行数据统计，各检测时间点人工肝组织内的细胞存活率约高于85％。
6、人工肝组织的功能检测
为了检测三维结构体中肝细胞的功能，采用免疫荧光染色检测了标记肝细胞功能的关键蛋白表达(如CYP3A4和ALB)，采用酶联免疫吸附试验(Elisa)检测构建三维组织的肝功能水平，采用qPCR技术检测成熟肝细胞标志性基因的转录水平。
1)免疫荧光染色：用磷酸缓冲液(PBS)(BI,02-024-1AC)洗涤三维结构体；4％多聚甲醛在室温下固定30分钟，用PBS洗涤3次，每次5分钟；含0.3％Triton-X(Sigma,X100)和5％牛血清白蛋白(bovine serum albumin,BSA)(Multicell,800-096-EG)的混合液封闭1小时；吸出封闭缓冲液，加入稀释后的一抗(含0.3％Triton-X和1％BSA)，CYP3A4(abcam，ab3572)和ALB(abcam，ab83465)，4℃过夜孵育。用PBS洗涤3次，每次5分钟；加入对应二抗Alexa
Figure PCTCN2021076789-appb-000008
594(abcam，ab150080)和Alexa
Figure PCTCN2021076789-appb-000009
488(abcam，ab150113)，室温避光孵育2小时后，用PBS洗涤3次，每次5分钟；接着加入DAPI染细胞核，室温避光孵育5分钟。用激光共聚焦显微镜(LSCM，Nikon，Z2)观察记录。由结果可知，CYP3A4和ALB蛋白均高表达。其中，CYP3A4是成熟肝细胞药物代谢功能的标志性蛋白；ALB是成熟肝细胞分泌功能的标志性蛋白。可以看出肝细胞离散且均匀的分布与结构微丝内，具有成熟的分泌和代谢功能。
2)采用白蛋白分泌检测试剂盒(Bethyl，E80-129、E101、E115)和尿素分泌检测试剂盒(BIO ASSAY SYSTEMS，DIUR-500)按照试剂盒说明书检测所得肝组织的白蛋白分泌和尿素分泌功能，结果如图12A所示，本实施例制备的肝组织与常规平面培养(所用培养基成分相同)的细胞相比，三维仿生肝组织的白蛋白分泌水平是平面培养细胞的6.3倍，尿素分泌水平是平面培养细胞的9.4倍。本实施例制备的人工肝组织结构体为直径3cm×高3cm的圆柱体，圆柱体中央均匀分布19个直径500μm的中空圆柱(图1)。结构体的杨氏模量为1KPa。人工肝组织结构体中的细胞表型高度一致，以小尺寸细胞团簇的形式均匀分散在整个结构中，每个小尺寸细胞团簇(10-50μm)中的细胞数量小于50个。该仿生人工肝组织包含直径大小约为400μm的微丝和内径大小约为1000mm的中空通道。
3)qPCR检测技术：
提取细胞RNA操作步骤：用PBS洗涤三维结构体1次，每个结构体加入1ml Trizol(Gibco,15596026)，反复吹打混匀，在室温静置10分钟，然后转移至1.5ml的EP管中，加入200ul氯仿，快速摇30秒，室温放置5分钟后，4℃ 12000g离心10分钟。弃上清，加入等体积异丙醇，4℃ 12000g离心10分钟。弃上清，用75％无水乙醇洗涤沉淀，风干后可获得RNA，使用DEPC水溶解。用spectrophotometer(Thermo Scientific)来检测RNA浓度及纯度。RNA反转录操作步骤：采用PrimeScript TM II 1st strand cDNA Synthesis Kit(TaKaRa，6210)，按照试剂盒说明书进行操作。RNA含量均调整为5ng。引物为Oligo dT Primer。反转录PCR程序为：42℃ 50min，95℃，5min，4℃保温，所用PCR仪(ABI，SimpliAmpTM热循环仪)。荧光定量PCR操作步骤：使用Applied Biosystems 60x基因检测试剂盒，按照试剂盒说明书进行操作。按要求加入反应液后，将反应板置于qPCR仪进行检测，反应程序为：95℃，10min，95℃ 15s，60℃ 30s，40个循环，72℃ 30s，72℃ 10min。获得基因在不同时间点的表达情况。 qPCR所用引物序列见表2：
表2
Figure PCTCN2021076789-appb-000010
以上基因编码的蛋白是成熟肝细胞解毒和药物代谢功能的标志性蛋白。检测结果如图12B和表2所示，人工肝组织各种基因的表达水平达到或高于人体肝细胞的基因表达水平，显著性高于平面培养的同种细胞基因表达水平，数据有显著性差异。
本发明提供的人工仿生人工肝组织，这种三维的仿生组织具有一定的结构、物质成分、物质排布和力学性能。细胞在组织中的生长及发育情况与常规的平面培养采用了完全不同的方式，而这正是能够成功诱导出特定三维结构的肝组织和生理功能的关键，为下游应用提供了更加接近人体组织的研究模型，添补了该领域的空白。
