Preparation method and application of cell culture liver based on microcarrier

Abstract

The invention discloses a preparation method and application of a cell culture liver based on a microcarrier. The method comprises the steps of obtaining liver seed cells, preparing the microcarrier, culturing cells in vitro based on the microcarrier, preparing the cell culture liver and the like. The preparation method of the cell culture liver based on the microcarrier is provided for the first time, and the preparation scheme that a cell-microcarrier compound serves as a structural unit and is assembled into the cell culture liver after being mixed with an edible material is adopted, so that on one hand, simulation of a real liver lobule structure can be achieved, and on the other hand, simulation of the liver lobule structure can be achieved; the whole structure relation of liver lobules and connective tissues in liver tissues can be simulated, full reduction of animal liver appearance and structural characteristics is achieved, edible materials used in the preparation process are low in price and good in biocompatibility, and the preparation method is suitable for large-scale industrial production. Liver seed cells can be subjected to efficient amplification and albumin synthesis through three-dimensional culture on a microcarrier prepared on the basis of edible materials, so that the cell culture liver is close to a real animal liver in the aspect of nutrition.

Description

Technical Field

[0001] The present invention belongs to the field of cultured meat, and particularly relates to a method for preparing a microcarrier-based cultured liver and its application. Background Art

[0002] Cultured meat is a new type of meat obtained by culturing animal cells and tissues in vitro based on the self-healing and regeneration ability of animal tissues. Compared with traditional livestock farming, cultured meat can significantly reduce energy consumption, water and soil resource occupation, and greenhouse gas emissions, and has attracted a research boom worldwide in recent years.

[0003] Microcarriers have the advantages of both suspension culture and adherent culture, and are commonly used in combination with bioreactors for vaccine production, cell amplification processes, etc. Due to the adherent characteristics of the seed cells for cultured meat and the industrial demand for large-scale amplification of seed cells, the application of microcarriers in the production of cultured meat has also attracted much attention, and some microcarrier-based cultured meatballs and block-shaped cultured meat products have been successively developed by researchers. Regarding microcarrier culture, the main problems are poor cell attachment, difficult cell harvesting, unsatisfactory cell amplification effect, and easy mechanical damage of cells on microcarriers.

[0004] Generally speaking, any animal tissue that can be used as human food can be called "meat". However, the current research focus of cultured meat mainly concentrates on the construction of skeletal muscle and adipose tissue, and little attention has been paid to the animal liver tissue that is also widely consumed by humans. The research on preparing cultured liver with microcarriers in the field is still blank.

[0005] Object of the Invention

[0006] Summary of the Invention: Aiming at the problems existing in the prior art, the present invention provides a method for preparing a microcarrier-based cultured liver. The cultured liver prepared by the present invention can not only simulate the true hepatic lobule structure of the liver, but also simulate the overall structural relationship between hepatic lobules and connective tissues in the liver tissue, fully restoring the shape and structural characteristics of the animal liver. The edible materials used in the preparation process are inexpensive and have good biocompatibility. The liver seed cells can be efficiently amplified and albumin can be synthesized during three-dimensional culture on the microcarriers prepared from the edible materials, making the cultured liver also close to the true animal liver in terms of nutrition, effectively filling the blank of cultured liver.

[0007] The present invention also provides the prepared microcarrier-based cultured liver and its application.

[0008] Technical Solution: In order to achieve the above object, the method for preparing a microcarrier-based cultured liver according to the present invention includes the following steps:

[0009] (1) Obtaining liver seed cells: Isolate liver seed cells from animal liver tissues, and purify and identify the seed cells;

[0010] (2) Preparation of microcarriers: Select edible materials with good gel-forming and biocompatibility, and process them into microcarriers for cell culture with uniform morphology and appropriate size;

[0011] (3) In vitro cell culture based on microcarriers: Inoculate liver seed cells onto the microcarriers, and perform in vitro proliferation and differentiation culture on the whole until they reach a mature state;

[0012] (4) Preparation of cell-cultured liver: Collect the mature cell-microcarrier complexes, mix them with edible materials, assemble and mold them, crosslink, and coat and cure them to obtain a microcarrier-based cell-cultured liver, or directly cook the crosslinked and molded cell-cultured liver without coating and curing.

[0013] Among them, in step (1), liver cells are initially isolated from animal liver tissues, and liver seed cells are further obtained based on the liver cells. The liver seed cells are derived from hepatocytes or non-parenchymal liver cells of pigs, cows, sheep, chickens, ducks, geese, rabbits, and fish. The non-parenchymal liver cells include one or more of bile duct cells, hepatic sinusoidal endothelial cells, hepatic stellate cells, and Kupffer cells.

[0014] Among them, the purification method of the liver seed cells in step (1) is one or more of chemical reprogramming method, flow cytometry sorting method, magnetic bead sorting method, and Percoll gradient centrifugation method. The liver seed cells are obtained from hepatocytes by the chemical reprogramming method, or from non-parenchymal liver cells by one or more of the flow cytometry sorting method, magnetic bead separation method, and Percoll gradient centrifugation method.

[0015] Preferably, the method for obtaining the liver seed cells is one or more of the chemical reprogramming method and the flow cytometry sorting method.

[0016] Among them, the flow cytometry sorting method is to label and separate liver non-parenchymal cells using specific antibody markers. The specific antibodies used are one or more of Lgr5, CD29, CD56, GFP, CD31, CD45, CD133, EPCAM, CD63, CD24, SOX9, PECAM, CD13, CD49f, Sca-1, ALDH+, c-Met; the chemical reprogramming method is obtained by inducing and culturing liver parenchymal cells using a chemical reprogramming medium. The components of the chemical reprogramming medium used include 77-99% basal medium, 0-20% fetal bovine serum, 1-3% penicillin-streptomycin, and 1-600 ng / mL growth factor; among them, the basal medium is one or more of DMEM / F-12, Advanced DMEM / F12, DMEM / F-12 GlutaMAX TM , DMEM, Advanced DMEM, DMEM / F-12 GlutaMAX TM , William's E, L-15, M199, HMM; the growth factor is one or more of dexamethasone (Dex), nicotinamide (NAM), epidermal growth factor (EGF), hepatocyte growth factor (HGF), insulin-transferrin-selenium-ethanolamine (ITS-X), β-mercaptoethanol, interleukin-6 (IL6), vitamin C, N-acetyl-cysteine (NAC), basic fibroblast growth factor (bFGF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), insulin-like growth factor-I (IGF-1), transforming growth factor-β (TGF-β), RSPO1, Y27632, CHIR99021, A83-01, N2, B27.

[0017] Preferably, the specific antibody markers used in the flow cytometry sorting method are one or more of CD31, CD45, CD133, and EPCAM.

