WO2021139124A1 - 多层次结构支架及其制备方法与应用 - Google Patents

多层次结构支架及其制备方法与应用 Download PDF

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WO2021139124A1
WO2021139124A1 PCT/CN2020/100525 CN2020100525W WO2021139124A1 WO 2021139124 A1 WO2021139124 A1 WO 2021139124A1 CN 2020100525 W CN2020100525 W CN 2020100525W WO 2021139124 A1 WO2021139124 A1 WO 2021139124A1
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cells
derivatives
stent
stent body
scaffold
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PCT/CN2020/100525
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English (en)
French (fr)
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姚睿
冯璐
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清华大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0062—General methods for three-dimensional culture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2535/00—Supports or coatings for cell culture characterised by topography

Definitions

  • the invention relates to the technical field of biological tissue engineering, in particular to a multi-layer structure scaffold and a preparation method and application thereof.
  • three-dimensional (3D) cell culture can reduce the difference between in vitro culture and natural tissues by reconstructing the interaction between cells and cells and between cells and matrix, which has great advantages.
  • microcarriers have been used as a platform for large-scale expansion of mesenchymal stem cells, embryonic stem cells or induced pluripotent stem cells.
  • 3D scaffolds composed of natural and/or synthetic biological materials have been used for the expansion of hematopoietic stem cells, mesenchymal stem cells and embryonic stem cells.
  • matrix materials alginate and gelatin are widely used due to their good biocompatibility, biodegradability and mild cross-linking conditions.
  • the embodiment of the present invention provides a multi-layered structure scaffold, which has high porosity and permeability, high cell load, good mechanical properties, and good biological performance, and can be used for three-dimensional (3D) cell culture, and the scaffold The inner cells can be recovered without damage.
  • a support with a multi-level structure including a support body, wherein,
  • the inside of the stent body has through large holes with an average pore diameter of 10 to 500 ⁇ m;
  • the porosity of the stent body is 10%-95%
  • the Young's modulus of the bracket body is 0.1 kPa-10 MPa.
  • the macrostructure of the stent body is columnar, block, sheet, capsule, tube, or any combination of shapes.
  • the bracket body is a cylinder, a cube, or a prism.
  • the inside of the stent body has through-holes with an average pore diameter of 80-200 ⁇ m.
  • the porosity of the stent body is 50%-95%.
  • the porosity of the stent body is, for example, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%.
  • the Young's modulus of the stent body is 30-500 kPa.
  • the Young's modulus of the stent body is 0.1kPa, 0.5kPa, 1kPa, 1.5kPa, 2kPa, 3kPa, 4kPa, 5kPa, 6kPa, 7kPa, 8kPa, 9kPa, 10kPa, 0.1MPa, 0.5MPa, 1MPa, 1.5MPa, 2MPa, 3MPa, 4MPa, 5MPa, 6MPa, 7MPa, 8MPa, 9MPa or 10MPa.
  • the stent body further has at least one hollow channel. Further, the hollow channel penetrates the top and bottom of the bracket body.
  • the diameter of the hollow channel is 0.1-5 cm, for example 2 cm.
  • the ratio of the height of the stent body to the diameter (referred to as the outer diameter) is (0.1-10): (10-0.1), for example, 1:1.
  • the height of the stent body is 0.1-8 cm, such as 6 cm; and/or the diameter (referred to as the outer diameter) of the stent body is 0.1-8 cm, such as 6 cm.
  • the porosity of the stent body is 75%-90%.
  • the stent body has a three-dimensional structure with an upper size of 1-50 cm. In some specific embodiments, the stent body has a three-dimensional structure with a size of 1 cm ⁇ 1 cm ⁇ 0.5 cm.
  • the stent body is composed of material microwires of about 50-800 ⁇ m.
  • the stent body has hollow channels with an interval of 0.1-1000 mm.
  • the multi-layer structure support has good elasticity.
  • the multi-layer structure stent exhibits a compressive strain of at least 20%-70% or higher when compressed without permanent deformation or mechanical damage.
  • the stent body is made of a biocompatible material.
  • the biocompatible material is selected from natural materials and/or artificial synthetic materials.
  • the natural material is selected from alginate, alginate derivatives, gelatin, gelatin derivatives, agar, matrigel, collagen, collagen derivatives, hyaluronic acid, hyaluronic acid derivatives , Cellulose, cellulose-derived materials, proteoglycans, proteoglycan derivatives, glycoproteins, glycoprotein-derived materials, chitosan, chitosan derivatives, laminin, fibronectin and fibrin, silk fibroin, At least one of silk fibroin derivatives, vitronectin, osteopontin, peptide hydrogel, DNA hydrogel, more preferably sodium alginate and/or gelatin.
  • the artificial synthetic material is selected from polyglycolic acid, polylactic acid, polylactic acid-glycolic acid copolymer, polyglutamic acid-polyethylene glycol, polycaprolactone, polytrimethylene carbonate Ester, polyglycolic acid, polyethylene glycol-polydioxanone, polyethylene glycol, polytetrafluoroethylene, polyethylene oxide, polyethylene vinyl acetate, polytrimethylene carbonate, polyparadioxane At least one of hexanone, polyetheretherketone, and derivatives and polymers of the above materials, more preferably polyglycolic acid or polylactic acid.
  • the cross-linking agent used in preparing the stent body is selected from one or more of the following: divalent cations, genipin, glutaraldehyde, adipic acid dihydrazide, epoxy chloride Propane, carbodiimide, thrombin and derivatives thereof are preferably calcium chloride.
  • the stent body is made of polyglycolic acid and fibrin, and the cross-linking agent used is thrombin.
  • the multi-level structure support structure of the present invention is controllable, has a controllable multi-level structure ranging from centimeters to micrometers, the macro structure can be customized and the micro pores can be adjusted.
  • the multi-layer structure scaffold prepared by the present invention is more suitable for culturing stem cells, such as liver stem cells (Life Technologies), embryonic stem cells (ATCC) and the like.
  • stem cells such as liver stem cells (Life Technologies), embryonic stem cells (ATCC) and the like.
  • the present invention also provides a method for preparing the above-mentioned multi-layer structure scaffold, which includes the following steps:
  • the biocompatible material has the same meaning as above, and is selected from natural materials and/or artificial synthetic materials, mainly some biocompatible hydrogel materials.
  • the mass percentage concentration of the biocompatible material is 0.1% to 80%, preferably 1% to 25%.
  • the crosslinking agent is selected from one or more of the following substances: divalent cations represented by calcium chloride, genipin, glutaraldehyde, adipic acid Dihydrazide, epichlorohydrin, carbodiimide, thrombin and its derivatives.
  • the crosslinking agent is calcium chloride.
  • the mass percentage concentration of the cross-linking solution used is 0.1 mM to 10M, preferably 1 mM to 100 mM.
  • the biocompatible material and the crosslinking agent solution are mixed in a volume ratio of 1000:1 to 1:1000, preferably 10:1 to 1:10.
  • the biocompatible material is made into a solution (the solvent is preferably sodium chloride solution), and then the crosslinking agent solution is made into a precursor solution.
  • the solvent is preferably sodium chloride solution
  • the biocompatible material is alginate and gelatin
  • the cross-linking liquid is calcium chloride
  • Alginate and gelatin are natural biological materials with good cell compatibility.
  • Alginate can be pre-crosslinked quickly after being mixed with calcium ions, and can be degraded under physiological conditions; gelatin has temperature-sensitive properties and can be reversibly crosslinked by adjusting the temperature.
  • a multi-layer structure scaffold is prepared with a precursor solution made of alginate, gelatin and calcium chloride.
  • a polyglycolic acid solution with a concentration of 1%-25% (the solvent is preferably a 0.1%-10% sodium chloride solution), a fibrinogen with a concentration of 1%-25%
  • the solution (the solvent is preferably 0.1%-10% sodium chloride solution) and the thrombin solution with a concentration of 1 ⁇ 2000mM are uniformly mixed to prepare a precursor solution to prepare a multi-layered structure scaffold, which has good cell compatibility and porosity Larger size is suitable for cell planting and growth, pore size is suitable for cell growth, mechanical properties are similar to natural tissues, and cells can be collected without damage.
  • the concentration of polyglycolic acid in the precursor solution is 0.1-21%
  • the concentration of fibrin is 0.1-21%
  • the concentration of thrombin is 0.1-1000 mM.