实施例5 利用双喷头生物3D打印技术制备人工肝结构体
本实施例提供一种可以通过生物3D打印设备构建的人工肝结构体，如图2所示。
1、肝细胞的平面培养
人正常肝细胞(L-02)购自普诺赛(Procell)公司，货号为HL-7702。使用扩增培养基对人正常 肝细胞进行平面贴壁培养，每2天更换培养液，当细胞达到85％汇合时按照1:3的比例传代。人正常肝细胞的培养基的成分为：Williams'Medium E培养液(Sigma,W4125)中添加10％FBS血清(Gibco,16000)，1％青链霉素(Gibco,15140122)，20ng/mL肝细胞生长因子(HGF,R&D Systems)和50μMβ巯基乙醇。
2、胆管上皮细胞的平面培养
人肝内胆管上皮细胞购自Procell公司(CP-H042)。使用扩增培养基对人肝内胆管上皮细胞进行平面贴壁培养，每2天更换培养液，当细胞达到85％汇合时按照1:3的比例传代。人肝内胆管上皮细胞的培养基的成分为：Vivo 15培养液(Lonza 04-418Q)中添加10％FBS血清(Gibco,16000)，1％上皮细胞生长添加剂(Procell，CP-H042)，5×10 -5mol/L氢化可的松琥珀酸钠(常州四药)，0.05％胰岛素(Sigma,I9278)，0.05％转铁蛋白(Transferrin，Sigma,T8158)，0.05％盐酸肾上腺素(Epinephrine hydrochloride，Sigma,E4642)，1％青链霉素(Gibco,15140122)。
3、打印墨水的制备
配制21％的聚乙醇酸(上海源叶生物科技有限公司，S26878，分子量1-2万Da)溶液和21％的纤维蛋白原溶液(Sigma-Aldrich，F3879)。
对增殖期间的人正常肝细胞，先加入PBS润洗表面，然后加入胰酶(Gibco，25200072)覆盖细胞表面，37℃条件下消化3min后收集，离心得到人正常肝细胞的沉淀，用基础培养基重悬，得到单细胞悬液。
对增殖期间的人肝内胆管上皮细胞，先加入PBS润洗表面，然后加入胰酶(Gibco，25200072)覆盖细胞表面，37℃条件下消化3min后收集，离心得到人肝内胆管上皮细胞的沉淀，用基础培养基(Vivo 15培养液，Lonza，04-418Q)重悬，得到单细胞悬液。
分别将两种细胞悬液与提前加热的聚乙醇酸溶液/纤维蛋白原溶液混合均匀，获得打印溶液A(单细胞悬液A)：人正常肝细胞浓度为0.5×10 5个细胞/mL，7％聚乙醇酸溶液和7％纤维蛋白原溶液；以及打印溶液B(单细胞悬液B)：人肝内胆管上皮细胞浓度为0.5×10 5个细胞/mL，7％聚乙醇酸溶液和7％纤维蛋白原溶液。
4、三维打印构建含有细胞的三维水凝胶结构体
使用捷诺飞生物科技股份有限公司的挤出式打印设备(Regenovo，Bio-architect X)构建三维结构体。分别将获得的可打印的单细胞悬液A和B装载至双喷头生物3D打印机上，控制打印机腔内温度、打印底板温度和喷头温度为20℃，按照设计好的CAD文件与计算机路径，可以构建多种具有复杂结构的三维立体结构。本实施例中使用双喷头打印机，构建了每层6根微丝(微丝的成分同打印溶液A、B)，一共6层，每根微丝长2cm，高0.5cm的立体六边形网格结构，结构示意图如图2所示。构建后使用200mM的凝血酶溶液进行浸泡交联完成稳定化后处理，得到三维水凝胶结构体。
5、三维水凝胶结构体的培养与功能成熟
人工肝结构体构建完成后，于37℃5％CO 2条件下采用共培养培养基培养10天，获得具有胆管组 织和肝组织的仿生人工结构体。共培养培养基的成分为Williams'Medium E培养液(Sigma,W4125)，10％FBS血清(Gibco,16000)，100ng/ml激活素A(Activin A，Gibco,PHG9014)，20ng/ml骨形态发生蛋白2(Gibco,PHC7146)，20ng/ml骨形态发生蛋白4(Gibco,PHC9533)，1％二甲基亚砜(Sigma,D2650)，20ng/ml肝细胞生长因子(R&D SYSTEMS，294-HGN-005)，5×10 -5M抑癌蛋白M(INVITROGEN，PHC5015)，20ng/ml角质细胞生长因子(R&D SYSTEMS，251-KG-010)，1.8×10 -6M丁酸钠(Sigma,6339)，1×10 -5M牛黄胆酸钠(Sigma，303410)，5×10 -5M表皮细胞生长因子(R&D SYSTEMS，236-EG-200)。
