Cattle liver organoid culture medium and cattle liver three-dimensional organoid culture method

Abstract

The invention discloses a bovine liver organoid culture medium and a bovine liver three-dimensional organoid culture method, and belongs to the technical field of cell culture. The bovine liver organoid culture medium comprises a bovine liver cell amplification culture medium and a bovine liver cell differentiation amplification culture medium. The three-dimensional organoid culture method comprises the following steps: dissociating bovine liver tissues by using a liver dissociation solution, and then incubating by using a red blood cell lysis buffer solution to obtain primary liver cells; preparing a bovine liver stem cell suspension from matrigel and the primary liver cells, planking, performing inverted culture to enable the matrigel to form a dome shape, performing upright culture, and sequentially performing multiplication culture through a bovine liver cell amplification culture medium and differentiation culture through a bovine liver cell differentiation amplification culture medium. According to the method disclosed by the invention, efficient acquisition, amplification and directional differentiation of the bovine liver primary cells are realized by customizing the culture medium, optimizing the cell separation process and adopting a 3D culture technology, and the blank of a large economic animal disease research model is filled.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of cell culture technology, and particularly relates to a bovine liver organoid culture medium and a bovine liver three-dimensional organoid culture method. BACKGROUND

[0002] Organoids are organ-like bodies extracted from tissues or stem cells and cultured in a special 3D structure. In recent years, organoids have played an important role in the establishment of animal disease models due to their high simulation of the physiological state of in vivo organs. Liver is the metabolic and immune regulation hub of animal body, and has important guiding significance in animal disease model research and drug screening. In 2013, Huch team first established a long-term culturable organoid model using adult rat liver cells, which promoted the rapid development of human and mouse liver organoid research. However, most of the current research on organoids is highly focused on human and mouse models, and the development of livestock organoid technology is obviously lagging behind. Livestock disease and drug research are also difficult to carry out due to limitations such as space, cost, and cycle.

[0003] Cattle, as large economic animals, have similar physiological structures and immune systems to humans, and bovine serum as an important biological product makes it an important animal resource with economic value, commercial value, and social value, playing an important role in livestock breeding. However, there is a lack of clinical research data on cattle compared to mouse models, and there is currently no systematic solution for bovine liver organoid culture. Culturing bovine liver organoids will also face the three major technical bottlenecks commonly encountered in organoid culture: low efficiency of primary liver cell isolation, lack of species-specific medium, and lack of mature culture system. These limitations have led most research in the livestock industry to rely on live animal experiments, which are not only limited by high costs and space requirements, but also raise ethical concerns, making it difficult to meet the urgent needs of veterinary drug development, feed safety evaluation, and disease prevention and control in intensive farming. The lack of species-specific medium is the first major problem faced by bovine liver organoid culture. Liver is a highly complex and species-specific organ. Liver cells of different species have formed unique metabolic networks, signal response systems, and microenvironment requirements during evolution. Culture medium is a key tool for simulating in vivo environment in vitro, and must meet the unique physiological, biochemical, and functional requirements of specific cell types and specific species as much as possible.

[0004] Therefore, developing a species-specific medium suitable for bovine liver stem cells not only provides a basis for three-dimensional organoid culture and fills the gap in large economic animal disease research models, but also provides key technical support for basic research and application transformation in the field of animal husbandry and veterinary medicine, which has important scientific value and practical significance. SUMMARY

[0005] In order to overcome the above technical deficiencies, the purpose of the present application is to provide a bovine liver organoid culture medium and a bovine liver three-dimensional organoid culture method, to solve the problem of the lack of species-specific culture medium in bovine three-dimensional organoid culture, and to provide key technical support for basic research and application transformation in the field of animal husbandry and veterinary medicine.

[0006] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: A bovine liver organoid culture medium, characterized in that: comprising a bovine liver cell expansion culture medium for obtaining bovine liver organoids by de-differentiation expansion culture of liver primary cells; the bovine liver cell expansion culture medium comprises DMEM / F12 culture medium, triple antibiotic, glutamine, HEPES buffer, B-27 serum substitute, N2 (serum substitute), nicotinamide, N-acetylcysteine, R-spondin-1 conditioned medium, ROCK inhibitor, forskolin, TGF-beta inhibitor, EGF, HGF, FGF10, gastrin, Noggin, Wnt3a, GSK-3 alpha / beta inhibitor; the triple antibiotic comprises penicillin, streptomycin and amphotericin B.

[0007] The key factors such as R-spondin-1, Wnt3a and Noggin are added in the bovine liver cell expansion culture medium to simulate the liver microenvironment and maintain the self-renewal ability of the liver organoid. Combined with Wnt pathway activator, GSK-3 alpha / beta inhibitor and ROCK inhibitor, the proliferation efficiency of the liver organoid is significantly improved and apoptosis is inhibited. Nicotinamide, N-acetylcysteine and other components resist oxidative stress and enhance cell survival rate.

