CN103756955B - The bionical composite construction of a kind of personalization and preparation thereof and for the method for drug screening - Google Patents

Abstract

A personalized bionic composite structure and its preparation and drug screening method relate to a bionic composite structure based on composite multi-nozzle three-dimensional printing technology and its preparation. The biomimetic composite structure includes a polymer shell, a branch blood vessel stent, a functional tissue area, a blood vessel tissue area and a polymer isolation layer. The functional tissue area is a hydrogel layer containing tissue cells; the vascular tissue area is a hydrogel layer containing vascular seed cells; the functional tissue area and the vascular tissue area are arranged alternately in space; the polymer isolation layer is arranged at intervals in the functional tissue area and the vascular tissue area, and divide the functional tissue area and the vascular tissue area into multiple small areas; the branch vascular stent includes arterial blood vessels and venous blood vessels. The present invention prepares bionic structures of tissues and organs with branched blood vessels through composite multi-nozzle 3D printing technology; the hydrogel structure does not need to be cross-linked or polymerized; it simulates the state of vascularized tissues and blood vessels in the body to the greatest extent, and provides vascularized organs and tissues for reference.

Description

A personalized biomimetic composite structure and its preparation and method for drug screening

technical field

The invention belongs to the field of bioengineering, specifically to the field of biology or enzymes and their compositions, and relates to a personalized biomimetic composite structure and a method for its preparation and drug screening.

Background technique

In recent years, with the increasingly close communication between disciplines, cell arrangement technology has emerged, laying the engineering foundation for tissue engineering, organ manufacturing and drug screening. Among them, the prominent 3D printing technology, also called Rapid Prototyping (RP) technology, uses computer-aided modeling and discrete-stacking principles to process materials [FedorovichNE, et al. Trends in biotechnology, 2011;29(12):601-606]. This technology can precisely position biomaterials and is highly valued in the field of in vitro assembly of complex organs. Tissues or organs formed in vitro have a wide range of uses, and can be used to study the basics of maintaining the normal shape of organs [GriffithLG, et al. Nature Reviews Molecular Cell Biology, 2006; 7(3): 211-224], cancer cells to evade chemotherapy and other treatment measures [YamadaKM, et al. Cell, 2007; 130(4): 601-610], transplantation of diseased organs [GriffithLG, et al. Science Signaling, 2002; 295(5557): 1009] and drug screening. Many foreign scientific research groups have completed the transformation of various RP technologies to realize the assembly or printing of 3D structures containing cells based on numerical control. The Center of Organ Manufacturing, Department of Mechanical Engineering, Tsinghua University in China has developed a series of rapid prototyping equipment, and used these equipment to successfully prepare simple vascular network, liver tissue and bone repair materials [WangXH, etal. Trends in Biotechnology, 2007 ;25:505; WangXH, et al. Tissue Engineering Part B, 2010;16:189; WangXH. Artificialorgans, 2012;36:591]. The three-dimensional structure of assembled cell/matrix materials generally requires cross-linking or polymerizing natural polymer materials to maintain the stability of the three-dimensional structure.

Since the 1990s, the development of new drugs has been mainly completed through molecular and cell-based bioinformatics detection platforms, mainly using single cells cultured in vitro in two dimensions. However, 2D planar culture cannot fully mimic the real-life 3D environment of corresponding multicellular (at least three) tissues in the human body. Most of the drugs screened using two-dimensional cultured cells as target cells have no activity in animals [KangXH, et al. Tissue Engineering, 2005; 11:456-458]. In recent years, with the micro-scale of biological research, more and more biologists have found that various indicators of cell survival, such as oxygen concentration and survival temperature, have a crucial impact on the growth state of cells, which also makes human tissues The in vitro simulation and cultivation of serotonin is particularly challenging. The development of combinatorial chemistry and genomics has led to a sharp increase in the types of compounds or drugs that can be used for screening, forcing the development of drug screening throughput to keep pace with the times. Previous verification methods have become no longer applicable. High-throughput drug screening ( HighthroughputScreening, HTS) was therefore proposed and rapidly promoted [RodriguesAD.Pharmaceuticalresearch, 1997; 4 (11): 1504-1510]. However, the HTS technology uses two-dimensional culture of a single cell, and the cell structure and function are incomplete, and the lead compound identified by this technology does not have the expected pharmacological activity. It has become a consensus in the field of drug research to use a new drug screening model that is more in line with human structure and has a high success rate - High Content Screening (High Content Screening, HCS) technology to determine lead compounds. Cells need to integrate the information provided by various substances such as DNA, RNA, protein, metabolites and ions, and display corresponding characteristics; and the interaction of ions, metabolites, macromolecules and organelles in time and space is a complex realization of organisms. Basis of life function. Therefore, in order to understand more clearly the role of the selected target in cell function, a tool that can automatically extract the information conveyed by the target in the cell is needed, and this tool is HCS technology [GiulianoKA , et al. Assay and drug development technologies, 2003; 1(4):565-577]. Compared with HTS technology, HCS technology can realize multi-target selection. HCS uses fluorescently labeled antibodies, DNA probes, biological ligands, or bioreactors to collect the effects of targets in cells on various aspects of cell morphology, growth, differentiation, and apoptosis, which can be obtained in one experiment. A wealth of information about the individual components of cells, DNA, proteins, and more. Compared with the HCS technology with orifice plate as the unit, HTS screening results are more accurate and biologically meaningful.

Patent (Application No. 200910079726.8) involves a partition-based vascularized adipose tissue and its construction method. Fat granules are wrapped in microcapsules to obtain a three-dimensional adipose tissue structure with a penetrating structure. The structure is mainly composed of fat areas and vascular areas . However, this method requires a large amount of cross-linking agent (such as CaCl ₂ ), which will damage the cell activity; the drug in this method is mainly released through microcapsules, and the drug effect is not easy to affect the entire structure. The patent (Application No. 200910155794.8) involves a high-content screening method for diabetes therapeutic drugs. The three-dimensional structure containing cells is obtained by rapid prototyping technology, and the three-dimensional structure containing islet cells is used for drug screening. However, this method does not include vascular branching system, and vascular endothelial cells are only distributed on the surface of the hydrogel. This method also requires a large amount of crosslinking agent to stabilize the shaped body.

Existing tissue engineering cell/scaffold composite structures can express the characteristics of tissues or organs in a certain dimension in vitro, but they cannot fully simulate the real structure of multicellular cells containing vascular system. The hydrogel material used in the present invention does not need to be cross-linked, because the polymer isolation layer and the outer shell have provided mechanical support for the structure; the isolation layer and the outer shell can be macro (micro) porous structure, which ensures the exchange of nutrients ; The three-dimensional vascularized tissue simulates the internal environment to a great extent; the branched vascular structure can be connected with the internal and external circulatory system, which is convenient for HCS drug screening. These factors prompted us to use composite multi-nozzle 3D printing technology to prepare personalized biomimetic composite structures with branched blood vessels to simulate the state of organs in vivo and use them for drug screening.

Contents of the invention

The purpose of the present invention is a personalized bionic composite structure and its preparation and method for drug screening, so that it can better simulate the state of arteries and veins in the body, the structure does not need to be cross-linked, and the cell density in the structure can Very high, the present invention can be used as a biomimetic composite structure to simulate the state of vascularized tissues in vivo, and then be used for drug screening.

A personalized biomimetic composite structure, characterized in that: the personalized biomimetic composite structure includes a polymer shell, a branch vascular stent, a functional tissue area, a vascular tissue area, and a polymer isolation layer; the functional tissue area is a tissue-containing The hydrogel layer of cells; the vascular tissue area is a hydrogel layer containing vascular seed cells; the functional tissue area and the vascular tissue area are alternately arranged in space; the polymer isolation layer is arranged at intervals in the functional In the tissue area and the vascular tissue area, the functional tissue area and the vascular tissue area are divided into multiple small areas; the branch vascular stent includes arterial blood vessels and venous blood vessels, arterial blood vessels contain at least one entrance and several branches, and venous blood vessels Contains at least one outlet and several branches; the branch vascular stent is made of synthetic polymer material, and the branch vascular stent runs through the polymer shell, the functional tissue area, the vascular tissue area and the polymer isolation layer; the polymer The shell adopts natural polymer or synthetic polymer material.