本发明的细胞培养液在已有研究的基础上进行了改良，增加了维持肝细胞功能的因子：激活素A，骨形态发生蛋白，二甲基亚砜，肝细胞生长因子和抑癌蛋白M，以及诱导分化产生胆管上皮细胞以及胆管细胞功能维持的因子：角质细胞生长因子，丁酸钠，牛黄胆酸钠和表皮细胞生长因子。
6、三维结构体的动态培养
静置培养10天后，再采用动态培养模式继续培养10天。
本实施例中采用脉动培养，脉动生物反应器参见ZL200910079726.8。
采用崇州市崇阳众诚不锈钢配件服务部生产的蠕动泵JD-200来提供相应的循环动力，设定其工作电压为12V，流速为60ml/min；直流电机为北京艾克斯电机有限公司生产的电机ZGB37RH52i，设定其工作电压为12V、转速为100r/min；采用100ml注射器；将自制导杆和滑块导轨，以及各部件，如直流电机、导杆、滑块导轨、注射器用自制的肝组织固定在底板上，连接各部件。
细胞培养液循环部分由培养液瓶、蠕动泵和培养盒构成，各部分由硅胶管连接，培养液经硅胶管由蠕动泵从培养液瓶泵入培养盒(内置工程化组织)，然后经硅胶管流回培养液瓶；导轨滑块、注射器和直流电机构成脉动部分，直流电机与导轨滑块连接推动注射器活塞往复运动，注射器与蠕动泵的出液端连接后和培养盒连接，由此形成脉动流；压力表设置在培养盒上，检测置于培养盒内的组织内的培养液压力。
在进行体外培养以前，先拆卸脉动生物反应器的连接管、注射器，利用高温高压灭菌。然后接通脉动生物反应器，蠕动泵和直流电机接通，先在培养液瓶中加入少量75％的酒精，利用酒精在脉动循环系统中流动灭菌；倒掉酒精然后在培养液瓶中加入一定量已灭菌的PBS溶液，利用该溶液冲洗残余酒精。
关掉电源，然后在培养液瓶中加入细胞培养液，用灭菌好的镊子夹住步骤1-5制备的工程化肝组织接到培养盒的接头上。为使工程化组织牢固地接在接头上，用已灭菌的细线固定工程化组织的两头。待脉动生物反应器系统完全连接好后，接通电源，调整蠕动泵的电压为12V，调节人造组织处所受压力到0.1MPa，即可可以持续运行脉动生物反应器对工程化组织进行脉动培养。
在培养过程中保持上述电压和组织的压力，使线性控制脉动培养过程中脉动频率在100次/分钟。
直流电机运行平稳后，带动滑块在导轨上推动注射器活塞往复运动，活塞拉出时从培养液瓶中吸取培养液，挤出时把吸入的培养液注入蠕动泵形成的循环系统中流经培养盒中的工程化组织流回培养 液瓶。通过调节注射器每次吸入、挤出的培养液的量可调节培养工程化组织处的压力。由此，蠕动泵和直流电机持续运动，脉动生物反应器就提供了一个脉动循环的培养液流实现了对工程化组织的脉动培养。
7、细胞形态观察与生物学检测
形态观察：第0天(打印后24小时以内)、第1天、第10天、第20天分别用光学显微镜(Olympus,CX40)每天观察细胞形态变化，并拍摄记录三维结构体内细胞生长形态和胆管网络形成情况(图14)。从第4天左右开始观察到明显的细胞团簇，且细胞团簇随着时间逐渐变大；在第20天左右开始观察到胆管网络结构的出现。此时所得三维仿生人体肝组织微观形貌如图14所示，获得了既有肝细胞团(星号所示)也有胆管网络(黑色箭头所示)的体外人工肝结构体显微形貌，从图中可以看出结构内部的贯穿通道(即中空通道)。
活死染色检测：使用2uM Calcein-AM(Dojindo，C326)和4.5uM PI(Dojindo，P346)的混合溶液分别对活(绿色)/死(红色)细胞进行染色，染色避光进行，持续15分钟。使用激光扫描共聚焦显微镜(LSCM，Nikon，Z2)观察记录。对活死染色的照片进行数据统计，打印结束Day0结构体内细胞存活率约95％左右。
生物学检测：为了检测三维结构体中胆管网络的形成和肝细胞功能维持情况，采用免疫荧光染色检测了肝细胞与肝内胆管上皮细胞特异性标记蛋白的表达(如CYP3A4、ALB和CK19)(图15)，采用酶联免疫吸附试验(Elisa)检测构建三维组织的肝功能水平，采用qPCR技术检测肝细胞与胆管上皮细胞相关基因的转录水平。
免疫荧光染色：用磷酸缓冲液(PBS)(BI,02-024-1AC)洗涤结构；4％多聚甲醛在室温下固定30分钟，用PBS洗涤3次，每次5分钟；含0.3％Triton-X(Sigma,X100)和5％牛血清白蛋白(bovine serum albumin,BSA)(Multicell,800-096-EG)的混合液封闭1小时；吸出封闭缓冲液，加入稀释后的一抗(含0.3％Triton-X和1％BSA)，CYP3A4(abcam，ab3572)、ALB(abcam，ab83465)和CK19(RD，MAB3506)，4℃过夜孵育。用PBS洗涤3次，每次5分钟；加入对应二抗Alexa