[0008] As a preferred scheme, the concentration of each component in the bovine liver cell expansion culture medium in the DMEM / F12 culture medium is as follows: triple antibiotic 0.8-1.2% (v / v), glutamine 0.8-1.2% (v / v), HEPES buffer 8-12 mM, B-27 serum substitute 1.8-2.2% (v / v), N2 0.8-1.2% (v / v), nicotinamide 8-12 mM, N-acetylcysteine 1-1.5 mM, R-spondin-1 conditioned medium 9-11% (v / v), ROCK inhibitor 8-12 μM, forskolin 8-12 μM, TGF-beta inhibitor 0.8-1.2 μM, EGF 95-105 ng / mL, HGF 23-27 ng / mL, FGF10 48-52 ng / mL, gastrin 8-12 nM, Noggin 95-105 ng / mL, Wnt3a 48-52 ng / mL, GSK-3 alpha / beta inhibitor 1.3-1.7 μM.

[0009] Further, the concentrations of each component in the bovine hepatocyte expansion medium in the DMEM / F12 medium are as follows: triple antibiotic 1% (v / v), glutamine 1% (v / v), HEPES buffer 10 mM, B-27 serum replacement 2% (v / v), N2 1% (v / v), nicotinamide 10 mM, N-acetyl cysteine 1.25 mM, R-spondin-1 conditioned medium 10% (v / v), ROCK inhibitor 10 μM, forskolin 10 μM, TGF-β inhibitor 1 μM, EGF 100 ng / mL, HGF 25 ng / mL, FGF10 50 ng / mL, gastrin 10 nM, Noggin 100 ng / mL, Wnt3a 50 ng / mL, GSK-3α / β inhibitor 1.5 μM.

[0010] As a preferred solution, the bovine liver organoid culture medium further comprises a bovine hepatocyte differentiation expansion medium for promoting differentiation of the bovine liver organoid; the bovine hepatocyte differentiation expansion medium comprises DMEM / F12 medium, triple antibiotic, glutamine, HEPES buffer, B-27 serum replacement, N2, N-acetyl cysteine, TGF-β inhibitor, EGF, HGF, FGF10, gastrin, Noggin, dexamethasone, γ-secretase inhibitor, BMP7.

[0011] The synergistic effect of dexamethasone, BMP7, γ-secretase inhibitor, etc. in the bovine hepatocyte differentiation expansion medium directionally induces differentiation of the liver organoid into functional hepatocytes, forming an organoid with a polar structure. Gradient regulation of EGF, HGF, FGF10 promotes differentiation of the bile duct-like cell and hepatocyte lineage, simulating the liver development process.

[0012] As a preferred solution, the concentrations of each component in the bovine hepatocyte differentiation expansion medium in the DMEM / F12 medium are as follows: triple antibiotic 0.8~1.2% (v / v), glutamine 0.8~1.2% (v / v), HEPES buffer 8~12 mM, B-27 serum replacement 1.8~2.2% (v / v), N2 0.8~1.2% (v / v), N-acetyl cysteine 1~1.5 mM, TGF-β inhibitor 0.8~1.2 μM, EGF 95~105 ng / mL, HGF 23~27 ng / mL, FGF10 48~52 ng / mL, gastrin 8~12 μM, Noggin 95~105 ng / mL, dexamethasone 28~32 μM, γ-secretase inhibitor 8~12 μM, BMP7 23~27 ng / mL.

[0013] Further, the concentrations of each component in the bovine liver cell differentiation expansion medium in the DMEM / F12 medium are as follows: triple antibiotic 1% (v / v), glutamine 1% (v / v), HEPES buffer 10 mM, B-27 serum replacement 2% (v / v), N2 1% (v / v), N-acetyl cysteine 1.25 mM, TGF-β inhibitor 1 μM, EGF 100 ng / mL, HGF 25 ng / mL, FGF1 50 ng / mL, gastrin 10 μM, Noggin 100 ng / mL, dexamethasone 30 μM, γ-secretase inhibitor 10 μM, and BMP7 25 ng / mL.

[0014] Further, the triple antibiotic includes 10 kU / mL penicillin, 10 mg / mL streptomycin, and 25 μg / mL amphotericin B; the glutamine is L-alanyl-L-glutamine; the TGF-β inhibitor is A83-01; the ROCK inhibitor is Y-27632; the GSK-3α / β inhibitor is CHIR99021; the γ-secretase inhibitor is DAPT; and the BMP7 is a recombinant BMP7 protein.

[0015] The application further provides a bovine liver three-dimensional organoid culture method, which is characterized by comprising the following steps: The bovine liver tissue is obtained, and liver primary cells are obtained from the bovine liver tissue. The bovine liver organoid culture medium is prepared. The liver cell suspension is prepared from the matrix glue and the liver primary cells and plated, and after plating, the matrix glue and the liver primary cells are inverted and cultured for 3-7 min to form a dome shape, and then the bovine liver cell expansion medium is added for dedifferentiation expansion culture after being cultured in a normal position for 25-35 min, so that the bovine liver three-dimensional organoid is obtained. The bovine liver three-dimensional organoid after dedifferentiation expansion culture is differentiated and cultured by using the bovine liver cell differentiation expansion medium, so that the differentiated bovine liver three-dimensional organoid is obtained.