In the above technical solution, the inner diameter of the branch vessel stent is 10 μm-10 mm, and the wall thickness is 100 μm-2 mm; the layer thickness of the polymer isolation layer and the polymer shell is 0.1 μm-2 mm.

The hydrogel layer containing tissue cells in the functional tissue area of the present invention and the hydrogel layer containing vascular seed cells in the vascular tissue area are natural polymer hydrogels, and the natural polymers are gelatin, sodium alginate, fiber At least one of protein, collagen, matrigel, carrageenan, chitosan, agar, hyaluronic acid, matrigel, elastin and laminin; the natural polymer hydrogel is compounded with cell growth factors , heparin, leptin, paclitaxel and at least one of astragalus anticancer powder.

The mass volume concentration of the natural polymer hydrogel in the present invention is 0.1-40%; the concentration of the vascular seed cells and tissue cells is 1×10 ³ to 1×10 ⁸ cells/mL; the functional tissue area Tissue cells are one of adult tissue cells, adult stem cells and cancer cells; the vascular seed cells in the vascular tissue area are vascular endothelial cells, fat stem cells, bone marrow mesenchymal stem cells, umbilical cord blood stem cells, bone marrow stem cells, embryonic stem cells and at least one of induced pluripotent stem cells.

A method for preparing a personalized bionic composite structure provided by the invention is characterized in that the method comprises the following steps:

1) Design a three-dimensional model of branch vascular stents, and divide them into two groups to simulate arterial vessels and venous vessels;

2) Load the synthetic polymer material, the prepared hydrogel containing tissue cells, the prepared hydrogel containing vascular seed cells, and the prepared synthetic polymer solution into different nozzles of the composite multi-nozzle 3D printing device ;

3) Preparation of branch vascular stents: according to the three-dimensional model in step 1), use a composite multi-nozzle three-dimensional printing device to extrude a synthetic polymer solution, and freeze-dry to remove organic solvents, or melt-extrude synthetic polymer materials to obtain branch vascular stents;

4) Preparation of functional tissue area: use the composite multi-nozzle 3D printing equipment to print the prepared hydrogel containing tissue cells layer by layer, and print at intervals along the branch vascular scaffold along the printing path to obtain a preliminary 3D structure;

5) Prepare the vascular tissue area: use a composite multi-nozzle 3D printing device to print layer by layer, fill the prepared hydrogel containing vascular seed cells in the gaps of the preliminary 3D structure, and obtain an intermediate 3D structure;

6) Preparation of polymer isolation layer and polymer shell: In the process of step 4) and step 5) layer by layer printing, after printing one or several layers, use a composite multi-nozzle 3D printing device to spray the synthetic polymer solution and extract the organic solvent , or melt-extruded synthetic polymer materials to form a polymer isolation layer to obtain an alternate structure of a cell-containing hydrogel layer and a polymer isolation layer; spray a synthetic polymer solution on the periphery of the alternate structure and extract an organic solvent, or Melt extrusion to synthesize polymer materials, form polymer shells, and obtain personalized biomimetic composite structure precursors;

7) Provide growth factors to the precursor of the above-mentioned personalized bionic composite structure, realize the vascularization of the vascular tissue area, make the functional tissue area form a tissue, and make the cells of the vascular tissue area and the functional tissue area connect, and finally obtain the personalized Biomimetic composite structures.

In the preparation method of the present invention, the composite multi-nozzle three-dimensional printing equipment adopts melt extrusion forming technology or low-temperature deposition forming technology to prepare branch vessel stents, polymer isolation layers and polymer shells; when using melt extrusion forming technology When preparing the branch vessel stent, the polymer isolation layer and the polymer shell, the synthetic polymer material is at least one of acrylonitrile-butadiene-styrene, polyurethane, polytetrafluoroethylene and carbohydrates; when using When the low-temperature deposition forming technology prepares the branch vessel stent, the polymer isolation layer and the polymer shell, the solute of the synthetic polymer solution is polyurethane, polycarbonate, polyurea ester, polyglycolide, polysuccinate, At least one of polyether ester, polyethylene glycol, polylactic acid, polycaprolactone, polylactic acid-glycolic acid copolymer, polyester and polyhydroxyester, the solvent of the synthetic polymer solution is tetraethylene glycol alcohol or 1,4-dioxane. The mass volume concentration of the synthetic polymer solution is 1%-30%; the synthetic polymer solution is compounded with an anticoagulant factor, and the anticoagulant factor is heparin or paclitaxel.

A method for drug screening with a personalized biomimetic composite structure provided by the present invention is characterized in that the method comprises the following steps:

1) If the tissue cells in the functional tissue area adopt adult tissue cells or adult stem cells, then supply the culture medium containing pathogenic drugs to the personalized biomimetic composite structure, so that the above-mentioned personalized bionic composite structure produces pathological symptoms; if the functional tissue area If the tissue cells are cancer cells, no pathogenic treatment is required;

2) Supply culture fluid containing different types and doses of therapeutic drugs to personalized biomimetic composite structures, and collect culture fluid in real time or later;

3) Analyze the above-mentioned collected culture fluid to obtain the effect of the type and dosage of the therapeutic drug on the personalized composite structure, and determine the effect of the drug by detecting and comparing biological indicators.

In the drug screening method of the present invention, the biological index is at least one of carbohydrate and lipid metabolism index, liver function index, kidney function index, cancer cell index and vascularization index. The pathological symptoms are diabetes, organ inflammation, organ failure or tumors.

Compared with the prior art, the present invention has the following advantages and outstanding technical effects: ①The polymer isolation layer of the present invention divides the hydrogel layer into several sub-districts, forming alternate arrangements of the hydrogel layer and the polymer isolation layer The structure; the polymer isolation layer provides mechanical support and biochemical protection for the hydrogel structure, which can ensure the formation of low-concentration hydrogel; the hydrogel does not need to be cross-linked, and the cell density can be large enough. ②The vascular tissue area and the functional tissue area of the present invention form cell connections, and the intervals are alternately arranged, simulating the state of vascularized tissue and blood vessels in the body, and providing a reference for vascularized organ tissues. ③ The polymer shell of the present invention provides mechanical support and biochemical protection for the internal structure, which is beneficial to the in vivo and in vitro cultivation of the personalized bionic structure. ④ The outlet and inlet of the branch vascular stent of the present invention can be connected with an in vitro pulsating culture system or an in vivo vascular system. ⑤ The drug screening method of the present invention can timely analyze the effect of drugs on the personalized bionic composite structure, which is beneficial to the drug screening work on metabolic diseases, organ diseases or cancer tumors.

Description of drawings

Figure 1 is a structural schematic diagram of a personalized biomimetic composite structure.

Fig. 2 is a schematic diagram of a branch vessel stent.

Figure 3a and Figure 3b are schematic diagrams of the printed functional tissue area and vascular tissue area, respectively.

Figure 4a and Figure 4b are respectively the alternate structure and exploded view of the polymer isolation layer, the functional tissue area and the vascular tissue area.