Figure PCTCN2021076789-appb-000011
594(abcam，ab150080)和Alexa
Figure PCTCN2021076789-appb-000012
488(abcam，ab150113)，室温避光孵育2小时后，用PBS洗涤3次，每次5分钟；接着加入DAPI染细胞核，室温避光孵育5分钟。用激光共聚焦显微镜(LSCM，Nikon，Z2)观察记录。
图15是对三维仿生肝组织的关键蛋白进行免疫荧光染色的结果，其中ALB、CYP3A4为成熟肝细胞的标志性蛋白，CK19是胆管上皮细胞的标记蛋白。其中，(A)是CYP3A4与CK19蛋白染色结果，(B)是ALB与CK19蛋白染色结果，(C)是CK19蛋白与DAPI染色结果，(D)是CK19蛋白在结构内的三维层扫图。由图可知，上述蛋白全部高表达。其中，CYP3A4是成熟肝细胞药物代谢功能的标志性蛋白；CK19是胆管上皮细胞的标记物，可以看出胆管上皮细胞在结构微丝内形成网络结构；ALB是成熟肝细胞分泌功能的标志性蛋白。可以看出肝细胞团具有成熟的分泌和代谢功能，胆管上皮细胞围绕肝细胞团形成网络结构，从(D)可以看出胆管细胞自组装形成三维中空管状结构(所述中空管状结构的内径约为800μm各管道之间的平均间隔距离约为500μm，管道分布密度约为50个/cm 2)，符合 人体肝脏生理结构。
采用白蛋白分泌检测试剂盒(Bethyl，E80-129、E101、E115)和尿素分泌检测试剂盒(BIO ASSAY SYSTEMS，DIUR-500)按照试剂盒说明书检测所得肝组织的白蛋白分泌和尿素分泌功能。结果显示本发明制备的含有胆管结构的肝组织与常规平面培养的细胞相比，三维仿生肝组织的白蛋白分泌水平是平面培养细胞(所用培养基成分相同)的7.9倍，尿素分泌水平是平面培养细胞的2.7倍。所得三维仿生肝组织为高2mm的立体六边形网格结构，一共10层，每根微丝长2cm。该仿生肝组织的杨氏模量为1.5KPa。该仿生肝组织包含直径大小约为500μm的微丝和内径大小约为800μm的中空通道。
注：平面培养方式一般在常规培养皿中，如6孔板中进行。
qPCR基因检测：提取细胞RNA操作步骤：用PBS洗涤结构1次，每个结构加入1ml Trizol(Gibco,15596026)，反复吹打混匀，在室温静置10分钟，然后转移至1.5ml的EP管中，加入200ul氯仿，快速摇30秒，室温放置5分钟后，在4℃以12000g条件离心10分钟。去除上清液，加入等体积异丙醇，在4℃以12000g条件离心10分钟。弃去上清，用75％无水乙醇洗涤沉淀，风干后可获得RNA，使用DEPC水溶解。用spectrophotometer(Thermo Scientific)检测RNA浓度及纯度。RNA反转录操作步骤：采用PrimeScript TM II 1st strand cDNA Synthesis Kit(TaKaRa，6210)，完全按照试剂盒说明书来进行操作。RNA含量均调整为5ng。引物为：Oligo dT Primer。反转录PCR程序为：42℃ 50min，95℃，5min，4℃保温，所用PCR仪(ABI，SimpliAmpTM热循环仪)。荧光定量PCR操作步骤：使用Maxima SYBR Green qPCR Master Mix(Thermo Scientific,K0251)试剂盒，完全按照试剂盒说明书进行操作。按要求加入反应液后，将反应板置于qPCR仪进行检测，反应程序为：95℃，10min，95℃ 15s，60℃ 30s，40个循环，72℃ 30s，72℃ 10min。获得基因在不同时间点的表达情况。
qPCR所用引物序列如下(5′-3′)：
CK19引物序列：
Forward：ATGGCCGAGCAGAACCGGAA
Reverse：CCATGAGCCGCTGGTACTTCC
ALB引物序列：
Forward：GCACAGAATCCTTGGTGAACAG
Reverse：ATGGAAGGTGAATGTTTCAGCA
CYP3A4引物序列：
Forward：TAACAGTCTTTCCATTCCTC
Reverse：GGACTCAGTTTCTTTTGAAT
检测结果显示，三维打印结构体各种基因的表达水平均高于二维培养基因表达水平，CYP3A4是成熟肝细胞药物代谢功能的标志性蛋白，其基因转录水平是同等条件下二维培养细胞的5.1倍，ALB是成熟肝细胞分泌功能的标志性蛋白，三维打印结构体中ALB基因转录水平是同等条件(采用相同的培养基，仅细胞培养方式不同，细胞培养在平面条件下，如6孔板中进行)下二维培养细胞的3.2倍； CK19是胆管上皮细胞的特异性表达的标记蛋白，三维打印结构体中CK19基因转录水平是同等条件下二维培养细胞的3.4倍，数据均有显著性差异。
