A method for long-term in vitro culture and expansion of primary hepatocytes and uses thereof

Abstract

The application provides a method for long-term in-vitro culture and expansion of primary hepatocytes and application thereof. The method for in-vitro culture and expansion of primary hepatocytes comprises using a culture medium containing interleukin-22 (IL-22), combined with hepatocyte growth factor (HGF) and / or epidermal growth factor (EGF) to induce primary hepatocytes to prepare proliferative liver precursor cells. The application combines interleukin-22 (IL-22), hepatocyte growth factor (HGF) and / or epidermal growth factor (EGF) to induce in-vitro mass expansion of primary hepatocytes, and through the method, mature-like hepatocytes with a gene expression profile extremely similar to that of primary hepatocytes and perfect functions can be obtained, and the hepatocytes expanded by the method have good in-vivo therapeutic effects.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for long-term and large-scale expansion of mouse or human primary hepatocytes in an in vitro environment and its application. Background Technology

[0002] The liver is the largest digestive organ in the human body. Its main functions include participating in the storage and regulation of nutrients and energy, removing toxins, producing bile, and synthesizing various proteins, clotting factors, and hormones in serum. Hepatocytes account for more than 80% of the liver's mass and are the most important functional cells in the liver. The liver is also the most important organ for drug metabolism, with 90% of drugs being metabolized there first. Therefore, in drug development, primary hepatocytes are one of the most important tools for evaluating the metabolic stability, safety, and toxicity of drugs. In clinical treatment, liver / hepatocyte transplantation is the only effective treatment for end-stage liver disease, viral or drug-induced acute liver failure, and liver metabolic diseases caused by genetic issues.

[0003] Hepatocytes possess irreplaceable practical value and powerful therapeutic effects, but they are difficult to culture in vitro and even more difficult to proliferate. The severe shortage of hepatocyte sources greatly limits their use in drug development and clinical treatment. Currently, besides directly isolating hepatocytes from donor tissues, other sources of hepatocytes mainly rely on stem cell differentiation or adult cell transdifferentiation. It is also possible to isolate and culture specific subpopulations of liver stem / precursor cells, induce their in vitro expansion, and then differentiate them into hepatocytes for use. Recent studies have found that using small molecule compounds, cytokines such as TNF-α or Wnt3a, hepatocytes can be reprogrammed into proliferating liver procuratorial cells, thereby achieving massive proliferation of hepatocytes. [1-4] Regarding methods for in vitro culture and expansion of hepatocytes, relevant domestic patents include CN112513252B, CN110093305B, CN108300688B, CN106754636B, and CN105296418B. However, the reported systems for expanding hepatocytes are all quite complex, involving the use of multiple small molecule compounds or various cytokines. The activation or inhibition of multiple molecular signaling pathways in cells may cause unnecessary changes in cell fate or pose potential mutation risks. Methods for obtaining hepatocytes through differentiation or transdifferentiation have long induction cycles and are cumbersome, and the resulting hepatocyte-like cells still differ significantly from primary hepatocytes in function and gene expression profile. Given the importance of hepatocytes in basic research and clinical applications, developing a convenient, efficient, economical, and stable method for expanding primary hepatocytes is of significant importance.

[0004] Existing scientific research has confirmed that hepatocyte growth factor (HGF) and epidermal growth factor (EGF) are key factors driving hepatocyte mitosis and are crucial for hepatocyte proliferation. When the signal transduction pathways of these two growth factors are simultaneously blocked, all mice undergoing hepatectomy die. Published studies on in vitro hepatocyte culture conditions indicate that adding HGF and EGF alone does not effectively promote hepatocyte proliferation in vitro. [5-7] .

[0005] [1].FU GB, HUANG WJ, ZENG M, et al. Expansion and differentiation of human hepatocyte-derived liver progenitor-like cells and their use for the study of hepatotropic pathogens[J]. Cell Res, 2019, 29(1): 8-22.

[0006] [2].ZHANG K, ZHANG L, LIU W, et al. In Vitro Expansion of Primary Human Hepatocytes with Efficient Liver Repopulation Capacity[J]. Cell Stem Cell, 2018, 23(6): 806-19e4.

[0007] [3]PENG WC, LOGAN CY, FISH M, et al. Inflammatory Cytokine TNFalphaPromotes the Long-Term Expansion of Primary Hepatocytes in 3D Culture[J]. Cell, 2018, 175(6): 1607-19e15.

[0008] [4]WU H, ZHOU

[0009] [5] Katsuda T, Kawamata M, Hagiwara K, et al. Conversion of TerminallyCommitted Hepatocytes to Culturable Bipotent Progenitor Cells withRegenerative Capacity[J]. Cell Stem Cell, 2016: 41.

[0010] [6] Kim Y, Kang K, Lee SB, et al. Small molecule-mediated reprogramming of human hepatocytes into bipotent progenitor cells [J]. Journal of hepatology, 2019, 70(1): 97-107.

[0011] [7]Guo R, Jiang M, Wang G, et al.IL6 supports long-term expansion of hepatocytes in vitro[J].Nature Communications. Summary of the Invention

[0012] In this invention, the inventors discovered that the combined use of interleukin-22 (IL-22), hepatocyte growth factor (HGF), and / or epidermal growth factor (EGF) can induce the large-scale expansion of primary mouse hepatocytes in vitro. Furthermore, this method can yield mature hepatocytes with gene expression profiles highly similar to primary hepatocytes and complete functional expression. Hepatocytes expanded using this method exhibit good in vivo therapeutic effects; after cell transplantation, they can be successfully transplanted into Fah... - / - It rapidly proliferates in the liver of mice and reconstructs Fah within 8 weeks. - / - The mouse's entire liver ultimately saved Fah. - / -The life of mice. More importantly, IL-22 combined with HGF, EGF, proliferative growth factors, and small molecule compounds that maintain stemness (such as Rho-associated kinase inhibitor Y-27632 (trans-4-[(R)-1-aminoethyl]-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride) and transforming growth factor-β receptor inhibitor A 83-01 ((3-(6-methyl-2-pyridyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-thiocarboxamide)) can significantly promote the in vitro expansion of human primary hepatocytes. This method for expanding human primary hepatocytes is simple, has a short cycle, and is highly efficient. The expanded hepatocytes have complete physiological functions.

[0013] Based on this, the present invention aims to provide a method for culturing and expanding primary hepatocytes in vitro, as well as hepatic progenitor cells and mature hepatocytes obtained by the method.

[0014] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0015] In a first aspect, a method for culturing and expanding primary hepatocytes in vitro is provided, comprising: inducing primary hepatocytes to prepare proliferating hepatic progenitor cells using a culture medium containing interleukin-22 (IL-22), in combination with hepatocyte growth factor (HGF) and / or epithelia growth factor (EGF).

[0016] In some embodiments, the concentration range of interleukin-22 (IL-22) is 0.1-1000 ng / mL, preferably 10-100 ng / mL.

[0017] In some embodiments, the concentration of hepatocyte growth factor (HGF) ranges from 0.1 to 1000 ng / mL, preferably from 10 to 100 ng / mL.

[0018] In some embodiments, the concentration of epidermal growth factor (EGF) ranges from 0.1 to 1000 ng / mL, preferably from 10 to 100 ng / mL.

[0019] The primary hepatocytes are mammalian primary hepatocytes, including but not limited to mouse primary hepatocytes and human primary hepatocytes.

[0020] In some embodiments, the primary hepatocytes are mouse primary hepatocytes, and the culture medium is a culture medium for inducing mouse primary hepatocytes to prepare proliferating liver progenitor cells and includes: liquid basal medium, cell culture nutrient supplements, interleukin-22 (IL-22), hepatocyte growth factor (HGF) and / or epithelial growth factor (EGF), and glucocorticoids.

[0021] In some embodiments, the concentration range of interleukin-22 (IL-22) is 0.1-1000 ng / mL, preferably 10-100 ng / mL.

[0022] In some embodiments, the concentration of hepatocyte growth factor (HGF) ranges from 0.1 to 1000 ng / mL, preferably from 10 to 100 ng / mL.

[0023] In some embodiments, the concentration of epidermal growth factor (EGF) ranges from 0.1 to 1000 ng / mL, preferably from 10 to 100 ng / mL.

[0024] In some embodiments, the culture medium also includes an antibacterial agent, such as 1% PS (Penicillin-Streptomycin solution).