[0018] Among them, the edible material described in step (2) is one or more of sodium alginate, chitosan, carboxymethyl chitosan, pectin, carrageenan, gellan gum, xanthan gum, guar gum, agar, agarose, locust bean gum, konjac gum, arabic gum, tamarind gum, cellulose, carboxymethyl cellulose, nanocellulose, collagen, recombinant collagen, gelatin, hyaluronic acid, silk fibroin, elastin, spider silk protein, fibrin, fibrinogen, lecithin, soy protein, pea protein, gluten protein, rice protein, peanut protein, yeast protein, fungal protein, barley protein, wheat protein, rye protein, buckwheat protein, potato protein, corn protein, chickpea protein, broad bean protein, black bean protein, kidney bean protein, mung bean protein, seaweed protein, almond protein, quinoa protein, sorghum protein, insect protein at a concentration of 1-100 mg / mL.

[0019] Preferably, the edible material is one or more of sodium alginate, gellan gum, collagen, gelatin, soy protein, pea protein, peanut protein, corn protein, insect protein at a concentration of 10-50 mg / mL.

[0020] Among them, the processing method of the microcarrier described in step (2) is one or more of the template method, stirring method, membrane emulsification method, emulsion shearing method, high-voltage electrospray method, droplet atomization method, spray drying method.

[0021] Preferably, the method for preparing the microcarrier is one or more of the stirring method, high-voltage electrospray method, emulsion shearing method.

[0022] Among them, the form of the microcarrier described in step (2) is one or more of solid, porous, "shell-core", hollow, multi-component microcarriers with a size of 50-3000 μm.

[0023] Among them, the cross-linking and shaping method of the microcarrier described in step (2) is temperature cross-linking; chemical cross-linking using calcium chloride, calcium sulfate, calcium lactate, genipin or glutaraldehyde; or enzyme cross-linking using transglutaminase, alkaline protease, neutral protease, flavor protease, tyrosinase, laccase, lysyl oxidase, polyphenol oxidase, catalase or thrombin.

[0024] Among them, when the liver seed cells in step (3) are inoculated onto the microcarrier, the cells grow by attaching to the surface of the microcarrier or being embedded inside the microcarrier, and the inoculation density is 1×10 4 -1×10 8 cells / mL of microcarrier.

[0025] Among them, the components of the culture medium used for proliferation and differentiation in step (3) include 77-99% basal medium, 0-20% fetal bovine serum, 1-3% penicillin-streptomycin, and 1-600 ng / mL growth factor; among them, the basal medium is one or more of DMEM, DMEM / F-12, Advanced DMEM, Advanced DMEM / F12, DMEM / F-12 GlutaMAX TM , William's E, Ham's F-12K, RPMI 1640, L-15, M199, HMM; the growth factor is one or more of dexamethasone (Dex), hepatocyte growth factor (HGF), epidermal growth factor (EGF), insulin-transferrin-selenium-ethanolamine (ITS-X), N-acetyl-cysteine (NAC), nicotinamide (NAM), gastrin (Leu15), forskolin (FSK), fibroblast growth factor 10 (FGF10), insulin (INS), β-mercaptoethanol, basic fibroblast growth factor (bFGF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), insulin-like growth factor-I (IGF-1), transforming growth factor-β (TGF-β), interleukin-6 (IL6), dimethyl sulfoxide (DMSO), oncostatin M (OSM), γ-secretase inhibitor (DAPT), fibroblast growth factor 19 (FGF19), bone morphogenetic protein 7 (BMP7), sodium pyruvate, L-glutamine, RSPO1, CHIR99021, A83-01, Y27632, Noggin, Wnt CM, HEPES, N2, B27. The proliferation and differentiation culture media only differ in the types and dosages of the growth factors used.

[0026] Among them, the edible materials in step (4) are one or more of sodium alginate, chitosan, carboxymethyl chitosan, pectin, carrageenan, gellan gum, xanthan gum, guar gum, agar, agarose, locust bean gum, konjac gum, arabic gum, tamarind gum, cellulose, carboxymethyl cellulose, nanocellulose, collagen, recombinant collagen, gelatin, hyaluronic acid, silk fibroin, elastin, spider silk protein, fibrin, fibrinogen, lecithin, soy protein, pea protein, gluten protein, rice protein, peanut protein, yeast protein, fungal protein, barley protein, wheat protein, rye protein, buckwheat protein, potato protein, corn protein, chickpea protein, fava bean protein, black bean protein, kidney bean protein, mung bean protein, seaweed protein, almond protein, quinoa protein, sorghum protein, insect protein at 1-100 mg / mL.

[0027] Preferably, the edible material in step (4) is one or more of sodium alginate, agar, agarose, gellan gum, peanut protein, pea protein, and carboxymethyl cellulose at a concentration of 10 - 50 mg / mL.

[0028] Among them, the method of assembling in step (4) is the mold forming method or the 3D printing method; the mold forming method is to mix the cell - microcarrier complex with the edible material and then transfer the whole to a three - dimensional mold for forming; the 3D printing method is to mix the cell - microcarrier complex with the edible material and then pump it into the extrusion nozzle of a 3D printer, and extrude and stack it for forming under the drive of the printer.

[0029] Among them, in the 3D mold forming method, the volume ratio of the cell - microcarrier complex to the edible material solution is 5 - 15:1, and the relevant parameters used in the 3D printing method are: the volume ratio of the cell - microcarrier complex to the edible material solution is 1 - 3:1; the diameter of the printing nozzle is 0.1 - 5 mm; the extrusion speed is 0.5 - 30 mL / h; the printing speed is 0.5 - 50 mm / s.

[0030] Preferably, in the 3D mold forming method, the volume ratio of the cell - microcarrier complex to the edible material solution is 6:1, and the relevant parameters used in the 3D printing method are: the volume ratio of the cell - microcarrier complex to the edible material solution is 1:1.

[0031] Among them, the cross - linking method of the cell - cultured liver in step (4) is temperature cross - linking; chemical cross - linking using calcium chloride, calcium sulfate, calcium lactate, genipin; or enzymatic cross - linking using one or more of transglutaminase, alkaline protease, neutral protease, flavor protease, tyrosinase, laccase, lysyl oxidase, polyphenol oxidase, catalase, thrombin.

[0032] Among them, the film - coating and curing method of the cell - cultured liver in step (4) is to use an edible forming material to coat the surface of the cross - linked cell - cultured liver with a "connective tissue" film.

[0033] The cell - cultured liver prepared by the preparation method of the present invention.

[0034] The application of the cell - cultured liver described in the present invention in the development of cell - cultured liver products.

[0035] Among them, the cell - cultured liver based on microcarriers obtained is processed by one or more of frying, braising, boiling, stir - frying, and cooking with oil to obtain a cell - cultured liver product.

[0036] Starting from the real liver structure, that is, the basic structural units of the liver - hepatic lobules exist independently of each other, and connective tissues are scattered among them to form a network and extend to the liver surface to form a layer of connective tissue membrane. On the one hand, microcarriers are used for in vitro culture of hepatocytes and finally loaded with a large number of mature liver parenchymal cells to restore the hepatic lobule structure. On the other hand, hepatocyte - microcarriers are mixed with simulated connective tissue materials, and the mixed materials are assembled and formed by 3D printing and the mold method, which can achieve the effect of uniform distribution of simulated hepatic lobules and dispersion of simulated connective tissues among them. Whether in terms of appearance, structure, cell composition or distribution, the real liver can be fully restored.

