Preparation method and application of cell culture liver based on three-dimensional mold

Abstract

The invention discloses a preparation method and application of a cell culture liver based on a three-dimensional mold, and the method comprises the following steps: obtaining liver seed cells, culturing the liver seed cells in vitro, preparing raw materials of the cell culture liver, and forming the cell culture liver based on the three-dimensional mold. The cell culture liver is produced based on the three-dimensional mold, the preparation condition is mild, the operation method is simple, and the forming process is rapid. The shape and the size of the developed three-dimensional mold can be flexibly customized, the three-dimensional mold is provided with a convex point microstructure, and the color and the flavor of a forming material are further adjusted, so that the produced cell culture liver achieves simulation of real animal liver tissues in various aspects such as the shape, the size, the structure, the texture, the color and the flavor.

Description

Technical Field

[0001] The present invention belongs to the field of cultured meat, and particularly relates to a preparation method and application of a cultured liver based on a three-dimensional mold. Background Art

[0002] With the increasing demand for meat consumption, the contradiction between the resource-intensive traditional meat production method relying on livestock farming and the environmental resource endowment has suddenly escalated, and it will also bring huge challenges to the meat supply and industrial development worldwide. Cultured meat is a new type of meat obtained by in vitro culture of relevant cells and tissues based on the self-healing and regeneration ability of animal tissues. Its production cycle is short, and it can greatly reduce the occupation of water and soil resources in meat production and the emission of greenhouse gases during the process, which has received extensive attention from all sectors of society.

[0003] Skeletal muscle and fat are two of the most common animal tissues used as raw materials for meat product production, and the in vitro construction of these two tissues is also the focus of the current breakthrough in cultured meat technology and product development. At present, some cultured muscle and fat products developed based on three-dimensional hydrogels, animal and plant protein scaffolds and other means have been successively disclosed. However, generally speaking, any animal tissue that can be used as human food can be called "meat". In addition to skeletal muscle and fat, animal internal organs, especially liver tissue, have a unique flavor, and the protein is a complete high-quality protein, and it is rich in nutrients such as B vitamins, vitamin A, vitamin D, and iron elements. It has long been active on the domestic and foreign diet menus, and French foie gras is even known as "edible soft gold" and "one of the world's three major delicacies". However, there is no report on the technology or application of cultured liver in the field of cultured meat.

[0004] Object of the Invention

[0005] Summary of the Invention: Aiming at the technical blank in the construction of cultured liver in the current field of cultured meat, the present invention provides a preparation method of a cultured liver based on a three-dimensional mold. The cultured liver prepared by the present invention simulates the real animal liver tissue in terms of shape, size, structure, texture, color and flavor, making the cultured liver close to the real animal liver, effectively filling the blank of cultured liver.

[0006] The present invention also provides the prepared cultured liver based on a three-dimensional mold and its application.

[0007] Technical Solution: To achieve the above object, a preparation method of a cultured liver based on a three-dimensional mold according to the present invention includes the following steps:

[0008] (1) Obtaining liver seed cells: Isolate liver seed cells from animal liver tissue, and purify and identify the seed cells;

[0009] (2) In vitro culture of liver seed cells: The liver seed cells are cultured in vitro for proliferation to obtain a large number of cells, and then differentiated to become mature hepatocytes for standby.

[0010] (3) Preparation of cell-cultured liver raw materials: The cultured mature hepatocytes are collected and mixed with an edible molding material to prepare cell-cultured liver raw materials, or mixed with an edible molding material and an edible coloring material to prepare cell-cultured liver raw materials; or mixed with an edible molding material, an edible coloring material and an edible flavor material to prepare cell-cultured liver raw materials.

[0011] (4) Molding of cell-cultured liver based on a three-dimensional mold: The cell-cultured liver raw materials are poured into a three-dimensional mold, demolded after preliminary cross-linking and molding, and then subjected to secondary cross-linking and molding. Finally, a "connective tissue" membrane is coated to obtain the cell-cultured liver, or the cross-linked and molded cell-cultured liver is directly cooked without coating.

[0012] 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.

[0013] Among them, the purification method of the liver seed cells in step (1) is one or more of chemical reprogramming, flow cytometry sorting, magnetic bead sorting, and Percoll gradient centrifugation. The liver seed cells are obtained from hepatocytes by chemical reprogramming, or from non-parenchymal liver cells by one or more of flow cytometry sorting, magnetic bead separation, and Percoll gradient centrifugation.