In the figure: 101—arterial blood inlet; 102—branch vessel stent; 103—functional tissue area; 104—vascular tissue area; 105—venous blood outlet; 106—polymer shell; 401—polymer isolation layer.

detailed description

The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

Fig. 1 is a structural schematic diagram of a personalized biomimetic composite structure, which includes a polymer shell 106, a branch vascular stent 102, a functional tissue area 103, a vascular tissue area 104 and a polymer isolation layer 401; the function The tissue area 103 is a hydrogel layer containing tissue cells; the vascular tissue area 104 is a hydrogel layer containing vascular seed cells; the functional tissue area 103 and the vascular tissue area 104 are alternately arranged in space; the The polymer isolation layer 401 is arranged at intervals in the functional tissue area 103 and the vascular tissue area 104, and divides the functional tissue area 103 and the vascular tissue area 104 into a plurality of small areas; the branch vascular stent 102 includes both arterial and venous Partly, the arterial blood vessel contains at least one inlet and several branches, and the venous blood vessel contains at least one outlet and several branches; the branch blood vessel stent 102 is made of synthetic polymer material, and the branch blood vessel stent 102 runs through the polymer shell 106, functional tissue Between the region 103, the vascular tissue region 104 and the polymer isolation layer 401; the polymer shell 106 is made of natural polymer or synthetic polymer material.

The inner diameter of the branch vessel stent is 10 μm-10 mm, and the wall thickness is 100 μm-2 mm; the layer thickness of the polymer isolation layer and the polymer shell is 0.1 μm-2 mm. Fig. 2 is a schematic diagram of a branch vascular stent, the hydrogel layer containing tissue cells in the functional tissue area and the hydrogel layer containing vascular seed cells in the vascular tissue area are natural polymer hydrogels, and the natural polymer is gelatin , sodium alginate, fibrinogen, collagen, matrigel, carrageenan, chitosan, agar, hyaluronic acid, at least one of matrigel, elastin and laminin; the natural polymer hydrogel At least one of cell growth factor, heparin, leptin, paclitaxel and astragalus anticancer powder is compounded in the medicine. The mass volume concentration of the natural polymer hydrogel is 0.1-40%; the concentration of the blood vessel seed cells and tissue cells is 1×10 ³ to 1×10 ⁸ cells/mL; the tissue cells in the functional tissue area It is one of adult tissue cells, adult stem cells and cancer cells; the vascular seed cells in the vascular tissue area are vascular endothelial cells, fat stem cells, bone marrow mesenchymal stem cells, umbilical cord blood stem cells, bone marrow stem cells, embryonic stem cells and induced multiple at least one of competent stem cells.

The preparation method of a personalized bionic composite structure provided by the present invention comprises the following steps: 1) designing a three-dimensional model of a branch vessel stent, and dividing into two groups to imitate arterial vessels and venous vessels respectively; The hydrogel of tissue cells, the prepared hydrogel containing vascular seed cells and the prepared synthetic polymer solution are loaded into different nozzles of the composite multi-nozzle 3D printing device; 3) Preparation of branch vascular scaffolds: according to step 1) The three-dimensional model of the composite multi-nozzle 3D printing equipment is used to extrude the synthetic polymer solution, and freeze-dried to remove the organic solvent, or the melt-extruded synthetic polymer material is obtained to obtain the branch vessel stent; 4) Preparation of functional tissue area: use the composite multi-nozzle The 3D printing equipment prints the prepared hydrogel containing tissue cells layer by layer, and the printing path is along the branch vascular stent, as shown in Figure 3a, printing at intervals to obtain a preliminary 3D structure; 5) Preparation of vascular tissue area: using a composite multi-nozzle The 3D printing equipment prints layer by layer, as shown in Figure 3b, the prepared hydrogel containing vascular seed cells is filled in the gaps of the preliminary 3D structure to obtain an intermediate 3D structure; 6) Prepare a polymer isolation layer and a high Molecular shell: In step 4) and step 5) layer-by-layer printing process, after printing one or several layers, use composite multi-nozzle 3D printing equipment to spray synthetic polymer solution and extract organic solvent, or melt extrude synthetic polymer material , forming a polymer isolation layer to obtain an alternating structure of a cell-containing hydrogel layer and a polymer isolation layer; spraying a synthetic polymer solution on the periphery of the alternating structure and extracting an organic solvent, or melt extruding a synthetic polymer material to form 7) Provide growth factors to the above-mentioned personalized biomimetic composite structure precursor to realize the vascularization of the vascular tissue area, make the functional tissue area form a tissue, and make the vascular tissue area and the functional The cells in the tissue area are connected, and finally the personalized biomimetic composite structure is obtained.

In the preparation method of the present invention, the composite multi-nozzle three-dimensional printing equipment adopts melt extrusion forming technology or low-temperature deposition forming technology to prepare branch vessel stents, polymer isolation layers and polymer shells; when using melt extrusion forming technology to prepare When the branch vessel stent, the polymer isolation layer and the polymer shell, the synthetic polymer material is at least one of acrylonitrile-butadiene-styrene, polyurethane, polytetrafluoroethylene and carbohydrates; When the deposition forming technology prepares the branch vessel stent, the polymer isolation layer and the polymer shell, the solute of the synthetic polymer solution is polyurethane, polycarbonate, polyurea ester, polyglycolide, polysuccinate, poly At least one of ether ester, polyethylene glycol, polylactic acid, polycaprolactone, polylactic acid-glycolic acid copolymer, polyester and polyhydroxyester, the solvent of the synthetic polymer solution is tetraethylene glycol or 1,4-dioxane. The mass volume concentration of the synthetic polymer solution is 1%-30%; the synthetic polymer solution is compounded with an anticoagulant factor, and the anticoagulant factor is heparin or paclitaxel.

A method of using a personalized biomimetic composite structure for drug screening provided by the present invention includes the following steps: 1) If the tissue cells in the functional tissue area adopt adult tissue cells or adult stem cells, supply the personalized biomimetic composite structure with pathogenicity. The culture solution of the drug will cause the above-mentioned personalized bionic composite structure to produce pathological symptoms; if the tissue cells in the functional tissue area are cancer cells, no pathogenic treatment will be performed; 2) the supply of personalized bionic composite structure contains different types and different doses The culture fluid of the therapeutic drug is collected in real time or subsequently; 3) The culture fluid collected above is analyzed to obtain the effect of the type and dose of the therapeutic drug on the personalized composite structure, and judge by detecting and comparing biological indicators The effect of the drug.

In the drug screening method of the present invention, the biological index is at least one of carbohydrate and lipid metabolism index, liver function index, kidney function index, cancer cell index and vascularization index. The pathological symptoms are diabetes, organ inflammation, organ failure or tumors.

Enumerate several specific embodiments below, to further understand the present invention.

Example 1: Preparation of a personalized bionic vascularized liver tissue and its application in hepatitis drug screening

1) Preparation of cell-containing hydrogel: extract rat adipose stem cells (ADSC) and hepatocytes (Hep), wherein ADSC is cultured and passaged to prepare cell suspension; natural polymer powder is cultured with (DMEM, dulbecco'smodifiedeaglemedium) solution to obtain a mass volume concentration of 10% gelatin solution, a mass volume concentration of 2% sodium alginate solution and a mass volume concentration of 1% fibrinogen solution, the above gelatin solution, sodium alginate solution and fibrin The volume ratio of the original solution was mixed at 2:2:1 to obtain a natural polymer solution for use; the ADSC and Hep suspensions were centrifuged and mixed with the natural polymer solution respectively to obtain ADSC-containing cells with a cell concentration of 1×10 ⁶ cells/mL. Natural polymer solution, and Hep-containing natural polymer solution with a cell concentration of 3×10 ⁶ cells/mL.

2) Preparation of branch vessel stents: Solidworks software designed a three-dimensional model of branch vessels, and after analysis by layering software, 3D printing technology was used to melt and extrude molten polytetrafluoroethylene (TEFLON) to obtain a synthetic polymer branch vessel stent.

3) Preparation of polymer shell and polymer isolation layer: 3D printing technology is also used to screw extrude molten TEFLON to prepare the shell and isolation layer; the thickness of the shell is 10 μm, and the thickness of the isolation layer is 0.5 μm.