[0037] In the prior art, there is currently no preparation of a liver cultured with cells. In the present invention, first, a self - made food - grade microcarrier is used for in vitro culture of hepatocytes to obtain a mature hepatocyte - microcarrier complex, and then the complex is mixed and assembled with simulated connective tissue materials to obtain a liver cultured with cells.

[0038] In the present invention, liver seed cells are inoculated on microcarriers for efficient in vitro amplification and differentiation. Further, the efficient amplification and differentiation of cells benefit from the three - dimensional environment provided by microcarriers for cells and the large specific surface area for cell spreading. On the other hand, the selection of microcarrier materials is also the key to efficient in vitro cell culture. The edible materials selected in the present invention have excellent cell compatibility and can support the efficient adhesion, proliferation and differentiation of liver seed cells. Through the preparation of the present invention, on the one hand, the simulation of the hepatic lobule structure of the real liver can be realized, and on the other hand, the overall structural relationship between hepatic lobules and connective tissues in the liver tissue can be simulated, fully restoring the shape and structural characteristics of the animal liver. Moreover, the edible materials used in the preparation process are inexpensive and have good biocompatibility. The liver seed cells can be efficiently amplified and albumin can be synthesized during three - dimensional culture on microcarriers prepared from edible materials, making the liver cultured with cells also close to the real animal liver in terms of nutrition.

[0039] The present invention uses edible materials, integrated microcarriers and 3D printing technology, mold method and other forming methods to highly simulate the real liver structure, that is, the basic structural units of the liver - hepatic lobules exist independently of each other. The surface of the cross - linked liver cultured with cells is coated with a "connective tissue" membrane using an edible forming material through film coating and curing. Connective tissues are scattered among them to form a network and extend to the liver surface to form a layer of connective tissue membrane. First, microcarriers can load a large number of liver parenchymal cells to restore the hepatic lobule structure. Second, mixing hepatocyte - microcarriers with simulated connective tissue materials and forming by 3D printing and the mold method can achieve the effect of uniform distribution of simulated hepatic lobules and dispersion of simulated connective tissues among them. Whether in terms of appearance, structure, cell composition or distribution, the real liver can be fully restored.

[0040] In an animal body, the main part of the liver is composed of hexagonal and three-dimensional hepatic lobules, and the hepatic lobules are connected by a network of connective tissue (white) to form a large mass of liver tissue. In the present invention, microcarriers are used to simulate the structure and morphology of hepatic lobules, and then the microcarriers are mixed with a small amount of edible white adhesive material, and the addition amount of the adhesive material is optimized so that it can be dispersed in the gaps between the spherical microcarriers without affecting the overall morphology of the liver formed by the microcarriers in cell culture. In addition to adhesion, the white adhesive material can also simulate the connective tissue network morphology of the animal liver in appearance, as in the present invention Figure 9 b. In addition, in the present invention, when the volume ratio of the microcarriers to the adhesive material is at a specific ratio (5-15:1 for 3D printing and 1-3:1 for the mold method), the microcarriers can be relatively evenly dispersed in the adhesive material without affecting the overall morphology; when the proportion of the adhesive material is low, the overall structure is loose and cannot be formed; when the proportion of the adhesive material is too high, there are many white areas in appearance, which is quite different from the real liver.

[0041] The preparation method of the microcarrier-based cell culture liver proposed by the present invention can achieve the efficient separation, purification and enrichment of animal liver seed cells and long-term efficient proliferation and differentiation in vitro. At present, there is no report on the application of cell culture liver tissue in the field of cultured meat. In the fields of cell engineering and tissue engineering, the liver seed cells isolated cannot maintain the proliferation and differentiation phenotypes in vitro for a long time, and the culturing ability is low. On the one hand, the present invention can reverse and restore the differentiated hepatocytes to the stem cell state by means of chemical reprogramming combined with a combination of multiple chemical small molecules, so that they have the ability of re-proliferation and differentiation in vitro, and the acquisition is efficient and stable; on the other hand, liver seed cells are purified and obtained from a variety of non-parenchymal liver cells isolated by flow cytometry, which have stable in vitro proliferation and differentiation and maturation abilities, and the acquisition is rapid, efficient and low-cost.

[0042] The non-parenchymal liver cells in the present invention that have not been sorted by flow cytometry contain a miscellaneous variety of cell types and the target liver seed cells cannot be distinguished. After being sorted by flow cytometry, the cells have a single morphology, high purity, and the ability to express specific marker proteins of liver seed cells, such as Figure 1 、 Figure 2 、 Figure 3 as shown.

[0043] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0044] (1) The present invention uses an edible material with good gel-forming property and biocompatibility to prepare microcarriers, which greatly reduces the food safety risk, and has a large throughput, a convenient method and low cost.

[0045] (2) The present invention is based on the three-dimensional culture of liver cells on microcarriers, which can largely restore the physiological state of liver cells in the hepatic lobule, has good biomimicry, is conducive to the maturation of liver seed cells and the synthesis of albumin, and makes the nutritional aspects of cell-cultured liver close to those of real animal livers.

[0046] (3) The implementation scheme of constructing a cell-cultured liver proposed by the present invention, with the mature cell-microcarrier complex as the structural unit, can deeply simulate the structural relationship between the basic structural unit of the animal liver - the hepatic lobule and the liver connective tissue, achieving true simulation.

[0047] (4) Coating and curing are carried out on the surface of the cell-cultured liver prepared by the present invention, which can not only prevent the loose structure of the formed cell-cultured liver, but also simulate the smooth touch of the animal liver surface.

[0048] (5) When preparing the cell-cultured liver of the present invention, only the microcarriers loaded with mature hepatocytes need to be collected as a whole, without the need to use trypsin to digest the hepatocytes or lyse the microcarriers to harvest the amplified mature hepatocytes. Then, the cell-microcarrier complex is directly mixed with edible materials for assembly and molding. The molding is rapid, the structural simulation degree is high, and high-precision equipment is not required. During the whole technological process, no toxic reagents and materials are involved, and the materials used are common animal and plant proteins used as food, which are safe to eat and low in cost.