[0025] In a further embodiment, the culture medium used to induce the preparation of proliferating hepatic progenitor cells from mouse primary hepatocytes includes:

[0026] The liquid basal culture medium is selected from one or more of the following: DMEM-F12 medium, DMEM medium, William's E medium, Advanced DMEM-F12 medium, HCM medium, RPMI1640 medium, etc.

[0027] The cell culture nutrient additives are selected from one or more of the following: insulin-transferrin-selenium (ITS), B27 additive, N2 additive, fetal bovine serum (FBS), and bovine albumin (BSA).

[0028] The glucocorticoids are selected from one or a combination of dexamethasone, hydrocortisone, etc.

[0029] In a further embodiment, the culture medium used to induce the preparation of proliferating hepatic progenitor cells from mouse primary hepatocytes comprises the following components: 1×DMEM-F12, 1% (v / v) insulin-transferrin-selenium culture medium additive (ITS), 0.1-1000 ng / mL interleukin-22 (IL-22) (preferably a concentration range of 10-100 ng / mL, more preferably a concentration of 30 ng / mL), 10 -7 The medium contains one or two of the following: M dexamethasone, 0.1% (v / v) bovine serum albumin (BSA), 1% (v / v) penicillin-streptomycin (PS), and 0.1-1000 ng / mL hepatocyte growth factor (HGF) (preferably 10-100 ng / mL, more preferably 20 ng / mL) and 0.1-1000 ng / mL epidermal growth factor (EGF) (preferably 10-100 ng / mL, more preferably 20 ng / mL). In a preferred embodiment, the culture medium contains both hepatocyte growth factor (HGF) and epidermal growth factor (EGF).

[0030] In some embodiments, the primary hepatocytes are human primary hepatocytes, and the culture medium is a culture medium for inducing human primary hepatocytes to prepare proliferating liver progenitor cells and includes: liquid basal medium, cell culture nutrient supplements, interleukin-22 (IL-22), hepatocyte growth factor (HGF), epidermal growth factor (EGF), stem cell maintenance factor, proliferative growth factor, and glucocorticoids.

[0031] In some embodiments, the concentration range of interleukin-22 (IL-22) is 0.1-1000 ng / mL, preferably 10-300 ng / mL.

[0032] In some embodiments, the concentration of hepatocyte growth factor (HGF) ranges from 0.1 to 1000 ng / mL, preferably from 10 to 100 ng / mL.

[0033] In some embodiments, the concentration of epidermal growth factor (EGF) ranges from 0.1 to 1000 ng / mL, preferably from 10 to 100 ng / mL.

[0034] In some embodiments, the concentration of the growth factor promoting proliferation ranges from 0.1 to 1000 ng / mL, preferably from 10 to 200 ng / mL.

[0035] In some embodiments, the concentration range of the cell stemness maintenance factor is preferably 0.1-50 μM, more preferably 1-20 μM.

[0036] In some embodiments, the culture medium also includes an antibacterial agent, such as 1% PS (Penicillin-Streptomycin solution).

[0037] In some embodiments, the culture medium used to induce the preparation of proliferating hepatic progenitor cells from human primary hepatocytes includes:

[0038] The liquid basal culture medium is selected from one or more of the following: DMEM-F12 medium, DMEM medium, William's E medium, Advanced DMEM-F12 medium, RPMI1640 medium, and commercially available media specifically for hepatocyte culture: Hepatocyte Culture Medium Bullet Kit (HCM Bullet Kit, Lonza, catalog number CC-3198) and Endothelial Cell Growth Medium Bullet Kit (EGM-2 Bullet Kit, Lonza, catalog number CC-3162). Both the Hepatocyte Culture Medium Bullet Kit and the Endothelial Cell Growth Medium Bullet Kit contain epidermal growth factor (EGF) and proliferative growth factors. When using these two media, additional or reduced amounts of epidermal growth factor (EGF) and proliferative growth factors may not be required.

[0039] The cell culture nutrient additives are selected from one or more of the following: insulin-transferrin-selenium (ITS), B27 additive, N2 additive, ascorbic acid, fetal bovine serum, and bovine serum albumin (BSA).

[0040] The cell stem maintenance factor is selected from one or more of the following: Transforming growth factor-β (TGFβ) receptor inhibitor A83-01, Rho-associated kinase (ROCK) inhibitor Y-27632, etc.

[0041] The growth factor that promotes proliferation is selected from one or more of the following: fibroblast growth factor 2, vascular endothelial growth factor, insulin-like growth factor 1, etc.

[0042] In some embodiments, the concentration of fibroblast growth factor 2 ranges from 0.1 to 300 ng / mL, preferably from 1 to 30 ng / mL.

[0043] In some embodiments, the concentration of vascular endothelial growth factor ranges from 0.1 to 300 ng / mL, with a preferred range of 10 to 50 ng / mL.

[0044] In some embodiments, the concentration of insulin-like growth factor 1 ranges from 0.1 to 300 ng / mL, preferably from 10 to 100 ng / mL.

[0045] In a further embodiment, the culture medium used to induce the preparation of proliferating hepatic progenitor cells from human primary hepatocytes comprises the following components: 1×DMEM-F12, 1×B-27 additive, 150 μM ascorbic acid, 10 ng / mL fibroblast growth factor 2, 20 ng / mL vascular endothelial growth factor, 50 ng / mL insulin-like growth factor 1, 5 μM hydrocortisone, 0.1-1000 ng / mL interleukin-22 (IL-22) (preferably 10-300 ng / mL, more preferably 100 ng / mL), 0.1-1000 ng / mL hepatocyte growth factor (HGF) (preferably 10-100 ng / mL, more preferably 20 ng / mL), 0.1-1000 ng / mL epidermal growth factor (EGF) (preferably 10-100 ng / mL, more preferably 20 ng / mL), 5 μM A83-01, 10 μM Y-27632, 0.1% (v / v) bovine serum albumin (BSA), 1% (v / v) penicillin-streptomycin (PS).

[0046] Alternatively, the culture medium used to induce the preparation of proliferating hepatic progenitor cells from human primary hepatocytes comprises the following components: 0.5× Hepatocyte Culture Medium Bullet Kit (HCM Bullet Kit, Lonza, catalog number CC-3198), 0.5× Endothelial Cell Growth Medium Bullet Kit (EGM-2 Bullet Kit, Lonza, catalog number CC-3162), 0.1-1000 ng / mL interleukin-22 (IL-22) (preferably 10-300 ng / mL, more preferably 100 ng / mL), 0.1-1000 ng / mL hepatocyte growth factor (HGF) (preferably 10-100 ng / mL, more preferably 20 ng / mL), 5 μM A83-01, 10 μM Y-27632, 0.1% (v / v) bovine serum albumin (BSA), 1% (v / v) penicillin-streptomycin (PS).

[0047] Furthermore, the method for in vitro culture and expansion of primary hepatocytes of the present invention further includes the step of inducing the hepatic progenitor cells to differentiate into mature hepatocytes using a hepatocyte maturation induction medium.

[0048] In a specific embodiment, the hepatocyte maturation induction culture medium includes: liquid basal culture medium, cell culture nutrient supplement, human oncostain-M (hOSM), and glucocorticoids.

[0049] The liquid basal culture medium is selected from one or more of the following: DMEM-F12 medium, DMEM medium, William's E medium, Advanced DMEM-F12 medium, HCM medium, and RPMI1640 medium;

[0050] The cell culture nutrient additives are selected from one or more of the following: insulin-transferrin-selenium (ITS), B27 additive, N2 additive, fetal bovine serum, and bovine serum albumin.

[0051] The glucocorticoid is selected from dexamethasone, hydrocortisone, or a combination thereof.

[0052] In a further embodiment, for mouse hepatocytes, the hepatocyte maturation induction medium comprises: 1×DMEM-F12, 1% (v / v) insulin-transferrin-selenium medium supplement (ITS), 20 ng / mL human inhibin-M (hOSM), 10 -6 M dexamethasone, 1% (v / v) Penicillin-Streptomycin (PS).

[0053] In a further embodiment, for human hepatocytes, the hepatocyte maturation induction medium comprises: 1×DMEM-F12, 1% (v / v) insulin-transferrin-selenium medium supplement (ITS), 20 ng / mL human inhibin-M (hOSM), 10 -6 M dexamethasone, 1% (v / v) Penicillin-Streptomycin (PS).

[0054] In the method for in vitro culture and expansion of primary hepatocytes described in this invention, mouse cells are cultured at 37°C and 5% CO2, and human cells are cultured at 37°C, 5% CO2, and 5% O2.

[0055] Secondly, the present invention provides a method for preparing liver progenitor cells by the method described in the first aspect.