[0016] As a preferred solution, the method for obtaining the liver primary cells comprises two-step dissociation of the bovine liver tissue; the two-step dissociation comprises: first step of dissociation: taking the bovine liver tissue, washing, and then dissociating by using a liver dissociation solution to obtain a tissue suspension; and second step of dissociation: taking the tissue suspension, filtering after terminating digestion, and incubating the filtrate by using a red blood cell lysis buffer to obtain the liver primary cells.

[0017] As a preferred solution, the bovine liver tissue is washed by using a liver tissue washing solution; the liver tissue washing solution comprises DMEM / F12 medium and triple antibiotic, and the volume percentage of the triple antibiotic in the DMEM / F12 medium is 0.8-1.2%.

[0018] As a preferred solution, the liver dissociation solution comprises Hanks solution, collagenase type IV and DNAase; the concentrations of collagenase type IV and DNAase in the liver dissociation solution in the Hanks solution are 2.5-3.5 mg / mL and 55-65 U / mL, respectively. Among them, collagenase type IV is an enzyme that can specifically decompose collagen, and when the concentration is too high, it will excessively degrade the collagen fibers and other extracellular matrix components in the liver tissue, and when the concentration is too low, it cannot fully degrade the collagen fibers in the liver tissue, resulting in incomplete dissociation of the tissue; DNAase is an enzyme that can catalyze the hydrolysis of DNA, and can be appropriately increased according to the size of the tissue, and too high a concentration of DNAase may have a potential impact on the function of some cells, and too low a concentration cannot effectively degrade the released DNA, resulting in the phenomenon that the cells still adhere.

[0019] Further, the method for obtaining the liver primary cells specifically comprises the following steps: 1) first dissociation: 1.1) prepare the bovine liver tissue washing solution and the dissociation solution, the liver tissue washing solution is prepared by adding 1% (volume percentage) triple antibiotic to DMEM / F12 medium, the triple antibiotic is composed of 10 kU / mL penicillin, 10 mg / mL streptomycin and 25 μg / mL amphotericin B; the liver dissociation solution is prepared by adding collagenase type IV and DNAase to Hanks solution, the concentrations of collagenase type IV and DNAase in the Hanks solution are 3 mg / mL and 60 U / mL, respectively; the bovine liver tissue washing solution is used to wash the bovine liver tissue twice to remove blood and other structures outside the liver to ensure the purity of the cultured organoids; 1.2) cut the washed liver tissue into a paste in a centrifuge tube containing the liver dissociation solution, then place it in a 37°C incubator for 15 min to obtain a tissue suspension; 2) second dissociation: 2.1) add the tissue suspension in step 1.2) to preheated fetal bovine serum at 37°C to terminate the digestion, then filter the tissue suspension through a 100 μm cell filter to obtain a first filtered filtrate; 2.2) wash the cell filter screen obtained in step 2.1) with Hanks solution and filter to reduce cell loss, complete the second filtration, and mix the first filtered filtrate and the second filtered filtrate to obtain a cell suspension; 2.3) to obtain pure liver primary cells, add red blood cell lysis buffer to the cell suspension obtained in step 2.2), incubate at 4°C for 15 min to obtain a mixture; 2.4) centrifuge the mixture obtained in step 2.3) at 80 g at 4°C for 5 min, discard the supernatant and collect the liver primary cells.