[0014] Preferably, the method for obtaining the liver seed cells is one or more of chemical reprogramming and flow cytometry sorting.

[0015] 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+, and 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, and B27.

[0016] Preferably, the specific antibody markers used in the flow cytometry sorting method are one or more of CD31, CD45, CD133, and EPCAM.

[0017] Among them, the components of the medium used for proliferation and differentiation culture in step (2) 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 DMEM, DMEM / F-12, Advanced DMEM, Advanced DMEM / F12, DMEM / F-12 GlutaMAX TM, one or more of William's E, Ham's F-12K, RPMI 1640, L-15, M199, HMM; the growth factors are 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 media only differ in the types and amounts of growth factors used.

[0018] Among them, the edible molding material described in step (3) 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 molding material is one or more of sodium alginate, agar, agarose, gelatin, soy protein, pea protein, insect protein at a concentration of 10-50 mg / mL.

[0020] Among them, the edible colorant material described in step (3) is one or more of monascus red, sunset yellow, carmine, allura red, wine red, caramel color, amaranth, lemon yellow, egg yolk, grape purple, brilliant blue at a concentration of 1-100 mg / mL.

[0021] Among them, the edible flavor material described in step (3) is one or more of ferrous lactate, hemoglobin, and succinic acid at a concentration of 1-100 mg / mL.

[0022] Preferably, the edible colorant is one or more of monascus red, carmine, caramel color, grape purple, and lemon yellow at a concentration of 1-30 mg / mL.

[0023] Preferably, the edible flavor material is one or more of ferrous lactate and hemoglobin at a concentration of 1-30 mg / mL.

[0024] Among them, the proportion of liver seed cells in the cell-cultured liver raw material described in step (3) is 30%-70% by volume fraction.

[0025] Among them, the three-dimensional mold described in step (4) has a bump structure, and the bumps are evenly distributed on the surface of the three-dimensional mold; the overall dimensions of the three-dimensional mold are 50 mm-200 mm in length, 30 mm-150 mm in width, and 10 mm-30 mm in height, and the bump dimensions of the three-dimensional mold are 1 mm-5 mm in length, 1 mm-3.5 mm in width, and 0.5 mm-2 mm in height; the size ratio between the mold size and the bump size can be adjusted accordingly according to the animal species and age of the animal liver simulated by the cell-cultured liver.

[0026] Among them, the production material of the three-dimensional mold described in step (4) is one or more of resin, soft rubber, PLA (polylactic acid), PET (polyethylene terephthalate), HDPE (high-density polyethylene), LDPE (low-density polyethylene), PP (polypropylene), PS (polystyrene), PC (polycarbonate), PVC (polyvinyl chloride), stainless steel, and nylon.

[0027] Among them, the forming method of the three-dimensional mold described in step (4) is one or more of injection molding, blow molding, casting molding, 3D printing molding, compression molding, extrusion molding, rotational molding, thermoforming, foaming molding, and slush molding.

[0028] Preferably, the forming method of the three-dimensional mold is one or more of injection molding and 3D printing molding.

[0029] Among them, the crosslinking forming method in step (4) is temperature crosslinking, chemical crosslinking using calcium chloride, calcium sulfate, calcium lactate, and genipin, or enzyme crosslinking using transglutaminase, alkaline protease, neutral protease, flavor protease, tyrosinase, laccase, lysyl oxidase, polyphenol oxidase, catalase, and thrombin.

[0030] Preferably, the cross-linking and forming method is temperature cross-linking, chemical cross-linking using calcium chloride or calcium lactate, or one or more of enzymatic cross-linking using transglutaminase, alkaline protease, neutral protease, or flavor protease.

[0031] Among them, in step (4), the coating of the "connective tissue" membrane means coating the surface of the cross-linked cell-cultured liver with an edible forming material.

[0032] Preferably, the edible forming material used for coating the "connective tissue" membrane is prepared into a milky white color.

[0033] The cell-cultured liver based on a three-dimensional mold prepared by the preparation method of the present invention.

[0034] The application of the cell-cultured liver based on a three-dimensional mold of the present invention in the development of cell-cultured liver products.