实施例6 利用铸模法制备人工肝结构体
本实施例提供一种通过铸模法制备人工肝结构体的方法，如图16和图7所示。包括如下步骤：
1、肝脏干细胞诱导
使用诱导性多能干细胞(iPSCs)进行诱导分化，获得肝脏干细胞。具体实施步骤为：将iPS细胞(安徽中盛溯源生物科技有限公司)使用细胞分散酶(Dispase,Roche Diagnostics)解离并接种于BD Matrigel Growth Factor Reduced Basement Membrane Matrix(Becton Dickinson)上。随后，将iPSCs置于L-WNT3A(CRL2647；ATCC)-expressing cell-conditioned RPMI 1640培养液中(Sigma)培养4天向限定性内胚层阶段进行分化，培养液成分包括：100ng/mL Activin A(R&D Systems),1％GlutaMAX(Thermo Fisher Scientific),1％青链霉素(Gibco)，0.2％FBS和1×B-27(Supplement，Thermo Fisher Scientific)，获得限定性内胚层阶段的细胞。随后，将限定性内胚层阶段的细胞培养于RPMI 1640基础培养液中培养8天，获得肝脏干细胞，培养液中包含：30ng/mL骨形态发生蛋白(BMP4,R&D Systems)和20ng/mL成纤维细胞生长因子(FGF4,R&D Systems),1％GlutaMAX和1×B-27(Thermo Fisher Scientific)。
2、铸模前体溶液的制备
配制16％的海藻酸钠溶液和30％的明胶溶液。
对增殖期间的肝脏干细胞，先加入PBS润洗表面，然后加入胰酶(Gibco，25200072)覆盖细胞表面，37℃条件下消化3min后收集，离心得到肝脏干细胞的沉淀，用基础培养基重悬，得到单细胞悬液，对细胞进行计数后按比例稀释，然后与提前加热的海藻酸钠/明胶混合溶液混合均匀，最终获得的前体溶液中细胞浓度为1×10 5个细胞/mL，4％海藻酸钠和15％明胶。
3、铸模法构建含有细胞的三维水凝胶结构体
将混好细胞的前体溶液倒入预先设计好的模具中。本实施例中所用模具的结构示意图如图13所示，形成体积为截面六边形直径6cm，中央圆柱直径2cm，高6cm的中空六面体样三维结构体。将含有细胞的生物墨水(前体溶液)倒入模具后，将模具于15℃进行交联15min，交联后将成型的结构取出，浸入5％(w/v)的戊二醛溶液进行交联，随后浸入300mM的氯化钙溶液进行交联然后于肝脏干细胞的双向分化培养基中进行培养，具体成分为：RPMI 1640培养液，含有20ng/mL肝细胞生长因子(HGF,R&D Systems),1％GlutaMAX(Thermo Fisher Scientific),1％青链霉素(Gibco),1×B27 Supplement Minus Vitamin A，1×10 -5M白介素6(Miltenyi)，1μM牛黄胆酸钠(Sigma)，1μM丁酸钠(Sigma)，培养15天，得到人工肝结构体。
最后，更换为肝细胞和胆管上皮细胞诱导成熟培养液培养15天，具体成分包括肝细胞培养基(HCM；Lonza)，25％人ES/iPS干细胞无血清培养基mTeSR TM1 medium(StemCell,05850)，20ng/ml骨形态发生蛋白2(BMP2,Gibco,PHC7146)，20ng/ml骨形态发生蛋白4(BMP4,Gibco,PHC9533)，30ng/ml成纤维细胞生长因子4(FGF4,R&D SYSTEMS，233-FB-025)，20ng/ml肝细胞生长因子(HGF,R&D  systems,294-HGN-005)，5×10 -5M抑癌蛋白M(OSM,INVITROGEN,PHC5015)，20ng/ml角质细胞生长因子(KGF,R&D,251-KG-010)，1.8×10 -6M丁酸钠(Sigma,6339)，1×10 -5M牛黄胆酸钠(Sigma，303410)，1％表皮细胞生长因子(EGF,R&D systems，236-EG-200)，1％上皮细胞生长添加剂(Gibco，PHG0367)，0.1M地塞米松(Sigma-Aldrich，D4902)，抗坏血酸(1:1,000)，1:1,000转铁蛋白(Sigma,T8158)，0.05％胰岛素(Sigma,I9278)，1％青链霉素(Gibco,15140122)，1.5％二甲基亚砜(Sigma,D2650)，1％GlutaMAX TM Supplement(Gibco,35050061),1％MEM Non-Essential Amino Acids Solution(Gibco,11140050)。