[0056] Thirdly, the present invention provides a method for preparing mature hepatocytes by the method described in the first aspect.

[0057] Fourthly, the present invention provides the use of proliferating hepatic progenitor cells or mature hepatocytes obtained by the method described in the first aspect in the preparation of products for treating liver diseases (e.g., hepatitis, cirrhosis, liver cancer, etc.), hepatocyte transplantation, or hepatocyte therapy.

[0058] The present invention also provides the application of proliferating hepatic progenitor cells or mature hepatocytes obtained by the method described in the first aspect in drug toxicity evaluation, new drug screening and basic research related to liver diseases.

[0059] The beneficial effects of this invention are as follows:

[0060] This invention relates to a method for inducing the in vitro proliferation of mouse and human primary hepatocytes based on a combination of cytokines. This method is highly efficient, easy to operate, uses a simple culture medium, is low in cost, and has good reproducibility.

[0061] For primary mouse hepatocytes, the method of this invention allows for continuous passage (IL22-td-iHPCs can be passaged for more than 30 generations), with a stable proliferation rate and no significant morphological changes during proliferation. Furthermore, the induced hepatocytes have a gene expression profile very similar to primary hepatocytes and possess complete mature hepatocyte function and therapeutic effects. The induced mouse hepatocytes can be used to alleviate Fah... - / - Liver damage in mice ultimately saved Fah - / - The life of the mouse.

[0062] For human primary hepatocytes, the conditions for in vitro expansion are more complex and influenced by various factors such as individual differences, making long-term stable expansion difficult to achieve at present. This invention adds IL-22 to the induction system, which, in combination with HGF and EGF, can further enhance the expansion efficiency of human primary hepatocytes, successfully achieving expansion exceeding 10⁻⁶ cells / year in vitro. 4 Amplification by a factor of two.

[0063] Furthermore, the high-quality hepatocytes prepared in this invention, with their complete preservation of biological characteristics and sufficient quantity, demonstrate broad application prospects. They can mimic the human liver's drug metabolism and excretion processes, making them suitable not only for drug toxicity evaluation but also for providing more accurate and reliable toxicity predictions for new drug development. Simultaneously, these hepatocytes are ideal for new drug screening, improving the success rate of drug development. In addition, these hepatocytes will play a crucial role in basic research related to liver diseases, contributing to the understanding of the pathogenesis of liver diseases and providing a scientific basis for the development of disease treatment strategies.

[0064] More importantly, the hepatocytes prepared in this invention show great potential in the fields of hepatocyte transplantation and cell therapy. Through advanced cell isolation, purification, expansion, and differentiation techniques, these cells are expected to become a novel cell source for treating liver diseases (such as hepatitis, cirrhosis, and liver cancer), providing new therapeutic avenues for liver damage repair and functional recovery. Furthermore, they may also be applied to the construction of liver tissue engineering, providing a richer donor resource for organ transplantation, alleviating the pressure of organ shortages, and improving the success rate of transplant surgeries and the quality of life for patients. Attached Figure Description

[0065] Figure 1 In Example 1, Albumin-Cre, R26R tdTomato The image shows the results of mouse primary hepatocytes induced into IL22-td-iHPCs for amplification. (A) Newly adherent tdTomato cells are shown. + Hepatocytes and the induction of tdTomato using HCM or IL22-HCM +(B) Cell morphology and fluorescence images of hepatocytes after 14 days of proliferation. (C) tdTomato cells cultured using HCM or IL22-HCM. + Hepatocyte proliferation curve after 14 days. (C) Induction of tdTomato cells using IL22-HCM. + Morphological and fluorescence images of hepatocytes after 30 passages of proliferation. (D) Induction of tdTomato cells with IL22-HCM. + Cumulative proliferation curves of hepatocytes after 30 passages. ***P<0.001. (E) Morphological images of newly adherent hepatocytes and hepatocytes induced by HCM, IL22+EGF, IL22+HGF, or IL22-HCM for 14 days. (F) Proliferation curves of hepatocytes in each experimental group after 14 days in Figure E.

[0066] Figure 2 This is a diagram showing the results of IL22-td-iHPCs inducing differentiation into mature hepatocytes (IL22-td-iMHs) in Example 1. (A) Cell morphology and fluorescence images of IL22-td-iMHs (P30). (B) Glycogen storage staining, indocyanine green uptake, lipid droplet staining images of IL22-td-iMHs (P30), and immunofluorescence staining of specific proteins albumin, Hnf4α, Cyp1a2, and Cyp2c9, with primary hepatocytes as a positive control. (C) Drug metabolism capacity of IL22-td-iMHs (P30), with primary hepatocytes as a positive control. (D) Levels of secreted albumin and urea from IL22-td-iMHs (P30), with primary hepatocytes as a positive control.

[0067] Figure 3 This is the IL22-td-iMHs reconstruction of Fah in Example 2. - / - Results of mouse liver transplantation. Among them, (A) Fah cells without transplanted hepatocytes after NTBC feeding was stopped. - / - Mice, Fah, transplanted with primary hepatocytes and transplanted with 30th generation IL22-td-iMHs - / - The weight change curve of mice. (B) Figure A shows the weight changes of each experimental group. - / - Survival curves of mice. (C) Normal mice, Fah without transplanted hepatocytes. - / - Mice (D35) transplanted with primary hepatocytes (Fah) - / - Mice (D63) and Fah mice transplanted with 30th generation IL22-td-iMHs - / -Bright field images of liver tissue, tdTomato fluorescence scan images of liver tissue, and fluorescence scan images of frozen sections of liver tissue from mice (D63). ***P<0.001, ns, no significance. (D) Blood biochemical function analysis of mice in each experimental group. ###P<0.001, ##P<0.01 indicates no difference compared to normal mice. - / - Group comparison. ***P<0.001 indicates a difference compared to Fah without transplanted hepatocytes. - / - Comparison of mice (D35). (E) Fah of each experimental group - / - HE staining image of mouse liver tissue.

[0068] Figure 4 This is the result of in vitro expansion of human primary hepatocytes induced by IL22-EHCM and the functional identification of the expanded human hepatocytes in Example 3. Among them, (A) morphological images of newly adhered human primary hepatocytes and those induced by EHCM or IL22-EHCM for 7 days; (B) proliferation curves of human hepatocytes cultured with EHCM or IL22-EHCM for 7 days; (C) morphological images of human primary hepatocytes after passages 2, 3, 4, and 5 induced by EHCM or IL22-EHCM; (D) cumulative proliferation curves of human primary hepatocytes after passages 5 induced by EHCM or IL22-EHCM; (E) morphological images of newly adhered human primary hepatocytes and those induced by IL22-EHCM after passages 1, 2, 3, 4, and 5 induced by IL22-EHCM; and (F) cumulative proliferation curves of human primary hepatocytes after passages 5 induced by IL22-EHCM. (G) Morphological diagram of IL22-hiHPCs differentiated into mature hepatocytes induced by HMM. (H) Immunofluorescence staining of A1bumin, AAT, and Cyp7a1 in IL22-hiMHs (P5). (I) The ability of IL22-hiMHs (P5) to metabolize testosterone and bupropion, with human primary hepatocytes as a positive control. Detailed Implementation

[0069] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, all percentages and parts are by weight.

[0070] Unless otherwise specified, the methods used in the following examples are conventional methods, and the reagents used are all commercially available.

[0071] Materials and Methods

[0072] 1. Laboratory animals:

[0073] (1) Albumin-Cre mice: Transgenic mice with albumin promoter-driven Cre enzyme gene expression purchased from The Jackson Laboratory in the United States, used to cross with Flox mice for hepatocyte-specific knockout of the target gene.

[0074] (2)Rosa26-LSL-tdTomato(R26R tdTomato Mice: Purchased from The Jackson Laboratory in the United States, a conditionally activated red fluorescent mouse strain carrying the red fluorescent protein reporter gene (tdTomato) expression element.

[0075] (3)Fah - / - Mice: Donated to the laboratory of Hui Lijian, Shanghai Institute of Cell Biology, Chinese Academy of Sciences, these mice have the gene of fumarylacetoacetate hydrolase (Fah) knocked out to evaluate the in vivo therapeutic effect on hepatocytes.

[0076] The laboratory mice were housed in an SPF-grade animal room with constant temperature (22±1℃), constant humidity (60±10%), and 12h light / 12h darkness. The animal room had professional cleaning and disinfection procedures and provided standard laboratory mouse feed and purified drinking water. The animal experiments involved in this project complied with the regulations and requirements of the Animal Ethics Committee of the Shanghai Institute of Materia Medica.