[0049] (6) The structure of the cell-cultured liver prepared by the present invention is close to that of real animal liver tissue. Microscopically, the cell-microcarrier complex in the cell-cultured liver simulates the basic structural unit of the hepatic lobule of animal liver tissue, and the edible adhesive material is dispersed therein to form a connective network structure to achieve adhesion to the complex. After further cross-linking and curing, it can ensure that the cell-cultured liver remains unchanged in its internal structure after high-temperature cooking and slicing. Its product, cell-cultured stir-fried pig liver, can achieve a high degree of similarity to real stir-fried pig liver in terms of taste, appearance, color, etc. Description of the Drawings

[0050] Figure 1 is the microscopic image of liver non-parenchymal cells obtained by the collagenase perfusion method of the present invention; (a) Picture of hepatocytes under a 10-fold microscope after 1 day of culture; (b) Picture of hepatocytes under a 20-fold microscope after 1 day of culture, and the scale bar is 100μm;

[0051] Figure 2 is the flow cytometry analysis picture of liver non-parenchymal cells obtained by the flow cytometry sorting method of the present invention; (a) Picture of separation of live and dead cells; (b) Picture of separation of single-cell adherent cells; (c) Picture of cell population separation based on hepatocyte markers; (d) Separation of target cells from miscellaneous cells and acquisition picture of target cells;

[0052] Figure 3 This is the immunofluorescence staining identification image of the liver seed cells purified by flow cytometry sorting method in the present invention. The scale bar is 25 μm;

[0053] Figure 4 This is the image of the microcarrier used for liver production by cell culture in the present invention. Among them, (a) is the physical picture of the microcarrier; (b) is the bright-field microscopic view of the microcarrier, and the scale is 400 μm;

[0054] Figure 5 This is the live / dead staining fluorescence image of the liver seed cells on the microcarrier in the present invention, and the scale is 400 μm;

[0055] Figure 6 This is the printing process image (a) and the printed finished product image (b) of the cell culture liver constructed based on 3D printing in the present invention, and the scale is 1 cm;

[0056] Figure 7 This is the three-dimensional mold model and physical picture in the present invention. (a) is the modeling drawing of the three-dimensional mold male mold; (b) is the physical pictures of the three-dimensional mold male mold (i) and female mold (ii), and the scale bar is 2 cm;

[0057] Figure 8 This is the finished product image of the cell culture liver prepared based on the edible adhesive material and mold in the present invention, and the scale is 1 cm;

[0058] Figure 9 This is the product image of the stir-fried cell culture liver developed in the present invention; (a) is the picture of the cell culture liver before stir-frying in slices, and (b) is the finished product picture of the stir-fried cell culture liver;

[0059] Figure 10 This is the live / dead staining fluorescence comparison image of the liver seed cells growing on the self-made microcarrier, common gelatin, and commercial Huakan microcarrier in the present invention. (a) is the gelatin microcarrier; (b) is the microcarrier constructed in the present invention; (c) is the commercial Huakan microcarrier, and the scale is 400 μm; Detailed implementation manners

[0060] The following further describes the present invention with reference to the drawings and embodiments.

[0061] The raw materials, reagents, etc. used in the embodiments are all commercially available.

[0062] Example 1

[0063] Isolation of liver non-parenchymal cells

[0064] Newborn large white pigs within one week of age were drowned in 75% alcohol and then soaked in 75% alcohol for 10 - 15 minutes before dissection. The abdomen was incised along the midline using surgical scissors, and the liver tissue was removed for perfusion digestion. The entire perfusion digestion process was carried out in two stages. A 50 mL syringe and catheter were used to perfuse the liver through the portal vein, and the liquid would flow out from the inferior vena cava. First, 1 L each of the first perfusion fluid and the second perfusion fluid, heated to 39 °C, were sequentially perfused at a rate of 30 mL / min to wash the liver tissue. The specific formula of the first perfusion fluid was: 9 g / L sodium chloride, 0.42 g / L potassium chloride, 2.1 g / L sodium bicarbonate, 0.9 g / L glucose, 4.78 g / L N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Sigma, H4034), 0.37 g / L ethylenediaminetetraacetic acid. The specific formula of the second perfusion fluid was: 9 g / L sodium chloride, 0.42 g / L potassium chloride, 2.1 g / L sodium bicarbonate, 0.9 g / L glucose, 4.78 g / L N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid. Perfusion was stopped when the liver tissue turned yellowish-white. 500 mL of the third perfusion fluid containing collagenase IV was used for the second-stage perfusion. The specific formula of the third perfusion fluid was: 9 g / L sodium chloride, 0.42 g / L potassium chloride, 2.1 g / L sodium bicarbonate, 0.9 g / L glucose, 4.78 g / L N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 0.5 g / L collagenase IV (Sigma, C5138), 0.55 g / L calcium chloride dihydrate. Perfusion digestion was completed when crack-like patterns appeared on the surface of the intact liver tissue in chunks.

[0065] After the collagenase perfusion was completed, the liver tissue was transferred to pre-cooled DMEM (containing 2% fetal bovine serum) medium. The liver tissue capsule was cut open, and the cells were released into the medium to obtain a suspension containing hepatocytes. After 10 minutes, the suspension containing hepatocytes was filtered through a 70 μm cell strainer. The undigested liver parts (especially the periportal area and bile duct parts) were minced and then subjected to secondary agitation digestion with the third perfusion fluid (37 °C, continuous agitation digestion for 30 minutes), and then filtered through a 70 μm cell strainer, and the two filtrates were combined. The combined cell filtrate was centrifuged at 100×g for 2 minutes, and this was repeated 1 - 2 times to remove the precipitate and collect the supernatant. Then, it was centrifuged at 300 g for 5 minutes, and the cells at the bottom of the centrifuge tube were collected. The centrifuged cells were resuspended in DMEM and washed 1 - 2 times, lysed on ice with red blood cell lysis buffer 1 - 2 times to obtain non-parenchymal liver cells; after centrifugation at 300×g for 5 minutes, the resulting non-parenchymal liver cell precipitate was resuspended in DMEM / F12. By cell counting, 1×10 6The cells were seeded into a 10-cm cell culture dish coated with 0.32 mg / mL rat tail collagen (Corning, 354236) and cultured. 8 mL of medium was added to each dish. The components of the medium were 89% DMEM / F12 (Gibco, C11330500BT) by volume fraction, 10% fetal bovine serum (Gibco, A5669701), and 1% penicillin-streptomycin (Gibco, 15140122), which were used for subsequent purification ( Figure 1 ). The remaining non-parenchymal liver cells were centrifuged at 300×g for 5 min. The cell pellet was resuspended in a cryopreservation solution containing 90% fetal bovine serum and 10% dimethyl sulfoxide DMSO (Sigma, D2650), aliquoted into cryotubes at 500 μL / tube, and then stored in liquid nitrogen. In this example, non-parenchymal liver cells were isolated, which contained cholangiocytes, hepatic sinusoidal endothelial cells, hepatic stellate cells, and Kupffer cells.

[0066] Example 2

[0067] Purification of liver seed cells from non-parenchymal liver cells

[0068] Flow cytometry was used to sort and purify non-parenchymal liver cells to obtain liver seed cells. The specific operation steps were as follows:

[0069] (1) Cell culture: The non-parenchymal liver cells seeded in the culture dish in Example 1 were placed in an incubator at 37 °C and 5% CO2. When the cells proliferated to 80-90% confluence, the cells were digested and harvested.