本发明的细胞培养液在已有研究的基础上进行了改良，增加了诱导肝细胞分化和维持肝细胞功能的因子：骨形态发生蛋白，成纤维细胞生长因子，肝细胞生长因子，二甲基亚砜和抑癌蛋白M，以及诱导胆管细胞分化因子：角质细胞生长因子，丁酸钠，牛黄胆酸钠和表皮细胞生长因子等多种因子。
4、细胞形态观察与生物学检测
形态观察：第0天(打印后24小时以内)、第1天、第10天、第20天分别用光学显微镜(Olympus,CX40)每天观察细胞形态变化，并拍摄记录三维结构体内细胞生长形态和胆管网络形成情况。从第4天左右开始观察到明显的细胞团簇，且细胞团簇随着时间逐渐变大；在第20天左右开始观察到胆管网络结构的出现。
活死染色检测：使用2uM Calcein-AM(Dojindo，C326)和4.5uM PI(Dojindo，P346)的混合溶液分别对活(绿色)/死(红色)细胞进行染色，染色避光进行，持续15分钟。使用激光扫描共聚焦显微镜(LSCM，Nikon，Z2)观察记录。对活死染色的照片进行数据统计，打印结束Day0结构体内细胞存活率约95％左右。
生物学检测：为了检测三维结构体中胆管网络的形成和肝细胞功能维持情况，采用免疫荧光染色检测了肝细胞与肝内胆管上皮细胞特异性标记蛋白的表达(如CYP3A4、ALB和CK19)，采用酶联免疫吸附试验(Elisa)检测构建三维组织的肝功能水平(图16A)，采用qPCR技术检测肝细胞与胆管上皮细胞相关基因的转录水平(图16B)。其中免疫荧光染色方法与实施例5相同。
采用白蛋白分泌检测试剂盒(Bethyl，E80-129、E101、E115)和尿素分泌检测试剂盒(BIO ASSAY SYSTEMS，DIUR-500)按照试剂盒说明书检测所得肝组织的白蛋白分泌和尿素分泌功能。结果显示(图16A)本实施例所得的含有胆管结构的肝组织与常规平面培养的细胞相比，三维仿生肝组织的白蛋白分泌水平是平面培养细胞的9.5倍，尿素分泌水平是平面培养细胞的1.7倍，数据均有显著性差异。
qPCR基因检测：提取细胞RNA操作步骤：用PBS洗涤结构1次，每个结构加入1ml Trizol(Gibco,15596026)，反复吹打混匀，在室温静置10分钟，然后转移至1.5ml的EP管中，加入200ul氯仿，快速摇30秒，室温放置5分钟后，在4℃以12000g条件离心10分钟。去除上清液，加入等体积异丙醇，在4℃以12000g条件离心10分钟。弃去上清，用75％无水乙醇洗涤沉淀，风干后可获得RNA，使用DEPC水溶解。用spectrophotometer(Thermo Scientific)来检测RNA浓度及纯度。RNA反转录操作步骤：采用PrimeScript TM II 1st strand cDNA Synthesis Kit(TaKaRa，6210)，完全按照试剂盒说明书来进行操作。 RNA含量均调整为5ng。引物为Oligo dT Primer。反转录PCR程序为：42℃ 50min，95℃，5min，4℃保温，所用PCR仪(ABI，SimpliAmpTM热循环仪)。荧光定量PCR操作步骤：使用Maxima SYBR Green qPCR Master Mix(Thermo Scientific,K0251)试剂盒，完全按照试剂盒说明书进行操作。按要求加入反应液后，将反应板置于qPCR仪进行检测，反应程序为：95℃，10min，95℃15s，60℃30s，40个循环，72℃ 30s，72℃ 10min。获得基因在不同时间点的表达情况(图16B)。
qPCR所用引物序列如下(5′-3′)：
CK19引物序列：
Forward：ATGGCCGAGCAGAACCGGAA
Reverse：CCATGAGCCGCTGGTACTTCC
ALB引物序列：
Forward：GCACAGAATCCTTGGTGAACAG
Reverse：ATGGAAGGTGAATGTTTCAGCA
CYP3A4引物序列：
Forward：TAACAGTCTTTCCATTCCTC
Reverse：GGACTCAGTTTCTTTTGAAT