4) The final establishment of the bionic model in vitro: use the composite multi-nozzle 3D printing technology to print the natural polymer containing Hep along the branch vascular stent, and reserve 50% space at intervals; use the composite multi-nozzle 3D printing technology to make the natural polymer containing ADSC Forming along the above-mentioned reserved space, pores can be reserved inside the forming body; the pulsation system is used to supply capillary growth factor (VEGF) and hepatocyte growth factor (HGF) to induce ADSC to differentiate into capillary endothelial cells or smooth muscle cells; the above-mentioned structure Perform pulsatile in vitro culture, alternately replace DMEM containing VEGF, DMEM containing HGF and general DMEM, to realize the stability of hepatocyte organization, the development of vascularization and the connection between the two.

5) Hepatitis induction and drug screening: Use pulsating system to supply DMEM containing pathogenic drugs (such as diacetate, methyldopa, furantidine, etc.) to induce drug-induced hepatitis, destroy liver cells, detect and record biology Indicators (such as albumin, glucose-6-phosphatase and tyrosine aminotransferase and vascularization expression); use the pulsatile system to supply therapeutic drugs (such as vitamins, reduced glutathione, glycyrrhizic acid preparations or new Developed drugs) in DMEM for treatment, record and analyze the above biological indicators, and obtain drug screening results.

Example 2: Preparation of a personalized bionic vascularized liver tissue and its application in hepatitis drug screening

1) Preparation of cell-containing hydrogel: extract rat adipose stem cells (ADSC) and embryonic stem cells (ESC), and prepare cell suspension after culture and passaging; mix natural polymer powder with DMEM culture medium to obtain mass volume concentration 20% gelatin solution, 4% mass volume concentration of sodium alginate solution and 2% mass volume concentration of fibrinogen solution, the volume ratio of the above gelatin solution, sodium alginate solution and fibrinogen solution is 1:2 : 1 mixed to obtain a natural polymer solution for use; the ADSC and ESC suspensions were centrifuged respectively, and mixed with the natural polymer solution respectively to obtain a cell concentration of 3 × 10 ⁶ natural polymer solutions containing ADSCs/mL, and ESC-containing natural polymer solution with a cell concentration of ¹ ×105 cells/mL.

2) Preparation of branched vascular stents: according to step 2) of Example 1, natural polymer collagen branched stents were prepared.

3) Preparation of polymer shell and polymer isolation layer: Step 3 of Example 1) preparing natural polymer collagen shell and isolation layer.

4) The final establishment of the in vitro bionic model: use the composite multi-nozzle 3D printing technology to 3D print the natural polymer containing ESC along the pre-designed branch blood vessel path or computer model, and leave 50% of the branch blood vessel space for cell assembly at intervals ; Then use the composite multi-nozzle 3D printing technology to print the natural polymer containing ADSC along the reserved path inside the above-mentioned branch blood vessels, and reserve holes on the inside of the printing layer, and use the CaCl ₂ solution with a mass volume concentration of 5% and 100 U /mL of thrombin solution for cross-linking for 2 minutes; use the pulsation system to supply inducers in stages to convert ESCs into hepatocytes, and the inducers include acidic FGF, HGF, OSM, dexamethasone, transferrin, etc.; use the pulsation system to supply capillaries Growth factor (VEGF) and hepatocyte growth factor (HGF) induced ADSCs to differentiate into capillary endothelial cells or smooth muscle cells; the above structures were cultured in vitro by pulsation, and DMEM containing VEGF, DMEM containing HGF, and staged liver cells were alternately replaced. DMEM as a cell inducer and general DMEM can realize the stability of hepatocyte organization, the development of vascularization and the connection between the two.

5) Carry out the induction of hepatitis and drug screening according to step 4) of Example 1.

Example 3: Preparation of a personalized bionic vascularized liver tissue and its application in hepatitis drug screening

1) Preparation of cell-containing hydrogels: extract rat adipose stem cells (ADSCs); introduce transcription factors (Oct3/4, Sox2, c-Myc and Klf4) into mouse fibroblasts to reprogram them into induced pluripotent stem cells (inducedpluripotentstemcell, iPSC), after culture and passaging, prepare cell suspension; mix natural polymer powder with DMEM culture solution or (phosphate buffered saline) PBS solution to obtain a gelatin solution with a mass volume concentration of 10%, and a mass volume concentration of 2% Sodium alginate solution and fibrinogen solution with a mass volume concentration of 1% are mixed with gelatin solution, sodium alginate solution and fibrinogen solution at a volume ratio of 2:2:1 to obtain a natural polymer for use; finally ADSC and The iPSC suspension was centrifuged separately to obtain a natural polymer solution containing ADSCs with a cell concentration of 2×10 ⁵ cells/mL and a natural polymer solution containing iPSCs with a cell concentration of 3×10 ⁶ cells/mL.

2) Preparation of branched vessel stents: Step 2 of Example 1) Preparation of synthetic polymer branched vessel stents.

3) Preparation of the polymer shell and the polymer isolation layer: the material is 1,4-dioxane solution of PLGA with a mass volume concentration of 5%, and the nozzle assembly is sprayed with a multi-nozzle, and the solution is sprayed onto the hydrogel layer In between, an alternating structure of hydrogel-PLGA layers is obtained, which is the isolation layer; finally, the solution is sprayed to the periphery of the structure to obtain a PLGA shell.

4) The final establishment of the bionic model in vitro: use the composite multi-nozzle 3D printing technology to 3D print the natural polymer containing iPSC along the pre-designed branch blood vessel path or computer model, and leave 50% of the branch blood vessel space for cell assembly at intervals ; Then use the composite multi-nozzle 3D printing technology to print the natural polymer containing ADSC along the reserved path inside the above-mentioned branch blood vessels, and reserve holes on the inside of the printing layer; immerse the formed body in PBS solution to extract and remove 1,4-dioxane Cyclic organic solvent; use the pulsating system to supply inducers in stages to transform iPSCs into hepatocytes, and the inducers include activin A, bFGF and HGF; use the pulsating system to supply capillary growth factor (VEGF) and hepatocyte growth factor (HGF), Induce ADSCs to differentiate into capillary endothelial cells or smooth muscle cells; culture the above structures in vitro by pulsating, alternately replace DMEM containing VEGF, DMEM containing HGF, DMEM with staged hepatocyte inducers and general DMEM to realize the organization of hepatocytes The stability of vascularization, the development of vascularization and the relationship between them.

5) Carry out the induction of hepatitis and drug screening according to step 4) of Example 1.

Example 4: Preparation of a personalized bionic vascularized liver cancer tissue and its application in liver cancer drug screening

1) Preparation of cell-containing hydrogel: extract rat adipose stem cells (ADSC), purchase liver cancer cells (HepG2), culture and passage, prepare cell suspension; mix natural polymer powder with PBS solution to obtain mass volume concentration Be 10% gelatin solution, mass volume concentration be 5% sodium alginate solution and mass volume concentration be the fibrinogen solution of 1%, with gelatin solution, sodium alginate solution and fibrinogen solution volume ratio 3:2: 1 After mixing, the natural polymer was obtained for use; finally, ADSC and HepG2 were centrifuged separately and mixed into the natural polymer solution to obtain a natural polymer solution containing ADSC with a cell concentration of 3×10 ⁵ cells/mL, and the cell concentration It is 3×10 ⁶ /mL natural polymer solution containing HepG2.

2) Preparation of branch vessel stents: Solidworks software designed a three-dimensional model of branch vessels, and after analysis by layering software, 3D printing technology was used to extrude molten ABS with a screw to obtain a branch vessel stent.

3) Preparation of polymer isolation layer and polymer shell: the material is a tetraethylene glycol solution of PU with a mass volume concentration of 5%, and the spray head assembly is sprayed with a multi-nozzle, and the solution is sprayed between the hydrogel layers to obtain water The alternating structure of gel-PU layer is the protective layer; finally, the solution is sprayed on the periphery of the structure to obtain the PU shell.