[0070] (2) Staining: The medium (with the same components as in Example 1), trypsin (Gibco, 25200-072), and PBS (Biosharp, BL302A) were pre-warmed in a 37 °C water bath. 2 mL of trypsin was added to each 10-cm dish to digest the cells, and then 2 mL of medium was added to terminate the digestion. The cell suspension was pipetted and transferred into a 15-mL centrifuge tube, centrifuged at 300×g for 5 min. After discarding the supernatant, the cells were resuspended in 1 mL of PBS and counted; 1% bovine serum albumin solution was prepared, filtered through a 0.22-μm filter membrane, and stored in a 4 °C refrigerator for later use. The dye was prepared on ice in the dark (antibody: 1% bovine serum albumin = 1:50 (v / v)). The antibodies used were CD31 (BIO-RAD, MCA1746APC), CD45 (BIO-RAD, MAC1333AC4), CD133 (NOVUS, NB120-16518PE), and EpCAM (Invitrogen, PA5-119097)); the digested cells were divided into two groups. The control group (NC group, CD31 monoclonal antibody group, CD45 monoclonal antibody group, CD133 monoclonal antibody group, EpCAM monoclonal antibody group) each had 2×10 5 cells in 1 mL of PBS suspension, and the experimental group contained 1×107 A 1 mL PBS suspension of cells was centrifuged at 300×g for 5 min. After discarding the supernatant, the dye was added in an ice bath and protected from light (except for the NC group, each of the other four monoclonal antibody groups was added with 50 μL of 1% bovine serum albumin and 1 μL of a single antibody, and the experimental group was added with 1 mL of 1% bovine serum albumin, 20 μL of CD31, 20 μL of CD45, 20 μL of CD133, and 20 μL of EpCAM). After resuspending and mixing evenly, the cells were incubated at room temperature for 30 min. After incubation, 3 mL of PBS was added to each tube, resuspended and mixed evenly, centrifuged at 300×g for 5 min, and then the supernatant was aspirated. The cells were resuspended with the medium (control group: 300 μL, experimental group: 500 μL), and the composition was 98% DMEM / F12 by volume fraction and 2% penicillin-streptomycin, and stored in an ice bath for cell sorting.

[0071] (3) Cell sorting: Prepare 40 mL of sorting receiving solution in advance (the composition is 97% DMEM / F12 by volume fraction and 3% penicillin-streptomycin), and 4 mL of the receiving solution was dispensed into each 15 mL centrifuge tube for receiving cells; the stained cells of each group were sorted by fluorescence-activated cell sorting method, and successively passed through the separation of live and dead cells and the separation of single-cell adhesion cells to obtain liver seed cells labeled with CD31-, CD45-, CD133+, and EpCAM+ ( Figure 2 ), and the antibodies used for sorting liver seed cells will gradually disappear during the later cell proliferation process. In this example, the purified cells are liver stem cells derived from cholangiocytes in liver non-parenchymal cells, that is, liver seed cells.

[0072] Example 3

[0073] Identification of liver seed cells

[0074] The liver seed cells purified in Example 2 can be identified by using liver cell-specific epithelial cell adhesion molecule (EpCAM), keratin 19 (CK19), alpha-fetoprotein (AFP), and albumin (ALB).

[0075] The specific steps are as follows: The purified liver seed cells in Example 2 were taken at 5×10 4Cells were seeded into 3.5-cm cell culture dishes, and 2 mL of culture medium (the same composition as in Example 1) was added to each dish. After the cells adhered and spread, the cells were fixed with 1 mL of 4% paraformaldehyde (Biosharp, BL539A). The fixed samples were permeabilized with 1 mL of 0.5% Triton X-100 for 30 min, and then blocked with 1 mL of 5% bovine serum albumin solution for 30 min. The primary antibody solution was prepared using 1% bovine serum albumin solution. The specific preparation ratio was 8 μL of epithelial cell adhesion molecule, 2.5 μL of keratin 19, 1.25 μL of alpha-fetoprotein, and 2.5 μL of albumin were added to 1 mL of 1% bovine serum albumin solution respectively. 1 mL of the primary antibody solution was added to each 3.5-cm dish and incubated overnight at 4°C. Subsequently, 1 mL of the corresponding secondary antibody solution was added to each dish. The specific preparation ratio was 2 μL of goat anti-rabbit 488 and 2 μL of goat anti-mouse 594 were added to 1 mL of 1% bovine serum albumin solution respectively. The samples were incubated at room temperature in the dark for 3 h. Finally, 100 μL of a mounting medium containing nuclear dye (Archimedes, H-1200) was added dropwise to the samples for mounting. The samples were observed and photographed using a laser confocal scanning microscope. The results are as Figure 3 shown. All four proteins were positive in the isolated cells, indicating that the purified hepatic progenitor cells had a high purity and were almost all hepatic progenitor cells.

[0076] Example 4

[0077] Proliferation and differentiation culture of hepatic progenitor cells

[0078] (1) Proliferation of hepatic progenitor cells

[0079] The hepatic progenitor cells purified in Example 2 were seeded at a density of 6000 cells / cm 2 on a 10-cm cell culture dish coated with rat tail collagen for proliferation. Each dish contained 8 mL of proliferation medium, which consisted of 89% DMEM / F-12 by volume, 10% fetal bovine serum, 1% penicillin-streptomycin, 10 mM nicotinamide (Sigma, N0636), 0.1 μM Dex (Sigma, D4902), 1×ITS-X (Gibco, 51500-056), 20 ng / mL EGF (Sigma, E9644), 20 ng / mL HGF (PeproTech, 100-39H). The cells were cultured at 37°C and 5% CO2, and the cell proliferation medium was changed every 2 days. When the cell confluence reached 80-90%, the cells were digested and passaged.

[0080] (2) Differentiation of hepatic progenitor cells

[0081] The hepatic progenitor cells digested and passaged in step (1) were seeded at a density of 6000 cells / cm 2Inoculate at a density on a 3.5 cm cell culture dish coated with rat tail collagen, culture at 37 °C and 5% CO2, and use a proliferation medium (89% DMEM / F-12, 10% fetal bovine serum, 1% penicillin-streptomycin, 10 mM nicotinamide, 0.1 μM Dex, 1×ITS-X, 20 ng / mL EGF, 20 ng / mL HGF) to culture until a certain confluence (50%-70%), then change to a differentiation medium. The differentiation step is carried out in two stages. In the first stage, culture for 3 days, and the components of the differentiation medium used are 89% DMEM / F-12 by volume fraction, 10% fetal bovine serum, 1% penicillin-streptomycin, 10 mM nicotinamide, 0.1 μM Dex, 1×ITS-X, 20 ng / mL OSM (PeproTech, 0421634). In the second stage, differentiate and culture for another 2 days, and the components of the medium used are 86.5% of the differentiation medium (the same components as above) by volume fraction, 1% DMSO, 12.5% phenol red-free growth factor-reduced basement membrane matrix (Corning, 356231). After the differentiation is completed, harvest mature hepatocytes.

[0082] Example 5

[0083] Preparation of microcarriers

[0084] (1) Preparation of high-voltage electrospray material

[0085] Measure 100 mL of ultrapure water with a pipette into a beaker, weigh 10 g of soy protein powder (Shansong Biology, SD-100) and pour it into the water, and use a magnetic stirrer to stir for 1 h to fully dissolve the soy protein, obtaining a 100 mg / mL soy protein solution. Centrifuge at 12000 rpm for 2 min and take the supernatant for standby.

