Culture solution and culture method for separating parenchymal hepatic cells from human hepatic tissue

Abstract

The invention discloses a culture solution and a culture method for separating parenchymal hepatic cells from human hepatic tissue, the culture solution comprises an AUTGC-HPM culture solution for promoting proliferation of precursor cells of the parenchymal hepatic cells and an AUTGC-HDM culture solution for inducing maturation of the precursor cells of the parenchymal hepatic cells, and the parenchymal hepatic cells which are not completely mature are obtained by mechanical shearing and enzymolysis digestion separation; according to the present invention, the proliferation of the parenchymal hepatic cell precursor cells is promoted by using the AUTGC-HPM, and the maturation of the parenchymal hepatic cell precursor cells is induced by using the AUTGC-HDM subsequently, such that the parenchymal hepatic cells highly similar to the source can be obtained in quantity by using the culture method, and the important basis is provided for the research such as drug research and development.

Description

Technical Field

[0001] The invention belongs to the technical field of biological tissue culture, and in particular relates to a culture medium and a culture method for separating hepatic parenchymal cells from human liver tissue. Background Art

[0002] The liver is one of the most important metabolic organs in the human body and has many important physiological functions, including synthesis, metabolism, decomposition and excretion of substances. The cell isolation and culture of liver tissue are of great significance for studying the physiological and pathological processes of the liver, as well as conducting drug metabolism and toxicity studies. The mainstream method for isolating hepatocytes is digestive enzyme perfusion. This type of method is efficient and feasible for experimental animals, but it is difficult to carry out human liver tissue samples for surgical cutting. At the same time, the in vitro proliferation and culture of hepatocytes is also a core bottleneck. Even if a large number of highly viable hepatocytes can be obtained during separation, it is difficult to carry out scientific research systematically, and usually there are no conditions for replication and reproduction. Therefore, it is of great significance to study the isolation of hepatocytes from human liver tissue and the proliferation and culture of hepatocytes. Summary of the invention

[0003] The object of the present invention is to provide a culture medium and a culture method for isolating hepatic parenchymal cells from human liver tissue, so as to solve the problems raised in the above-mentioned background technology.

[0004] To achieve the above object, the present invention provides the following technical solutions: a culture medium for isolating hepatocytes from human liver tissue, comprising an AUTGC-HPM culture medium for promoting the proliferation of hepatocyte precursor cells and an AUTGC-HDM culture medium for inducing the maturation of hepatocyte precursor cells;

[0005] 50 ml of AUTGC-HPM culture medium was composed of the following components: 10 μL of 125 μg / mL HGF, 10 μL of 125 μg / mL FGF10, 10 μL of 250 μg / mL EGF, 8 μL of 200 μg / mL Wnt3a, 25 μL of 10 mM A8301, 10 μL of 10 mM Y27632, 5 μL of 100 μM Gastrin I, 500 μL of penicillin-streptomycin, and the rest was AUTGC-SBM;

[0006] 50 ml of AUTGC-HDM culture medium was composed of the following components: 10 μL of 125 μg / mL HGF, 5 μL of 250 μg / mL EGF, 100 μL of 10 mM Forskolin, 25 μL of 10 mM A8301, 50 μL of 10 mM SB431542, 25 μL of 1 mM IWP2, 25 μL of 100 μM DAPT, 5 μL of 100 μM LDN193189, 500 μL of penicillin-streptomycin, and the rest was AUTGC-SBM;

[0007] 500 ml of AUTGC-SBM is composed of the following components: 10 ml of inorganic salt mother solution, 10 mL of amino acid mother solution, 10 mL of vitamin mother solution, 4.46 g of glucose, 5 ml of basic mother solution, and the rest is ddH2O;

[0008] The EGF is epidermal growth factor, DAPT is a γ-secretase inhibitor, IWP2 is a membrane-targeted O-acyltransferase, SB431542 is a TGF-β receptor kinase inhibitor, Forskolin is an adenylate cyclase activator, Wnt3a is a Wnt3a protein, A8301 is a small molecule inhibitor with a chemical formula of C6H6N2O, Y27632 is an ATP-competitive ROCK-I and ROCK-II inhibitor, and LDN193189 is an effective selective BMP type I receptor inhibitor.