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

[0036] The present invention starts from obtaining animal liver seed cells, performs in vitro proliferation and differentiation culture on the seed cells, and then mixes the obtained animal liver cells with an edible forming material, an edible color-enhancing material, and an edible flavor material to prepare a raw material for cell-cultured liver. After preliminary molding with a mold, post-cross-linking and forming, and coating the "connective tissue" membrane, a cell-cultured liver is obtained. The present invention first proposes a preparation method for a cell-cultured liver based on a three-dimensional mold, which to a certain extent makes up for the problems of single type of cell-cultured meat and cumbersome molding steps at the present stage; on the other hand, the specific preparation method of the present invention includes key designs for forming the raw material of the cell-cultured liver based on a mold with bumps and coating the "connective tissue" membrane. Among them, the mold forming method is convenient and fast. By specially designing the bumps on the mold, the surface convex texture caused by liver lobules in the animal liver can be restored; in addition, when the milky white "connective tissue" membrane material is coated on the surface of the cell-cultured liver, the flowing membrane material fills the grooves between adjacent bumps, fully restoring the connective tissue network structure of the animal liver tissue, making the appearance closer to that of a real liver.

[0037] The present invention uses mature animal liver seed cells and edible materials as raw materials. After mixing the above two, a cell-cultured liver tissue is prepared by the simplest method of casting. The three-dimensional mold is designed in a bumpy pattern. The surface of the cell-cultured liver prepared accordingly has a bumpy morphology, which to a certain extent simulates the hepatic lobule structure. After the cell-cultured liver is initially formed and post-crosslinked in the mold, a "connective tissue" membrane material is coated, so that the final cell-cultured liver product has a smooth film structure similar to that of a real animal liver and a similar connective tissue network morphology. The preparation of the cell-cultured liver based on the three-dimensional mold is simple, convenient to operate, does not rely on high-precision equipment, and has a large throughput. Its texture, color, and flavor are similar to those of animal liver, and it fully restores the bumpy microstructure and connective tissue network morphology of animal liver.

[0038] The present invention is the first to propose the preparation of cell-cultured liver based on a three-dimensional mold, or in other words, the first to propose the preparation of cell-cultured animal viscera in the field of cell-cultured meat. The three-dimensional mold for preparing the cell-cultured liver of the present invention has a bumpy structure, which can better simulate the bumpy tissue structure of animal liver caused by hepatic lobules. By coating the "connective tissue" membrane material, the surface of the cell-cultured liver has a smooth film structure similar to that of a real animal liver. Since the membrane material is opaque, when it fills the grooves between adjacent bumps and forms, it fully restores the connective tissue network structure of animal liver tissue.

[0039] The present invention conducts overall modeling through three-dimensional modeling software with reference to the three-dimensional shape and size of a real animal liver. Then, irregular polygon textures are applied to the model surface and raised, forming a model with a bumpy structure on the surface. Based on this model, mold forming operations such as 3D printing and injection molding are carried out to obtain a three-dimensional mold that can be used for the preparation of cell-cultured liver. Based on this three-dimensional mold, the simulation of the shape, size, and structure of animal liver can be realized. By adjusting the types and concentrations of edible molding materials and combining crosslinking methods, the simulation of the texture of real animal liver can be achieved. By adjusting the types and concentrations of edible flavor materials, the whole has the smell of real animal liver such as fishy smell and metallic taste. On the other hand, the amount of liver seed cells used can be increased to make the whole have the flavor of real animal liver.

[0040] The preparation method of cell-cultured liver based on a three-dimensional mold proposed by the present invention can achieve the efficient separation, purification and enrichment of animal liver seed cells, as well as long-term efficient proliferation and differentiation in vitro. At present, there is no report on the application of cell-cultured liver tissue in the field of cell-cultured meat. Moreover, liver seed cells isolated in fields such as cell engineering and tissue engineering cannot maintain the proliferation and differentiation phenotypes in vitro for a long time, and their culturing ability is low. On the one hand, the present invention can reverse and restore differentiated hepatocytes to a stem cell state through chemical reprogramming means combined with a variety of chemical small molecule combinations, endowing them with 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 from a variety of isolated non-parenchymal liver cells by flow cytometry sorting, which have stable in vitro proliferation and differentiation and maturation abilities, and the acquisition is rapid, efficient and low-cost.

[0041] The non-parenchymal liver cells in the present invention without flow cytometry sorting contain a miscellaneous variety of cell types and the target liver seed cells cannot be distinguished. After flow cytometry sorting, 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.