[0077] 2. Cells:

[0078] (1) Primary Albumin-Cre, R26R tdTomato Mouse hepatocytes: In our laboratory, Albumin-Cre transgenic mice were compared with R26R cells. tdTomato Transgenic mouse hybridization, from adult Albumin-Cre, R26R tdTomato Hepatocytes were isolated from mice.

[0079] (2) Primary human hepatocytes: Cryopreserved primary human hepatocytes were purchased from Liwo Biotechnology Co., Ltd. The donor was derived from the transplanted liver waste of a 29-year-old adult male, and the batch number was Lot#386545.

[0080] 3. Reagent preparation:

[0081] (1) Preparation of EGTA solution: Weigh out 43.875g of NaCl (National Pharmaceutical Group Co., Ltd., 10019308), 1.862g of KCl (National Pharmaceutical Group Co., Ltd., 10016308), 10.5g of NaHCO3 (National Pharmaceutical Group Co., Ltd., 10018960), 4.5g of glucose (National Pharmaceutical Group Co., Ltd., 63005518), 23.83g of HEPES (National Pharmaceutical Group Co., Ltd., SH4034500G), and 1.9g of EGTA (National Pharmaceutical Group Co., Ltd., SE388903) in sequence. Make up to 5L with distilled water and stir thoroughly on a magnetic stirrer until the solution is clear. Adjust the pH to 7.3-7.4. Filter through a 0.22μm filter membrane and store at 4℃.

[0082] (2) Preparation of enzyme buffer solution: Weigh out 43.875g of NaCl (National Pharmaceutical Group Co., Ltd., 10019308), 1.862g of KCl (National Pharmaceutical Group Co., Ltd., 10016308), 10.5g of NaHCO3 (National Pharmaceutical Group Co., Ltd., 10018960), 4.5g of glucose (National Pharmaceutical Group Co., Ltd., 63005518), and 11.9g of HEPES (National Pharmaceutical Group Co., Ltd., SH4034500G). Make up the volume to 5L with distilled water and stir thoroughly on a magnetic stirrer for 30 minutes. Then slowly add 1.4g of calcium chloride (National Pharmaceutical Group Co., Ltd., SC330602) and adjust the pH to 7.3-7.4 to obtain the enzyme buffer solution. Filter through a 0.22μm filter membrane and store at 4℃. During hepatocyte isolation, 25 mL of enzyme buffer was taken from each mouse, and 15 mg of type I collagenase (Worthington, LS004197) was added. The buffer was preheated in a 42°C water bath for 20 min before use. The buffer was prepared and used immediately.

[0083] (3) Preparation of insulin-transferrin-selenium (ITS) medium additive: Weigh out 0.1g of insulin (MedChemExpress, HY-P0035), 55mg of transferrin (MedChemExpress, HY-P3267), and 50μg of sodium selenite (MedChemExpress, HY-W686381) in sequence, and make up to 100mL with (1×) PBS buffer solution to fully dissolve them. After filtering through a 0.22μm filter membrane, store at 4℃.

[0084] (4) Preparation of penicillin-streptomycin solution (PS): Weigh 10g of penicillin G sodium salt (Yifei, P3032) and 16.5g of streptomycin (Yifei, 0382) in sequence, dissolve them thoroughly in 825μL of distilled water, filter through a 0.22μm filter membrane and store at 4℃.

[0085] (5) Preparation of trypsin solution: Weigh 2.5g of trypsin (Gibco, 27250-018) and 3.82g of disodium ethylenediaminetetraacetate dihydrate (Guoyao, 10009717) in sequence, make up to 1L with (1×) PBS buffer solution and stir at low speed until fully dissolved. Place in a refrigerator at 4℃ and let stand until the solution is clear. Adjust the pH to 7.2-7.4, filter with a 0.22μm filter membrane and store at 4℃.

[0086] (6) Preparation of PBS (10×) buffer: Weigh out 40g of NaCl (Guoyao, 10019308), 2g of KCl (Guoyao, 10016308), 14.2g of Na2HPO4 (Guoyao, 20040618), and 2.7g of KH2PO4 (Guoyao, 100176008) in sequence. Make up the volume to 1L with distilled water and place it on a magnetic stirrer to stir it thoroughly until the solution is clear. Filter it through a 0.22μm filter membrane and store it at 4℃.

[0087] Example 1: Induction, expansion, and differentiation of primary mouse hepatocytes into mature hepatocytes

[0088] 1. The classic two-step perfusion method for isolating primary mouse hepatocytes

[0089] The specific operating procedure is as follows:

[0090] (1) Preheat mouse primary hepatocytes to EGTA solution and enzyme buffer in a 37℃ water bath.

[0091] (2) Anesthesia of healthy, moderately weighted 8-12 week old Albumin-Cre, R26R tdTomato After the mouse was emptied, the skin and muscle layers were sequentially cut to expose the liver, inferior vena cava, and portal vein. A cannula was inserted and fixed parallel to the inferior vena cava, the portal vein was cut, and the perfusion pump was started.

[0092] (3) EGTA perfusion: Perfuse approximately 5 mL of EGTA solution (to remove blood from the liver) at a rate of 3 mL / min, approximately 5 mL per mouse.

[0093] (4) Digestion: The mice were perfused with a buffer containing 0.6 mg / mL collagenase-I, and each mouse was perfused with 25 mL.

[0094] (5) Liver grinding: After perfusion, the liver was removed and placed in a culture dish. It was then transferred to a clean bench, and an appropriate amount of M199 medium (Gibco, 11150059) was added to the culture dish for rinsing. After removing the gallbladder, the liver was transferred to a new culture dish and digestion termination medium (M199 + 10% FBS) was added. The liver was ground with curved forceps. After thorough grinding, the liver tissue was found to be diffuse. The separated liver tissue was then filtered through a 70 μm filter to obtain the cell suspension.

[0095] (6) Centrifugation: Collect the filtered cell suspension in a 50mL centrifuge tube, centrifuge at 200G for 5min, and discard the supernatant.

[0096] (7) Density gradient centrifugation: Add 4.5 mL of M199 medium, 5 mL of Percoll density gradient centrifugation buffer (Sigma, P4937) and 0.5 mL of (10×) PBS buffer in sequence, resuspend the cells, mix thoroughly by inverting, centrifuge at 400G for 15 min, and the cell pellet at the bottom is the primary hepatocyte.

[0097] 2. IL22-HCM induces expansion of primary mouse hepatocytes

[0098] The specific operating procedure is as follows:

[0099] (1) Cell seeding: Primary hepatocytes isolated in the above steps were seeded in adherent culture medium (DMEM / F12 + 10% FBS + 1% PS) at a concentration of 5 × 10⁻⁶ cells / mL. 4 They were seeded at a density of 1 / mL in pre-coated gelatin-coated cell culture plates.

[0100] (2) Inducing mouse primary hepatocytes (td-Hepa) to reprogram into hepatic progenitor cells (IL22-td-iHPCs) and expanding them in vitro: After the cells adhered, they were cultured in mouse primary hepatocyte expansion medium containing IL-22 (named IL22-HCM, containing IL-22+HGF+EGF), or mouse primary hepatocyte expansion medium without IL-22 (HCM, containing HGF+EGF, as a control), or IL-22+HGF mouse primary hepatocyte expansion medium, or IL-22+EGF mouse primary hepatocyte expansion medium, and IL-22+EGF mouse primary hepatocyte expansion medium. After 14 days of induction culture, proliferating hepatic progenitor cells (IL22-td-iHPCs) were obtained (expanded by about 4 times). The IL22-HCM culture medium consisted of: 1×DMEM-F12 (Gibco, 11320033), 1% (v / v) Insulin-Transferrin-Selenium (ITS), 20 ng / mL hepatocyte growth factor (ABclonal, RP01584), 20 ng / mL epidermal growth factor (Sigma, E9644), 30 ng / mL IL-22 (ABclonal, RP02942), 10 - 7 M dexamethasone (MedChemExpress, HY-14648), 1% (v / v) penicillin-streptomycin (PS); HCM medium was identical to IL22-HCM medium except for the absence of IL-22. Culture conditions: incubated at 37°C and 5% CO2.