[0086] (2) Preparation of cross-linking agent

[0087] Weigh 5 g of transglutaminase powder (Solarbio, T9482) into a centrifuge tube, add 50 mL of ultrapure water to dissolve it, and obtain a 100 mg / mL transglutaminase solution for standby.

[0088] (3) Preparation of microcarriers by high-voltage electrospray method

[0089] The soy protein supernatant solution and the transglutaminase solution were mixed in a ratio of 9:1 to prepare a premixed solution, which was then filled into a syringe. A polyethylene plastic tube was then used to connect the syringe outlet and a single-channel capillary (inner diameter 260um). The syringe was mounted on a peristaltic pump, and the positive and negative electrodes were respectively connected to the syringe outlet and the iron ring at the receiving end. The peristaltic pump was started and adjusted to a suitable flow rate (6mL / h). After uniform liquid flowed out of the single-channel capillary orifice, a high-voltage power supply was started, and the voltage was gradually increased to an appropriate value (5kV) so that the liquid at the capillary orifice was in the shape of a Taylor cone, and the liquid from the capillary orifice to the receiving end was in the shape of uniform and continuous liquid beads. Liquid nitrogen was placed at the receiving end for reception, and the soy protein liquid beads were instantly frozen and solidified when in contact with the liquid nitrogen, so that preformed spherical microcarriers with uniform size and complete morphology were obtained, such as Figure 4 (b) shows a solid microcarrier of about 250 μm, which is then placed in a -20°C refrigerator for slow crosslinking to obtain a finished microcarrier ( Figure 4 (a)).

[0090] Example 6

[0091] Cultivation of mature hepatocyte microtissues on microcarriers

[0092] The finished microcarriers of Example 5 that were completely cross-linked were collected and washed with water, and then sterilized by soaking in 75% alcohol. Then, they were washed 2-3 times with DMEM basal medium (C11995500CP, Gibco) and soaked for 3-5 hours. After soaking, the basal medium was removed, and 2 mL of microcarriers were mixed with 2 mL of liver seed cells at the proliferation stage in step (1) of Example 4 at a density of 5×10 5 / mL cell suspension, shake slightly to disperse initially; after inoculation, add proliferation medium to 8mL, the proliferation and differentiation medium components are consistent with those in Example 4, and place the culture dish in a 37°C, 5% CO2 incubator for culture. The cell growth is as shown in Figure 5 As shown, the hepatocytes proliferate on the microcarriers for about 3-5 days to completely cover the surface of the microcarriers. The proliferation medium is replaced with a differentiation medium, and mature hepatocyte microtissues (mature cell-microcarrier complexes) are harvested after 7 days of differentiation culture.

[0093] In addition, from Figure 5 It can also be seen that the hepatocytes isolated independently can be efficiently amplified in three-dimensional culture on food-grade homemade microcarriers, and their proliferation can reach about 90% microcarrier coverage after three days, and the live-dead staining fluorescence image shows that the cells have high vitality. The soy protein microcarriers in Example 5 were replaced with gelatin microcarriers (Sigma, V900863) and commercial Huakan microcarriers (Huakan Biology, P01-100), and cultured in the same way as above, with higher cell attachment and proliferation activity ( Figure 10 ).

[0094] Example 7

[0095] Preparation of 3D Printed Cell-Cultured Liver

[0096] The mature cell-microcarrier complex obtained in Example 6 was thoroughly washed with ultrapure water to remove the residual growth factors in the culture medium, and then mixed with a 1.5 mg / mL sodium alginate (sigma, 71238) solution in a volume ratio of 1:1 and stirred evenly to form a blended ink. The blended ink was filled into the feed inlet of a printer (UnionTech, Lite800). After the fibers were generated at the print head, 3D printing was started. The diameter of the print head was 0.3 mm, the ink extrusion speed was 2.5 mL / h, and the printing speed was 5 mm / s. Subsequently, the print head movement system of the 3D printer moved on the x and z axes, and the printed sample was moved on the y axis by the stage. The movement speed of each optical axis was 5 mm / s, so that the generated composite system fibers were deposited on the stage and stacked and formed along the path of the G-code printing instruction ( Figure 6 (a)). Before crosslinking and forming, the fibers would fuse and connect with each other, forming a connective tissue structure similar to that of a real liver with internal and external connections. Moreover, the microcarriers loaded with mature hepatocytes were evenly and orderly arranged, simulating the distribution of real liver lobules. After printing, a preformed cell-cultured liver was obtained. A 10 mg / mL calcium chloride solution was dropped on the surface of the printed liver to fully infiltrate the entire printed liver, and crosslinked for 20 min to solidify and form.

[0097] Example 8

[0098] Fabrication of 3D Mold

[0099] (1) Fabrication of male mold

[0100] The 3D mold - male mold was modeled using Blender software, and the model file was converted into the STL 3D printing file format. The designed 3D mold - male mold had a groove-like appearance, with an overall size of 50 mm in length, 40 mm in width, and 13 mm in height; there were some protrusions inside the groove, and the protrusion shape was like that of a liver, with a smooth surface and no special structure ( Figure 7 a). The obtained STL model file was processed by slicing software for layer slicing to obtain a G-code motion control instruction file that could be recognized by a 3D printer (UnionTech, Lite800). Then, the G-code printing instruction was imported into the 3D printer, and resin (Future 8200Pro resin) was used as the material. The printer was started to print to obtain the 3D mold - male mold ( Figure 7 i) in b).

[0101] (2) Fabrication of female mold

[0102] Dow Corning SYLGARD TMSYLGARD in the 184 Silicone Elastomer Kit TM 184 Silicone Elastomer Base and SYLGARD TM Mix 184 Silicone Elastomer Curing Agent and 184 Silicone Elastomer Base at a mass ratio of 10:1, stir evenly, and then place them in a vacuum drying kettle for vacuum treatment to remove most of the bubbles generated by stirring. After preliminary degassing, pour the mixture into a three-dimensional mold - male mold, make the material liquid level flush with the top edge of the three-dimensional mold - male mold, and then place it in the vacuum drying kettle again for vacuum degassing to remove the remaining bubbles. The procedures for both degassing operations are "maintain a vacuum degree of 0.08 MPa for 2 min - release air to burst the bubbles - maintain a vacuum degree of 0.08 MPa for 2 min", and repeat the above procedures 8 - 12 times. Place the degassed mold - material as a whole in an oven at 60 °C for 6 h. After molding, take out the material part to obtain a three-dimensional mold - female mold ( Figure 7 ii) in b.