  • the precursor solution can be prepared into a three-dimensional structure according to a pre-designed structure by the following methods: casting method (or process), lost foam method (or process), biological 3D printing Method (or process), inkjet printing method (or process), fused deposition modeling method (or process), electrostatic spinning method (or process), electrostatic drive printing method (or process), particle leaching method (or process), Gas foaming technology method (or process), stereo lithography technology method (or process), laser sintering technology method (or process).
  • a casting method (or process) is used.
  • the lost foam method (or process) is used.
  • a biological 3D printing method (or process) is used.
  • step (3) freezes the three-dimensional structure to obtain a solid three-dimensional structure.
  • the three-dimensional structure is frozen in a gradient manner, preferably, incubated at 4°C for 0.5-24 hours, then at -20°C for 0.5-48 hours, and then at -80°C for 0.5-48 hours.
  • the method can obtain large pores with permeability, which is convenient for cell planting and long-term culture; at the same time, it can improve the mechanical properties of the scaffold and is convenient for operation and transportation.
  • the aforementioned method step (4) drying the frozen three-dimensional three-dimensional structure, thereby obtaining a stent with a multi-layer structure.
  • the frozen three-dimensional structure is dried in a vacuum freeze-drying manner, preferably under the conditions of -4°C to -80°C and 1 to 1000 Pa.
  • the macroscopic size of the multi-layer structure bracket can be adjusted by methods such as the size and structure of the mold cavity, and computer modeling. It can be made into block, sheet, capsule, tube or any combination of shapes as required.
  • the present invention also provides a multi-level structure stent prepared according to the foregoing method.
  • the present invention also provides the application of the above-mentioned multi-layer structure scaffold in at least one of the following aspects: 1) cell culture and/or large-scale expansion; 2) drug development, drug screening, drug testing or drug testing; 3) construction of pharmacological models, Pathological model, tissue/organ model; 4) Preparation of materials for tissue repair or regeneration in vivo; 5) Preparation of corrective or plastic implants.
  • the present invention also provides a three-dimensional cell culture method, which includes seeding cells or a mixture of cells and biocompatible materials on the above-mentioned multi-layer structure scaffold for three-dimensional culture. Further, it also includes the steps of cell collection and/or detection.
  • the cell is selected from one or more of the following cells: embryonic stem cells from various sources, pluripotent stem cells, induced pluripotent stem cells, stem cells from various organs, progenitor cells from various organs, mesenchymal Stem cells, cells derived from induced differentiation of various stem cells, fibroblasts derived from various organs, epithelial cells derived from various organs, epidermal cells derived from various organs, endothelial cells derived from various organs, muscles derived from various organs Cells, amniotic membrane cells, cone cells, nerve cells, blood cells, red blood cells, white blood cells, platelets, vascular cells, phagocytes, immune cells, lymphocytes, eosinophils, basophils, plasma cells, mast cells, antigens Presenting cells, cells of the mononuclear phagocyte system, melanocytes, chondrocytes, bone-derived cells, smooth muscle cells, skeletal muscle cells, cardiomyocytes, secretory cells, adip
  • the cells are particularly preferably stem cells, and more preferably embryonic stem cells or liver stem cells.
  • the biocompatible material is alginate, alginate derivatives, gelatin, gelatin derivatives, agar, matrigel, collagen, collagen derivatives, hyaluronic acid, hyaluronic acid derivatives, cellulose, Cellulose-derived materials, proteoglycans, proteoglycan derivatives, glycoproteins, glycoprotein-derived materials, chitosan, chitosan derivatives, laminin, fibronectin and fibrin, silk fibroin, silk fibroin derivatives At least one of phytochemicals, vitronectin, osteopontin, peptide hydrogel, DNA hydrogel, preferably collagen and its derivatives.
  • the obtained cell-loaded multi-layer structure scaffold can be used in a static or dynamic culture system, for example, by means of various forms of bioreactor, pulse culture, chip, perfusion and other culture systems.
  • the aforementioned method realizes the collection of cells/cell clusters inside the multi-layer structure scaffold under physiological conditions according to the characteristics of the selected biological material, and the process of collecting cells from the multi-layer structure scaffold affects the cells/cell clusters.
  • the morphology, phenotype and function of cell clusters are not affected.
  • the harvested cells/cell clusters can be used for cell biology research, tissue repair, cell transplantation therapy, new drug development, drug screening, drug testing, pathology/pharmacological model construction, and Construction of various tissue chip models.
  • the aforementioned method, the obtained cell-loaded multi-layer structure scaffold, in vitro research applications include but not limited to cell culture, cell expansion, cell biology research, drug development, drug screening, drug testing, drug testing, construction Pathological models, construction of pharmacological models, tissue/organ models, tissue repair or regeneration and implants for correction or plastic surgery.
  • the present invention also includes the three-dimensional cell culture obtained by culturing the above method.
  • the present invention has at least the following advantages and beneficial effects:
  • the multi-layer structure scaffold of the present invention has the characteristics of individualization, customization, high cell load, high porosity and permeability, adjustable pore size, high elastic modulus, and injectable transplantation.
  • the scaffold with a multilayer structure in the present invention has a high cell loading rate. Because of the multi-layer structure and macroporous characteristics of the stent with multi-layer structure of the present invention, cells can be evenly distributed in the stent and have a high loading rate, and can load drugs and/or cells to be used as drug carriers and / Or therapeutic implants;
  • the stent with a multilayer structure in the present invention has good biocompatibility.
  • the stent with multi-level structure of the present invention uses biocompatible materials as the matrix material, has very good biocompatibility, and can be used for implantation in the body;
  • the stent with a multi-level structure in the present invention has good mechanical properties.
  • the stent with multi-level structure of the present invention exhibits higher mechanical stability than conventional/same component gel stents;
  • the scaffold with a multi-level structure in the present invention has good biological properties.
  • the scaffold with a multi-layer structure of the present invention can load a variety of cells, and significantly promotes cell proliferation, cell aggregation, cell activity, and maintains and improves cell functions;
  • the scaffold with a multi-level structure of the present invention can realize the non-destructive collection of cells.
  • the scaffold with a multilayer structure in the present invention uses a biocompatible material as a matrix material, which can be hydrolyzed under physiological conditions to achieve non-destructive collection of cells/cell clusters in the scaffold.
  • Fig. 1 is a schematic diagram of a multi-layer structure stent according to an embodiment of the present invention.
  • Fig. 2 is a schematic diagram of a multi-layer structure stent according to some embodiments of the present invention.
  • Figure 3 shows the culture of liver stem cells on a multi-layer structure scaffold in Example 1 of the present invention.
  • Figure 3A shows the distribution and agglomeration state of liver stem cells after 7 days of proliferation in the scaffold;
  • Figure 3B shows the liver stem cells cultured in a plane under the same conditions, the liver stem cells on a 3D scaffold with a multi-layer structure, and the liver harvested after hydrating the scaffold Transcription level of liver-specific genes of stem cells.
  • FIG. 4 is a schematic diagram of a single-jet three-dimensional printing grid-like structure used in Embodiment 2 of the present invention.
  • Fig. 5 is a morphological characterization of a three-dimensional printed scaffold with a multilayer structure prepared in Example 2 of the present invention.
  • 5A is a schematic diagram of a grid-like three-dimensional structure formed by three-dimensional printing;
  • FIG. 5B is a top view of a multi-level structure stent prepared by three-dimensional printing technology;
  • FIG. 5C is a side view of a multi-level structure stent prepared by three-dimensional printing technology;
  • 5D is the microscopic morphology of the stent with multi-level structure observed by scanning electron microscope.
  • Figure 6 shows the culture of embryonic stem cells in a scaffold with a multilayer structure in Example 2 of the present invention.
  • Figure 6A shows the distribution and clustering of embryonic stem cells in the multi-layer structure scaffold under the light microscope for 4 days;
  • Figure 6B shows the embryonic stem cells cultured on the plane and the embryonic stem cells in the multi-layer structure scaffold and Day0 phase after 4 days of culture.
  • Figure 6C shows the pluripotency gene transcription levels of embryonic stem cells cultured under the same conditions, embryonic stem cells on a 3D scaffold with a multilayer structure, and embryonic stem cells harvested after hydrating the scaffold.
  • the percentage sign "%" involved in the present invention refers to mass percentage unless otherwise specified; but the percentage of the solution, unless otherwise specified, refers to the number of grams of solute contained in 100 mL of the solution.