[0009] The present invention also provides a culture method for isolating hepatocytes from human liver tissue, comprising the following steps:

[0010] Primary Isolation and Processing of Human Liver Tissue

[0011] S1. Human liver tissue is a sample with cell activity obtained from non-thermal surgery, or a sample with cell activity away from the thermal incision, preferably without preoperative embolization treatment, and the liver tissue sample is primary isolated within 8 hours after ex vivo;

[0012] S2. After removal from the body, the liver tissue sample should be placed in liver tissue preservation solution (containing sodium heparin / Y27632) at a temperature of 2-8°C. Use 4°C liver tissue cleaning solution to clean the blood, shed red blood cells and dead cells on the surface.

[0013] S3. Use sterile forceps, scissors, scalpels and other tools to remove the bile ducts and blood vessels visible in the liver tissue in the biosafety cabinet. After removing the bile ducts and blood vessels, continue to use 4°C liver tissue cleaning solution to clean the blood, shed red blood cells and dead cells remaining on the surface;

[0014] S4. Use tissue scissors or comprehensive scissors to cut liver tissue into 0.5-3 mm3 For tissue particles of different sizes, resuspend the liver tissue particles with digestive enzymes and digest them in a 37°C water bath with shaking (200 rpm) for 18 min;

[0015] S5. Use a Pasteur pipette to blow the liver tissue digestion enzyme suspension to accelerate the shedding of surface cells. Continue digestion for 7 minutes and then use a Pasteur pipette to blow the liver tissue digestion enzyme suspension to fully detach the cells. Use a 100 μm cell sieve to filter the liver tissue digestion enzyme suspension to remove incompletely digested tissue particles, and then centrifuge the filtrate.

[0016] S6. After centrifugation in the previous step, remove the supernatant, resuspend the precipitate with 4°C DPBS buffer, centrifuge the suspension again, resuspend the precipitate with 1mL red blood cell lysis buffer in advance, add 5mL red blood cell lysis buffer after the precipitate is fully dissolved, and let stand at 4°C for 2min;

[0017] S6. Centrifuge to recover the precipitate, wash the precipitate twice with 10 mL DPBS, centrifuge again, and then inoculate. Use AUTGC-HPM as the culture medium. The culture medium volume is 4 mL. The culture conditions are 37°C and 5% CO. 2。

[0018] Primary induction culture of hepatocyte progenitor cells

[0019] S7. Replace the AUTGC-HPM culture medium every 48 hours. Primary cells usually need to be cultured for 8 to 12 days to form typical hepatocyte precursor cell clones. Whether to subculture is determined based on the adhesion area of ​​the clones and the size of the single clones.

[0020] S8: When the total adherent area of ​​the primary clones exceeds 40% of the bottom area of ​​the culture flask, subculture is required, or when the diameter of a single clone in the primary clones exceeds 3 mm, subculture is required;

[0021] S9, remove AUTGC-HPM, add 2 mL of PBS buffer preheated to 37°C, wash the bottom of the culture flask, remove AUTGC-HPM residues, dead cells and immune cells;

[0022] S10. Add 7 mL of PBS buffer preheated to 37°C to neutralize TryplE and resuspend the cells using a pipette. Transfer the cell suspension to a new 15 mL centrifuge tube and recover the cell pellet by centrifugation. Then, wash the cell pellet with PBS buffer at 18-25°C and recover the pellet by centrifugation.