由图16B可知，本实施例制备的人工肝结构体各种基因的表达水平均高于二维培养(所用培养基成分相同)中基因表达水平，CYP3A4是成熟肝细胞药物代谢功能的标志性蛋白，其基因转录水平是同等条件下二维培养细胞的2.3倍，ALB是成熟肝细胞分泌功能的标志性蛋白，三维打印结构体中ALB基因转录水平是同等条件下二维培养细胞的36.2倍；CK19是胆管上皮细胞的特异性表达的标记蛋白，三维打印结构体中CK19基因转录水平是同等条件下二维培养细胞的18.7倍。**和***表示数据有显著性差异。
所得三维人工肝组织的截面是外切圆直径为6cm的六边形，高6cm；中央含有直径2cm的圆柱结构和6根辐条支撑结构(图13)。细胞密度约为8×10 6个/cm 3，杨氏模量为1.5KPa。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
工业实用性
本发明提供的仿生人工肝组织可用于药物临床前检测、再生医学和体内移植、生物人工肝和肝功能代偿、肝脏疾病研究与治疗、药物测试与新药开发等领域。本发明提供含有胆管和肝脏组织的人工肝结构体是一种具有广泛用途的肝脏模型，用于药物临床前检测、环境监测、毒理检测、组织工程、再生医学、新药研发、研究肝组织发育、研究疾病发生和发展等领域。

Claims (12)

  1. 仿生人工肝组织，其特征在于，所述仿生人工肝组织的尺寸大小为0.1～50cm，其宏观结构为柱状、块状、片状、囊状、管状、网格状、编织状或任意形状组合；
    所述仿生人工肝组织包含直径大小为50～2000μm的微丝和内径大小为0.01～300mm的中空通道；其中，所述微丝是由生物相容性材料和细胞通过铸模法或3D打印工艺形成的，呈丝状或圆柱状结构；所述中空通道是由相邻的数根微丝围绕形成的；
    所述仿生人工肝组织的杨氏模量为0.1-150Kpa；
    所述仿生人工肝组织还具有以下任一特征：
    ①所述仿生人工肝组织具有接近人体天然肝组织的极高的细胞密度，细胞密度达到甚至超过10 7～10 8个/mL量级，阳性表达成熟肝组织的标志性基因和蛋白，具有仿生的生理功能，具有白蛋白分泌、氮代谢、尿素合成、解毒和药物代谢的一种或多种肝组织生理功能；
    ②所述仿生人工肝组织为含有类胆管和类肝脏组织结构的人工肝结构体，具有胆管上皮细胞连接形成的类胆管网络结构，表达胆管上皮细胞标志物，阳性表达成熟肝组织的标志性基因和蛋白，具有白蛋白分泌、氮代谢、尿素合成、解毒和药物代谢的一种或多种肝组织生理功能；
    ③所述仿生人工肝组织中细胞以小尺寸团簇的形式均匀分散在整个结构中，细胞密度为10 5～10 7个/mL量级，具有仿生的生理功能，阳性表达成熟肝组织的标志性基因和蛋白。
  2. 根据权利要求1所述的仿生人工肝组织，其特征在于，所述细胞来源于胚胎干细胞、诱导多能干细胞、肝脏干细胞、肝脏祖细胞、内胚层细胞、肝脏内胚层细胞、肝母细胞、间充质干细胞或成体干细胞，以及这些细胞分化得到的肝细胞；人体各种组织来源的肝细胞及其细胞系；以及上述所有细胞经过基因编辑、病毒包装或改造获得的相关细胞；优选肝脏干细胞及其细胞系、诱导多能干细胞分化得到的肝脏细胞。
  3. 根据权利要求2所述的仿生人工肝组织，其特征在于，所述细胞还包括胆管上皮细胞、肝星状细胞、肝窦内皮细胞、内皮细胞、成纤维细胞、枯否细胞中的一种或多种，包括上述细胞及其细胞系，以及上述细胞经过基因编辑、病毒包装或改造获得的相关细胞；细胞来源于诱导多能干细胞、胚胎干细胞、肝脏干细胞、肝脏祖细胞、内胚层细胞、肝脏内胚层细胞、间充质干细胞或成体干细胞，由多种细胞分化得到，或人体各种组织获得；优选成纤维细胞和/或内皮细胞。
  4. 根据权利要求1-3任一项所述的仿生人工肝组织，其特征在于，所述生物相容性材料选自天然水凝胶材料和/或人工合成的水凝胶材料；
    其中，所述天然水凝胶材料选自壳聚糖、壳聚糖衍生物、明胶、明胶衍生物、藻酸盐、藻酸盐衍生物、琼脂、基质胶、胶原、胶原衍生物、透明质酸、透明质酸衍生物、纤维素、纤维素衍生材料、蛋白多糖、蛋白多糖衍生物、糖蛋白、糖蛋白衍生材料、层连接蛋白、纤连接蛋白、纤维蛋白、丝素蛋白、丝素蛋白衍生物、玻连蛋白、骨桥蛋白、肽段水凝胶、DNA水凝胶中的至少一种；优选胶原、壳聚糖、壳聚糖衍生物、藻酸盐、藻酸盐衍生物、纤维蛋白、明胶和/或明胶衍生物；
    所述人工合成的水凝胶材料选自聚乙醇酸、聚乳酸、聚乳酸-羟基乙酸共聚物、聚谷氨酸-聚乙二醇、聚己内酯、聚三亚甲基碳酸酯、聚乙醇酸、聚乙二醇-聚二氧六环酮、聚乙二醇、聚四氟乙烯、聚氧化乙烯、聚乙烯醋酸乙烯酯、聚三亚甲基碳酸酯、聚对二氧环己酮、聚醚醚酮，以及它们的衍生物或聚合物中的至少一种；优选聚乙醇酸、聚乳酸-羟基乙酸共聚物或聚乳酸。
  5. 仿生人工肝组织的制备方法，其特征在于，包括：