[0103] Example 9

[0104] Preparation of Cell-Cultured Liver Based on the Mold Method

[0105] (1) Preparation of Edible Adhesive Material

[0106] Weigh 3 g of peanut protein powder (Tianrui), dissolve it in 150 mL of ultrapure water to make a 20 mg / mL peanut protein solution, centrifuge at 3000×g for 5 min, and take the supernatant for standby. Take 50 mL of the supernatant of the peanut protein solution, weigh 0.4 g of agarose powder (Biofrog, 1110GR100) and add it to the solution. After magnetic stirring and mixing evenly, place it in a water bath at 95 °C and heat it. During this period, take it out and stir magnetically every 10 min until there is no undissolved agarose powder in the solution to obtain a composite solution. Then, weigh 1 g of sodium alginate powder and add it to the above composite solution, stir magnetically and mix evenly; then weigh 0.25 g of sodium carboxymethylcellulose powder (Aladdin, C104981) and add it to the above composite solution, stir magnetically and mix evenly. Use a glass rod to crush the lumpy powder that is not easily dispersed and continue magnetic stirring until it is all dispersed and dissolved, that is, obtain the edible adhesive material for the preparation of cell-cultured liver.

[0107] (2) Preparation of Cell-Cultured Liver

[0108] Take 30 mL of the cell - microcarrier complex that has matured after 7 - day differentiation culture in Example 6 and place it in a 50 - mL beaker. Transfer the whole to a 60°C water bath. Add 5 mL of edible binding material to the beaker, gently stir and mix evenly, then pour it into the pre - fabricated negative mold with a liver shape in Example 8, and let it stand at room temperature for 2 h. After the whole solidifies, demold it and soak it in a 10 mg / mL calcium chloride solution, and cross - link overnight at 4°C to further cross - link and form the whole.

[0109] In Examples 7 - 9, a cell - cultured liver is obtained by mixing microcarriers loaded with mature hepatocytes with a simulated connective tissue material and then assembling and forming it using 3D printing or the mold method, which can achieve a high - simulation effect with uniform and independent distribution of microcarriers, and connective tissue dispersed among them to form a network structure.

[0110] Example 10

[0111] Coating curing of the cell - cultured liver

[0112] Weigh 0.8 g of sodium alginate, dissolve it in 50 mL of ultrapure water, and mix it evenly with magnetic stirring to prepare a 16 mg / mL sodium alginate coating solution. Take out the cell - cultured liver cross - linked and formed in Example 9 from the calcium chloride solution, blot the excess calcium chloride on the surface with paper, and then evenly apply the sodium alginate coating solution to the surface of the cell - cultured liver. Use the residual calcium chloride to cross - link sodium alginate to form a protective film. On the one hand, it simulates the connective tissue and smooth touch on the surface of the liver tissue, and on the other hand, it uses the coating to protect the overall shape of the cell - cultured liver from falling apart. Finally, the cell - cultured liver ( Figure 6 (b), Figure 8 ) is obtained. As Figure 8 shown, the cell - microcarrier complex simulates the hepatic lobule structure, is mixed with edible materials and assembled and formed. Among them, the arrangement of the simulated hepatic lobule structure is uniform and clearly visible, and the edible binding material is dispersed among them to form a connective network, fully restoring the external shape and structural characteristics of the animal liver.

[0113] Example 11

[0114] Stir - frying the production of the cell - cultured liver

[0115] Harvest the cell - cultured liver formed in Example 9, wash it repeatedly with PBS and clear water, and place it on a plate for standby after draining the water. Cut the washed cell - cultured liver into 1 - mm - thick slices ( Figure 9a), Take 150 g and add 5 g of cooking wine, 3 g of salt, 3 g of oyster sauce, 3 g of starch, and 1 g of pepper powder. Mix well with your hands and marinate for 5 - 10 minutes. Set aside. While marinating, prepare the sauce for stir-frying the liver. Take a disposable paper cup, add 5 g of granulated sugar, 3 g of salt, 1 g of pepper powder, 5 g of water, 5 g of light soy sauce, and 3 g of dark soy sauce. Mix well and set aside. Add 80 g of soybean oil to the pot. After the oil is heated, add 5 g of green onions, 5 g of garlic, 5 g of ginger, 25 g of green peppers, and 30 g of garlic sprouts. Stir-fry for 15 - 20 s, then pour in the pre-marinated cell-cultured liver and stir-fry for 10 - 15 s. Then add the prepared sauce and stir-fry for 5 - 10 s before taking it out of the pot. Figure 9 b).

[0116] In summary, the cell-cultured liver prepared by the present invention fully restores the real liver in terms of appearance, structure, cell composition, and distribution, and various indicators, nutritional components, and tastes are also almost the same as those of the real liver.

Claims

1. A method for preparing a cell culture liver based on microcarriers, characterized in that: The following steps are involved: (1) Obtaining liver seed cells: isolating liver seed cells from animal liver tissue, and purifying and identifying the seed cells; (2) Preparation of microcarriers: Select edible materials with good gelling properties and biocompatibility, and process them into microcarriers with uniform shape and appropriate size for cell culture; (3) In vitro cell culture based on microcarriers: Liver seed cells are inoculated onto microcarriers and the whole is cultured in vitro for proliferation and differentiation to a mature state; (4) Preparation of cell culture liver: collecting mature cell-microcarrier complexes, mixing them with edible materials, assembling them into a shape, cross-linking them, and coating and curing them to obtain a microcarrier-based cell culture liver, or directly cooking the cross-linked cell culture liver without coating and curing it.

2. The method for preparing a microcarrier-based cell culture liver according to claim 1, characterized in that: In step (1), liver cells are initially separated from animal liver tissue, and liver seed cells are further obtained based on the liver cells. The liver seed cells are hepatic parenchymal cells or hepatic non-parenchymal cells derived from pigs, cattle, sheep, chickens, ducks, geese, rabbits, and fish. The hepatic non-parenchymal cells include one or more of bile duct cells, hepatic sinusoidal endothelial cells, hepatic stellate cells, and hepatic Kupffer cells.

3. The method for preparing a microcarrier-based cell culture liver according to claim 1, characterized in that: The liver seed cell purification method in step (1) is preferably one or more of chemical reprogramming, flow cytometry, magnetic bead separation, and Percoll gradient centrifugation. The liver seed cells are obtained from liver parenchymal cells by chemical reprogramming, or from liver non-parenchymal cells by flow cytometry, magnetic bead separation, and Percoll gradient centrifugation.

4. The method for obtaining liver seed cells according to claim 3, characterized in that: The flow cytometry sorting method is to use specific antibody markers to mark and separate liver non-parenchymal cells, and the specific antibodies used are one or more of Lgr5, CD29, CD56, GFP, CD31, CD45, CD133, EPCAM, CD63, CD24, SOX9, PECAM, CD13, CD49f, Sca-1, ALDH+, and c-Met; the chemical reprogramming method is to use chemical reprogramming medium to induce and culture liver parenchymal cells, and the components of the chemical reprogramming medium used include 77-99% basal medium, 0-20% fetal bovine serum, 1-3% penicillin-streptomycin, and 1-600ng / mL growth factor; wherein the basal medium is DMEM / F-12, AdvancedDMEM / F12, DMEM / F-12GlutaMAX TM , DMEM, AdvancedDMEM, DMEM / F-12GlutaMAX TM , William's E, L-15, M199, HMM, one or more; growth factors are dexamethasone (Dex), nicotinamide (NAM), epidermal growth factor (EGF), hepatocyte growth factor (HGF), insulin-transferrin-selenoethanolamine (ITS-X), β-mercaptoethanol, interleukin-6 (IL6), vitamin C, N-acetyl-cysteine ​​(NAC), basic fibroblast growth factor (bFGF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), insulin-like growth factor-I (IGF-1), transforming growth factor-β (TGF-β), RSPO1, Y27632, CHIR99021, A83-01, N2, B27, one or more.