[0023] Differentiation and induction culture of hepatocyte precursor cells

[0024] S11. Replace the AUTGC-HPM culture medium with AUTGC-HDM and culture for 72 hours. Replace AUTGC-HDM every 24 hours to complete the induced activation. Continuous culture can promote the hepatocytes to restore their functions in vivo.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention uses mechanical shearing and enzymatic digestion to separate and obtain immature hepatocytes, and promotes the proliferation of hepatocyte precursor cells through AUTGC-HPM, and subsequently uses AUTGC-HDM to induce the maturation of hepatocyte precursor cells. This culture method can obtain a large number of hepatocytes that are highly similar to the source, providing an important basis for research such as drug development. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a picture of the culture process of the experimental samples;

[0027] Figure 2 Diagram of the cell states of culture for the isolation and acquisition of hepatic parenchymal precursor cells;

[0028] Figure 3 This is a diagram of the cell state after differentiation induced by AUTGC-HDM. DETAILED DESCRIPTION

[0029] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0030] 1. The present invention provides a culture medium for isolating hepatocytes from human liver tissue, including an AUTGC-HPM culture medium for promoting the proliferation of hepatocyte precursor cells and an AUTGC-HDM culture medium for inducing the maturation of hepatocyte precursor cells;

[0031] The composition of AUTGC-HPM culture medium is shown in Table 1 below.

[0032]

[0033]

[0034] The composition of AUTGC-HDM culture medium is shown in Table 2 below.

[0035] Components Mother liquor 50mL required volume AUTGC-SBM 1X Make up to 50mL HGF 125 μg / mL 10μL EGF 250 μg / mL 5μL Forskolin 10mM 100μL A8301 10mM 25μL SB431542 10mM 50μL IWP2 1mM 25μL DAPT 100μM 25μL LDN193189 100μM 5μL Penicillin-Streptomycin (optional) 100X 500μL

[0036] The formulation composition of AUTGC-SBM is shown in Table 3 below.

[0037] 50X Inorganic Salt Stock Solution 10mL 50X amino acid stock solution 10mL 50X Vitamin Stock Solution 10mL glucose 4.46g 100X Basic Stock Solution 5mL <![CDATA[ddH2O]]> Make up to 500mL

[0038] The composition ratio of 50X amino acid stock solution is shown in Table 301 below:

[0039]

[0040]

[0041] The composition ratio of 50X inorganic salt mother solution is shown in Table 302 below:

[0042]

[0043]

[0044] The formulation of 50x vitamin stock solution is as follows in Table 303:

[0045] 1L Reagents type mg Ascorbyl phosphate Vitamins 125 Biotin Vitamins 200 Choline chloride Vitamins 449 D-Calcium Pantothenate Vitamins 200 Folic acid Vitamins 200 Niacinamide Vitamins 200 Pyridoxine Hydrochloride Vitamins 200 Riboflavin (B2) Vitamins 20 Thiamine Hydrochloride Vitamins 200 Vitamin B12 Vitamins 34 i-Inositol Vitamins 360

[0046] The proportioning components of 100x basic mother solution are shown in Table 304 below:

[0047] 1L Reagents type mg AlbuMAXII Base 40000 Human transferrin (holo) Base 750 Recombinant insulin full chain Base 1000 Glutathione, monosodium Base 100 Ammonium metavanadate Base 0.03 Manganese Dichloride Base 0.005 Sodium selenite Base 0.5 Ethanolamine Base 190 Sodium hypoxanthine Base 239 Linoleic acid Base 4.2 Lipoic Acid Base 10.5 Phenol red Base 1500 Putrench2HCl Base 8.1 Sodium pyruvate Base 11000 Thymidine Base 36.5

[0048] EGF: epidermal growth factor, manufacturer Merck, product number: N0636-500G, solvent: Dhank's stock solution, concentration: 300 μg / mL.

[0049] Y27632: ATP-competitive ROCK-I and ROCK-II inhibitor (chemical formula: C 14 H 21 N3O.2HCl), relative molecular mass: 247.34, manufacturer MCE, item number: HY-10071, solvent: DMSO, mother liquor concentration: 10mmol / L.