4) Use the composite multi-nozzle 3D printing technology to 3D print the natural polymer containing HepG2 along the pre-designed branch blood vessel path or computer model, leaving 50% of the branch blood vessel space for cell assembly; then use the composite multi-nozzle 3D The printing technology prints the natural polymer containing ADSC along the reserved path inside the above-mentioned branch blood vessels, and reserves channels on the inside of the printing layer; immerses the formed body in PBS solution, extracts and removes the tetraethylene glycol organic solvent; uses the pulsation system to supply MEM (minimumessentialmedium ) culture medium to stabilize liver cancer cells to organize them; use the pulsating system to supply capillary growth factor (VEGF) and hepatocyte growth factor (HGF) to induce ADSC to differentiate into capillary endothelial cells or smooth muscle cells; continue the above structure Pulsating in vitro culture, alternate replacement of VEGF-containing MEM, HGF-containing MEM and general MEM, to achieve the stability of the organization of liver cancer cells, the development of vascularization and the connection between the two.

5) Liver cancer drug screening: detect and record the biological indicators of the formed body (such as jaundice level, albumin level, indocyanine green retention rate, glucose-6-phosphatase and tyrosine aminotransferase and vascularization expression); Use the pulsation system to supply therapeutic drugs such as vitamins, chemotherapeutic drugs, sorafenib or newly developed drugs, record and analyze biological indicators, and perform drug screening.

Example 5: Preparation of a personalized bionic vascularized pancreatic tissue and its application in drug screening for diabetes

1) Preparation of cell-containing hydrogel: extract rat adipose stem cells (ADSC) and pancreatic beta cells (Panβ) to prepare cell suspension; mix natural polymer powder with DMEM culture medium to obtain a mass volume concentration of 10% The gelatin solution is ready for use; finally, the ADSC and Panβ suspensions are centrifuged separately and mixed into the natural polymer solution to obtain 3×10 ⁶ /mL natural polymer solution containing ADSC, and 3×10 ⁶ /mL Natural polymer solution containing Panβ.

2) Preparation of branch vessel stents: according to step 2) of Example 1, synthetic polymer PU stents were prepared.

3) Preparation of shell and isolation layer: step 3 of embodiment 4) preparing PU shell and isolation layer.

4) Use the composite multi-nozzle 3D printing technology to 3D print the natural polymer containing Panβ along the pre-designed branch blood vessel path or computer model, leaving 50% of the branch blood vessel space for cell assembly; then use the composite multi-nozzle 3D The printing technology prints the natural polymer containing ADSC along the reserved path on the inner side of the above-mentioned branch blood vessels, and reserves holes on the inner side of the printing layer; uses the pulsating system to supply the culture medium to stabilize the islet β cells to organize them; uses the pulsating system to supply capillary growth Hepatocyte growth factor (VEGF) and hepatocyte growth factor (HGF) induce ADSCs to differentiate into capillary endothelial cells or smooth muscle cells; continue to culture the above structures in vitro by pulsating, and alternately replace DMEM containing VEGF, DMEM containing HGF and general DMEM to achieve Stability of organization, development of vascularization of pancreatic β-cells and the relationship between them.

5) Diabetes induction and drug screening: use the pulsating system to supply streptozotocin (Streptozotocin, STZ) to directly destroy pancreatic β cells to induce type 2 diabetes, and detect and record biological indicators at this time (such as glucose consumption, insulin release, C-peptide and vascularization expression); use the pulsatile system to supply therapeutic drugs such as sulfonylureas, biguanides, glinides or newly developed drugs, record and analyze biological indicators, and conduct drug screening.

Example 6: Preparation of a personalized bionic vascularized pancreatic tissue and its application in drug screening for diabetes

1) Preparation of cell-containing hydrogel: extract rat adipose stem cells (ADSC) and embryonic stem cells (ESC), and prepare cell suspension after culture and passaging; mix natural polymers with DMEM culture medium to obtain a mass volume concentration of 10% gelatin solution, 2% sodium alginate solution by mass volume concentration and 1% fibrinogen solution by mass volume concentration, the volume ratio of gelatin solution, sodium alginate solution and fibrinogen solution is 2:2:1 After mixing, the natural polymer was obtained for use; finally, ADSC and ESC were centrifuged separately, and mixed into the natural polymer solution respectively to obtain a natural polymer solution containing ADSC with a cell concentration of 3×10 ⁶ cells/mL, and a cell concentration of 3×10 ⁶ /mL natural polymer solution containing ESC.

2) Preparation of branched vascular stents: according to step 2) of Example 1, synthetic polymer PEG branched vascular stents were prepared.

3) Preparation of the outer shell and the isolation layer: step 3 of Example 4) preparing the outer shell and the isolation layer.

4) The final establishment of the in vitro bionic model: use the composite multi-nozzle 3D printing technology to 3D print the natural polymer containing ESC along the pre-designed branch blood vessel path or computer model, and leave 50% of the branch blood vessel space for cell assembly at intervals ; Then use the composite multi-nozzle 3D printing technology to print the natural polymer containing ADSC along the reserved path inside the above-mentioned branch blood vessels, and reserve channels inside the printing layer; use the pulsation system to supply inducers in stages to transform ESC into pancreatic β cells The inducer contains ActivinA, FGF, CYC, RA, CYC, Extendin-4, IGF and Nicotinamide, etc.; the pulsating system is used to supply capillary growth factor (VEGF) and hepatocyte growth factor (HGF) to induce ADSC to differentiate into capillary endothelium Cells or smooth muscle cells; continue to culture the above structure in vitro by pulsating, alternately replace DMEM containing VEGF, DMEM containing HGF, DMEM containing staged islet β cell inducers and general DMEM, to realize the stability of the organization of islet β cells, The development of vascularization and the link between the two.

5) According to step 4) of Example 5, the induction of diabetes and drug screening were carried out.

Example 7: Preparation of a personalized bionic vascularized pancreatic tissue and its application in drug screening for diabetes

1) Preparation of cell-containing hydrogels: extract rat adipose stem cells (ADSCs); introduce transcription factors (Oct3/4, Sox2, c-Myc and Klf4) into mouse fibroblasts to reprogram them into induced pluripotent stem cells (inducedpluripotentstemcell, iPSC), after culture and passaging, prepare cell suspension; mix natural polymer powder with PBS solution to obtain sodium alginate solution with a mass volume concentration of 2%, and finally centrifuge ADSC and iPSC suspension respectively to obtain cells A natural polymer solution containing ADSCs at a concentration of 3×10 ⁶ cells/mL, and a natural polymer solution containing iPSCs at a cell concentration of 3×10 ⁶ cells/mL.

2) Preparation of branch vascular stent: according to step 2) of Example 1, a synthetic polymer PCL branch vascular stent was prepared.

3) Preparation of the outer shell and the isolation layer: step 3) of the embodiment 1 to prepare the outer shell and the isolation layer.

4) The final establishment of the bionic model in vitro: use the composite multi-nozzle 3D printing technology to 3D print the natural polymer containing iPSC along the pre-designed branch blood vessel path or computer model, and leave 50% of the branch blood vessel space for cell assembly at intervals ; Then use the composite multi-nozzle 3D printing technology to print the natural polymer containing ADSC along the reserved path on the inside of the above-mentioned branch blood vessels, and reserve holes on the inside of the printing layer; use the pulsation system to supply inducers in stages to transform iPSC into pancreatic β cells The inducer contains ActivinA, FGF, CYC, RA, CYC, Extendin-4, IGF and Nicotinamide, etc.; the pulsating system is used to supply capillary growth factor (VEGF) and hepatocyte growth factor (HGF) to induce ADSC to differentiate into capillary endothelium Cells or smooth muscle cells; continue to culture the above structure in vitro by pulsating, alternately replace DMEM containing VEGF, DMEM containing HGF, DMEM containing staged islet β cell inducers and general DMEM, to realize the stability of the organization of islet β cells, The development of vascularization and the link between the two.