[0042] Advantageous effects: Compared with the prior art, the present invention has the following advantages:

[0043] (1) The present invention discloses a preparation method and application of cell-cultured liver based on a three-dimensional mold, filling the blank of research and application of cell-cultured visceral products in the field of cell-cultured meat, and broadening the research scope of the field of cell-cultured meat;

[0044] (2) The present invention uses a three-dimensional mold to produce cell-cultured liver, with mild preparation conditions, simple operation methods, rapid forming process, and does not rely on high-cost and high-precision equipment, having good industrial application prospects;

[0045] (3) The cell-cultured liver produced by the present invention using a three-dimensional mold simulates real animal liver tissue in terms of shape, size, texture, color and flavor;

[0046] (4) The inner surface of the three-dimensional mold used in the present invention for producing cell-cultured liver has a bump structure, so that the surface of the formed cell-cultured liver has bumps, better simulating the lobular protrusion morphology of real animal liver;

[0047] (5) The cell-cultured liver prepared by the present invention is coated with a "connective tissue" membrane on the surface, which can not only reinforce the overall structure of the cell-cultured liver, but also interact with the divided areas between the bumps to restore the connective tissue network of the liver tissue, simulate the connective tissue morphology of the animal liver, and endow the cell-cultured liver with a smooth surface touch.

[0048] (6) When preparing the cell-cultured liver of the present invention, only the liver seed cells obtained by culture need to be mixed with edible materials, poured into a three-dimensional mold for preliminary shaping, demolded and then secondarily shaped, and coated to obtain the cell-cultured liver. During the whole technological process, the materials used are all common animal and plant proteins used as food, which are safe, edible and low in cost; the mixing and casting process is simple in operation, rapid in shaping, and does not rely on redundant high-cost and high-precision equipment; the crosslinking and shaping conditions after casting are mild and do not involve toxic and harmful reagents.

[0049] (7) The shape of the cell-cultured liver prepared by the present invention is close to that of real animal liver tissue. Microscopically, the cell-cultured liver has bumpy structures, simulating the basic structural unit of the hepatic lobule of animal liver tissue. Whether it is the overall size or the bump size, it can be flexibly adjusted by three-dimensional modeling according to production needs. Thanks to the presence of edible color-enhancing materials, after preparation, the color of the cell-cultured liver is blood-red and purplish, close to the actual color of the liver after animal slaughter and bloodletting. Based on the shape, size, and color characteristics, the cell-cultured liver prepared by the present invention is close to real animal liver tissue in appearance. In addition, by analyzing the flavor source of animal liver and selecting corresponding edible flavor-enhancing materials for addition, the final product has the special flavor of animal liver.

[0050] In summary, the cell-cultured liver product prepared by the present invention is similar to real animal liver tissue in terms of shape, size, structure, texture, color, and flavor, effectively filling the gap in cell-cultured liver. Description of the Drawings

[0051] Figure 1 is the microscopic image of non-parenchymal liver 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;

[0052] Figure 2 is the flow cytometry analysis picture of non-parenchymal liver 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 cells and adherent cells; (c) Picture of cell population separation based on hepatocyte markers; (d) Separation of target cells and miscellaneous cells, and picture of obtaining target cells;

[0053] Figure 3 is the immunofluorescence staining identification image of the liver seed cells purified by the flow cytometry sorting method of the present invention, and the scale bar is 25μm;

[0054] Figure 4These are the 3D mold model and physical pictures in the present invention. (a) 3D modeling drawing of the male mold of the 3D mold; (b) Physical pictures of the male mold (i) and female mold (ii) of the 3D mold, with a scale bar of 2 cm.

[0055] Figure 5 These are the physical pictures of the cell-cultured porcine liver with bumps (a), the cell-cultured porcine liver without bumps (b), and the cell-cultured porcine liver with bumps coated with "connective tissue" membrane (c) prepared based on the 3D mold of the present invention, with a scale bar of 2 cm.

[0056] Figure 6 These are the pictures of the developed braised cell-cultured liver products. (a) Image of the braised cell-cultured liver before slicing; (b) Image of the braised cell-cultured liver after slicing. Detailed implementation manners

[0057] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0058] The raw materials, reagents, etc. used in the embodiments are all commercially available.

[0059] Example 1

[0060] Isolation of non-parenchymal liver cells

[0061] Newborn Duroc 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 solution and the second perfusion solution were heated to 39°C and then perfused successively at a rate of 30 mL / min to wash the liver tissue. The specific formula of the first perfusion solution 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 solution 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 solution containing collagenase IV was used for the second-stage perfusion. The specific formula of the third perfusion solution 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.