[0050] A8301: small molecule inhibitor (chemical formula: C6H6N2O), relative molecular mass: 421.52, manufacturer MCE, item number: N0636-500G, solvent: DMSO, mother liquor concentration: 10mmol / L.

[0051] Wnt3a: Wnt3a protein; manufacturer: MCE, product number: HY-P70453B, solvent: Dhank's, stock solution concentration: 300 μg / mL.

[0052] hGF10: recombinant human fibroblast growth factor, manufacturer: OrganRegen, product number: 816-FGF-0100, solvent: Dhank's, stock solution concentration: 1 mg / mL.

[0053] Forskolin: It is an effective adenylate cyclase activator and an inducer of intracellular cAMP formation. Manufacturer: MCE, Product No.: HY-15371, Solvent: DMSO, Stock solution concentration: 10mmol / L.

[0054] SB431542: It is a TGF-β receptor kinase inhibitor (TRKI) that inhibits the activity of ALK4, ALK5, and ALK7, and can also inhibit TGF-β-induced transcription, gene expression, apoptosis, and growth inhibition. Manufacturer: MCE, item number: HY-10431, solvent: DMSO, mother liquor concentration: 10mmol / L.

[0055] IWP2: Targets the membrane-bound O-acyltransferase porcupine (Porcn), thereby preventing the palmitosylation of key Wnt ligands. Relative molecular weight: 466.60, manufacturer MCE, item number: HY-13912, solvent: DMSO, mother liquor concentration: 10mmol / L.

[0056] DAPT: It is a γ-secretase inhibitor that inhibits Notch 1 signaling and induces cell differentiation, as well as cell autophagy and apoptosis. Relative molecular weight: 432.46, manufacturer: MCE, item number: HY-13027, solvent: DMSO, mother liquor concentration: 10mmol / L.

[0057] LDN193189: It is an effective and selective BMP type I receptor inhibitor, which effectively inhibits the transcriptional activity of BMP type I receptors ALK2 and ALK3. Relative molecular weight: 406.48, manufacturer MCE, item number: HY-12071, solvent: DMSO.

[0058] Second, the present invention also provides a culture method for isolating hepatocytes from human liver tissue, comprising the following steps:

[0059] (1) Primary isolation and processing of human liver tissue

[0060] 1) Human liver tissue should be a sample with cell activity obtained by non-thermal surgery, or a sample with cell activity away from the thermal incision, preferably without preoperative embolization therapy.

[0061] 2) The primary culture of the sample should be separated within 8 hours after ex vivo separation.

[0062] 3) After removal from the body, liver tissue samples should be placed in liver tissue preservation solution (containing sodium heparin / Y27632) and stored at 2-8°C.

[0063] 4) The samples must be tested in a P2 or above laboratory, and the patients must not have liver-related infectious diseases or other blood-related infectious diseases.

[0064] 5) Use 4°C liver tissue cleaning solution to clean the blood, detached red blood cells and dead cells on the surface of liver tissue.

[0065] 6) Use sterile forceps, scissors, scalpels and other tools to remove the bile ducts and blood vessels visible in the liver tissue in a biosafety cabinet.

[0066] 7) Remove the bile duct and blood vessels from the liver tissue and continue to use 4°C liver tissue cleaning solution to clean the blood, shed red blood cells and dead cells remaining on the surface.

[0067] 8) Use tissue scissors or comprehensive scissors to cut liver tissue into 0.5-3 mm 3 Size of tissue particles.

[0068] 9) Resuspend the liver tissue pellet with digestive enzymes and digest in a 37°C water bath with shaking (200 rpm) for 18 min.

[0069] 10) Use a Pasteur pipette to blow the liver tissue digestion enzyme suspension to accelerate the shedding of surface cells.

[0070] 11) Continue digestion for 7 min.

[0071] 12) Continue to use the Pasteur pipette to blow the liver tissue digestion enzyme suspension to fully detach the cells.

[0072] 13) Filter the liver tissue digestion enzyme suspension through a 100 μm cell sieve to remove incompletely digested tissue particles.