5) According to step 4) of Example 5, the induction of diabetes and drug screening were carried out.

Example 8: Preparation of a personalized bionic vascularized pancreatic cancer tissue and its application in drug screening of pancreatic cancer

1) Preparation of cell-containing hydrogel: extract rat adipose stem cells (ADSC), purchase pancreatic cancer cells (PANC-1), culture and passage, prepare cell suspension; mix natural polymer powder with PBS solution to obtain The fibrinogen solution with a mass volume concentration of 1%, centrifuged ADSC and PANC-1 respectively, and mixed them into the natural polymer solution respectively to obtain a natural polymer solution containing ADSC with a cell concentration of 3×10 ⁶ cells/mL , and a natural polymer solution containing PANC-1 with a cell concentration of 3×10 ⁶ cells/mL.

2) Preparation of branched vascular stents: according to step 2) of Example 1, synthetic polymer PGA branched vascular stents were prepared.

3) Preparation of the shell and the isolation layer: step 3 of Example 4) preparing the shell and the isolation layer.

4) The final establishment of the in vitro bionic model: use the composite multi-nozzle 3D printing technology to 3D print the natural polymer containing PANC-1 along the pre-designed branch blood vessel path or computer model, leaving 50% of the branch blood vessel space at intervals. Cell assembly; then use the composite multi-nozzle 3D printing technology to print the natural polymer containing ADSC along the reserved path on the inside of the above-mentioned branch blood vessels, and reserve holes on the inside of the printing layer, and use the pulsation system to supply the MEM culture medium to stabilize the pancreatic cancer cells. Organization: Use the pulsating system to supply capillary growth factor (VEGF) and hepatocyte growth factor (HGF) to induce ADSCs to differentiate into capillary endothelial cells or smooth muscle cells; continue to culture the above structures in pulsating in vitro, and alternately replace the MEM containing VEGF , HGF-containing MEM and general MEM, to achieve the stability of the tissue of pancreatic cancer cells, the development of vascularization and the connection between the two.

5) Pancreatic cancer drug screening: detect and record the biological indicators of the formed body (such as alanine aminotransferase, glucose, CEA, CA19-9 and vascularization expression); use the pulsating system to supply therapeutic drugs such as gemcitabine, 5-Fu, substitute Gio et al. or newly developed drugs, record and analyze biological indicators, and conduct drug screening.

Example 9: Preparation of a personalized bionic vascularized kidney tissue and its application in drug screening for nephritis

1) Preparation of cell-containing hydrogel: extract rat adipose stem cells (ADSC) and neonatal rat kidney cells (NKC), and prepare cell suspension after culture and passaging; mix natural polymer powder with DMEM culture medium to obtain mass The gelatin solution with a volume concentration of 20%, centrifuged the ADSC and NKC suspensions, and mixed them with the natural polymer solution respectively to obtain a natural polymer solution containing ADSC with a cell concentration of 1×10 ⁶ cells/mL, and a cell concentration of 2 ×10 ⁷ /mL natural polymer solution containing NKC. indivual

2) Preparation of branch vessel stents: according to step 2) of Example 1, a synthetic polymer PHB branch vessel stent was prepared.

3) Preparation of the shell and the isolation layer: step 3 of Example 4) preparing the shell and the isolation layer.

4) The final establishment of the bionic model in vitro: use the composite multi-nozzle 3D printing technology to 3D print the natural polymer containing NKC along the pre-designed branch blood vessel path or computer model, and leave 50% of the branch blood vessel space for cell assembly at intervals ; Then use the composite multi-nozzle 3D printing technology to print the natural polymer containing ADSC along the reserved path inside the above-mentioned branch blood vessels, and reserve holes on the inside of the printing layer; use the pulsation system to supply the culture medium to stabilize the kidney cells to make them organized; The pulsating system supplies capillary growth factor (VEGF) and hepatocyte growth factor (HGF) to induce ADSCs to differentiate into capillary endothelial cells or smooth muscle cells; combine and connect synthetic polymer branch scaffolds, and continue to culture the above structures pulsatingly in vitro, and replace them alternately DMEM containing VEGF, DMEM containing HGF and general DMEM can realize the stability of renal cell organization, the development of vascularization and the connection between them.

5) Induction of nephritis and drug screening: Use the pulsating system to supply antibiotics or non-steroidal anti-inflammatory drugs to destroy NKC, induce acute interstitial nephritis, and detect and record biological indicators at this time (such as glucose, urea, urine protein creatinine, etc.) and vascular expression); use the pulsatile system to supply therapeutic drugs such as hydrochlorothiazide, furosemide, hormones or newly developed drugs, record and analyze biological indicators, and perform drug screening.

Example 10: Preparation of a personalized bionic vascularized kidney tissue and its application in drug screening for nephritis

1) Preparation of cell-containing hydrogel: extract rat adipose stem cells (ADSC) and embryonic stem cells (ESC), and prepare cell suspension after culture and passaging; mix natural polymer powder with DMEM culture medium to obtain mass volume concentration 10% gelatin solution, 2% sodium alginate solution by mass volume concentration and 1% fibrinogen solution by mass volume concentration, the volume ratio of the above gelatin solution, sodium alginate solution and fibrinogen solution is 2:2 :1 mixed to obtain the natural polymer solution for use; the ADSC and ESC suspensions were centrifuged and mixed with the natural polymer solution respectively to obtain the natural polymer solution containing ADSC with a cell concentration of 1×10 ⁵ cells/mL, and the cells ESC-containing natural polymer solution at a concentration of 1× ¹⁰ cells/mL.

2) Preparation of branched vascular stent: according to step 2) of Example 1, a synthetic polymer PU branched stent was prepared.

3) Preparation of polymer shell and polymer isolation layer: Step 3 of Example 4) Preparation of shell and isolation layer.

4) The final establishment of the in vitro bionic model: use the composite multi-nozzle 3D printing technology to 3D print the natural polymer containing ESC along the pre-designed branch blood vessel path or computer model, and leave 50% of the branch blood vessel space for cell assembly at intervals ; Then use the composite multi-nozzle 3D printing technology to print the natural polymer containing ADSC along the reserved path inside the above-mentioned branch blood vessels, and reserve holes on the inside of the printing layer; use the pulsation system to supply inducers in stages to transform ESC into kidney cells, Inducers include FGF-2 and LIF, etc.; use the pulsating system to supply capillary growth factor (VEGF) and hepatocyte growth factor (HGF), and induce ADSCs to differentiate into capillary endothelial cells or smooth muscle cells; continue to culture the above structures in vitro with pulsation , Alternately replace DMEM containing VEGF, DMEM containing HGF, DMEM containing renal cell inducer and general DMEM to realize the stability of renal cell organization, the development of vascularization and the connection between the two.

5) According to step 4) of Example 9, the induction of nephritis and drug screening for nephritis were carried out.

Example 11: Preparation of a personalized bionic vascularized kidney tissue and its application in drug screening for nephritis

1) Preparation of cell-containing hydrogels: extract rat adipose stem cells (ADSCs); introduce transcription factors (Oct3/4, Sox2, c-Myc and Klf4) into mouse fibroblasts to reprogram them into induced pluripotent stem cells (inducedpluripotentstemcell, iPSC), after culture and passage, prepare cell suspension; mix natural polymer powder with PBS solution to obtain gelatin solution with mass volume concentration of 10%, sodium alginate solution with mass volume concentration of 2%, and mass volume The fibrinogen solution with a concentration of 1% was mixed with gelatin solution, sodium alginate solution and fibrinogen solution at a volume ratio of 1:2:2 to obtain a natural polymer for use; finally, the ADSC and iPSC suspensions were centrifuged separately to obtain A natural polymer solution containing ADSCs at a cell concentration of 1×10 ⁷ cells/mL, and a natural polymer solution containing iPSCs at a cell concentration of 3×10 ⁷ cells/mL.