[0020] As a preferred solution, the above-mentioned bovine liver three-dimensional organoid culture method specifically comprises the following steps: (1) A bovine liver cell expansion medium (BEM) is configured, wherein the BEM is prepared by adding triple antibiotics, glutamine, HEPES buffer, B-27 serum substitute, N2, nicotinamide, N-acetylcysteine, R-spondin-1 conditioned medium, ROCK inhibitor, forskolin, TGF-β inhibitor, EGF, HGF, FGF10, gastrin, Noggin, Wnt3a, and GSK-3α / β inhibitor into DMEM / F12 medium; the concentrations of the triple antibiotics, glutamine, HEPES buffer, B-27 serum substitute, N2, nicotinamide, N-acetylcysteine, R-spondin-1 conditioned medium, ROCK inhibitor, forskolin, TGF-β inhibitor, EGF, HGF, FGF10, gastrin, Noggin, Wnt3a, and GSK-3α / β inhibitor in the DMEM / F12 medium are 1% (v / v), 1% (v / v), 10 mM, 2% (v / v), 1% (v / v), 10 mM, 1.25 mM, 10% (v / v), 10 μM, 10 μM, 1 μM, 100 ng / mL, 25 ng / mL, 50 ng / mL, 10 nM, 100 ng / mL, 50 ng / mL, and 1.5 μM, respectively; (2) The liver primary cells obtained by the above-mentioned method are taken out by using a pre-cooled gun head, and are mixed in the pre-thawed and pre-dispensed Matrigel to obtain a liver cell suspension; (3) The liver cell suspension in step (2) is uniformly spread in a 48-well cell culture plate at a volume of 25 μL per well, and is immediately inverted and placed in a cell culture incubator at 37°C and 5% CO2 for 5 min; after the Matrigel forms a dome shape, the plate is placed vertically and cultured for 30 min to promote Matrigel gelation and stabilize the structure; (4) The cell culture plate is taken out, the BEM pre-heated at 37°C is added, and the cell culture plate is placed in a cell culture incubator at 37°C and 5% CO2 for de-differentiation expansion culture; the BEM is replaced every 2-3 days, and after 7-14 days of culture, a bovine liver three-dimensional organoid is obtained, which can be passaged, cryopreserved, or subjected to differentiation treatment; (5) configure bovine liver cell differentiation expansion medium (Bovine Differentiation Medium, BDM), the BDM is prepared by adding triple antibiotic, glutamine, HEPES buffer, B-27 serum substitute, N2, N-acetyl cysteine, TGF-beta inhibitor, EGF, HGF, FGF10, gastrin, noggin, dexamethasone, gamma-cleavage enzyme inhibitor, BMP7 to DMEM / F12 medium;The concentration of triple antibiotic, glutamine, HEPES buffer, B-27 serum substitute, N2, N-acetyl cysteine, TGF-β inhibitor, EGF, HGF, FGF10, gastrin, noggin, dexamethasone, gamma-cleavage enzyme inhibitor, BMP7 in DMEM / F12 medium is 1% (v / v), 1% (v / v), 10 mM, 2% (v / v), 1% (v / v), 1.25 mM, 1 μM, 100 ng / mL, 25 ng / mL, 50 ng / mL, 10 μM, 100 ng / mL, 30 μM, 10 μM, 25 ng / mL respectively; (6) replace BEM in bovine liver three-dimensional organoid cultured for 7-14d in step (4) with the same volume of BDM preheated at 37 DEG C, promote the differentiation of liver primary cells into mature liver cells, replace BDM every 2-3d, and collect the differentiated bovine liver three-dimensional organoid after 7d for subsequent test.

[0021] Compared with the prior art, the present application has the following advantages: The bovine liver organoid culture medium and the bovine liver three-dimensional organoid culture method provided by the present application adopt dedifferentiation expansion and differentiation expansion medium, which is based on the physiological needs of bovine liver and a large number of test screening, and the nutrient ratio and concentration are sufficient to meet the growth needs of bovine liver organoids according to the growth status of organoids. The bovine liver organoid culture medium can successfully culture organoids, solve the problem of the lack of species-specific culture medium in bovine three-dimensional organoid culture, and provide key technical support for basic research and application transformation in the field of animal husbandry and veterinary medicine.

[0022] The three-dimensional organoid culture method of the present application adopts the matrix glue dome culture method combined with the inverted / orthogonal static step to form a stable 3D structure, which more truly simulates the in-vivo microenvironment of bovine liver.

[0023] The bovine liver primary cell separation method in the present application is based on a two-step enzyme dissociation method combined with red blood cell lysis buffer treatment to separate bovine liver primary cells, which can effectively remove impurity cells and significantly improve the purity and activity of liver primary cells.

[0024] The application adopts an optimized dissociation solution formula to maximize the protection of cell integrity while mildly digesting tissues, and avoid cell damage caused by excessive digestion.

[0025] The organoids obtained by the application can be subcultured for a long time (7-14 days per generation) and maintain genomic stability, are suitable for large-scale drug screening or disease modeling, and can be used for bovine liver disease mechanism research or drug toxicity testing, fill the gap of bovine liver organoid culture technology, and provide a new tool for veterinary medicine and comparative medicine research.

[0026] In summary, the application realizes efficient acquisition, expansion and directional differentiation of bovine liver primary cells by customizing the culture medium, optimizing 3D culture technology and cell separation process, provides a reliable platform for liver biology research and clinical application, and promotes the mechanism and drug screening research of organoids in large livestock. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Fig. 3 is a growth morphology diagram of bovine liver organoids cultured for 1 week in Example 3 and a growth morphology diagram after subculture for 2 weeks; Figure 2 Fig. 4 is an immunofluorescence staining diagram of bovine liver organoid markers in Example 3; Figure 3 Fig. 5 is an oil red O staining diagram of bovine liver organoid fatty liver model establishment in Example 3. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the application will be described below in detail with reference to the drawings of the embodiments of the application. Obviously, the described embodiments are only part of the embodiments of the application, not all embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of the application.