[0073] 14) Centrifuge the filtrate: 500 rpm, 4°C, ramp up 4, ramp down 6, centrifuge for 5 min.

[0074] 15) Remove the supernatant and resuspend the pellet in 4°C DPBS buffer.

[0075] 16) Centrifuge the suspension at 200 rpm, 4°C, ramp up 4, ramp down 6, and centrifuge for 5 min.

[0076] 17) Red blood cell lysis: Use 1 mL of red blood cell lysis buffer to resuspend the precipitate in advance. After the precipitate is fully dissolved, add 5 mL of red blood cell lysis buffer and let it stand at 4°C for 2 minutes.

[0077] 18) Centrifugation: 200 rpm, 4°C, speed up 4, speed down 6, centrifuge for 5 min, and recover the precipitate.

[0078] 19) Wash the pellet twice with 10 mL DPBS and centrifuge at 200 rpm, 4°C, speed up 4, speed down 6, and centrifuge for 5 min.

[0079] 20) Inoculation: 5E5 per T12.5 bottle. AUTGC-HPM was used as culture medium, culture medium volume: 4 mL, culture conditions: 37°C, 5% CO2.

[0080] (2) Primary induction culture of hepatocyte precursor cells.

[0081] 1) Medium change frequency: AUTGC-HPM culture medium is replaced every 48 hours.

[0082] 2) Primary cells usually need to be cultured for 8 to 12 days to form typical hepatocyte precursor cell clones. The size of the clones determines whether to pass them on, based on the adherent area of ​​the clones.

[0083] 3) When the entire adherent area of ​​the primary clones accounts for more than 40% of the bottom area of ​​the culture flask, subculture is required, or when the diameter of a single clone in the primary clones is greater than 3 mm, subculture is required.

[0084] 4) The PBS buffer, TryplE digestion solution, and new AUTGC-HPM culture medium used for subculturing need to be preheated to 37°C.

[0085] 5) Invert the culture flask to remove the AUTGC-HPM, and add 2 mL of PBS buffer preheated to 37°C to clean the bottom of the culture flask to remove the AUTGC-HPM residue, dead cells and immune cells.

[0086] 6) Remove the PBS buffer and add 3 mL of TrypE digestion solution preheated to 37°C, and digest at 37°C for 210 seconds.

[0087] 7) Add 7 mL of PBS buffer preheated to 37°C to neutralize the TryplE and resuspend the cells using a pipette.

[0088] 8) Transfer the cell suspension to a new 15 mL centrifuge tube and recover the cell pellet by centrifugation. The centrifugation parameters are: 4°C, 700 rpm, speed increase 4, speed decrease 5, 5 min.

[0089] 9) Wash the cell pellet with PBS buffer at 18-25°C and recover the pellet by centrifugation. The centrifugation parameters are: 4°C, 700 rpm, speed increase 4, speed decrease 5, 5 min.

[0090] 10) Adjust the cell density to 5E5 / T12.5 flask.

[0091] (3) Differentiation and induction culture of hepatocyte precursor cells.

[0092] 1) Replace the AUTGC-HPM culture medium with AUTGC-HDM and culture for 72 hours, replacing AUTGC-HDM every 24 hours to complete the induced activation.

[0093] 2) Continuous culture using AUTGC-HDM can promote hepatocytes to restore their functions in vivo.