[0062] 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 incised, 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 solution (37°C, continuous agitation digestion for 30 minutes), and then filtered through a 70 μm cell strainer. 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. After lysing on ice with red blood cell lysate 1 - 2 times, liver non-parenchymal cells were obtained. After centrifuging at 300×g for 5 minutes, the precipitate of the obtained liver non-parenchymal cells was resuspended in DMEM / F12. By cell counting, 1×10 6Cells 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 medium components 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, and the cell pellet was resuspended in a cryopreservation solution containing 90% fetal bovine serum and 10% dimethyl sulfoxide DMSO (Sigma, D2650). After aliquoting 500 μL per tube into cryotubes, they were 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.

[0063] Example 2

[0064] Purification of liver seed cells from non-parenchymal liver cells

[0065] Flow cytometry was used to sort and purify non-parenchymal liver cells to obtain liver seed cells. The specific operation steps were as follows:

[0066] (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 for culture. When the cells proliferated to 80 - 90% confluence, the cells were digested and harvested.

[0067] (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. A 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 1 mL of PBS suspension containing 2×10 5 cells, 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, it was 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 components were 98% DMEM / F12 by volume fraction and 2% penicillin-streptomycin, and stored under ice bath conditions for sorting.

[0068] (3) Cell sorting: Prepare 40 mL of sorting receiving solution in advance (the components are 97% DMEM / F12 by volume fraction and 3% penicillin-streptomycin), and 4 mL of the receiving solution was aliquoted into each 15 mL centrifuge tube for receiving cells; the stained cells of each group were sorted using 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 antibody 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..

[0069] Example 3

[0070] Identification of liver seed cells

[0071] 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).

[0072] 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 components 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. Incubate 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 onto the samples for mounting. Observation and photography were performed 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 almost all the cells were hepatic progenitor cells.

[0073] Example 4

[0074] Proliferation and differentiation culture of hepatic progenitor cells

[0075] (1) Proliferation of hepatic progenitor cells

[0076] The hepatic progenitor cells purified in Example 2 were seeded at a density of 6000 cells / cm 2 on 10-cm cell culture dishes 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). Cultured at 37°C and 5% CO2, and the cell proliferation medium was changed every 2 days. When the cells reached 80-90% confluence, they were digested and passaged.

[0077] (2) Differentiation of hepatic progenitor cells

[0078] 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 and culture at 37 °C and 5% CO2. 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%) and then change to a differentiation medium. The differentiation step is carried out in two stages. In the first stage, culture for 3 days. 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. 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 matrix gel (Corning, 356231). After the differentiation is completed, harvest the mature hepatocytes.

[0079] Example 5

[0080] Fabrication of a three-dimensional mold

[0081] (1) Fabrication of the male mold

[0082] Use Blender software to model the three-dimensional mold - male mold, and convert the model file into the STL 3D printing file format. The designed three-dimensional mold - male mold has a groove-like appearance, with an overall size of 80 mm in length, 60 mm in width, and 18 mm in height; there are some protrusions inside the groove, and the protrusion shape is in the form of a liver, with convex dot structures ([ Figure 4 a) evenly distributed on the surface. Each convex dot is 1.5 mm in length, 1 mm in width, and 0.5 mm in height. Use slicing software to perform layer slicing on the obtained STL model file to obtain a G-code motion control instruction file that can be recognized by a 3D printer, and then import the G-code printing instruction into a 3D printer (UnionTech, Lite800). Using resin (Future 8200Pro resin) as the material, start the printer to print to obtain the three-dimensional mold - male mold ([ Figure 4 i) in b).

[0083] (2) Fabrication of the female mold

[0084] Dow Corning SYLGARD TM 184 Silicone Elastomer Kit's SYLGARD TM 184 Silicone Elastomer Base and SYLGARD TM184Silicone Elastomer Curing Agent is mixed at a mass ratio of 10:1. After stirring evenly, it is placed in a vacuum drying kettle for vacuum treatment to remove most of the bubbles generated by stirring. After preliminary degassing, the mixture is poured into a three-dimensional mold - male mold, making the material liquid level flush with the top edge of the three-dimensional mold - male mold. Then it is placed 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 minutes - release air to burst the bubbles - maintain a vacuum degree of 0.08 MPa for 2 minutes", and the above procedures are repeated 8 - 12 times. The degassed mold - material as a whole is placed in an oven at 60 °C for incubation for 6 hours. After molding, the material part is taken out to obtain a three-dimensional mold - female mold ( Figure 4 ii) in b.