[0101] Furthermore, to further compare the effects of different added components on inducing the reprogramming of mouse primary hepatocytes (td-Hepa) into hepatic progenitor cells (IL22-td-iHPCs) and their in vitro expansion, the methods described in steps (1) and (2) were followed, with the only difference being the mouse primary hepatocyte expansion medium used in step (2). The media used included: IL22-HCM medium, HCM medium, IL-22+HGF medium, or IL-22+EGF medium. Among them, the IL-22+HGF medium was identical to the IL22-HCM medium except that it did not contain EGF; the IL-22+EGF medium was identical to the IL22-HCM medium except that it did not contain HGF.

[0102] tdTomato (newly pasted on the wall) + Hepatocytes (D0) and tdTomato cells induced by HCM, IL22-HCM, IL-22+EGF or IL-22+HGF culture medium.+ Cell morphology and fluorescence images of hepatocytes after 14 days of proliferation are shown below. Figure 1 Figures (A and E) show that HCM medium cannot induce hepatocyte expansion in vitro. During induction with IL-22-HCM, IL-22+EGF, or IL-22+HGF media, hepatocyte morphology underwent significant changes, with smaller cell volume and increased nucleocytoplasmic ratio. The proliferation curves after 14 days of induction are shown in Figures (A and E). Figure 1 (Figures B and F show different batches of cell experiments), indicating that mouse primary hepatocytes could be increased from 2x10⁻¹⁰ cells within 14 days. 4 Amplification exceeding 8x10 4 Furthermore, compared to IL-22+EGF or IL-22+HGF culture media, IL22-HCM is more conducive to achieving efficient expansion of hepatocytes in vitro.

[0103] (3) Induction of stable long-term in vitro expansion of IL22-td-iHPCs: When IL22-td-iHPCs reached 90% or more confluence (approximately 14 days), the cells were digested with trypsin solution, resuspended in twice the volume of digestion stop solution (DMEM / F12 + 10% FBS + 1% PS), centrifuged at 200G for 3 min, and the supernatant was discarded. The cells were resuspended in IL22-HCM medium and counted using an automated cell counter. Next, cells were expanded at 1×10⁻⁶ cells / year. 5 Cells per well were added to 6-well plates pretreated with 1% collagen I for subculturing. Long-term expansion was continued using IL22-HCM medium, with the medium changed every 2 days and subculturing approximately every 5-7 days, allowing for over 30 passages. IL22-HCM was used to induce tdTomato... + Cell morphology and fluorescence images of hepatocytes after 30 passages of proliferation are shown below. Figure 1 In the middle C, it was shown that the morphology of cells cultured continuously for more than 30 generations did not change significantly; IL22-HCM was used to induce tdTomato + The cumulative proliferation curve of hepatocytes after 30 passages is shown below. Figure 1 As shown in Figure D, the proliferation rate of IL22-td-iHPCs is stable, with passage approximately every 5-7 days, and can expand to approximately 10^ ... 25 Times, that is, from 2.5 x 10 4 Amplified to 2x10 30 .

[0104] 3. IL-22-td-iHPCs induce differentiation into mature hepatocytes (IL-22-induced mature hepatocytes, IL22-td-iMHs).

[0105] The specific operating procedure is as follows:

[0106] When IL22-td-iHPCs, after 30 passages of long-term in vitro culture, reached 70-80% confluence, they were induced to differentiate into mature hepatocytes (IL22-td-iMHs) using hepatocyte maturation medium (HMM). The differentiation time was 7 days, with the culture medium being changed every two days, ultimately yielding L22-td-iMHs. The HMM medium consisted of: 1×DMEM-F12 (Gibco, 11320033), 1% (v / v) Insulin-Transferrin-Selenium (ITS), 20 ng / mL human inhibin-M (hOSM) (Novoprotein, C099), 10 -6 M dexamethasone (MedChemExpress, HY-14648), 1% (v / v) penicillin-streptomycin (PS). Culture conditions: Incubate at 37°C and 5% CO2.

[0107] like Figure 2 As shown in the cell morphology diagram, the IL22-td-iMHs (P30) cells exhibit a tightly packed, cobblestone-like morphology, which is essentially similar to freshly isolated primary hepatocytes.

[0108] The obtained IL22-td-iMHs were subjected to glycogen storage staining, indocyanine green uptake assay, lipid droplet staining, and immunofluorescence staining for specific proteins Albumin, Hnf4α, Cyp1a2, and Cyp2c9, respectively. The specific methods are as follows.

[0109] Glycogen storage staining experiments were performed using the Periodic Acid-Schiffstain (PAS) staining kit (product number: G1008-100mL) from Sewell Biotech. The specific steps were as follows: Cells were fixed in 4% paraformaldehyde (PFA) solution for 10 min and washed three times with ddH2O. Cells were then stained with PAS staining solution B (0.5% periodic acid solution) for 15 min and washed three times with ddH2O. Cells were then stained with PAS staining solution A (Schiff reagent, stored at 4℃, brought to room temperature before use) in the dark for 30 min and washed three times with ddH2O. Cell nuclei were stained with PAS staining solution C (hematoxylin stain) for 30 s and washed three times with ddH2O. After the experiment, cells were observed under a bright-field microscope. White balance was corrected using a blank well, and a suitable field of view was selected for photographing.

[0110] Indocyanine green uptake (ICG uptake) assay was performed using indocyanine green (product number: 1340009) from Sigma-Aldrich, USA. The specific steps were as follows: Indocyanine green stock solution (100 mg / mL, stored at 4°C) was diluted to 1 mg / mL with fresh cell culture medium. The cell culture medium was replaced with the diluted stain, and the cells were incubated at 37°C for 1 hour. The stain was discarded, and the cells were fixed with 4% PFA solution for 10 minutes, followed by washing three times with PBS for 5 minutes each time. After the experiment, the cells were observed and photographed under a microscope.

[0111] Oil Red staining was performed using Oil Red O (product number: O0625) from Sigma-Aldrich. The specific steps were as follows: Oil Red stock solution (0.25 g Oil Red O powder dissolved in 50 mL isopropanol, stored at room temperature and protected from light) and ddH2O were mixed at a ratio of 3:2 (v / v) and filtered through a 0.45 μm filter. Cells were fixed with 4% PFA for 10 min, washed three times with PBS, with 5 min intervals between each wash. After washing once with 60% isopropanol, Oil Red dye was added, and the cells were incubated at room temperature and protected from light for 10 min, followed by three washes with PBS. Hematoxylin staining solution was added, and the stain was immediately aspirated, followed by three washes with PBS. After the experiment, the cells were observed and photographed under a microscope.

[0112] For protein immunofluorescence staining, cells were first fixed with 4% PFA solution for 10 min, followed by washing three times with PBS, with a 5-min interval between each wash. For cytoplasmic or nuclear staining, the cell membrane was permeated with PBS containing 0.33% Triton (Sinopharm, 30188928) for 30 min, followed by washing three times with PBS, with a 5-min interval between each wash. Cells were then blocked with PBS containing 5% BSA (to prevent binding of the primary antibody to non-target proteins) for 1 h, followed by washing three times with PBS, with a 5-min interval between each wash. The primary antibody was diluted with PBS and added to the cells, then incubated overnight at 4°C. The next day, cells were removed and washed three times with PBS, with a 5-min interval between each wash. The corresponding secondary antibody was diluted with PBS and incubated at room temperature in the dark for 1 h, followed by washing three times with PBS. 5 mg / mL Hoechst 33342 (Invitrogen, 62249) staining solution was diluted 1:500 (v / v) with PBS, incubated at room temperature in the dark for 30 min, followed by washing three times with PBS, with a 5-min interval between each wash. After the experiment, the cells were observed and images were acquired under a fluorescence microscope. The antibody information used is as follows:

[0113]

[0114]

[0115] The results are as follows Figure 2The results showed that IL22-td-iMHs possess hepatocyte-related functions such as glycogen storage, indocyanine green uptake, and lipid droplet production, and can stably and highly express hepatocyte-specific proteins albumin (Albumin), hepatocyte nuclear factor (Hnf4α), and cytochrome P450 enzymes Cyp1a2 and Cyp2c9. IL22-td-iMHs also exhibit typical hepatocyte drug metabolism capabilities, as well as the ability to secrete albumin and urea, such as... Figure 2 C and Figure 2 As shown in D.

[0116] The obtained IL22-td-iMHs were subjected to drug metabolism tests and functional tests for albumin and urea secretion, as follows.