5. The method for preparing a microcarrier-based cell culture liver according to claim 1, characterized in that: The edible material in step (2) is 1-100 mg / mL of sodium alginate, chitosan, carboxymethyl chitosan, pectin, carrageenan, gellan gum, xanthan gum, guar gum, agar, agarose, locust bean gum, konjac gum, gum arabic, tamarind gum, cellulose, carboxymethyl cellulose, nanocellulose, collagen, recombinant collagen, gelatin, hyaluronic acid, fibroin, elastin, spider silk protein, fibrin, fibrinogen, lecithin, soy protein, pea protein, gluten protein, rice protein, peanut protein, yeast protein, fungal protein, barley protein, wheat protein, rye protein, buckwheat protein, potato protein, corn protein, chickpea protein, broad bean protein, black bean protein, kidney bean protein, mung bean protein, seaweed protein, almond protein, quinoa protein, sorghum protein, and insect protein. One or more.

6. The method for preparing a microcarrier-based cell culture liver according to claim 1, characterized in that: The processing method of the microcarrier in step (2) is one or more of the following: template method, stirring method, membrane emulsification method, emulsion shearing method, high-voltage electrospraying method, droplet atomization method, and spray drying method.

7. The method for preparing a microcarrier-based cell culture liver according to claim 1, characterized in that: The microcarrier in step (2) is in the form of one or more of a solid type, a porous type, a "shell-core" type, a hollow type, and a multi-component type microcarrier with a size of 50-3000 μm.

8. The method for preparing a microcarrier-based cell culture liver according to claim 1, characterized in that: The cross-linking molding method of the microcarrier in step (2) is temperature cross-linking; chemical cross-linking using calcium chloride, calcium sulfate, calcium lactate, genipin or glutaraldehyde; or one or more of enzyme cross-linking using transglutaminase, alkaline protease, neutral protease, flavor protease, tyrosinase, laccase, lysyl oxidase, polyphenol oxidase, catalase or thrombin.

9. The method for preparing a microcarrier-based cell culture liver according to claim 1, characterized in that: In step (3), the liver seed cells are inoculated onto the microcarriers by cells adhering to the surface of the microcarriers for growth or cells embedded in the microcarriers for growth, and the inoculation density is 1×10 4 -1×10 8 cells / mL microcarriers.

10. The method for preparing a microcarrier-based cell culture liver according to claim 1, characterized in that: The culture medium used in the proliferation and differentiation culture in step (3) includes 77-99% basal culture medium, 0-20% fetal bovine serum, 1-3% penicillin-streptomycin, and 1-600 ng / mL growth factor; wherein the basal culture medium is DMEM, DMEM / F-12, AdvancedDMEM, AdvancedDMEM / F12, DMEM / F-12GlutaMAX TM , William's E, Ham's F-12K, RPMI1640, L-15, M199, HMM or one or more; growth factors are dexamethasone (Dex), hepatocyte growth factor (HGF), epidermal growth factor (EGF), insulin-transferrin-seleno-ethanolamine (ITS-X), N-acetyl-cysteine ​​(NAC), nicotinamide (NAM), gastrin (Leu15), forskolin (FSK), fibroblast growth factor 10 (FGF10), insulin (INS), β-mercaptoethanol, basic fibroblast growth factor (bFGF), neurotrophic factor (NGF), One or more of growth factor (NGF), vascular endothelial growth factor (VEGF), insulin-like growth factor-I (IGF-1), transforming growth factor-β (TGF-β), interleukin-6 (IL6), dimethyl sulfoxide (DMSO), oncostatin M (OSM), γ-secretase inhibitor (DAPT), fibroblast growth factor 19 (FGF19), bone morphogenetic protein 7 (BMP7), sodium pyruvate, L-glutamine, RSPO1, CHIR99021, A83-01, Y27632, Noggin, Wnt CM, HEPES, N2, and B27; the proliferation and differentiation medium differ only in the type and amount of growth factor used.

11. The method for preparing a microcarrier-based cell culture liver according to claim 1, characterized in that: In step (4), the edible material is 1-100 mg / mL of sodium alginate, chitosan, carboxymethyl chitosan, pectin, carrageenan, gellan gum, xanthan gum, guar gum, agar, agarose, locust bean gum, konjac gum, gum arabic, tamarind gum, cellulose, carboxymethyl cellulose, nanocellulose, collagen, recombinant collagen, gelatin, hyaluronic acid, fibroin, elastin, spider silk protein, fibrin, fibrinogen, lecithin, soy protein, pea protein, gluten protein, rice protein, peanut protein, yeast protein, fungal protein, barley protein, wheat protein, rye protein, buckwheat protein, potato protein, corn protein, chickpea protein, broad bean protein, black bean protein, kidney bean protein, mung bean protein, seaweed protein, almond protein, quinoa protein, sorghum protein, and insect protein. One or more.

12. The method for preparing a microcarrier-based cell culture liver according to claim 1, characterized in that: The assembly method described in step (4) is a mold molding method or a 3D printing method; the mold molding method is to mix the cell-microcarrier complex with the edible material and then transfer the whole to a three-dimensional mold for molding; the 3D printing method is to mix the cell-microcarrier complex with the edible material and then pump it into the extrusion nozzle of a 3D printer so that it is extruded and stacked under the drive of the printer.

13. The method for preparing a microcarrier-based cell culture liver according to claim 11, characterized in that: In the 3D mold forming method, the volume ratio of the cell-microcarrier complex to the edible material solution is 5-15:1, and the relevant parameters used in the 3D printing method are: the volume ratio of the cell-microcarrier complex to the edible material solution is 1-3:1; the printing nozzle diameter is 0.1-5mm; the extrusion speed is 0.5-30mL / h; the printing speed is 0.5-50mm / s.

14. The method for preparing a microcarrier-based cell culture liver according to claim 1, characterized in that: The cell culture liver in step (4) is cross-linked by temperature cross-linking; chemical cross-linking using calcium chloride, calcium sulfate, calcium lactate, or genipin; or enzymatic cross-linking using transglutaminase, alkaline protease, neutral protease, flavor protease, tyrosinase, laccase, lysyl oxidase, polyphenol oxidase, catalase, or thrombin.

15. The method for preparing a microcarrier-based cell culture liver according to claim 1, characterized in that: The coating curing in step (4) is to use an edible molding material to coat the surface of the cross-linked cell culture liver with a "connective tissue" membrane.

16. A cell culture liver prepared by the preparation method according to claim 1.

17. Use of the cell cultured liver according to claim 16 in developing cell cultured liver products.

18. The use according to claim 17, characterized in that The microcarrier-based cell cultured liver is processed by one or more of frying, braising, boiling, stir-frying, and cooking to obtain a cell cultured liver product.

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