2) Preparation of branched vascular stents: according to step 2) of Example 1, synthetic polymer PLGA branched vascular stents were prepared.

3) Preparation of polymer shell and polymer isolation layer: step 3) of Example 1 to prepare the shell and isolation layer.

4) The final establishment of the bionic model in vitro: use the composite multi-nozzle 3D printing technology to 3D print the natural polymer containing iPSC along the pre-designed branch blood vessel path or computer model, and leave 50% of the branch blood vessel space for cell assembly at intervals ; Then use the composite multi-nozzle 3D printing technology to print the natural polymer containing ADSC along the reserved path inside the above-mentioned branch blood vessels, and reserve holes on the inside of the printing layer; use the pulsation system to supply inducers in stages to convert iPSC into kidney cells, The inducer includes FGF-2 and LIF, etc.; the pulsating system is used to supply capillary growth factor (VEGF) and hepatocyte growth factor (HGF) to induce ADSC to differentiate into capillary endothelial cells or smooth muscle cells; combined connection to synthesize polymer branch scaffolds, Continue to culture the above structure in vitro by pulsating, and alternately replace DMEM containing VEGF, DMEM containing HGF, DMEM containing kidney cell inducer and general DMEM, so as to realize the stability of tissue organization of kidney cells, the development of vascularization and the relationship between the two. contact.

5) According to step 4) of Example 9, the induction of nephritis and drug screening for nephritis were carried out.

Example 12: Preparation of a personalized bionic vascularized renal cell carcinoma tissue and its application in drug screening of renal cell carcinoma

1) Preparation of cell-containing hydrogel: extract rat adipose stem cells (ADSC), purchase renal cancer cells (RCC), culture and passage, and prepare cell suspension; mix natural polymer powder with PBS solution to obtain mass volume The gelatin solution with a concentration of 10% and the fibrinogen solution with a mass volume concentration of 1% are mixed with the gelatin solution and fibrinogen solution at a volume ratio of 1:1 to obtain a natural polymer for use; finally, ADSC and RCC are centrifuged separately, and mixed into the natural polymer solution respectively to obtain a natural polymer solution containing ADSC with a cell concentration of 3×10 ⁶ cells/mL and a natural polymer solution containing RCC with a cell concentration of 3×10 ⁶ cells/mL.

2) Preparation of branched vascular stents: according to step 2) of Example 1, natural polymer sodium alginate branched stents were prepared and cross-linked with CaCl2.

3) Preparation of polymer shell and polymer isolation layer: Step 3 of Example 1 to prepare natural polymer sodium alginate shell and isolation layer, and cross-link with CaCl2.

4) The final establishment of the bionic model in vitro: use the composite multi-nozzle 3D printing technology to 3D print the natural polymer containing RCC along the pre-designed branch blood vessel path or computer model, and leave 50% of the branch blood vessel space for cell assembly at intervals ; Then use the composite multi-nozzle 3D printing technology to print the natural polymer containing ADSC along the reserved path inside the above-mentioned branch blood vessels, and reserve holes on the inside of the printing layer; use the pulsation system to supply the MEM culture solution to stabilize the renal cancer cells and make them organized ; Utilize the pulsating system to supply capillary growth factor (VEGF) and hepatocyte growth factor (HGF) to induce ADSCs to differentiate into capillary endothelial cells or smooth muscle cells; continue the pulsating in vitro culture of the above structure, alternately replace the MEM containing VEGF, containing The MEM of HGF and the general MEM realize the stability of the organization of renal cancer cells, the development of vascularization and the connection between the two.

5) Kidney cancer drug screening: detect and record the biological indicators of the formed body (such as urea nitrogen, creatinine, alkaline phosphatase, lactate dehydrogenase and vascularization expression); use the pulsating system to supply therapeutic drugs such as sorafenib , bevacizumab or newly developed drugs, record and analyze biological indicators, and conduct drug screening.

Example 13 Preparation of a personalized bionic vascularized lymphoma tissue and its application in drug screening of lymphoma

1) Preparation of cell-containing hydrogel: extract rat adipose stem cells (ADSC), purchase lymphoma cell (EL4), culture and passage, prepare cell suspension; mix natural polymer powder with PBS solution to obtain mass volume The fibrinogen solution with a concentration of 1%, centrifuged ADSC and EL4 respectively, and mixed them into the natural polymer solution respectively to obtain a natural polymer solution containing ADSC with a cell concentration of 2×10 ⁵ cells/mL, and the cell concentration It is 2×10 ⁵ /mL natural polymer solution containing EL4.

2) Preparation of branched vessel stent: According to step 2) of Example 1, a synthetic natural polymer fibrinogen branched vessel stent was prepared, and polymerized with 100 U/mL thrombin.

3) Preparation of polymer shell and polymer isolation layer: step 3 of embodiment 1) Prepare natural polymer fibrinogen shell and isolation layer, polymerize with 100 U/mL thrombin.

4) Use the composite multi-nozzle 3D printing technology to 3D print the natural polymer containing EL4 along the pre-designed branch blood vessel path or computer model, leaving 50% of the branch blood vessel space for cell assembly; then use the composite multi-nozzle 3D The printing technology prints the natural polymer containing ADSC along the reserved path on the inner side of the above-mentioned branch blood vessels, and reserves holes on the inner side of the printing layer; uses the pulsation system to supply the MEM culture solution to stabilize the lymphoma cells to organize them; uses the pulsation system to supply the capillary Vascular growth factor (VEGF) and hepatocyte growth factor (HGF) induce ADSCs to differentiate into capillary endothelial cells or smooth muscle cells; continue to culture the above structures in pulsating in vitro, alternately replace DMEM containing VEGF, DMEM containing HGF and general DMEM , to achieve the stability of the organization of lymphoma cells, the development of vascularization and the connection between the two.

5) Drug screening for lymphoma: detect and record the biological indicators of the formed body (such as lactate dehydrogenase, alkaline phosphatase, tumor giant cells and vascularized expression); use the pulsating system to supply therapeutic drugs such as doxorubicin , bleomycin, vinblastine, methazepam or newly developed drugs, record and analyze biological indicators for drug screening.

Example 14: Preparation of a Personalized Biomimetic Vascularized Microtia Tissue

1) Preparation of cell-containing hydrogel: extract rat adipose stem cells (ADSC) and chondrocytes (CHO) to prepare cell suspension; mix natural polymer powder with DMEM culture medium to obtain a hydrogel with a mass volume concentration of 5%. Gelatin solution, centrifuge the ADSC and CHO suspensions respectively, and mix them into the natural polymer solution to obtain 3×10 ⁶ /mL natural polymer solution containing ADSC and 3×10 ⁶ /mL CHO-containing Natural polymer solution.

2) Preparation of branched vascular stents: according to step 2) of Example 1, synthetic polymer ABS branched vascular stents were prepared.

3) Preparation of polymer shell and polymer isolation layer: the material is 1,4-dioxane solution of PU with a mass volume concentration of 3%, heparin and leptin are mixed in the solution, and the nozzle assembly is sprayed with multi-nozzles. The solution is sprayed between the hydrogel layers; the PBS solution is sprayed with a spray nozzle to extract and remove tetraethylene glycol to obtain a porous PU isolation layer; the alternating structure of the hydrogel-PU layer is obtained, which is the protective layer; finally The solution is sprayed to the periphery of the structure to obtain a PU shell.