[0117] The albumin secretion assay was performed using the Mouse Albumin ELISA Kit (product number: E99-134) provided by Bethyl Laboratories. The specific steps were as follows: The test cells and newly adherent primary hepatocytes were replaced with fresh DMEM-F12 + 10% FBS medium and cultured for 24 hours. The culture medium was then collected, centrifuged at 12000G for 10 minutes, and the culture supernatant was collected. Cells were fixed with 4% PFA solution for 10 minutes, and cell nuclei were counted using Hoechst 33342 staining solution. The procedure was performed according to the instructions provided with the Mouse Albumin ELISA Kit. The absorbance of the standards and test samples was measured at 450 nm and 570 nm using a microplate reader. A standard curve was generated based on the concentration of the standards and the corresponding absorbance, allowing the albumin concentration in the original sample to be calculated from the absorbance of the test sample. The albumin secretion concentration (unit: μg / day / million cells) was calculated based on the number of hepatocytes corresponding to each sample.

[0118] The urea level assay was performed using the Urea Assay Kit (product number: MAK006) provided by Sigma-Aldrich, USA. The specific steps were as follows: The test cells and newly adherent primary hepatocytes were replaced with fresh culture medium and cultured for 24 hours. The culture medium was then collected, centrifuged at 12000G for 10 minutes, and the culture supernatant was collected. Cells were fixed with 4% PFA solution for 10 minutes, and cell nuclei were stained with Hoechst 33342 for cell counting. The reaction mixture was prepared according to the instructions of the Urea Assay Kit and incubated at 37°C in the dark for 60 minutes. The absorbance of the standards and test samples was measured at 570 nm using a microplate reader. A standard curve was generated based on the concentration of the standards and the corresponding absorbance, allowing the calculation of the albumin concentration in the original sample from the absorbance of the test sample. The concentration of urea synthesis (unit: μg / day / million cells) was calculated based on the number of hepatocytes corresponding to each sample.

[0119] The results are as follows Figure 2 As shown in C and D, IL22-td-iMHs (P30) have drug metabolism capabilities, albumin secretion, and urea secretion functions similar to those of primary hepatocytes.

[0120] Example 2. Treatment of Fah with IL22-td-iMHs - / - Liver injury in mice

[0121] Fah - / - In mice, the absence of the fumarylacetoacetate hydrolase (Fah) gene leads to the accumulation of toxic tyrosine metabolites in hepatocytes, resulting in widespread and persistent damage to hepatocytes. - / - Mice need to be given the drug niticinone (2-[2-nitro-4-trifluoromethylbenzoyl]-1,3-cyclohexanedione, NTBC) to maintain normal survival. After NTBC is withdrawn, their lives can be saved through hepatocyte transplantation.

[0122] The specific operating procedure is as follows:

[0123] (1) td-Hepa cells were induced to proliferate for 14 days with IL22-HCM medium, and then the cells were cultured at 5×10⁻⁶ cells / day. 4 / mL was seeded into cell culture dishes pre-coated with Collagen-I, and cultured for 30 generations using IL22-HCM. When the IL22-td-iHPCs reached 70-80% confluence, differentiation was induced using HMM medium for 7 days to obtain IL22-td-iMHs.

[0124] (2) Cell preparation: Freshly isolated hepatocytes (td-Hepa) or IL22-td-iMHs hepatocyte suspension obtained after digestion were added to deoxyribonuclease I (DNase I) (Sigma, 10104159001), mixed, and allowed to stand for 5 min. Impurities and residual digestive enzymes were removed by low-speed centrifugation (800 rpm, 3 min). The cell concentration was adjusted to 1×10⁻⁶ cells / mL. 7 (cells / mL) were suspended in 0.9% sodium chloride solution and transferred on ice to an ultra-clean bench in an SPF-grade animal facility.

[0125] (3) Cell transplantation: This experiment selected 8-12 week old Fah cells. - / - Hepatocyte transplantation experiments were conducted on mice. Before the experiment, mice were fed drinking water containing 7.5 mg / L NTCb and ensured they showed no signs of disease. - / - Mice require the addition of the drug NTBC to their drinking water to maintain normal survival. This is achieved by using 1.25% tribromoethanol to treat Fah... - / - After anesthetizing the mice, an incision was made under the left rib to expose the spleen, and the distal end of the spleen was ligated with surgical suture approximately 1 cm. A 2.5 × 10⁻⁶ splenic tube was injected using a micro-syringe. 6 A hepatocyte suspension (approximately 0.25 mL) was slowly and evenly injected into the spleen parenchyma of the mice, avoiding puncture of the spleen capsule. After injection, the spleen was ligated again to ensure the hepatocyte suspension remained within the spleen. Finally, the abdominal wound of the mice was sutured layer by layer using sterile sutures. A control group was established, which was identical to the experimental group in all aspects except that it did not receive hepatocyte transplantation.

[0126] (4) After surgery, the mice were returned to the SPF-grade animal facility for rearing, maintaining a constant temperature, humidity, and sterile environment. NTBCs were removed from the drinking water, and the mice's survival status was observed weekly, with changes in body weight recorded. Fah was harvested 8-10 weeks after transplantation. - / - Mouse liver samples were analyzed to detect Fah by observing the expression of tdTomato fluorescence signal in transplanted hepatocytes. - / - The remodeling status of mouse liver was assessed using multiple serum biochemical indicators and HE staining to detect Fah. - / - The extent of liver damage in mice.

[0127] Fah after hepatocyte transplantation - / -The specific method for determining liver injury indicators in mice is as follows: Prepare blood collection tubes containing an appropriate amount of anticoagulant (1% heparin sodium) in advance, and then... (The sentence is incomplete and requires more context to translate accurately.) - / - Blood was collected from Fah- / - mice in the non-hepatocyte transplanted group on day 35 post-experiment, and blood was collected from Fah- / - mice in the hepatocyte transplanted group on day 63 post-experiment. - / - Mouse blood. Sterile capillary glass tubes were used as blood collection tubes. The mice were immobilized and the sides of their necks were gently compressed to obstruct venous return, causing the eyeballs to protrude fully. The tip of the blood collection tube was inserted between the inner corner of the eye and the eyeball, gently protruding towards the fundus. The tube was rotated to cut the venous plexus, allowing blood to flow out. Approximately 200 μL of blood was collected and immediately inverted to thoroughly mix the anticoagulant with the blood. The blood was centrifuged at 12000g for 10 minutes at 4°C. The supernatant was collected as the plasma to be tested and stored at -80°C. After all blood samples were collected, they were sent to the Drug Safety Evaluation Research Center of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, for blood biochemical analysis, including the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and total bilirubin (TBIL).

[0128] The specific method for immunohistochemical HE staining of liver tissue is as follows: After ex vivo, liver tissue is rapidly fixed in 4% polyfluoroalkoxy (PFA) solution for 24 hours. The tissue is then dehydrated sequentially with ethanol solutions of increasing concentrations (50%, 75%, 85%, 95%, 100%) and treated three times with xylene. Paraffin wax is then used for impregnation with paraffin wax having a melting point of 52-60℃. Sections with a thickness of 5 μm are cut at 15 μm intervals after each treatment and stored at room temperature after preparation.

[0129] For staining, paraffin sections were pre-dewaxed in a 65℃ oven for 2 hours. The sections were then quickly transferred and immersed in a paraffin section storage box containing xylene for 20 minutes, followed by sequential immersion in xylene for 20 minutes, 50% xylene and 50% ethanol for 15 minutes, 100% ethanol for 15 minutes, 100% ethanol for 5 minutes, 95% ethanol for 5 minutes, 90% ethanol for 5 minutes, 85% ethanol for 5 minutes, 80% ethanol for 5 minutes, 75% ethanol for 5 minutes, 50% ethanol for 5 minutes, 30% ethanol for 5 minutes, and pure water for 5 minutes. Hematoxylin was then stained for 10 minutes, followed by dehydration treatment with sequential immersion in 80% ethanol for 5 minutes, 90% ethanol for 5 minutes, and 95% ethanol for 5 minutes. Finally, eosin was stained for 5 minutes, and the sections were washed with pure water to remove the stain. The paraffin sections were then sequentially placed in 90% ethanol for 5 minutes, 95% ethanol for 5 minutes, 100% ethanol for 5 minutes, 50% xylene and 50% ethanol for 10 minutes, and xylene for 10 minutes to dehydrate and make them transparent. Finally, they were mounted with neutral resin and allowed to air dry naturally at room temperature in the dark to obtain the finished product.

[0130] The results are as follows Figure 3 As shown, after 8 weeks, IL22-td-iMHs(P30) can completely reconstruct Fah. - / - Mouse liver, Fah - / - The mice regained their pre-transplant weight, Fah - / - The survival rate of mice can reach over 70%, while that of mice without transplanted hepatocytes is significantly higher. - / - The mice continued to lose weight and all died after about a month. Figure 3 (A and B in the middle).