  • crosslinking solution refers to a solution that has a crosslinking effect with a biocompatible material in the solution the day before preparation. It can be known to those skilled in the art and can be used to crosslink the biocompatible material.
  • a material with a certain viscosity solution such as a calcium chloride solution, is preferably 1-100 mM, for example, a calcium chloride solution with a concentration of 5 mM.
  • three-dimensional printing refers to the three-dimensional precise deposition using materials compatible with three-dimensional printing via a method matched with an automatic or semi-automatic, computer-aided three-dimensional molding device (such as a three-dimensional printer).
  • Figures 1 and 2 are schematic diagrams of a multi-layer structure stent according to an embodiment of the present invention.
  • the multi-level structure stent includes a stent body, wherein the inside of the stent body has a through hole with an average pore diameter of about 100 ⁇ m, and the pores of the stent body The rate is 75%, the Young’s modulus of the stent body is 220kPa; the height of the stent body is 6cm, the diameter (referring to the outer diameter) is 6cm, the stent body also has a hollow channel, the diameter of the channel It is 2cm.
  • This embodiment provides a method for preparing the above-mentioned multi-level structure stent, which includes the following steps:
  • polyglycolic acid solution mix polyglycolic acid powder (Sigma-Aldrich) with 0.9% sodium chloride solution at a mass ratio of 21:100, stir with a magnetic stirrer for about 5 minutes, and heat at 100°C until it reaches its mass Dissolve uniformly, pack it after cooling, and store at 4°C.
  • fibrin solution mix fibrinogen powder (Sigma-Aldrich) with 0.9% sodium chloride solution in a mass ratio of 21:100, and heat at 37°C until it is uniformly dissolved.
  • 600mM thrombin solution Dissolve thrombin powder in deionized water to make 600mM thrombin solution as the cross-linking solution.
  • the 21% polyglycolic acid solution, 21% fibrinogen solution, and 600 mM thrombin solution prepared as described above are uniformly mixed to obtain a precursor solution with a final concentration of 7% polyglycolic acid, 7% fibrin, and 200 mM thrombin.
  • the stent was sterilized under UV irradiation for 2 hours and then stored under aseptic conditions.
  • liver stem cells into a scaffold with a hierarchical structure
  • liver stem cells (Life Technologies) at a density of 10 4 cells / mL uniformly dispersed in a cell culture medium which is formed a cell suspension, the cell suspension was added dropwise 1mL cells in three-dimensional scaffold, the cells are left to stand in an incubator 24h.
  • the scaffold of the inoculated cells is given a sufficient amount of cell culture medium and placed under conventional cell culture conditions (37° C., 5% CO 2 incubator) for culture, and fresh culture medium is replaced every 2 to 3 days.
  • liver stem cells in the example 1 on the multilayer structure scaffold constructed by the casting method is shown in Fig. 3.
  • Figure 3A shows the morphology of liver stem cells after 7 days of culture in a scaffold with a multilayer structure. Under the light microscope, it can be observed that the cells are evenly distributed in the scaffold, forming clusters of uniform size, as shown by the arrows in the figure.
  • the present invention uses a mixed solution of 2 uM Calcein-AM (Dojindo, C326) and 4.5 uM PI (Dojindo, P346) to stain live (green)/dead (red) cells respectively, and staining is performed in the dark for 15 minutes.
  • the record was observed using a laser scanning confocal microscope (LSCM, Nikon, Z2). After printing, the cell survival rate in the Day0 structure is about 98%.
  • the proliferation of hepatic stem cells in the multi-layer structure scaffold was detected on the 3rd and 7th day respectively.
  • the conventional two-dimensional culture under the condition that the initial load cell number, culture environment, culture medium and culture conditions are exactly the same, it can be identified by the commonly used cell metabolic activity detection kit ( Cell Viability Assay, Promega), each detection time point showed that culturing liver stem cells in the scaffold with a multilayer structure prepared by the present invention and two-dimensional culturing made no significant difference in the metabolic activity of the cultured cells.
  • liver stem cells In order to detect the function of liver stem cells on the scaffold, immunofluorescence staining was used to detect the expression of mature liver cell-specific marker proteins (such as ALB and MRP2).
  • mature liver cell-specific marker proteins such as ALB and MRP2.
  • Immunofluorescence staining Wash the structure with phosphate buffered saline (PBS) (BI, 02-024-1AC); fix with 4% paraformaldehyde for 30 minutes at room temperature and wash 3 times with PBS for 5 minutes each time; containing 0.3% Triton -X (Sigma, X100) and 5% bovine serum albumin (bovine serum albumin, BSA) (Multicell, 800-096-EG) mixed solution for 1 hour; aspirate the blocking buffer, add the diluted primary antibody (containing 0.3% Triton-X and 1% BSA), ALB (Abcam, ab83465) and MRP2 (Abcam, ab3373), incubated overnight at 4°C.
  • PBS phosphate buffered saline
  • BSA bovine serum albumin
  • the scaffold with multi-layer structure in this experiment is composed of hydrolyzable natural materials, which can be hydrolyzed under physiological conditions to achieve non-destructive collection of cells in the scaffold.
  • the qPCR technology was used to detect the transcription levels of mature hepatocyte-related genes in the cell clusters in the planar culture, the cell clusters in the scaffold, and the cell clusters harvested after hydrolysis of the scaffold.
  • the results are shown in Figure 3B.
  • the gene transcription level of the cells in the 3D scaffold was significantly higher than that of the cells in the planar culture.
  • the ALB expression level of the cells in the 3D scaffold was 15 times that of the cells in the planar culture.
  • the expression level of MRP2 in cells was 4 times that of cells in flat culture. This indicates that after 7 days of culture on the scaffold, liver stem cells differentiated significantly into mature hepatocytes.
  • the gene expression levels of ALB and MRP2 of the cell clusters that can be harvested after hydrolyzing the structure are not different from those of the cells in the 3D scaffold. It shows that the process of hydrolyzing the scaffold to obtain cells has no effect on the morphology, phenotype and function of the cells.
  • Steps for extracting cellular RNA Wash the structures once with PBS, add 1ml Trizol (Gibco, 15596026) to each structure, mix by pipetting repeatedly, let stand at room temperature for 10 minutes, then transfer to a 1.5ml EP tube and add 200ul chloroform , Shake quickly for 30 seconds, leave it at room temperature for 5 minutes, and centrifuge at 12000g for 10 minutes at 4°C. Remove the supernatant, add an equal volume of isopropanol, and centrifuge at 12000g for 10 minutes at 4°C. The supernatant was discarded, the precipitate was washed with 75% absolute ethanol, and RNA was obtained after air-drying, which was dissolved in DEPC water. Use spectrophotometer (Thermo Scientific) to detect RNA concentration and purity.
  • RNA reverse transcription operation steps use PrimeScript TM II 1st strand cDNA Synthesis Kit (TaKaRa, 6210) to operate completely in accordance with the kit instructions. The RNA content was adjusted to 5ng. The primer is: Oligo dT Primer. The reverse transcription PCR program is: 42°C 50min, 95°C, 5min, 4°C incubation, the PCR machine used (ABI, SimpliAmpTM thermal cycler).
  • Fluorescence quantitative PCR operation steps use Maxima SYBR Green qPCR Master Mix (Thermo Scientific, K0251), and operate the kit completely in accordance with the instructions of the kit. After adding the reaction solution as required, put the reaction plate in a qPCR instrument for detection. The reaction procedure 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 ( Figure 3B).
  • primer sequences used in qPCR are as follows (5′-3′):
  • the multi-level structure stent includes a stent body, wherein the inside of the stent body has a through hole with an average pore diameter of about 100 ⁇ m, and the stent The porosity of the body is 95%, the Young's modulus of the stent body is 30kPa; the stent body has a three-dimensional structure with a size of 1cm ⁇ 1cm ⁇ 0.5cm, and the stent body is composed of material microwires of about 300 ⁇ m.
  • the stent body has hollow channels with an interval of about 1 mm.
  • the stent body is composed of multiple hierarchical structures.
  • This embodiment provides a method for preparing the above-mentioned multi-level structure stent, which includes the following steps:
  • a single-jet extrusion printer is used to construct a three-dimensional three-dimensional structure.
  • the schematic diagram of the single-jet 3D printer is shown in Figure 4. Collect the precursor solution into a sterile syringe, and load the sterile syringe into a bio-architect X (Regenovo, Bio-architect X).