4) Use the composite multi-nozzle 3D printing technology to 3D print the natural polymer containing CHO along the pre-designed branch blood vessel path or computer model, leaving 50% of the branch blood vessel space for cell assembly; then use the composite multi-nozzle 3D The printing technology prints the natural polymer containing ADSC along the reserved path on the inner side of the above-mentioned branch blood vessels, and reserves channels on the inner side of the printing layer; immerses the formed body in PBS solution, extracts and removes the tetraethylene glycol organic solvent; uses the pulsation system to supply the culture medium to stabilize Chondrocytes were organized by CHO; the pulsating system was used to supply capillary growth factor (VEGF) and hepatocyte growth factor (HGF) to induce ADSCs to differentiate into capillary endothelial cells or smooth muscle cells; Replace DMEM containing VEGF, DMEM containing HGF and general DMEM to realize the stability of CHO cell organization, the development of vascularization and the connection between them.

Claims (9)

1. A personalized biomimetic composite structure, characterized in that: the personalized biomimetic composite structure comprises a polymer shell (106), a branch vascular support (102), a functional tissue area (103), a vascular tissue area (104) and Polymer isolation layer (401); the functional tissue area (103) is a hydrogel layer containing tissue cells; the vascular tissue area (104) is a hydrogel layer containing vascular seed cells; the function The tissue area (103) and the vascular tissue area (104) are alternately arranged in space; the polymer isolation layer (401) is arranged at intervals in the functional tissue area (103) and the vascular tissue area (104), and the functional tissue area The area (103) and the vascular tissue area (104) are divided into multiple small areas; the branch vessel stent (102) includes two parts, the arterial vessel and the venous vessel, the arterial vessel contains at least one entrance and several branches, and the venous vessel contains at least one outlet and several branches; the branch vascular stent (102) adopts synthetic polymer material, and the branch vascular stent (102) runs through the polymer shell (106), functional tissue area (103), vascular tissue area (104) and Between the polymer isolation layers (401); the polymer shell (106) adopts natural polymer or synthetic polymer material; the hydrogel layer containing tissue cells in the functional tissue area and the blood vessel-containing layer in the vascular tissue area The hydrogel layer of the seed cells is a natural polymer hydrogel, and cell growth factors are compounded in the natural polymer hydrogel; The inner diameter of the branch vessel stent is 10 μm-10 mm, and the wall thickness is 100 μm-2 mm; the layer thickness of the polymer isolation layer and the polymer shell is 0.1 μm-2 mm. 2. A kind of personalized biomimetic composite structure as claimed in claim 1, is characterized in that: this natural polymer hydrogel is gelatin, sodium alginate, fibrinogen, collagen, matrigel, carrageenan, chitosan , at least one of agar, hyaluronic acid, elastin and laminin; and at least one of heparin, leptin, paclitaxel and astragalus anticancer powder are compounded in the natural polymer hydrogel. 3. A kind of personalized biomimetic composite structure as claimed in claim 2, is characterized in that: the mass volume concentration of described natural polymer hydrogel is 0.1～40%; The concentration of described blood vessel seed cell and tissue cell is 1×10 ³ ～1×10 ⁸ cells/mL; the tissue cells in the functional tissue area are one of adult tissue cells, adult stem cells and cancer cells; the vascular seed cells in the vascular tissue area are vascular endothelial cells, At least one of adipose stem cells, bone marrow mesenchymal stem cells, umbilical cord blood stem cells, bone marrow stem cells, embryonic stem cells and induced pluripotent stem cells. 4. A preparation method of individualized biomimetic composite structure as claimed in claim 1, characterized in that the method may further comprise the steps: 1) Design a three-dimensional model of branch vascular stents, and divide them into two groups to simulate arterial vessels and venous vessels; 2) Load the synthetic polymer material, the prepared hydrogel containing tissue cells, the prepared hydrogel containing vascular seed cells, and the prepared synthetic polymer solution into different nozzles of the composite multi-nozzle 3D printing device ; 3) Preparation of branch vascular stents: according to the three-dimensional model in step 1), use a composite multi-nozzle three-dimensional printing device to extrude a synthetic polymer solution, and freeze-dry to remove organic solvents, or melt-extrude synthetic polymer materials to obtain branch vascular stents; 4) Preparation of functional tissue area: use the composite multi-nozzle 3D printing equipment to print the prepared hydrogel containing tissue cells layer by layer, and print at intervals along the branch vascular scaffold along the printing path to obtain a preliminary 3D structure; 5) Prepare the vascular tissue area: use a composite multi-nozzle 3D printing device to print layer by layer, fill the prepared hydrogel containing vascular seed cells in the gaps of the preliminary 3D structure, and obtain an intermediate 3D structure; 6) Preparation of polymer isolation layer and polymer shell: In the process of step 4) and step 5) layer by layer printing, after printing one or several layers, use a composite multi-nozzle 3D printing device to spray the synthetic polymer solution and extract the organic solvent , or melt-extruded synthetic polymer materials to form a polymer isolation layer to obtain an alternate structure of a cell-containing hydrogel layer and a polymer isolation layer; spray a synthetic polymer solution on the periphery of the alternate structure and extract an organic solvent, or Melt extrusion to synthesize polymer materials, form polymer shells, and obtain personalized biomimetic composite structure precursors; 7) Provide growth factors to the precursor of the above-mentioned personalized bionic composite structure, realize the vascularization of the vascular tissue area, make the functional tissue area form a tissue, and make the cells of the vascular tissue area and the functional tissue area connect, and finally obtain the personalized Biomimetic composite structures. 5. The preparation method of a kind of personalized bionic composite structure as claimed in claim 4, it is characterized in that: the composite multi-nozzle three-dimensional printing equipment adopts melt extrusion forming technology or low temperature deposition forming technology to prepare branch vessel stent, high Molecular isolation layer and polymer shell; when adopting melt extrusion forming technology to prepare branch vessel stent, polymer isolation layer and polymer shell, described synthetic polymer material is acrylonitrile-butadiene-styrene, polyurethane, At least one of polytetrafluoroethylene and sugars; when low-temperature deposition forming technology is used to prepare branch vessel stents, polymer isolation layers and polymer shells, the solute of the synthetic polymer solution is polyurethane, polycarbonate, At least one of polyurea ester, polyglycolide, polysuccinate, polyether ester, polyethylene glycol, polylactic acid, polycaprolactone, polylactic acid-glycolic acid copolymer and polyhydroxyester, The solvent of the synthetic polymer solution is tetraethylene glycol or 1,4-dioxane. 6. A method for preparing a personalized bionic composite structure as claimed in claim 4, characterized in that: the mass volume concentration of the synthetic polymer solution is 1% to 30%; the synthetic polymer solution is compounded with Anticoagulant factor, the anticoagulant factor is heparin or paclitaxel. 7. A method for drug screening of a personalized biomimetic composite structure as claimed in claim 1, characterized in that the method comprises the steps: 1) If the tissue cells in the functional tissue area adopt adult tissue cells or adult stem cells, then supply the culture medium containing pathogenic drugs to the personalized biomimetic composite structure, so that the above-mentioned personalized bionic composite structure produces pathological symptoms; if the functional tissue area If the tissue cells are cancer cells, no pathogenic treatment is required; 2) Supply culture fluid containing different types and doses of therapeutic drugs to the personalized biomimetic composite structure, and collect the culture fluid in real time or subsequently; 3) Analyze the above-mentioned collected culture fluid to obtain the effect of the type and dosage of the therapeutic drug on the personalized composite structure, and determine the effect of the drug by detecting and comparing biological indicators. 8. A method for drug screening with a personalized bionic composite structure as claimed in claim 7, characterized in that: said biological indicators are carbohydrate and lipid metabolism indicators, liver function indicators, kidney function indicators, cancer cell At least one of index and vascularization index. 9. The method for using a personalized biomimetic composite structure for drug screening according to claim 7, wherein the pathological symptoms are diabetes, organ inflammation, organ failure or tumor.