    (1)将生物相容性材料与细胞均匀混合得到含有细胞的前体溶液；
    (2)将所述前体溶液按照预先设计的结构制备成三维水凝胶结构体；
    (3)对三维水凝胶结构体进行后处理；
    (4)三维水凝胶结构体的体外培养和/或细胞诱导分化获得仿生人工肝组织；
    其中，所述细胞同权利要求2或3中所述，所述生物相容性材料同权利要求4中所述。
  6. 根据权利要求5所述的方法，其特征在于，步骤(2)采用如下方法将所述前体溶液按照预先设计的结构制备成三维水凝胶结构体：铸模法、消失模法、生物3D打印法、喷墨打印法、熔融沉积成型法、静电纺丝法、静电驱动打印法、立体光刻法或激光烧结法；
    所述方法是通过控制温度使三维结构成型，温度控制范围在0℃～37℃，优选4℃～36℃；和/或
    所述方法是通过光处理使三维结构成型，优选白光或紫外光。
  7. 根据权利要求5所述的方法，其特征在于，步骤(3)所述后处理方法包括稳定化处理和/或牺牲材料处理；
    其中，对三维水凝胶结构体进行稳定化处理所用的交联试剂选自二价阳离子、京尼平、戊二醛、已二酸二酰肼、环氧氯丙烷、碳化二亚胺、凝血酶及其衍生物中的至少一种；优选二价阳离子和/或凝血酶；所述交联试剂的浓度为0.1mM～10M，优选10mM～500mM；
    对三维水凝胶结构体进行牺牲材料处理，包括去除多余材料，所述多余材料包括三维水凝胶结构体中的温敏材料、交联试剂。
  8. 根据权利要求5-7任一项所述的方法，其特征在于，步骤(4)对三维水凝胶结构体进行体外培养，包括静置培养和/或动态培养；
    优选地，静置培养在培养皿、多孔板中进行；动态培养在生物反应器、脉动培养装置、微重力培养装置、搅拌培养装置、波浪式培养装置、芯片或灌注培养系统中进行；和/或
    体外培养所用细胞培养液是在基础培养液的基础上添加了诱导肝细胞分化和维持肝细胞功能的因子；其中，所述诱导肝细胞分化和维持肝细胞功能的因子选自骨形态发生蛋白、成纤维细胞生长因子、肝细胞生长因子、二甲基亚砜、抑癌蛋白M中的至少一种；所述诱导胆管细胞分化因子选自角质细胞生长因子、丁酸钠、牛黄胆酸钠、表皮细胞生长因子中的至少一种；
  9. 根据权利要求8所述的方法，其特征在于，所述细胞培养液包含50-120ng/ml激活素A，10-50ng/ml骨形态发生蛋白2，10-50ng/ml骨形态发生蛋白4，10-50ng/ml成纤维细胞生长因子4，0.1％-2％v/v二甲基亚砜，10-50ng/ml肝细胞生长因子，1×10 -5-1×10 -4M抑癌蛋白M，1mM抗坏血酸，0.2mM N- 乙酰半胱氨酸酰胺和1×10 -7M地塞米松；和/或
    体外培养条件为：35℃～38℃，5％CO 2。
  10. 根据权利要求8所述的方法，其特征在于，所述细胞培养液包含100-200ng/ml激活素A，100-300ng/ml骨形态发生蛋白2，100-300ng/ml骨形态发生蛋白4，100-500ng/ml成纤维细胞生长因子4，0.1％-5％v/v二甲基亚砜，100-300ng/ml肝细胞生长因子，1×10 -5-1×10 -4M抑癌蛋白M和1mM抗坏血酸；和/或
    体外培养条件为：35℃～38℃，5％CO 2。
  11. 根据权利要求10所述的方法，其特征在于，所述细胞培养液包含50-120ng/ml激活素A，10-50ng/ml骨形态发生蛋白2，10-50ng/ml骨形态发生蛋白4，10-50ng/ml成纤维细胞生长因子4，0.1％-2％v/v二甲基亚砜，10-50ng/ml肝细胞生长因子，1×10 -5-1×10 -4M抑癌蛋白M，10-50ng/ml角质细胞生长因子，1×10 -6-5×10 -6M丁酸钠，1×10 -6-5×10 -6M牛黄胆酸钠和1×10 -6-5×10 -6M表皮细胞生长因子；和/或
    体外培养条件为：35℃～38℃，5％CO 2。
  12. 权利要求1-4任一项所述的仿生人工肝组织或按照权利要求5-11任一项所述方法制备的仿生人工肝组织的以下任一应用：
    1)药物临床前检测；
    2)用作再生医学和体内移植的材料；
    3)用作生物人工肝和肝功能代偿的研究；
    4)肝脏疾病病理学研究；
    5)新药研发；
    6)肝组织发育研究；
    7)肝组织再生研究；
    8)药物毒理学检测和研究；
    9)环境、大气、水体污染物和毒理学检测和研究。
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