  • the printer is equipped with a non-destructive optical coherence tomography (OCT) system that can be used in Non-destructive monitoring during the printing process to ensure sample quality and reduce intra-batch and inter-batch variation.
  • OCT optical coherence tomography
  • the printer supports three-dimensional printing on a sterile temperature-controllable bottom platform under the parameters of supporting speed, contour speed, grid speed and extrusion speed of 50mm/s, 50mm/s, 50mm/s, and 50 ⁇ L/s respectively.
  • the temperature of the bottom platform is set to 0°C to form a three-dimensional hydrogel structure with a volume of 3cm/3cm/1cm, as shown in Figure 5A.
  • a stent with a hierarchical structure Dry the three-dimensional structure for 24 hours at -80°C, 500Pa low temperature and high vacuum conditions to form a stent with a hierarchical structure.
  • the stent was sterilized under UV irradiation for 2 hours and then stored under aseptic conditions.
  • the macrostructure of the multi-layered stent after drying and freezing is shown in Fig. 5B (top view) and Fig. 5C (side view).
  • the microscopic macroporous structure of the stent was observed with a scanning electron microscope, and the diameter of the large-hole penetrating through the stent was 100-300 ⁇ m, as shown in Figure 5D.
  • the method of embryonic stem cells (Life Technologies) at a density of 10 4 cells / mL uniformly dispersed in a cell culture medium which is formed a cell suspension, the cell suspension was added dropwise 1mL cells in three-dimensional scaffold, dynamic planted, will join
  • the scaffold of the cell suspension was cultured on a horizontal shaker (WD-9405F, Beijing Hinsr Technology Co., Ltd.) at a speed of 5000 RPM, under cell culture conditions (37° C., 5% CO 2 incubator) for 12 hours.
  • Fig. 6A shows the morphology of embryonic stem cells cultured in a scaffold with a multilayer structure for 7 days, and the arrow points to the embryonic stem cell clusters. Under the light microscope, it can be observed that the cells are evenly distributed in the scaffold, forming clusters of uniform size.
  • the present invention uses a mixed solution of 2 uM Calcein-AM (Dojindo, C326) and 4.5 uM PI (Dojindo, P346) to stain live (green)/dead (red) cells respectively, and staining is performed in the dark for 15 minutes.
  • a laser scanning confocal microscope (LSCM, Nikon, Z2) was used to observe and record. After printing, the cell survival rate in the Day0 structure is about 99%.
  • Figure 6B shows the proliferation of embryonic stem cells in a three-dimensional printed scaffold with a multilayer structure.
  • the conventional two-dimensional culture under the condition that the initial load cell number, culture environment, culture medium and culture conditions are exactly the same, it can be identified by the commonly used cell metabolic activity detection kit ( Cell Viability Assay, Promega), each detection time point showed that culturing embryonic stem cells in the three-dimensional printed scaffold with a multilayer structure prepared by the present invention significantly improved the metabolic activity of the cultured cells compared to two-dimensional culture.
  • immunofluorescence staining was used to detect the expression of classic pluripotency marker proteins (such as OCT4 and Ecad).
  • Immunofluorescence staining Wash the structure with phosphate buffered saline (PBS) (BI, 02-024-1AC); fix with 4% paraformaldehyde for 30 minutes at room temperature and wash 3 times with PBS for 5 minutes each time; containing 0.3% Triton -X (Sigma, X100) and 5% bovine serum albumin (bovine serum albumin, BSA) (Multicell, 800-096-EG) mixed solution for 1 hour; aspirate the blocking buffer, add the diluted primary antibody (containing 0.3% Triton-X and 1% BSA), OCT4 (Abcam, ab19857) and E-cadherin (Abcam, ab231303), incubated overnight at 4°C.
  • PBS phosphate buffered saline
  • BSA bovine serum albumin
  • the scaffold with multi-layer structure in this experiment is composed of hydrolyzable natural materials, which can be hydrolyzed under physiological conditions to achieve non-destructive collection of cells in the scaffold.
  • the qPCR technology was used to detect the transcription levels of classical pluripotency-related genes in planar cultured cell clusters in the scaffold and cell clusters harvested after hydrolysis of the scaffold.
  • the results are shown in Fig. 6C, there is no significant difference in the transcription levels of pluripotency genes in the planar cultured cells, the cells in the 3D scaffold, and the cell clusters harvested after hydrolyzing the structure. It shows that the processes of culturing and hydrolyzing the scaffold to obtain cells on our multi-layered scaffold have no effect on the morphology, phenotype and pluripotency of the cells.
  • qPCR technology extract cellular RNA. Operation steps: wash the structures once with PBS, add 1ml Trizol (Gibco, 15596026) to each structure, pipette and mix repeatedly, let stand at room temperature for 10 minutes, and then transfer to a 1.5ml EP tube. Add 200ul chloroform, shake quickly for 30 seconds, leave it at room temperature for 5 minutes, and centrifuge at 12000g for 10 minutes at 4°C. Remove the supernatant, add an equal volume of isopropanol, and centrifuge at 12000g for 10 minutes at 4°C. The supernatant was discarded, the precipitate was washed with 75% absolute ethanol, and RNA was obtained after air-drying, which was dissolved in DEPC water. Use spectrophotometer (Thermo Scientific) to detect RNA concentration and purity.
  • RNA reverse transcription operation steps use PrimeScript TM II 1st strand cDNA Synthesis Kit (TaKaRa, 6210) to operate completely in accordance with the kit instructions. The RNA content was adjusted to 5ng. The primer is: Oligo dT Primer. The reverse transcription PCR program is: 42°C 50min, 95°C, 5min, 4°C incubation, the PCR machine used (ABI, SimpliAmpTM thermal cycler).
  • Fluorescence quantitative PCR operation steps use Maxima SYBR Green qPCR Master Mix (Thermo Scientific, K0251), and operate the kit completely in accordance with the instructions of the kit. After adding the reaction solution as required, put the reaction plate in a qPCR instrument for detection. The reaction procedure is: 95°C, 10min, 95°C 15s, 60°C 30s, 40 cycles, 72°C 30s, 72°C 10min. The expression of genes at different time points was obtained ( Figure 6B).
  • primer sequences used in qPCR are as follows (5′-3′):
  • the invention provides a multi-layer structure bracket and a preparation method and application thereof.
  • the multi-level structure scaffold of the present invention has a structure ranging from centimeters to micrometers, and is used for three-dimensional cell culture, in vitro large-scale expansion, in vitro tissue construction, tissue engineering and regenerative medicine, pathological model research, new drug development, drug toxicology research, etc. field.
  • the multi-layer structure scaffold has customizable macrostructure, adjustable hierarchical structure, adjustable pore size, high porosity and permeability, high cell load, high elastic modulus, good mechanical properties, good cell function, and non-destructive cells The characteristics of the collection have good economic value and application prospects.