[0085] Example 6

[0086] Preparation of cell-cultured pig liver

[0087] Weigh 2.5 g of agarose and place it in a 500 mL beaker. Measure 250 mL of ultrapure water with a measuring cylinder and pour it into the beaker. After mixing evenly with a magnetic stirrer, place it in a water bath at 95 °C for heating. During this period, take it out and mix evenly with a magnetic stirrer every 10 minutes. Wait until the solution is clear and transparent to obtain a 10 mg / mL agarose solution. Then, based on the above agarose solution, weigh 12.5 g of soy protein powder (Shansong Biology, SD-100), add it to the 10 mg / mL agarose solution, mix evenly with a magnetic stirrer, and place it in a water bath at 85 °C for heating. During this period, take it out and mix evenly with a magnetic stirrer every 10 minutes. Wait until no undissolved soy protein powder can be observed in the solution system to obtain a double composite solution system (10 mg / mL agarose + 50 mg / mL soy protein).

[0088] Adjust the color of the composite solution system. During magnetic stirring, add 5 mL of 100 mg / mL monascus red solution, 2 mL of 10 mg / mL grape purple solution, and 0.3 g of caramel color to the 250 mL double composite solution system. Then weigh sodium alginate powder and add it to the composite solution system after color adjustment. Stir magnetically until the sodium alginate is completely dissolved to obtain a colored triple composite solution system (10 mg / mL agarose + 50 mg / mL soy protein + 8 mg / mL sodium alginate). Finally, pipette 15 mL of the triple composite solution system into a 50 mL beaker, and add 10 mL of porcine hepatocytes that have been differentiated and cultured for 7 days (4 days of differentiation in the second stage) and matured by the method in Example 4 to the system (ensuring that the cells account for 40% of the total volume, calculated based on the volume of the cell pellet after centrifugation). After stirring and mixing evenly, the cell culture liver raw material for casting is obtained and set aside. In addition, edible flavoring materials such as 10 mg / mL ferrous lactate, hemoglobin, or succinic acid can be added to the cell culture liver raw material prepared in this example to enhance the flavor.

[0089] Prepare a three-dimensional mold - female mold with overall length, width, and height dimensions of 80 mm × 60 mm × 18 mm and bump dimensions of 1.5 mm × 1 mm × 0.5 mm according to the method of Example 5. Slowly pour the prepared cell culture liver raw material into the female mold and let it stand at room temperature for 2 h. After the whole solidifies, demold after preliminary cross-linking and shaping, and soak it in a 10 mg / mL calcium chloride solution to cross-link overnight at 4°C to further cross-link and shape the whole. Then, soak the shaped cell culture liver in ultrapure water to remove the excess calcium chloride cross-linking solution to obtain the cell culture liver ( Figure 5 a), which is the cell culture liver without coating the "connective tissue" membrane at this time. It can be seen that it is similar to a real liver in shape, wedge-shaped, dark purple-red in color, and has bumpy textures on the surface. While the cell culture liver without the dot structure has a smooth surface and no "connective tissue" network morphology, which is very different from a real animal liver. Figure 5 b).

[0090] Example 7

[0091] Coat the "connective tissue" membrane on the cell culture liver

[0092] Weigh 0.4 g of peanut protein powder (Tianrui), dissolve it in 200 mL of ultrapure water to prepare a 2 mg / mL peanut protein solution. Centrifuge at 3000×g for 3 min to remove the supernatant and reserve it. Then, weigh 0.5 g of agarose, add it to the supernatant of the 2 mg / mL peanut protein solution, and heat it in a 95°C water bath. During this period, take it out every 10 min and mix it evenly with a magnetic stirrer until the solution becomes clear. Finally, weigh sodium alginate, add it to the above composite solution, and mix it evenly with a magnetic stirrer to obtain a coating solution (a 2 mg / mL peanut protein solution containing 5 mg / mL agarose and 30 mg / mL sodium alginate).

[0093] Take a 15-cm cell culture dish, add 10 mL of 10 mg / mL calcium chloride to it, evenly spread it on the bottom of the dish, and then suck it out with a suction device. Next, add 20 mL of the coating solution to the culture dish, evenly spread it on the bottom of the dish, and then place the cell-cultured pig liver prepared in Example 6 into the culture dish. Then, quickly pour the coating solution onto the cell-cultured pig liver to evenly coat its surface. Finally, quickly suck out the excess coating solution in the culture dish, put the whole thing into a 4°C refrigerator for preliminary shaping for 10 min, and then soak it in 10 mg / mL calcium chloride for crosslinking for 20 min to obtain a cell-cultured pig liver coated with a "connective tissue" membrane, as Figure 5 (c) shows. At this time, it is a cell-cultured liver coated with a "connective tissue" membrane, and the membrane material used is light milky white. After membrane coating, the surface of the cell-cultured liver is smooth and reflective, and the grooves between the convex points are filled and connected by the light milky white membrane material, forming a structural morphology similar to a connective tissue network.