[0029] Example 1 The bovine liver organoid culture medium of the application includes a bovine liver cell expansion culture medium for de-differentiation expansion culture of liver primary cells to obtain bovine liver organoids, and a bovine liver cell differentiation expansion culture medium for promoting differentiation of bovine liver organoids.

[0030] The bovine hepatocyte expansion medium comprises DMEM / F12 medium and the following components in the following concentrations in the DMEM / F12 medium: triple antibiotic 0.8-1.2% (v / v), glutamine 0.8-1.2% (v / v), HEPES buffer 8-12 mM, B-27 serum replacement 1.8-2.2% (v / v), N2 0.8-1.2% (v / v), nicotinamide 8-12 mM, N-acetyl cysteine 1-1.5 mM, R-spondin-1 conditioned medium 9-11% (v / v), ROCK inhibitor 8-12 mM, forskolin 8-12 mM, TGF-beta inhibitor 0.8-1.2 mM, EGF 95-105 ng / mL, HGF 23-27 ng / mL, FGF10 48-52 ng / mL, gastrin 8-12 nM, Noggin 95-105 ng / mL, Wnt3a 48-52 ng / mL, GSK-3a / b inhibitor 1.3-1.7 mM. Preferably, the concentrations of the components are: triple antibiotic 1% (v / v), glutamine 1% (v / v), HEPES buffer 10 mM, B-27 serum replacement 2% (v / v), N2 1% (v / v), nicotinamide 10 mM, N-acetyl cysteine 1.25 mM, R-spondin-1 conditioned medium 10% (v / v), ROCK inhibitor 10 mM, forskolin 10 mM, TGF-beta inhibitor 1 mM, EGF 100 ng / mL, HGF 25 ng / mL, FGF10 50 ng / mL, gastrin 10 nM, Noggin 100 ng / mL, Wnt3a 50 ng / mL, GSK-3a / b inhibitor 1.5 mM.

[0031] The bovine liver cell differentiation expansion medium comprises DMEM / F12 medium and the following components in DMEM / F12 medium respectively: triple antibiotic 0.8-1.2% (v / v), glutamine 0.8-1.2% (v / v), HEPES buffer 8-12 mM, B-27 serum replacement 1.8-2.2% (v / v), N2 0.8-1.2% (v / v), N-acetyl cysteine 1-1.5 mM, TGF-β inhibitor 0.8-1.2 μM, EGF 95-105 ng / mL, HGF 23-27 ng / mL, FGF10 48-52 ng / mL, gastrin 8-12 μM, Noggin 95-105 ng / mL, dexamethasone 28-32 μM, γ-secretase inhibitor 8-12 μM, and BMP7 23-27 ng / mL. The preferred concentrations of the components are: triple antibiotic 1% (v / v), glutamine 1% (v / v), HEPES buffer 10 mM, B-27 serum replacement 2% (v / v), N2 1% (v / v), N-acetyl cysteine 1.25 mM, TGF-β inhibitor 1 μM, EGF 100 ng / mL, HGF 25 ng / mL, FGF10 50 ng / mL, gastrin 10 μM, Noggin 100 ng / mL, dexamethasone 30 μM, γ-secretase inhibitor 10 μM, and BMP7 25 ng / mL.

[0032] wherein: the DMEM / F12 medium does not contain fetal bovine serum; the triple antibiotic is composed of 10 kU / mL penicillin, 10 mg / mL streptomycin, and 25 μg / mL amphotericin B; the glutamine is L-alanyl-L-glutamine; the TGF-β inhibitor is A83-01; the ROCK inhibitor is Y-27632; the GSK-3α / β inhibitor is CHIR99021; the γ-secretase inhibitor is DAPT; the BMP7 is a recombinant BMP7 protein; and the Wnt3a is a Wnt replacement protein.

[0033] Example 2 The method for obtaining liver primary cells of the present application comprises two-step dissociation of bovine liver tissue and obtaining liver primary cells; the two-step dissociation comprises: first step of dissociation: taking bovine liver tissue, washing, and then dissociating using a liver dissociation solution to obtain a tissue suspension; second step of dissociation: taking the tissue suspension, terminating digestion, filtering, and then incubating the filtrate using a red blood cell lysis buffer to obtain liver primary cells. The specific operation method is as follows: Table 1: Composition and ratio of bovine liver tissue washing solution

[0034] Table 2: Composition and ratio of bovine liver dissociation solution

[0035] 1) First step dissociation: 1.1) Preparation before experiment: Prepare the liver tissue washing solution and liver dissociation solution in advance according to Table 1 and Table 2, and store them at 4°C for standby; sterilize the spring scissors and tweezers in advance for standby; prepare the bovine liver tissue washing solution and liver dissociation solution, the liver tissue washing solution is prepared by adding 1% (volume percentage) triple antibiotic solution (containing 10 kU / mL penicillin, 10 mg / mL streptomycin and 25 μg / mL amphotericin B) to DMEM / F12 medium. The liver dissociation solution is prepared by adding type IV collagenase and DNAase to Hanks solution, and the concentrations of type IV collagenase and DNAase in Hanks solution are 3 mg / mL and 60 U / mL respectively; prepare sterile centrifuge tubes with specifications of 1.5 mL, 15 mL and 50 mL for standby; prepare 200 μL pre-cooled sterile gun heads for standby.