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Abstract

一种多层次结构支架及其制备方法与应用。多层次结构支架具有从厘米到微米尺度的结构，用于细胞三维培养、体外大规模扩增、体外类组织构建、组织工程与再生医学、病理模型研究、新药研发和药物毒理学研究等领域。

Description

多层次结构支架及其制备方法与应用
交叉引用
本申请要求2020年1月6日提交的专利名称为“多层次结构支架及其制备方法与应用”的第202010011513.8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
本发明涉及生物组织工程技术领域，具体涉及一种多层次结构支架及其制备方法与应用。
背景技术
一般来说，成人细胞移植的单次剂量必须达到10 8～10 9个细胞才能实现有效的功能。同时，干细胞治疗产品必须要满足安全性、有效性和最小批次间差异，以确保稳定的治疗效果。因此，能够实现干细胞的扩增、分化和/或功能维护以及无害的收获并具有成本效益的策略亟待开发。
与平面培养相比，三维(3D)细胞培养可以通过重建细胞与细胞、细胞与基质之间的相互作用来减少体外培养与天然组织之间的差异，具有很大的优势。对于微米等级的培养系统，微载体已被用于间充质干细胞、胚胎干细胞或诱导多能干细胞的大规模扩增平台。对于尺寸更大一些的扩增平台，由天然和/或合成生物材料组成的3D支架已被用于造血干细胞、间充质干细胞和胚胎干细胞的扩增。在基质材料中，海藻酸盐和明胶由于具有良好的生物相容性、生物可降解性和交联条件温和的特点而被广泛使用。此外，藻酸盐的水化特性和明胶在细胞培养温度下实现可逆交联的特性，使得在生理条件下的细胞的无损收集成为可能。然而，大规模体外扩增系统的发展仍然存在挑战，即目前仍缺乏一个可以同时满足以下条件的扩增系统：1)大量细胞被吸收并均匀分布在整个培养系统；2)有充足的营养成分的运输，机械稳定性可支持长期培养；3)收获后的细胞/细胞集群保持其表型和功能。
发明内容
本发明实施例提供一种多层次结构支架，其具有较高的孔隙率和通透率，且细胞负载高，力学性能好，生物学性能好，能够用于三维(3D)细胞培养，且支架内细胞可无损回收。
一种多层次结构支架，包括支架本体，其中，
所述支架本体的内部具有平均孔径10～500μm的贯穿大孔；
所述支架本体的孔隙率为10％-95％；
所述支架本体的杨氏模量为0.1kPa-10MPa。
在本发明一些实施例中，所述支架本体的宏观结构为柱状、块状、片状、囊状、管状或任意形状组合。
在本发明一些实施例中，所述支架本体为圆柱体、正方体或棱柱体。
在本发明一些实施例中，所述支架本体的内部具有平均孔径80～200μm级别的贯穿大孔。
在本发明一些实施例中，所述支架本体的孔隙率为50％-95％。
在本发明一些实施例中，所述支架本体的孔隙率为例如20％、30％、40％、50％、60％、70％、80％、90％或95％。
在本发明一些实施例中，所述支架本体的杨氏模量为30-500kPa。
在本发明一些实施例中，所述支架本体的杨氏模量为0.1kPa、0.5kPa、1kPa、1.5kPa、2kPa、3kPa、4kPa、5kPa、6kPa、7kPa、8kPa、9kPa、10kPa、0.1MPa、0.5MPa、1MPa、1.5MPa、2MPa、3MPa、4MPa、5MPa、6MPa、7MPa、8MPa、9MPa或10MPa。
在本发明一些实施例中，所述支架本体还具有至少一个中空通道。进一步地，所述中空通道贯穿所述支架本体的顶部和底部。
在本发明一些实施例中，所述中空通道为两个、三个、四个或四个以上。
在本发明一些实施例中，所述中空通道的直径为0.1-5cm，例如2cm。
在本发明一些实施例中，所述支架本体的高度与直径(指外径)的比 例为(0.1-10):(10-0.1)，例如1:1。
在本发明一些实施例中，所述支架本体的高度为0.1～8cm，例如6cm；和/或，所述支架本体的直径(指外径)为0.1～8cm，例如6cm。
在本发明一些实施例中，所述支架本体的孔隙率为75％-90％。
在本发明一些实施例中，所述支架本体具有上尺寸为1～50cm的三维结构。在一些具体实施例中，所述支架本体具有尺寸为1cm×1cm×0.5cm的三维结构。
在本发明一些实施例中，所述支架本体所述支架本体由50～800μm左右的材料微丝组成。
在本发明一些实施例中，所述支架本体具有间隔0.1～1000mm的中空通道。
在本发明一些实施例中，所述多层次结构支架具有较好的弹性。
在本发明一些实施例中，所述多层次结构支架在压缩时表现出至少20％-70％或更高的压缩应变而不发生永久形变或机械破坏。
在本发明一些实施例中，所述支架本体由生物相容性材料制成。
在本发明一些实施例中，所述生物相容性材料选自天然材料和/或人工合成材料。
在本发明一些实施例中，所述天然材料选自藻酸盐、藻酸盐衍生物、明胶、明胶衍生物、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、壳聚糖、壳聚糖衍生物、层连接蛋白、纤连接蛋白和纤维蛋白、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，更优选海藻酸钠和/或明胶。
在本发明一些实施例中，所述人工合成材料选自聚乙醇酸、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、以及以上材料的衍生物和聚合物中的至少一种，更优选聚乙醇酸或聚乳酸。
在本发明一些实施例中，制备所述支架本体所用的交联剂选自以下的一种或多种：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，优选为氯化钙。
在本发明一些实施例中，所述支架本体由聚乙醇酸和纤维蛋白制成，所用的交联剂为凝血酶。
本发明多层次结构支架结构可控，具有从厘米到微米尺度的可控的多层次结构，宏观结构可定制且微观孔隙可调节。
本发明所制得的多层次结构支架更适合于培养干细胞，例如肝脏干细胞(Life Technologies)、胚胎干细胞(ATCC)等。
本发明还提供上述多层次结构支架的制备方法，包括以下步骤：
1)将生物相容性材料与相应的交联剂制成前体溶液；
2)以所述前体溶液为材料制备成三维结构体；
3)冷冻所述三维结构体；
4)干燥冷冻后的所述三维结构体，从而得到多层次结构支架。
研究发现，将所述前体溶液制备成三维结构体，并进一步冷冻和干燥后，不仅可以形成宏观空隙，而且还能够形成具有多层次的结构支架。
根据本发明所述多层次结构支架的制备方法，所述生物相容性材料与上文含义相同，选自天然材料和/或人工合成材料，主要是一些具有生物兼容性的水凝胶材料。
在本发明一些具体实施方式中，所述生物相容性材料的质量百分比浓度为0.1％～80％，优选为1％～25％。
根据本发明多层次结构支架的制备方法，所述交联剂选自包含以下的一种或多种物质：以氯化钙为代表的二价阳离子、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。在本发明一些具体实施方式中，所述交联剂为氯化钙。
在本发明一些具体实施方式中，所用交联溶液的质量百分比浓度为0.1mM～10M，优选1mM～100mM。
在本发明一些具体实施方式中，所述生物相容性材料与交联剂溶液按1000:1-1:1000的体积比混合，优选10:1～1:10。
在本发明一些具体实施方式中，是将所述生物相容性材料制成溶液(其溶剂优选为氯化钠溶液)，然后与交联剂溶液制成前体溶液。
在本发明一些具体实施方式中，所述生物相容性材料为海藻酸盐和明胶，所述交联液为氯化钙。
海藻酸盐和明胶均为天然生物材料，细胞相容性好。海藻酸盐可与钙离子混合后可以迅速实现预交联，并可以在生理条件下被降解；明胶具有温敏特性，可通过调节温度实现可逆交联。以含有由海藻酸盐、明胶和氯化钙制成的前体溶液，制备多层次结构支架。
在本发明一些具体实施方式中，是将浓度为1％～25％的聚乙醇酸溶液(其溶剂优选为0.1％～10％氯化钠溶液)、浓度为1％～25％的纤维蛋白原溶液(其溶剂优选为0.1％～10％氯化钠溶液)和浓度为1～2000mM的凝血酶溶液均匀混合制成前体溶液，制备多层次结构支架，其具有细胞相容性好、孔隙率较大适合细胞种植和生长、孔隙尺寸合适细胞生长、力学性能与天然组织相似、可无损收集细胞等优点。
在本发明一些具体实施方式中，所述前体溶液中聚乙醇酸的浓度为0.1-21％，纤维蛋白的浓度为0.1-21％，凝血酶的浓度为0.1-1000mM。