[0094] Example 8

[0095] Preparation of cell-cultured pig liver for marinated pig liver products

[0096] Weigh 2.5 g of agarose and place it in a 500-mL beaker. Measure 250 mL of ultrapure water with a measuring cylinder and pour it into the beaker. After mixing it evenly with a magnetic stirrer, heat it in a 95°C water bath. During this period, take it out every 10 min and mix it evenly with a magnetic stirrer until the solution is clear and transparent to obtain a 10 mg / mL agarose solution. Then, based on the above agarose solution, weigh 12.5 g of pea protein powder (Yu Wang), add it to the 10 mg / mL agarose solution, mix it evenly with a magnetic stirrer, and heat it in an 85°C water bath. During this period, take it out every 10 min and mix it evenly with a magnetic stirrer until no undissolved pea protein powder can be observed in the solution system to obtain a double composite solution system (10 mg / mL agarose + 50 mg / mL pea protein).

[0097] Then, weigh the sodium alginate powder and add it to the compound solution system. Stir magnetically until the sodium alginate is completely dissolved to obtain a triple compound solution system (10 mg / mL agarose + 50 mg / mL pea protein + 8 mg / mL sodium alginate). In this example, no coloring material needs to be added, and the pea protein will make the solution system show the color of cooked liver. Finally, suck 15 mL of the triple compound solution system into a 50 mL beaker, and add 10 mL of the mature porcine hepatocytes differentiated and cultured for 7 days (4 days of differentiation in the second stage) by the method in Example 4 to the system (ensuring that the cells account for 40% of the total volume, calculated according to the volume of the cell precipitate after centrifugation) (the cells account for 40% of the total volume). After stirring and mixing evenly, the cell culture liver raw material for casting is obtained and reserved for use.

[0098] Prepare a three-dimensional mold - female mold with dimensions of 80 mm × 60 mm × 18 mm in length, width, and height and bump dimensions of 1.5 mm × 1 mm × 0.5 mm according to the method of Example 5. Slowly pour the prepared cell culture liver raw material into the mold and let it stand at room temperature for 2 h. After the whole solidifies, demold after preliminary cross-linking and shaping, and soak it in a 10 mg / mL calcium chloride solution to cross-link overnight at 4°C to further cross-link and shape the whole. Then, soak the shaped cell culture liver in ultrapure water to remove the excess calcium chloride cross-linking solution, and the cell culture liver for braised and marinated liver products is obtained. Without applying connective tissue coating, it is directly used for subsequent cooking.

[0099] Example 9

[0100] Production of braised and marinated cell culture liver

[0101] Harvest the shaped cell culture liver in Example 8, wash it repeatedly with PBS and clean water. Place the whole liver in a pot and blanch it for 2 min, then take it out, wash the surface with warm water and reserve it. Prepare the marinade in a disposable plastic plate, including 30 g of scallions, 10 g of ginger slices, 5 g of dried chili peppers, 3 g of star anises, 3 g of cinnamon, 2 g of cinnamon leaves, 2 g of Chinese prickly ash, and 10 g of rock sugar. Add clean water to the pot and bring it to a boil over high heat. After the water boils, add 3 g of monascus red to adjust the soup color, and pour in the prepared marinade and stir evenly. After boiling over high heat for 5 - 10 s, add 300 g of the processed cell culture liver, then add 15 g of light soy sauce, 10 g of cooking wine, and 10 g of salt to the pot. After stirring and mixing evenly, simmer over low heat for 10 - 15 min and then take it out of the pot. Cut the braised and marinated cell culture liver into slices of 1 - 2 mm and place them on a plate for standby ( Figure 6 b), Figure 6 Figure a shows a physical picture of the braised and marinated cell culture liver product. The whole is the color of the marinade. After cutting, it can be observed that the internal and external colors are heterogeneous. The inside of the braised and marinated cell culture liver is light pink and slightly yellow, which is very similar to the braised liver based on real animal liver in the market.

[0102] 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 taste are also almost the same as those of the real liver.