[0131] The in vivo therapeutic effect of IL22-td-iMHs (P30) is comparable to that of primary hepatocytes. IL22-td-iMHs (P30) can remodel Fah - / - In mice, over 95% of the liver was reconstructed, and the liver morphology remained largely normal. Figure 3 (C)

[0132] IL22-td-iMHs (P30) can significantly reduce Fah - / - The levels of liver injury markers such as alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), and total bilirubin (TBIL) in mouse liver were measured, and the normal tissue structure of the liver was restored. Figure 3 (D and E).

[0133] Example 3. Induction, expansion, and differentiation of primary human hepatocytes into mature hepatocytes

[0134] 1. Resuscitation and culture of primary human hepatocytes

[0135] The specific operating procedure is as follows:

[0136] (1) Cell resuscitation: The primary human hepatocytes used in this invention are cryopreserved hepatocytes obtained from discarded liver transplant tissues of healthy adult males from regular hospitals. All materials have clear origins, and informed consent was obtained from the patient or their family. The specific steps for human hepatocyte resuscitation are as follows: 10 mL of resuscitation medium (Liver Biotechnology (Shenzhen), Co., Ltd., LV-Rec001) is added to a 15 mL centrifuge tube, preheated in a 37°C water bath for 20 min, and then transferred to a biosafety cabinet. The plate culture medium is preheated in a 37°C water bath. After removing the cryopreserved hepatocytes from liquid nitrogen, they are quickly transferred to a 37°C water bath and thawed by rotating clockwise for approximately 90 seconds. At this point, only small pieces of ice float in the cryopreservation tube. The cryopreservation tube is disinfected with 75% alcohol and then transferred to a biosafety cabinet. The cells are added dropwise to a centrifuge tube containing resuscitation medium, and gently inverted 2-3 times to mix. Centrifuged at 800 rpm for 5 min, and the supernatant is removed.

[0137] (2) Cell seeding: Add 4 mL of plating medium: 1×DMEM-F12 (Gibco, 11320033), 10% (v / v) Fetal Bovine Serum (FBS) (Gibco, 10099-141C), 10 μM Y-27632 (MedChemExpress, HY-10071), 1% (v / v) Penicillin-Streptomycin (PS). Resuspend and count the cells. Spread hepatocytes at 5×10⁻⁶ cells / mL. 4 Add cells per well to a density of 1% collagen I pretreated 24-well plates, mix well, and incubate at 37°C in a 5% CO2, 5% O2 incubator.

[0138] (3) Inducing human primary hepatocytes to reprogram into hepatic progenitor cells for in vitro expansion:

[0139] After 24 hours of cell adhesion, the culture medium was changed to human hepatocyte expansion medium containing IL-22 (named IL22-EHCM) or human hepatocyte expansion medium without IL-22 (EHCM, as a control) for continued culture. The culture medium was changed every 2 days to induce human primary hepatocytes into proliferating human hepatic progenitor cells (IL-22-induced human hepatic progenitor cells, IL22-hiHPCs). The IL22-EHCM medium consisted of: 0.5×HCM Bullet Kit (Lonza, CC-3198) (containing epidermal growth factor), 0.5×EGM-2 Bullet Kit (Lonza, CC-3162) (containing epidermal growth factor), 20 ng / mL hepatocyte growth factor (ABclonal, RP01602), 100 ng / mL IL-22 (ABclonal, RP01616), 5 μM A83-01 (MedChemExpress, HY-10432), 10 μM Y-27632 (MedChemExpress, HY-10071), 0.1% (v / v) Bovine Serum Albumin (BSA) (MP Biomedicals, 02199896), and 1% (v / v) Penicillin-Streptomycin (PS); the EHCM medium consisted of: 0.5×HCM Bullet Kit... Kit (Lonza, CC-3198) (containing epidermal growth factor), 0.5×EGM-2 Bullet Kit (Lonza, CC-3162) (containing epidermal growth factor), 20 ng / mL hepatocyte growth factor (ABclonal, RP01602), 0.1% (v / v) Bovine Serum Albumin (BSA) (MP Biomedicals, 02199896), 1% (v / v) Penicillin-Streptomycin (PS).

[0140] Alternatively, after 24 hours of adhesion, the culture medium is replaced with human hepatocyte expansion medium containing IL-22 (named IL22-OEHCM), and cultured for another 2 days, changing the culture medium every 2 days, in order to induce human primary hepatocytes into proliferating human hepatic progenitor cells (IL-22-induced human hepatic progenitor cells, IL22-hiHPCs). The IL22-OEHCM medium consisted of: 1×DMEM-F12 (Gibco, 11320033), 1×B-27 supplement (Gibco, 17504044), 150 μM ascorbic acid (MedChemExpress, HY-B0166), 20 ng / mL epidermal growth factor (Sigma, E9644), 10 ng / mL fibroblast growth factor 2 (R&D Systems, 3718-FB), 20 ng / mL vascular endothelial growth factor (R&D Systems, 293-VE), 50 ng / mL insulin-like growth factor 1 (R&D Systems, 291-G1), 5 μM hydrocortisone (MedChemExpress, HY-N0583), 5 μM A83-01 (MedChemExpress, HY-10432), and 10 μM... Y-27632 (MedChemExpress, HY-10071), 100 ng / mL IL-22 (ABclonal, RP01616), 20 ng / mL hepatocyte growth factor (ABclonal, RP01602), 0.1% (v / v) Bovine Serum Albumin (BSA), 1% (v / v) Penicilin-Streptomycin (PS).

[0141] The morphology of newly adherent human primary hepatocytes and human primary hepatocytes induced by EHCM or IL22-EHCM proliferation for 7 days is as follows: Figure 4 As shown in Figure A, IL22-EHCM induced a significant morphological change in hepatocytes, with smaller cell volume and an increased nucleocytoplasmic ratio. After 7 days of induction, the number of hepatocytes increased from 1 x 102 4 Amplified to 10x10 4 It increased by about 10 times. Figure 4 (B)

[0142] (4) When the IL22-hiHPCs have reached 90% or more confluence, digest the cells with trypsin, resuspend them in twice the volume of digestion stop solution (DMEM / F12 + 10% FBS + 1% PS), centrifuge at 200G for 5 min, and discard the supernatant. Resuspend the cells in IL22-EHCM medium and count them using an automated cell counter. Then, divide the cells into groups of 1×10⁻⁶ cells. 5 Cells were added at a density of 100 cells / well to 6-well plates pretreated with 1% collagen I for subculturing and continued to culture in IL22-EHCM medium. The culture medium was changed every two days, and the cells were subcultured every 7 days.

[0143] The amplified IL22-hiHPCs can be continuously passaged for more than 5 generations without significant changes in cell morphology. Figure 4 In C), IL22-hiHPCs amplify approximately 10 within about 35 days. 4 Times, that is, from 1.5 x 10 4 Amplified to 1.8x10 8 ( Figure 4 (D). Further experiments using IL22-OEHCM to induce newly adherent human primary hepatocytes also showed similar proliferative effects. Figure 4 (E and F in the middle).

[0144] 2. IL22-hiHPCs are induced to differentiate into IL22-hiMHs

[0145] The specific operating procedure is as follows:

[0146] When the IL22-hiHPCs, after being expanded to the 5th generation, reached a confluence of 70-80%, differentiation was induced using hepatocyte maturation medium (HMM). Differentiation took 7 days, with the culture medium changed every two days, ultimately yielding matured hepatocytes (IL22-hiMHs). The HMM medium consisted of: 1×DMEM / F12 (Gibco, 11320033), 1×B-27 additive, 20 ng / mL human inhibin-M (hOSM) (Novoprotein, C099), and 10... -6 MDexamethasone (MedChemExpress, HY-14648), 1% (v / v) Penicillin-Streptomycin (PS); Culture conditions: cultured at 37℃, 5% CO2, and 5% O2.

[0147] like Figure 4As shown in the morphology diagram of G cells, the cells redifferentiated from IL22-hiHPCs after five generations of expansion using HMM medium are closely arranged cobblestone-like cells, consistent with the morphology of mature hepatocytes.