[0094] 3. Cultivation Experiment

[0095] Three surgical specimens of intrahepatic hemangioma and four surgical specimens of hepatocellular carcinoma were selected to isolate and culture hepatocellular progenitor cells. The sample information is shown in Table 4 below:

[0096] Sample No. Patient age gender patient weight Sample weight Out of body time Intrahepatic hemangioma surgery sample 1 47 years old male 73Kg 2.3g 7h Intrahepatic hemangioma surgery sample 2 66 years old male 68Kg 3.4g 6.5h Intrahepatic hemangioma surgery sample 3 67 years old female 54Kg 6g 7h Hepatocellular carcinoma surgical specimen 1 38 years old male 77Kg 1.8g 7h Hepatocellular carcinoma surgical specimen 2 65 years old male 70Kg 3.5g 6h Hepatocellular carcinoma surgical specimen 3 73 years old female 49Kg 7.1g 8h Hepatocellular carcinoma surgical specimen 4 71 years old female 43Kg 6.3g 7.5h

[0097] Each sample was cultured according to the culture method of isolating hepatocytes from human liver tissue mentioned above. The culture process is shown in the figure Figure 1 shown.

[0098] The bright field microscope image of the sample is as follows: Figure 2 As shown, Figure 2 The culture state of hepatic parenchymal progenitor cells was obtained by isolating samples of intrahepatic hemangioma. After primary treatment, a good clone cluster of hepatic parenchymal progenitor cells was formed within 6 days. After continuous culture for up to 12 days, the hepatic parenchymal progenitor cells in the clone cluster grew well and could be proliferated in large quantities within 4-6 days after passage. In this study, we recorded the proliferation cycle of 7 samples for 5 consecutive passages and summarized their proliferation rates. Two of the 7 samples showed attenuation of proliferation ability after the fifth passage, and 5 samples showed attenuation of proliferation ability after the third passage. Combined with the information of cell sample donors, it was found that the donor age was negatively correlated with the proliferation ability of hepatic parenchymal progenitor cells. The proliferation ability of isolated hepatic parenchymal progenitor cells was less correlated with the cause and severity of the donor. The severity of the sample donor's disease only determines the number of hepatic parenchymal progenitor cells that can be obtained by primary treatment.

[0099] In order to obtain hepatocytes, AUTGC-HDM was used to induce differentiation of hepatocyte progenitor cells. Figure 3 It can be seen that 4 days after the culture medium of the hepatocyte precursor cell clones was replaced with AUTGC-HDM, the cells began to expand, the distance between cells in the clone cluster increased, and after 8 days of differentiation culture, about 80% of the hepatocyte precursor cells developed into hepatocytes, and the cells no longer proliferated.

[0100] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A culture medium for isolating hepatocytes from human liver tissue, characterized in that: It includes AUTGC-HPM culture medium for promoting the proliferation of hepatocyte precursor cells and AUTGC-HDM culture medium for inducing the maturation of hepatocyte precursor cells; 50 ml of AUTGC-HPM culture medium was composed of the following components: 10 μL of 125 μg / mL HGF, 10 μL of 125 μg / mL FGF10, 10 μL of 250 μg / mL EGF, 8 μL of 200 μg / mL Wnt3a, 25 μL of 10 mM A8301, 10 μL of 10 mM Y27632, 5 μL of 100 μM Gastrin I, 500 μL of penicillin-streptomycin, and the rest was AUTGC-SBM; 50 ml of AUTGC-HDM culture medium was composed of the following components: 10 μL of 125 μg / mL HGF, 5 μL of 250 μg / mL EGF, 100 μL of 10 mM Forskolin, 25 μL of 10 mM A8301, 50 μL of 10 mM SB431542, 25 μL of 1 mM IWP2, 25 μL of 100 μM DAPT, 5 μL of 100 μM LDN193189, 500 μL of penicillin-streptomycin, and the rest was AUTGC-SBM; 500 ml of AUTGC-SBM is composed of the following components: 10 ml of inorganic salt mother solution, 10 mL of amino acid mother solution, 10 mL of vitamin mother solution, 4.46 g of glucose, 5 ml of basic mother solution, and the rest is ddH2O; The EGF is epidermal growth factor, DAPT is a γ-secretase inhibitor, IWP2 is a membrane-targeted O-acyltransferase, SB431542 is a TGF-β receptor kinase inhibitor, Forskolin is an adenylate cyclase activator, Wnt3a is a Wnt3a protein, A8301 is a small molecule inhibitor with a chemical formula of C6H6N2O, Y27632 is an ATP-competitive ROCK-I and ROCK-II inhibitor, and LDN193189 is an effective selective BMP type I receptor inhibitor.