Claims

1. A method for preparing a cell culture liver based on a three-dimensional mold, 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) In vitro culture of liver seed cells: In vitro proliferation culture of liver seed cells is performed to obtain a large number of cells, and then differentiation culture is performed to make them mature hepatocytes for future use; (3) Preparation of cell culture liver raw materials: collecting the cultured mature liver cells, mixing them with edible molding materials to prepare cell culture liver raw materials, or mixing them with edible molding materials and edible color enhancement materials to prepare cell culture liver raw materials; or mixed with edible forming materials, edible coloring materials and edible flavoring materials to prepare raw materials for cell culture liver; (4) Cell culture liver molding based on three-dimensional molds: The raw materials of the cell culture liver are cast in a three-dimensional mold, demolded after initial cross-linking molding, and then subjected to secondary cross-linking molding. Finally, a "connective tissue" membrane is coated to obtain the cell culture liver, or the cross-linked cell culture liver is directly cooked without coating.

2. The method for preparing a cell cultured liver based on a three-dimensional mold 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 cell cultured liver based on a three-dimensional mold 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 cell cultured liver based on a three-dimensional mold according to claim 1, characterized in that: The culture medium used in the proliferation and differentiation culture in step (2) includes 77-99% basal culture medium, 0-20% fetal bovine serum, 1-3% penicillin-streptomycin, and 1-600ng / mL growth factor; wherein the basal culture medium is DMEM, DMEM / F-12, Advanced DMEM, Advanced DMEM / F12, DMEM / F-12GlutaMAX TM , William's E, Ham's F-12K, RPMI 1640, 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), 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, one or more of B27; the proliferation and differentiation medium differ only in the type and amount of growth factor used.

6. The method for preparing a cell cultured liver based on a three-dimensional mold according to claim 1, characterized in that: The edible molding material in step (3) is 1-100 mg / mL of one or more 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.

7. The method for preparing a cell cultured liver based on a three-dimensional mold according to claim 1, characterized in that: The edible color-enhancing material in step (3) is one or more of 1-100 mg / mL of red yeast rice, sunset red, carmine, allura red, wine red, caramel color, amaranth red, lemon yellow, egg yolk, grape purple, and brilliant blue.

8. The method for preparing a cell culture liver based on a three-dimensional mold according to claim 1, characterized in that: The edible flavor material in step (3) is one or more of ferrous lactate, hemoglobin, and succinic acid at a concentration of 1-100 mg / mL.

9. The method for preparing a cell cultured liver based on a three-dimensional mold according to claim 1, characterized in that: In step (3), the liver seed cells account for 30%-70% of the raw material for cell culture of liver.

10. The method for preparing a cell cultured liver based on a three-dimensional mold according to claim 1, characterized in that: The three-dimensional mold described in step (4) has a convex point structure, and the convex points are evenly distributed on the surface of the three-dimensional mold; the overall size of the three-dimensional mold is 50mm-200mm in length, 30mm-150mm in width, and 10mm-30mm in height, and the convex point size of the three-dimensional mold is 1mm-5mm in length, 1mm-3.5mm in width, and 0.5mm-2mm in height; the size ratio between the mold size and the convex point size can be adjusted accordingly according to the animal species and age of the animal liver simulated by the cell culture liver.

11. The method for preparing a cell cultured liver based on a three-dimensional mold according to claim 1, characterized in that: The three-dimensional mold in step (4) is made of one or more of resin, soft glue, PLA (polylactic acid), PET (polyethylene terephthalate), HDPE (high-density polyethylene), LDPE (low-density polyethylene), PP (polypropylene), PS (polystyrene), PC (polycarbonate), PVC (polyvinyl chloride), stainless steel, and nylon.

12. The method for preparing a cell cultured liver based on a three-dimensional mold according to claim 1, characterized in that: The molding method of the three-dimensional mold in step (4) is one or more of injection molding, blow molding, casting molding, 3D printing molding, compression molding, extrusion molding, rotational molding, vacuum molding, foaming molding, and slush molding.

13. The method for preparing a cell cultured liver based on a three-dimensional mold according to claim 1, characterized in that: The cross-linking molding method in step (4) is temperature cross-linking, chemical cross-linking using calcium chloride, calcium sulfate, calcium lactate, genipin, or enzyme cross-linking using one or more of transglutaminase, alkaline protease, neutral protease, flavor protease, tyrosinase, laccase, lysyl oxidase, polyphenol oxidase, catalase, and thrombin.

14. The method for preparing a cell cultured liver based on a three-dimensional mold according to claim 1, characterized in that: The "connective tissue" membrane coating in step (4) is to coat the cross-linked cell culture liver surface with an edible molding material.

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

16. Use of the cell cultured liver according to claim 15 in developing cell cultured liver products.

17. The use according to claim 16, characterized in that The 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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