[0148] The expression of hepatocyte secretory proteins (A1bumin), α-trypsin (AAT), and cytochrome P450 (Cyp7a1) in induced mature IL22-hiMHs, as well as drug metabolism (testosterone and bupropion), were tested. The specific method was as follows: First, cells were fixed with 4% paraformaldehyde (PFA) solution for 10 min, followed by washing three times with PBS, with 5 min intervals between each wash. For cytoplasmic or nuclear staining, the cell membrane was permeated with PBS solution containing 0.33% Triton for 30 min, followed by washing three times with PBS, with 5 min intervals between each wash. Cells were blocked with PBS solution containing 5% BSA (to prevent the primary antibody from binding to non-target proteins) for 1 h, followed by washing three times with PBS, with 5 min intervals between each wash. The primary antibody was diluted with PBS and added to the cells, and incubated overnight at 4°C. The next day, the cells were removed, washed three times with PBS, with 5 min intervals between each wash. The corresponding secondary antibody was diluted with PBS and incubated at room temperature in the dark for 1 h, followed by washing three times with PBS. Hoechst 33342 (5 mg / mL) staining solution was diluted 1:500 (v / v) with PBS solution and incubated at room temperature in the dark for 30 min. The solution was then washed three times with PBS, with 5 min intervals between each wash. After the experiment, the solution was observed and images were acquired under a fluorescence microscope.

[0149]

[0150] The results are as follows Figure 4 In the HI, IL22-hiMHs showed high expression of hepatocyte-specific proteins Hnf4α, Albumin, Cyp7a1, and AAT (e.g., ...). Figure 4 (H). IL22-hiMHs also possess the typical drug metabolism capabilities of hepatocytes (H). Figure 4 Middle I).

Claims

1. A method for culturing and expanding primary hepatocytes in vitro, comprising: Proliferating hepatic progenitor cells were prepared by inducing primary hepatocytes with a culture medium containing interleukin-22, combined with hepatocyte growth factor and / or epidermal growth factor.

2. The method according to claim 1, characterized in that, The primary hepatocytes are mammalian primary hepatocytes, preferably including mouse primary hepatocytes and human primary hepatocytes.

3. The method according to claim 2, characterized in that, The primary hepatocytes are mouse primary hepatocytes, and the culture medium is a culture medium for inducing mouse primary hepatocytes to prepare proliferating liver progenitor cells and includes: liquid basal medium, cell culture nutrient supplements, interleukin-22, hepatocyte growth factor and / or epidermal growth factor, and glucocorticoids. Preferably, the culture medium further includes an antibacterial agent, such as a 1% penicillin-streptomycin solution by volume; More preferably, in the culture medium used to induce the preparation of proliferating hepatic progenitor cells from mouse primary hepatocytes: The liquid basal medium is selected from one or more of the following: DMEM-F12 medium, DMEM medium, William's E medium, Advanced DMEM-F12 medium, HCM medium, and RPMI 1640 medium; and / or The cell culture nutrient additive is selected from one or more of the following: insulin-transferrin-selenium medium additive, B27 additive, N2 additive, fetal bovine serum, and bovine serum albumin; and / or The glucocorticoid is selected from: dexamethasone, hydrocortisone, or a combination thereof; More preferably, the culture medium used to induce the preparation of proliferating hepatic progenitor cells from mouse primary hepatocytes comprises the following components: 1×DMEM-F12, 1% (v / v) insulin-transferrin-selenium culture medium additive, 0.1-1000 ng / mL interleukin-22 (IL-22) (preferably 10-100 ng / mL, more preferably 30 ng / mL), 10 -7 M dexamethasone, 0.1% bovine serum albumin (V / v), 1% penicillin-streptomycin solution, and one or two of the following: 0.1-1000 ng / mL hepatocyte growth factor (preferably 10-100 ng / mL, more preferably 20 ng / mL) and 0.1-1000 ng / mL epidermal growth factor (EGF) (preferably 10-100 ng / mL, more preferably 20 ng / mL); preferably, the culture medium contains both hepatocyte growth factor and epidermal growth factor.

4. The method according to claim 2, characterized in that, The primary hepatocytes are human primary hepatocytes, and the culture medium is a culture medium for inducing human primary hepatocytes to prepare proliferating liver progenitor cells and includes: liquid basal culture medium, cell culture nutrient supplements, interleukin-22, hepatocyte growth factor, epidermal growth factor, cell stemness maintenance factor, proliferative growth factor, and glucocorticoids. Preferably, the culture medium further includes an antibacterial agent, such as a 1% (v / v) penicillin-streptomycin solution; and / or More preferably, in the culture medium used to induce the preparation of proliferating hepatic progenitor cells from human primary hepatocytes: The liquid basal medium is selected from one or more of the following: DMEM-F12 medium, DMEM medium, William's E medium, Advanced DMEM-F12 medium, RPMI1640 medium, and commercially available media specifically for hepatocyte culture: Hepatocyte Culture Medium Bullet Kit, Endothelial Cell Growth Medium Bullet Kit; and / or The cell culture nutrient additives are selected from one or more of the following: insulin-transferrin-selenium medium additive, B27 additive, N2 additive, ascorbic acid, fetal bovine serum, and bovine serum albumin; and / or The cell stemness maintenance factor is selected from one or more of the following: transforming growth factor-β receptor inhibitor A 83-01 and Rho kinase inhibitor Y-27632; The growth factor that promotes proliferation is selected from one or more of the following: fibroblast growth factor 2, vascular endothelial growth factor, and insulin-like growth factor 1. More preferably, the culture medium used to induce the preparation of proliferating hepatic progenitor cells from human primary hepatocytes comprises the following components: 1×DMEM-F12, 1×B-27 additive, 150μM ascorbic acid, 10ng / mL fibroblast growth factor 2, 20ng / mL vascular endothelial growth factor, 50ng / mL insulin-like growth factor 1, 5μM hydrocortisone, 0.1-1000ng / mL interleukin-22 (preferably 10-300ng / mL, more preferably 100ng / mL), 0.1-1000ng / mL hepatocyte growth factor (preferably 10-100ng / mL, more preferably 20ng / mL), 0.1-1000ng / mL epidermal growth factor (preferably 10-100ng / mL, more preferably 20ng / mL), 5μM A83-01, 10μM Y-27632, 0.1% bovine serum albumin by volume, and 1% penicillin-streptomycin solution by volume; Alternatively, the culture medium used to induce the preparation of proliferating hepatic progenitor cells from human primary hepatocytes comprises the following components: 0.5×Hepatocyte Culture Medium Bullet Kit, 0.5×Endothelial Cell Growth Medium Bullet Kit, 0.1-1000 ng / mL interleukin-22 (IL-22) (preferably 10-300 ng / mL, more preferably 100 ng / mL), 0.1-1000 ng / mL hepatocyte growth factor (HGF) (preferably 10-100 ng / mL, more preferably 20 ng / mL), 5 μM A83-01, 10 μM Y-27632, 0.1% bovine serum albumin (v / v), and 1% penicillin-streptomycin solution (v / v).

5. The method according to any one of claims 1-4, characterized in that, The method for culturing and expanding primary hepatocytes in vitro further includes the step of inducing the hepatic progenitor cells to differentiate into mature hepatocytes using a hepatocyte maturation induction medium.

6. The method according to claim 5, characterized in that, The hepatocyte maturation induction culture medium includes: liquid basal culture medium, cell culture nutrient supplements, human inhibin-M, and glucocorticoids; Preferably, the liquid basal culture medium is selected from one or more of the following: DMEM-F12 medium, DMEM medium, William's E medium, Advanced DMEM-F12 medium, HCM medium, and RPMI 1640 medium; The cell culture nutrient additives are selected from one or more of the following: insulin-transferrin-selenium (ITS), B27 additive, N2 additive, fetal bovine serum, and bovine serum albumin. The glucocorticoid is selected from dexamethasone, hydrocortisone, or a combination thereof.

7. The method according to claim 6, characterized in that, For human hepatocytes, the hepatocyte maturation induction medium comprises: 1×DMEM-F12, 1% (v / v) insulin-transferrin-selenium medium additive, 20 ng / mL human inhibin-M, 10 -6 M dexamethasone, 1% penicillin-streptomycin solution (by volume).

8. Liver progenitor cells prepared by the method according to any one of claims 1-4, or mature hepatocytes prepared by the method according to any one of claims 5-7.

9. The use of hepatic progenitor cells prepared by the method of any one of claims 1-4, or mature hepatocytes prepared by the method of any one of claims 5-7, in the preparation of products for the treatment of liver diseases (e.g., hepatitis, cirrhosis, liver cancer), hepatocyte transplantation, or hepatocyte therapy.

10. The use of hepatic progenitor cells prepared by the method of any one of claims 1-4, or mature hepatocytes prepared by the method of any one of claims 5-7, in drug toxicity evaluation, new drug screening, and basic research related to liver diseases.

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