根据本发明多层次结构支架的制备方法，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结构体：铸模法(或工艺)、消失模法(或工艺)、生物3D打印法(或工艺)、喷墨打印法(或工艺)、熔融沉积成型法(或工艺)、静电纺丝法(或工艺)、静电驱动打印法(或工艺)、颗粒浸出法(或工艺)、气体发泡技术法(或工艺)、立体光刻技术法(或工艺)、激光烧结技术法(或工艺)。
在本发明一些具体实施方式中，采用铸模法(或工艺)。
在本发明一些具体实施方式中，采用消失模法(或工艺)。
在本发明一些具体实施方式中，采用生物3D打印法(或工艺)。
根据本发明多层次结构支架的制备方法，步骤(3)冷冻所述三维结构体，从而得到固态的立体三维结构。其中以梯度方式冷冻所述三维结构体，优选地，在4℃下孵育0.5～24h，然后在-20℃下孵育0.5～48h，然后-80℃下孵育0.5～48h。该方法可以得到具有贯通性的大孔，便于细胞种植和长期培养；同时能提高支架的力学性能，方便操作和运输。
根据本发明多层次结构支架的制备方法，前述的方法，步骤(4)干燥所述冷冻的立体三维结构，从而得到具有多层级结构的支架。其中以真空冷冻干燥的方式干燥所述冷冻的三维结构体，优选地在-4℃～-80℃、1～1000Pa的条件下进行真空冷冻干燥。
根据本发明多层次结构支架的制备方法，可通过模具内腔尺寸和结构、计算机建模等方法调节所述多层次结构支架的宏观尺寸。并可根据需要制成块状、片状、囊状、管状或任意形状的组合。
本发明还提供根据前述方法制得的多层级结构支架。
本发明还提供上述多层次结构支架在至少如下一个方面的应用：1)细胞体外培养和/或大规模扩增；2)药物开发、药物筛选、药物检测或药物测试；3)构建药理模型、病理模型、组织/器官模型；4)制备体内组织修复或再生的材料；5)制备矫正或整形的植入物。
本发明还提供一种细胞三维培养方法，包括将细胞或细胞与生物相容性材料的混合物接种于上述多层次结构支架上进行三维培养。进一步地，还包括进行细胞收集和/或检测的步骤。
其中，所述细胞选自以下一种或多种细胞：各种来源的胚胎干细胞、多能干细胞、诱导性多能干细胞、各种器官来源的干细胞、各种器官来源的祖细胞、间充质干细胞、各种干细胞诱导分化得到的细胞、各种器官来源的成纤维细胞、各种器官来源的上皮细胞、各种器官来源的表皮细胞、各种器官来源的内皮细胞、各种器官来源的肌细胞、羊膜细胞、视锥细胞、 神经细胞、血细胞、红细胞、白细胞、血小板、血管细胞、吞噬细胞、免疫细胞、淋巴细胞、嗜酸性粒细胞、嗜碱性粒细胞、浆细胞、肥大细胞、抗原呈递细胞、单核吞噬细胞系统的细胞、黑色素细胞、软骨细胞、骨来源细胞、平滑肌细胞、骨骼肌细胞、心肌细胞、分泌细胞、脂肪细胞、纤毛细胞、胰腺细胞、肾细胞、肠粘膜细胞、肝细胞、肝来源的干细胞或祖细胞、肝巨噬细胞、枯否细胞、星状细胞、胆管上皮细胞、肝窦内皮细胞和其他各种组织和器官来源细胞，以及各种肿瘤细胞、各种用于免疫治疗的细胞、各种经过基因编辑、病毒包装或改造的细胞与细胞系。
基于如上所述的理由，所述细胞特别优选为干细胞，更优选为胚胎干细胞或肝脏干细胞。
其中，所述生物相容性材料为藻酸盐、藻酸盐衍生物、明胶、明胶衍生物、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、壳聚糖、壳聚糖衍生物、层连接蛋白、纤连接蛋白和纤维蛋白、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，优选胶原及其衍生物。
具体地，前述的方法，制得的负载细胞的多层次结构支架可用采用静态或动态培养系统，例如借助各种形式的生物反应器、脉动培养、芯片、灌注等培养系统。
具体地，前述的方法，根据所选生物学材料的特性，实现在生理条件对多层级结构支架的内部的细胞/细胞团簇的收集，且从多层结构支架内收集细胞的过程对细胞/细胞团簇的形态、表型和功能均无影响，收获的细胞/细胞团簇可用于细胞生物学研究、组织修复、细胞移植治疗、新药研发、药物筛选、药物检测、病理/药理模型构建以及各种组织芯片模型的构建。
前述的方法，所获得的负载细胞的多层次结构支架，在体外研究中的应用包括但不限于细胞培养、细胞扩增、细胞生物学研究、药物开发、药 物筛选、药物检测、药物测试、构建病理模型、构建药理模型、组织/器官模型、组织修复或再生以及矫正或整形的植入物。
本发明还包括上述方法培养所得的细胞三维培养物。
有益效果：
籍由上述技术方案，本发明至少具有下列优点及有益效果：
本发明所述多层级结构支架具有个性化、可定制、细胞负载高、孔隙率和通透率高、孔径大小可调节、弹性模量高以及可注射移植的特点。
1)本发明中的具有多层级结构的支架细胞负载率高。本发明的具有多层级结构的支架因其具有的多层级结构及大孔特性，细胞可均匀的分布于支架内且具有较高的负载率，能够负载药物和/或细胞以用作药物载体和/或治疗性植入物；
2)本发明中的具有多层级结构的支架具有良好的生物相容性。本发明的具有多层级结构的支架采用生物相容性材料作为基质材料，具有非常好的生物相容性，可用于体内植入；
3)本发明中的具有多层级结构的支架机械性能好。本发明的具有多层级结构的支架相比于常规/相同组分的凝胶支架表现出更高的机械稳定性；
4)本发明中的具有多层级结构的支架具有良好的生物学性能。本发明的具有多层级结构的支架可以负载多种细胞，并显著地促进了细胞的增殖、细胞聚集成团、细胞活性，维持与提高细胞功能；
5)本发明中的具有多层级结构的支架可以实现细胞的无损收集。本发明中具有多层级结构的支架由生物相容性材料作为基质材料，可以在生理条件下被水解而实现对支架内细胞/细胞团的无损收集。
附图说明
图1为本发明一个实施例多层级结构支架的示意图。
图2为本发明一些实施例多层级结构支架的示意图。
图3为本发明实施例1中肝脏干细胞在多层级结构支架上的培养情 况。图3A为肝脏干细胞在支架内增殖7天后的分布情况与成团状态；图3B为相同条件下平面培养的肝脏干细胞、具有多层级结构的3D支架上的肝脏干细胞以及水化支架后收获的肝脏干细胞的肝脏特异性基因的转录水平。
图4为本发明实施例2中使用的单喷头三维打印网格样结构的示意图。
图5为本发明实施例2中制备的三维打印具有多层级结构支架的形貌表征。其中，图5A为三维打印形成的网格状三维结构体的示意图；图5B为三维打印技术制备的多层级结构支架的俯视图；图5C为三维打印技术制备的多层级结构支架的侧视图；图5D为扫描电镜观察的具有多层级结构支架的微观形貌。
图6为本发明实施例2中胚胎干细胞在具有多层级结构支架中的培养情况。其中，图6A为光镜下胚胎干细胞在多层级结构支架中培养4天后的分布与成团情况；图6B为培养4天后，平面培养的胚胎干细胞与多层级结构支架内的胚胎干细胞与Day0相比的增殖情况；图6C为相同条件下平面培养的胚胎干细胞、具有多层级结构的3D支架上的胚胎干细胞以及水化支架后收获的胚胎干细胞的全能性基因的转录水平。
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。
本发明中涉及到的百分号“％”，若未特别说明，是指质量百分比；但溶液的百分比，除另有规定外，是指100mL溶液中含有溶质的克数。
除非另有定义，本文使用的所有科技术语具有本领域普通技术人员所理解的相同含义。
本文中使用的术语“交联溶液”是指在配制前天溶液中与生物相容性材料起到交联作用的溶液，其可以是本领域技术人员公知可用于使得生物 相容性材料发生交联从而形成具有一定黏度溶液的材料，例如氯化钙溶液，优选1～100mM，例如5mM浓度的氯化钙溶液。
本文中使用的术语“三维打印”是指：经由与自动的或半自动的、计算机辅助的三维成型装置(例如三维打印机)相匹配的方法，利用三维打印相容的原料进行的三维精确沉积。
图1、图2分别为本发明实施例多层级结构支架的示意图。
实施例1 通过铸模法制备具有多层级结构支架
本实施例提供一种多层级结构支架，如图1所示，该多层级结构支架包括支架本体，其中，所述支架本体的内部具有平均孔径100μm左右的贯穿大孔，所述支架本体的孔隙率为75％，所述支架本体的杨氏模量为220kPa；所述支架本体的高度为6cm，直径(指外径)为6cm，所述支架本体还具有中空的通道，所述通道的直径为2cm。
本实施例提供上述多层级结构支架的制备方法，包括如下步骤：
1.生物材料溶液的制备
21％聚乙醇酸溶液：将聚乙醇酸粉末(Sigma-Aldrich)与0.9％氯化钠溶液按照21:100的质量比混合，用磁力搅拌器搅拌约5分钟，在100℃条件下加热直至其均匀溶解，冷却后分装，置于4℃保存。
21％纤维蛋白溶液：将纤维蛋白原粉末(Sigma-Aldrich)与0.9％氯化钠溶液按照21:100的质量比混合，，在37℃条件下加热直至其均匀溶解。
2.交联溶液的制备