2. A method for culturing hepatocytes isolated from human liver tissue, characterized in that: The steps include: Primary Isolation and Processing of Human Liver Tissue S1. Human liver tissue is a sample with cell activity obtained from non-thermal surgery, or a sample with cell activity away from the thermal incision, preferably without preoperative embolization treatment, and the liver tissue sample is primary isolated within 8 hours after ex vivo; S2. After removal from the body, the liver tissue sample should be placed in liver tissue preservation solution (containing sodium heparin / Y27632) at a temperature of 2-8°C. Use 4°C liver tissue cleaning solution to clean the blood, shed red blood cells and dead cells on the surface. S3. Use sterile forceps, scissors, scalpels and other tools to remove the bile ducts and blood vessels visible in the liver tissue in the biosafety cabinet. After removing the bile ducts and blood vessels, continue to use 4°C liver tissue cleaning solution to clean the blood, shed red blood cells and dead cells remaining on the surface; S4. Use tissue scissors or comprehensive scissors to cut liver tissue into 0.5-3 mm 3 For tissue particles of different sizes, resuspend the liver tissue particles with digestive enzymes and digest them in a 37°C water bath with shaking (200 rpm) for 18 min; S5. Use a Pasteur pipette to blow the liver tissue digestion enzyme suspension to accelerate the shedding of surface cells. Continue digestion for 7 minutes and then use a Pasteur pipette to blow the liver tissue digestion enzyme suspension to fully detach the cells. Use a 100 μm cell sieve to filter the liver tissue digestion enzyme suspension to remove incompletely digested tissue particles, and then centrifuge the filtrate. S6. After centrifugation in the previous step, remove the supernatant, resuspend the precipitate with 4°C DPBS buffer, centrifuge the suspension again, resuspend the precipitate with 1mL red blood cell lysis buffer in advance, add 5mL red blood cell lysis buffer after the precipitate is fully dissolved, and let stand at 4°C for 2min; S6. Centrifuge to recover the precipitate, wash the precipitate twice with 10 mL DPBS, centrifuge again, and then inoculate. Use AUTGC-HPM as the culture medium. The culture medium volume is 4 mL. The culture conditions are 37°C and 5% CO. 2。 Primary induction culture of hepatocyte progenitor cells S7. Replace the AUTGC-HPM culture medium every 48 hours. Primary cells usually need to be cultured for 8 to 12 days to form typical hepatocyte precursor cell clones. Whether to subculture is determined based on the adhesion area of ​​the clones and the size of the single clones. S8: When the total adherent area of ​​the primary clones exceeds 40% of the bottom area of ​​the culture flask, subculture is required, or when the diameter of a single clone in the primary clones exceeds 3 mm, subculture is required; S9, remove AUTGC-HPM, add 2 mL of PBS buffer preheated to 37°C, wash the bottom of the culture flask, remove AUTGC-HPM residues, dead cells and immune cells; S10. Add 7 mL of PBS buffer preheated to 37°C to neutralize TryplE and resuspend the cells using a pipette. Transfer the cell suspension to a new 15 mL centrifuge tube and recover the cell pellet by centrifugation. Then, wash the cell pellet with PBS buffer at 18-25°C and recover the pellet by centrifugation. Differentiation and induction culture of hepatocyte precursor cells S11. Replace the AUTGC-HPM culture medium with AUTGC-HDM and culture for 72 hours. Replace AUTGC-HDM every 24 hours to complete the induced activation. Continuous culture can promote the hepatocytes to restore their functions in vivo.

3. The method for culturing hepatocytes isolated from human liver tissue according to claim 2, characterized in that: The PBS buffer, TryplE digestion solution, and new AUTGC-HPM culture medium used for subculturing in S8 need to be preheated to 37°C.

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