[0036] 1.2) Take 0.5 cm 3 of fresh liver tissue sample of a newborn calf (freshly taken within 1 h), rinse twice with 1 mL of liver tissue washing solution and discard the supernatant.

[0037] First add 0.5 mL of liver dissociation solution described in Table 2, finely chop the liver tissue with the spring scissors sterilized in step 1.1); after the liver tissue is chopped into paste, add the remaining 0.5 mL of liver dissociation solution and mix well by blowing, incubate the mixture at 37°C for 15 min to obtain a tissue suspension; during the digestion process, gently blow the sample every 5 min to promote cell dissociation.

[0038] 2) Second step dissociation: 2.1) Add 1 mL of preheated fetal bovine serum at 37°C to the tissue suspension obtained in step 1.2) to terminate enzyme digestion, filter the digested tissue suspension through a 100 μm cell filter to remove undigested tissue fragments, and directly filter into the 50 mL sterile centrifuge tube prepared in step 1.1) to obtain the first step filtered filtrate.

[0039] 2.2) Wash the cell filter screen obtained in step 2.1) with 1 mL of Hanks solution for 2-3 times and filter to reduce cell loss, complete the second step filtration, mix the first step and second step filtered filtrates to obtain a cell suspension.

[0040] 2.3) Add 3 times the volume of red blood cell lysis buffer to the cell suspension obtained in step 2.2), incubate at 4°C for 15 min to obtain a mixture; 2.4) Centrifuge the mixture obtained in step 2.3) at 80g for 5 min at 4°C, collect the hepatocyte pellet after discarding the supernatant, and obtain the primary hepatocytes.

[0041] Example 3 The present application is a method for culturing bovine liver three-dimensional organoids, which is carried out by culturing the primary hepatocytes obtained in Example 2, comprising the following steps: obtaining the primary hepatocytes by the method in Example 2; preparing the bovine hepatocyte expansion medium (a medium designed to maintain the stem cell properties of hepatocytes, which can enable them to regain proliferative and differentiation potential in a specific culture environment) and the bovine hepatocyte differentiation expansion medium (a medium that can promote the differentiation of liver progenitor cells into mature hepatocytes) in Example 1; preparing a hepatocyte suspension with Matrigel and primary hepatocytes and plating, adding the bovine hepatocyte expansion medium for expansion culture, and obtaining bovine liver three-dimensional organoids; using the bovine hepatocyte differentiation expansion medium to differentiate the bovine liver three-dimensional organoids after expansion culture, and obtaining differentiated bovine liver three-dimensional organoids.

[0042] Table 3: BEM components, sources and proportions

[0043] The specific operation of the above three-dimensional organoid culture method is as follows: (One) Three-dimensional culture of bovine liver primary cells (dedifferentiation): the steps for culturing bovine liver organoids using BEM are as follows, and all operations are carried out in a sterile clean bench: (1) Prepare BEM according to Table 3 and filter through a 0.22 μm filter, take the filtrate and store it at 4°C, prepare it for use, store it for no more than one week, and preheat it in a 37°C water bath for at least 30 min before use; thaw Matrigel on ice in advance; pre-cool 200 μL of gun tip in the refrigerator at 4°C in advance; (2) Take the hepatocyte pellet obtained in Example 2 with the pre-cooled gun tip, resuspend it with the thawed Matrigel at a volume ratio (v / v) of 1:25, blow it gently, mix it thoroughly, and pay attention not to generate bubbles, and obtain a hepatocyte suspension; (3) Slowly add the Matrigel-cell mixture (hepatocyte suspension) to the central part of the 48-well plate at a volume of 25 μL per well, so that it forms a semicircular dome-like structure, pay attention not to generate bubbles, and continue to plate the remaining cells in the same way; Place the plated 48-well plate upside down in a cell culture incubator at 37°C, 5% CO2 for 5 min, let the Matrigel form a full dome-like structure to provide enough space for the formation of liver organoids; then, restore the culture plate to the upright state and continue to culture it in a 37°C, 5% CO2 incubator for 30 min to promote gel formation and stabilize the structure of Matrigel; (4) Take out the solidified 48-well plate, add 300 μL of BEM prepared in advance and preheated, and then return to the cell incubator at 37°C, 5% CO2. Observe the growth of the organoids every day, and replace the BEM every 2-3 days. The bovine liver three-dimensional organoids can be subcultured or differentiated cultured 7-14 days later.