600mM凝血酶溶液：将凝血酶粉末溶于去离子水中制成600mM的凝血酶溶液作为交联液。
3.前体溶液的制备
将如上所述制备的21％聚乙醇酸溶液、21％纤维蛋白原溶液、600mM凝血酶溶液均匀混合，获得最终浓度为7％聚乙醇酸、7％纤维蛋白以及200mM凝血酶的前体溶液。
4.铸模法制备三维结构体
将上述前体溶液倒入预设好的模具中，如图1所示，形成体积为外圆直径6cm，中空直径2cm，高6cm的中空圆柱体样三维结构体。
5.冷冻制备的三维结构体
梯度冷却预凝胶三维结构体，具体步骤为将三维结构体在4℃保存24h，-20℃保存48h。
6.干燥冷冻的三维结构体
在-80℃，500Pa的低温、高真空度条件下干燥三维结构体24h，形成具有层级结构的支架。为了支架后续的生物学应用，支架在紫外照射下灭菌2h后无菌条件下保存。
利用本实施例1构建的多层级结构支架对肝脏干细胞进行培养，具体如下：
7.将肝脏干细胞接种到具有层级结构支架内
将肝脏干细胞(Life Technologies)以10 4个/mL的密度均匀分散在其细胞培养基中形成细胞悬液，将1mL细胞混悬液滴加在三维细胞支架中，细胞培养箱中静置24h。
8.检测支架内细胞的分布、增殖、成团和代谢活性
给予接种后细胞的支架足量细胞培养基，置于常规细胞培养条件下(37℃，5％CO 2孵箱)进行培养，每2～3天更换新鲜培养基。
本实施例1中肝脏干细胞在铸模法构建的多层级结构支架上的培养情况见图3。
图3A为肝脏干细胞在具有多层级结构支架中培养7天后的形貌。光镜下可以观察到细胞在支架内均匀分布，形成大小均一的团簇，如图中箭头所示。
第0天、第7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。打印结束Day0结构体内细胞存活率约98％左右。
分别在第3天、第7天检测肝脏干细胞在具有多层级结构支架中的增殖情况。与常规二维培养相比，在初始负载细胞数量、培养环境、培养液和培养条件等完全相同的情况下，通过常用的细胞代谢活性检测试剂盒鉴定(
Figure PCTCN2020100525-appb-000001
Cell Viability Assay，Promega)，各个检测时间点都显示出在本发明所制备的具有多层级结构的支架中培养肝脏干细胞与二维培养使得所培养细胞的代谢活性无显著差异。
9.检测支架上肝脏干细胞的功能
为了检测支架上肝脏干细胞的功能，采用免疫荧光染色检测了成熟肝细胞特异性标记蛋白的表达(如ALB和MRP2)。
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)，ALB(Abcam，ab83465)和MRP2(Abcam,ab3373)，4℃过夜孵育。用PBS洗涤3次，每次5分钟；加入对应二抗Alexa
Figure PCTCN2020100525-appb-000002
594(abcam，ab150080)和Alexa
Figure PCTCN2020100525-appb-000003
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。
10.支架内细胞团簇的无损收集与收获细胞团簇的表型、功能维持
本实验中的具有多层级结构支架是由可水解的天然材料构成，可以在生理条件下被水解而实现支架内细胞的无损收集。
采用qPCR技术分别检测平面培养的、支架内的细胞团簇、以及水解支架后收获的细胞团簇的成熟肝细胞相关基因的转录水平。结果如图3B所示，3D支架内的细胞的基因转录水平显著高于平面培养的细胞水平，其中3D支架内的细胞的ALB的表达水平是平面培养的细胞的15倍，而 3D支架内的细胞的MRP2的表达水平是平面培养的细胞的4倍。这说明在支架上培养了7天后，肝脏干细胞显著向成熟肝细胞分化。而水解结构后可以收获的细胞团簇，其ALB和MRP2的基因表达水平与3D支架内的细胞没有差异。表明水解支架获得细胞这一过程对细胞的形态、表型和功能均没有影响。
qPCR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。获得基因在不同时间点的表达(图3B)。
qPCR所用引物序列如下(5′-3′)：
ALB引物序列：
Forward：GCACAGAATCCTTGGTGAACAG
Reverse：ATGGAAGGTGAATGTTTCAGCA
MRP2引物序列：
Forward：TGAGCAAGTTTGAAACGCACAT
Reverse：AGCTCTTCTCCTGCCGTCTCT
实施例2 通过单喷头三维打印制备具有多层级结构的支架
本实施例提供一种多层级结构支架，如图4、图5所示，该多层级结构支架包括支架本体，其中，所述支架本体的内部具有平均孔径100μm左右的贯穿大孔，所述支架本体的孔隙率为95％，所述支架本体的杨氏模量为30kPa；所述支架本体具有尺寸为1cm×1cm×0.5cm三维结构，所述支架本体由300μm左右的材料微丝组成，所述支架本体具有间隔约为1mm的中空通道。
进一步次，所述支架本体由多个层级的结构组成。
本实施例提供上述多层级结构支架的制备方法，包括如下步骤：
1.按与实施例1相同的方法制备浓度为7％聚乙醇酸、7％纤维蛋白原以及200mM凝血酶的前体溶液。
2.通过单喷头三维打印制备具有多层级结构的支架
使用单喷头挤压式打印机构建立体三维结构，单喷头3D打印机的示意图如图4所示。将前体溶液收集至无菌注射器内，无菌注射器装载到生物三维打印设备中(Regenovo,Bio-architect X)，该打印机配备了非破坏性光学相干层析成像(OCT)系统，可以实现在打印过程中的无损监测，以保证样品质量，减少批次内和批次间差异。打印机以支持速度、轮廓速度、网格速度和挤出速度分别为50mm/s,50mm/s,50mm/s,50μL/s的参数条件下，在无菌的可温控的底面平台上三维打印，底面平台温度设置为0℃，形成体积为3cm/3cm/1cm的水凝胶三维结构体，示意图如图5A所示。
3.冷冻制备的三维结构体
梯度冷却预凝胶三维结构体，具体步骤为将三维结构体在4℃保存24h，-20℃过夜保存。
4.干燥冷冻的三维结构体
在-80℃，500Pa的低温、高真空度条件下干燥三维结构体24h，形成具有层级结构的支架。为了支架后续的生物学应用，支架在紫外照射下灭菌2h后无菌条件下保存。干燥冷冻后的具有多层级结构支架的宏观结构如图5B(俯视图)和图5C(侧视图)所示。采用扫描电镜观察了支架微观的大孔结构，支架内贯穿大孔直径在100～300μm，如图5D所示。
利用本实施例构建的多层级结构支架对胚胎干细胞进行培养，具体如下：
5.将胚胎干细胞接种到具有层级结构支架内
将胚胎干细胞(Life Technologies)以10 4个/mL的密度均匀分散在其细胞培养基中形成细胞悬液，将1mL细胞混悬液滴加在三维细胞支架中，采用动态种植的方法，将加入细胞悬液的支架在水平振动筛(WD-9405F,Beijing Hinsr Technology Co.，Ltd.)上以5000RPM速度旋转培养，在细胞培养条件下(37℃，5％CO 2孵箱)持续12h。
6.检测支架内细胞的分布、增殖、成团和代谢活性
给予接种后细胞的支架足量细胞培养基，置于常规细胞培养条件下(37℃，5％CO2孵箱)进行培养，每2～3天更换新鲜培养基。图6A为胚胎干细胞在具有多层级结构支架中培养7天后的形貌，箭头所指为胚胎干细胞团簇。光镜下可以观察到细胞在支架内均匀分布，形成大小均一的团簇，
第0天、第7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。打印结束Day0结构体内细胞存活率约99％左右。
图6B图为胚胎干细胞在三维打印具有多层级结构支架中增殖情况。与常规二维培养相比，在初始负载细胞数量、培养环境、培养液和培养条件等完全相同的情况下，通过常用的细胞代谢活性检测试剂盒鉴定 (
Figure PCTCN2020100525-appb-000004
Cell Viability Assay，Promega)，各个检测时间点都显示出在本发明所制备的三维打印的具有多层级结构支架中培养胚胎干细胞相比于二维培养使得所培养细胞的代谢活性显著提高。
7.检测支架上胚胎干细胞的全能性
为了检测支架上胚胎干细胞的全能性，采用免疫荧光染色检测了经典的全能性标记蛋白的表达(如OCT4和Ecad)。
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)，OCT4(Abcam,ab19857)和E-cadherin(Abcam,ab231303)，4℃过夜孵育。用PBS洗涤3次，每次5分钟；加入对应二抗Alexa
Figure PCTCN2020100525-appb-000005