[0044] Table 4: BDM components, sources and proportions

[0045] (B) Three-dimensional culture (differentiation) of bovine liver primary cells: the steps of using BDM to culture bovine liver organoids are as follows, and all the following operations are carried out in a sterile clean bench: (5) Prepare BDM according to Table 4 in advance and filter through a 0.22 μm filter. The filtrate is stored at 4°C for standby. It is best to prepare it immediately before use and store it for no more than one week. Before use, preheat it in a 37°C water bath for at least 30 minutes. (6) After the bovine liver three-dimensional organoids in step (4) are maintained in BEM for 7-14 days, the liver organoids grow to about 80% of the Matrigel. At this time, replace the culture medium with the same volume (300 μL) of BDM to promote the differentiation of liver primary cells into mature hepatocytes. Replace the BDM every 2-3 days. After 7 days of culture, the bovine liver three-dimensional organoids can be collected for related model establishment to carry out subsequent experiments.

[0046] Results verification I. Optical microscope observation The organoids cultured in BEM medium for one week in step (4) of Example 3 (the first row in Figure 1 ) and the organoids obtained after the organoids cultured in BEM medium for one week were subcultured for two weeks (the second row in Figure 1 ) were photographed by optical microscope to obtain the growth morphology, and the results are shown in Figure 1 . As can be seen from Figure 1 , some small round or oval cell aggregates can be seen in the initial stage of the organoids, and the morphology is still irregular. Some spherical structures begin to form. With the increase of culture time, the organoids gradually grow larger, and the structure changes from loose to dense, and the morphology changes from irregular to spherical. After 14 days of subculture, the organoids reach a large size, with regular shape and clear edge, which is the typical morphology of mature organoids, suitable for subsequent functional experiments (such as transcriptome analysis, drug screening, etc.), indicating that the organoids are successfully cultured.

[0047] II. Immunofluorescence staining Immunofluorescence staining was performed on the organoids cultured in BDM for 7 days in step (6) of Example 3.

[0048] Using the method of frozen section immunofluorescence staining, the cell nucleus was stained by DAPI, and the specific primary antibody of tight junction protein ZO-1 and liver cell specific markers albumin (ALB) and hepatocyte nuclear factor 4α (HNF-4α) in bovine liver organoids were stained by CY3 labeled secondary antibody, respectively. After staining, the observation and photography were carried out under the excitation wavelength of 358 nm (DAPI) and the excitation wavelength of 550 nm (CY3) under the fluorescence microscope, and the red signal presented by CY3 and the blue signal presented by DAPI were synthesized, and the results are shown in Figure 2 As can be seen from Figure 2 The tight junction protein ZO-1 in the organoid showed positive fluorescence, indicating that the tight junction structure was formed in the organoid; the liver cell specific markers albumin (ALB) and hepatocyte nuclear factor 4α (HNF-4α) showed positive fluorescence, confirming that the liver stem cells successfully differentiated into hepatocytes. In summary, the experimental results confirmed that the organoid model expressed key hepatocyte markers and cell junction proteins, and had hepatocyte characteristics.

[0049] III. Establishment of bovine liver organoid fatty liver model The bovine liver organoid fatty liver model was established using the organoids cultured for 7 days by BDM in step (6) of Example 3.

[0050] The bovine liver organoid fatty liver model was established by treating the organoids with palmitic acid (PA) and oleic acid (OA) for 24 hours, and the model was subjected to oil red O staining, and the staining results are shown in Figure 3 As can be seen from Figure 3 Compared with the control group, the lipid accumulation significantly increased after the establishment of the fatty liver model, indicating that the model was successfully established.

[0051] The above only describes the preferred embodiments of the present application, and does not limit the patent scope of the present application, and any equivalent structural transformation made under the inventive concept of the present application, or direct / indirect application in other related technical fields is included in the patent protection scope of the present application.

Claims

1. A bovine liver organoid culture medium, characterized in that: The medium includes bovine hepatocyte expansion medium; the bovine hepatocyte expansion medium includes DMEM / F12 medium, triple antibiotics, glutamine, HEPES buffer, B-27 serum substitute, N2, nicotinamide, N-acetylcysteine, R-spondin-1 conditioned medium, ROCK inhibitor, forskolin, TGF-β inhibitor, EGF, HGF, FGF10, gastrin, Noggin, Wnt3a, and GSK-3α / β inhibitor; the triple antibiotics include penicillin, streptomycin, and amphotericin B.

2. The bovine liver organoid culture medium according to claim 1, characterized in that: The concentrations of each component in the bovine hepatocyte expansion medium in DMEM / F12 medium are as follows: triple antibiotics 0.8–1.2% (v / v), glutamine 0.8–1.2% (v / v), HEPES buffer 8–12 mM, B-27 serum substitute 1.8–2.2% (v / v), N2 0.8–1.2% (v / v), nicotinamide 8–12 mM, N-acetylcysteine ​​1–1.5 mM, R-spondin-1 conditioned medium 9–11% (v / v), ROCK inhibitor 8–12 μM, forskolin 8–12 μM, TGF-β inhibitor 0.8–1.2 μM, EGF 95–105 ng / mL, HGF 23–27 ng / mL, FGF 1048–52 ng / mL, gastrin 8–12 nM, Noggin 95–105 ng / mL, Wnt3a 48~52 ng / mL, GSK-3α / β inhibitor 1.3~1.7 μM.