594(abcam，ab150080)和Alexa
Figure PCTCN2020100525-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。
8.支架内细胞团簇的无损收集与收获细胞团簇的表型、功能维持
本实验中的具有多层级结构支架是由可水解的天然材料构成，可以在生理条件下被水解而实现支架内细胞的无损收集。
采用qPCR技术分别检测平面培养的、支架内的细胞团簇、以及水解支架后收获的细胞团簇的经典全能性相关基因的转录水平。结果如图6C所示，平面培养的细胞、3D支架内的细胞以及水解结构后收获的细胞团簇的全能性基因转录水平没有显著差异。表明在我们的具有多层级结构的支架上培养以及水解支架获得细胞这些过程对细胞的形态、表型和全能性均没有影响。
qPCR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。获得基因在不同时间点的表达(图6B)。
qPCR所用引物序列如下(5′-3′)：
OCT4引物序列：
Forward：GAAGCAGAAGAGGATCACCTTG
Reverse：TTCTTAAGGCTGAGCTGCAAG
Nanog引物序列：
Forward：CCTCAGCCTCCAGCAGATGC
Reverse：CCGCTTGCACTTCACCCTTTG
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
本发明提供一种多层次结构支架及其制备方法与应用。本发明多层次结构支架具有从厘米到微米尺度的结构，用于细胞三维培养、体外大规模扩增、体外类组织构建、组织工程与再生医学、病理模型研究、新药研发和药物毒理学研究等领域。所述多层次结构支架具有宏观结构可定制、层级结构可调控、孔径大小可调节、孔隙率和通透率高、细胞负载高、弹性模量高、机械性能好、细胞功能好以及细胞可无损收集的特点，具有较好的经济价值和应用前景。

Claims (10)

  1. 一种多层次结构支架，包括支架本体，其中，
    所述支架本体的内部具有平均孔径10～500μm的贯穿大孔；
    所述支架本体的孔隙率为10％-95％；
    所述支架本体的杨氏模量为0.1kPa-10MPa。
  2. 根据权利要求1所述的多层次结构支架，其中，
    所述支架本体的宏观结构为柱状、块状、片状、囊状或管状；和/或，
    所述支架本体为圆柱体、正方体或棱柱体；和/或，
    所述支架本体的内部具有平均孔径80～200μm级别的贯穿大孔；和/或，
    所述支架本体的孔隙率为50％-95％；和/或，
    所述支架本体的杨氏模量为30-500kPa。
  3. 根据权利要求1或2所述的多层次结构支架，其中，
    所述支架本体还具有至少一个中空通道；优选地，所述中空通道贯穿所述支架本体的顶部和底部；进一步优选地，所述中空通道为两个、三个、四个或四个以上；和或，进一步优选地，所述中空通道的直径为0.1-5cm；
    和/或，所述支架本体的高度与直径的比例为(0.1-10):(10-0.1)，优选为1:1；
    和/或，所述支架本体的高度为0.1-8cm，优选1cm；和/或，所述支架本体的直径为0.1-8cm，优选1cm；
    和/或，所述支架本体的孔隙率为75％-95％。
  4. 根据权利要求1或2所述的多层次结构支架，其中，
    所述支架本体具有上尺寸为0.5～50cm的三维结构；优选所述支架本体具有尺寸为1cm×1cm×0.5cm的三维结构；和/或，
    所述支架本体所述支架本体由50～800μm左右的材料微丝组成；和/或，
    所述支架本体具有间隔0.1～1000mm的中空通道。
  5. 根据权利要求1-4任一项所述的多层次结构支架，其中，所述多层次结构支架在压缩时表现出至少20％-70％或更高的压缩应变而不发生永久形变或机械破坏。
  6. 根据权利要求1-5任一项所述的多层次结构支架，其中，所述支架本体由生物相容性材料制成；
    优选地，所述生物相容性材料选自天然材料和/或人工合成材料；
    进一步优选地，所述天然材料选自藻酸盐、藻酸盐衍生物、明胶、明胶衍生物、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、壳聚糖、壳聚糖衍生物、层连接蛋白、纤连接蛋白和纤维蛋白、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，更优选海藻酸钠和/或明胶；和/或，
    进一步优选地，所述人工合成材料选自聚乙醇酸、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、以及以上材料的衍生物和聚合物中的至少一种，更优选聚乙醇酸或聚乳酸；和/或，
    进一步优选地，制备所述支架本体所用的交联剂选自以下的一种或多种：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，更优选为氯化钙；
    进一步优选地，所述支架本体由聚乙醇酸和纤维蛋白制成，所用的交联剂为凝血酶。
  7. 权利要求1-6任一项所述多层次结构支架的制备方法，包括以下步骤：
    1)将生物相容性材料与相应的交联剂制成前体溶液；
    2)以所述前体溶液为材料制备成三维结构体；
    3)冷冻所述三维结构体；
    4)干燥冷冻后的所述三维结构体，从而得到多层次结构支架；
    优选地，所述生物相容性材料的质量百分比浓度为0.1％～80％，更优选为1％～25％；和/或，
    优选地，所用交联溶液的质量百分比浓度为0.1mM～10M，更优选1mM～100mM；和/或，
    优选地，所述生物相容性材料与交联剂溶液按1000:1-1:1000的体积比混合，优选10:1～1:10；和/或，
    优选地，所述前体溶液是由浓度为1％～25％的聚乙醇酸溶液、浓度为1％～25％的纤维蛋白原溶液和浓度为1～2000mM的凝血酶溶液制成；和/或，
    优选地，其中以梯度方式冷冻所述三维结构体，更优选地，在4℃下孵育0.5～24h，然后在-20℃下孵育0.5～48h，然后-80℃下孵育0.5～48h；和/或，
    优选地，以真空冷冻干燥的方式干燥所述冷冻的三维结构体，更优选在-4℃～-80℃、1～1000Pa的条件下进行真空冷冻干燥。
  8. 权利要求7所述方法制备的所述多层次结构支架。
  9. 权利要求1-6、8任一项所述多层次结构支架在至少如下一个方面的应用：1)细胞体外培养和/或大规模扩增；2)药物开发、药物筛选、药物检测或药物测试；3)构建药理模型、病理模型、组织/器官模型；4)制备体内组织修复或再生的材料；5)制备矫正或整形的植入物。
  10. 一种细胞三维培养方法，包括将细胞或细胞与生物相容性材料的混合物接种于权利要求1-6、8任一项所述多层次结构支架上进行三维培养；或者，进一步地，还包括进行细胞收集和/或检测的步骤；
    优选地，所述细胞选自以下一种或多种细胞：各种来源的胚胎干细胞、多能干细胞、诱导性多能干细胞、各种器官来源的干细胞、各种器官来源的祖细胞、间充质干细胞、各种干细胞诱导分化得到的细胞、各种器官来 源的成纤维细胞、各种器官来源的上皮细胞、各种器官来源的表皮细胞、各种器官来源的内皮细胞、各种器官来源的肌细胞、羊膜细胞、视锥细胞、神经细胞、血细胞、红细胞、白细胞、血小板、血管细胞、吞噬细胞、免疫细胞、淋巴细胞、嗜酸性粒细胞、嗜碱性粒细胞、浆细胞、肥大细胞、抗原呈递细胞、单核吞噬细胞系统的细胞、黑色素细胞、软骨细胞、骨来源细胞、平滑肌细胞、骨骼肌细胞、心肌细胞、分泌细胞、脂肪细胞、纤毛细胞、胰腺细胞、肾细胞、肠粘膜细胞、肝细胞、肝来源的干细胞或祖细胞、肝巨噬细胞、枯否细胞、星状细胞、胆管上皮细胞、肝窦内皮细胞和其他各种组织和器官来源细胞，以及各种肿瘤细胞、各种用于免疫治疗的细胞、各种经过基因编辑、病毒包装或改造的细胞与细胞系；进一步优选地，所述细胞为干细胞，更优选为胚胎干细胞或肝脏干细胞；
    和/或，优选地，所述生物相容性材料为藻酸盐、藻酸盐衍生物、明胶、明胶衍生物、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、壳聚糖、壳聚糖衍生物、层连接蛋白、纤连接蛋白和纤维蛋白、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，更优选胶原及其衍生物。
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