3. The bovine liver organoid culture medium according to claim 1, characterized in that: The concentrations of each component in the bovine hepatocyte expansion medium in DMEM / F12 medium are as follows: triple antibiotic 1% (v / v), glutamine 1% (v / v), HEPES buffer 10mM, B-27 serum substitute 2% (v / v), N2 1% (v / v), nicotinamide 10mM, N-acetylcysteine ​​1.25mM, R-spondin-1 conditioned medium 10% (v / v), ROCK inhibitor 10μM, forskolin 10μM, TGF-β inhibitor 1μM, EGF 100ng / mL, HGF 25ng / mL, FGF10 50ng / mL, gastrin 10nM, Noggin 100ng / mL, Wnt3a 50ng / mL, and GSK-3α / β inhibitor 1.5μM.

4. The bovine liver organoid culture medium according to claim 1, characterized in that: The bovine liver organoid culture medium also includes bovine hepatocyte differentiation and expansion medium; the bovine hepatocyte differentiation and expansion medium includes DMEM / F12 medium, triple antibiotics, glutamine, HEPES buffer, B-27 serum substitute, N2, N-acetylcysteine, TGF-β inhibitor, EGF, HGF, FGF10, gastrin, Noggin, dexamethasone, γ-cleavage enzyme inhibitor, and BMP7.

5. The bovine liver organoid culture medium according to claim 4, characterized in that: The concentrations of each component in the bovine hepatocyte differentiation and expansion medium in DMEM / F12 medium are as follows: triple antibiotics 0.8-1.2% (v / v), glutamine 0.8-1.2% (v / v), HEPES buffer 8-12 mM, B-27 serum substitute 1.8-2.2% (v / v), N2 0.8-1.2% (v / v), N-acetylcysteine ​​1-1.5 mM, TGF-β inhibitor 0.8-1.2 μM, EGF 95-105 ng / mL, HGF 23-27 ng / mL, FGF 1048-52 ng / mL, gastrin 8-12 μM, Noggin 95-105 ng / mL, dexamethasone 28-32 μM, γ-cleavage enzyme inhibitor 8-12 μM, and BMP7 23-27 ng / mL.

6. The bovine liver organoid culture medium according to claim 4, characterized in that: The concentrations of each component in the bovine hepatocyte differentiation and expansion medium in DMEM / F12 medium are as follows: triple antibiotic 1% (v / v), glutamine 1% (v / v), HEPES buffer 10mM, B-27 serum substitute 2% (v / v), N2 1% (v / v), N-acetylcysteine ​​1.25mM, TGF-β inhibitor 1μM, EGF 100ng / mL, HGF 25ng / mL, FGF10 50ng / mL, gastrin 10μM, Noggin 100ng / mL, dexamethasone 30μM, γ-cleavage enzyme inhibitor 10μM, and BMP7 25ng / mL.

7. A method for culturing three-dimensional bovine liver organoids, characterized in that: Includes the following steps: Obtain bovine liver tissue and then obtain primary liver cells from the bovine liver tissue; Configure the bovine liver organoid culture medium as described in any one of claims 4 to 6; A hepatocyte suspension was prepared using matrix gel and primary liver cells and plated. After plated, the cells were inverted and cultured for 3-7 minutes to allow the matrix gel and primary liver cells to form a dome. Then, the cells were incubated upright for 25-35 minutes and then bovine hepatocyte expansion medium was added for dedifferentiation and expansion culture to obtain three-dimensional bovine liver organoids. Bovine liver three-dimensional organoids were differentiated and expanded using bovine hepatocyte differentiation and expansion medium to obtain differentiated bovine liver three-dimensional organoids.

8. The method for culturing bovine liver three-dimensional organoids according to claim 7, characterized in that: The method for obtaining primary liver cells includes a two-step dissociation of bovine liver tissue. The two-step dissociation includes: a first step of dissociation: taking bovine liver tissue, washing it, and then dissociating it using liver dissociation solution to obtain a tissue suspension; and a second step of dissociation: taking the tissue suspension, terminating digestion, filtering it, and incubating the filtrate with red blood cell lysis buffer to obtain primary liver cells.

9. The method for culturing bovine liver three-dimensional organoids according to claim 8, characterized in that: The bovine liver tissue was cleaned with a liver tissue cleaning solution, which included DMEM / F12 culture medium and triple antibiotics.

10. The method for culturing bovine liver three-dimensional organoids according to claim 8 or 9, characterized in that: The liver dissociation solution includes Hanks' solution, type IV collagenase, and DNase; the concentrations of type IV collagenase and DNase in the liver dissociation solution in Hanks' solution are 2.5~3.5 mg / mL and 55~65 U / mL, respectively.

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