Preparation method of liver organ containing interstitial substance

Abstract

The invention discloses a method for inducing a liver organoid containing interstitial substance, and belongs to the technical field of organoid culture. The method comprises the following steps: (1) pretreating human embryonic stem cells WIBR3; (2) inducing liver organs and mesenchymal cells by using the human embryonic stem cells WIBR3; and (3) co-culturing the mesenchymal cells and liver organ cells to obtain the mesenchymal-containing liver organs. The method has the beneficial effect that the culture mode and method of the liver organ containing the interstitial substance are changed. The mesenchymal-containing liver organ induction method provided by the invention is simple, interstitial cells and other liver cells have the same genetic background source, and the mesenchymal-containing liver organ induction method is suitable for safety evaluation of chemical substances such as drugs, modeling of hepatic fibrosis and non-alcoholic hepatitis and efficacy evaluation.

Description

Technical Field

[0001] The present invention belongs to the technical field of organoid culture, and in particular relates to a method for preparing a liver organoid containing stroma. Background Art

[0002] The lack of suitable animal or cell models has hampered theoretical understanding, drug and treatment discovery of liver diseases. Primary human hepatocytes retain many of the characteristics of hepatocytes, but can only survive for a few days in culture, so fresh tissue is constantly required and they lack the complex structure of the human liver. Cell lines from liver cancer and hepatocellular carcinoma can proliferate indefinitely, but they are cancerous and lack key features of normal liver cells. Therefore, drug discovery for diseases such as liver fibrosis and non-alcoholic liver disease has always been difficult, and organoids are expected to serve as a powerful tool for constructing liver fibrosis or non-alcoholic liver disease.

[0003] Since the occurrence and development of these diseases are inseparable from the presence of hepatic mesenchymal cells (such as stellate cells), unlike hepatocytes and bile duct cells, which originate from the endoderm, hepatic mesenchymal cells originate from the lineage differentiation of the mesoderm. However, the source of mesenchymal cells is unstable, and the huge developmental differences with liver organoids hinder the co-culture, resulting in poor morphology of liver organoids containing mesenchyme and low culture efficiency.

[0004] The Chinese patent application document with publication number CN117701492A discloses a method for constructing liver organoids and their application. The liver organoids constructed in this patent contain hepatocytes and non-parenchymal cells, and can maintain typical liver functions, have the ability to transplant, survive and repair damaged liver tissue in mice, can well simulate fatty degeneration and inflammatory response, and simulate the pathological process and mechanism of ALD, and have broad application prospects in clinical practice. However, the efficiency and stability of the cultivation of liver organoids in this patent are poor, so it needs to be further improved. Summary of the invention

[0005] The technical problem to be solved by the present invention is how to solve the problems of poor stability and low culture efficiency in the existing culture of mesenchymal liver organoids.

[0006] The present invention solves the above technical problems through the following technical means:

[0007] The present invention provides a method for preparing a liver organoid containing mesenchyme, comprising the following steps:

[0008] (1) Human embryonic stem cell WIBR3 pretreatment:

[0009] First, the WIBR3 cells were cultured until they could be passaged, and then digested with Accutase digestion solution. After digestion, the digestion was terminated with DMEM / F12 medium, the cell suspension was collected and centrifuged, the supernatant was discarded, and the cell pellet was collected.

[0010] The collected cell pellets were resuspended in stem cell culture medium, plated in a well plate, and apoptosis inhibitors were added and mixed;

[0011] (2) Inducing liver organoids and mesenchymal cells using human embryonic stem cell WIBR3:

[0012] Taking a portion of the WIBR3 cells treated by (1), and culturing them in a liver organoid culture medium to obtain liver organoid cells; taking another portion of the WIBR3 cells treated by (1), and inducing differentiation in a mesenchymal cell induction culture medium to obtain mesenchymal cells;

[0013] (3) Co-culturing mesenchymal cells and liver organoid cells to obtain liver organoids containing mesenchyme.

[0014] Furthermore, in step (1), WIBR3 cells are pre-cultured in a six-well plate with growth factor-reduced Matrigel (a basement membrane matrix extracted from mouse EHS sarcoma), wherein the growth factor-reduced Matrigel is diluted in DMEM / F12 at a ratio of 1: (50-200), evenly spread on the bottom of the well, and added at 1 mL per 6-well plate (0.8 mL per 12-well plate; 0.5 mL per 24-well plate); the culture conditions are: the culture medium is the stem cell culture medium mTeSR1, and the cells are cultured in a 37°C incubator for 1 day.

[0015] Furthermore, in step (1), the digestion conditions are as follows: 500 μL of Accutase digestion solution is added to a well of a six-well plate, and digestion is carried out in a 37° C. incubator for 3 to 10 minutes.

[0016] Furthermore, in step (1), when terminating the digestion with DMEM-F12, the volume added is 3 to 4 times the volume of the Accutase digestion solution.

[0017] Furthermore, in step (1), the centrifugation condition is: centrifugation at 1000-1200 rpm for 3-5 minutes.

[0018] Furthermore, in step (1), after resuspending in stem cell culture medium mTeSR1, the mixture was heated to (1-5)×10 5 The cells were plated at a density of 100 cells / well in a 6-well plate.

[0019] Furthermore, in step (1), the apoptosis inhibitor is Y-27632 (a ROCK inhibitor) at a concentration of 2 to 10 μM.

[0020] Furthermore, in step (2), the step of culturing using a liver organoid culture medium comprises:

[0021] After 1 to 2 days of culture, the medium was changed and adherent culture was performed using liver organoid medium S1;

[0022] After 2 to 4 days of culture, the medium was changed and adherent culture was performed using liver organoid medium S2;

[0023] After 5 to 7 days of culture, the culture medium was replaced and the liver organoid culture medium S3 was used for adherent culture.

[0024] Furthermore, the liver organoid culture medium S1-S3 includes a basal culture medium RPMI 1640 or a Hepatocyte culture medium, B27 without vitamin A, 100 to 200 ng / mL of Activin A, 50 to 100 ng / mL of Wnt-3a, 10 to 50 ng / mL of FGF2, and 20 to 50 ng / mL of BMP4, wherein 100x B27 without vitamin A is used in the liver organoid culture medium S1, and 50x B27 without vitamin A is used in the liver organoid culture medium S2.

[0025] Furthermore, in step (2), the mesenchymal cell induction medium includes a basal medium high-glucose DMEM, 15-20% KSR, 200 mM Glutamine, 100x NEAA, 0.1-0.5 μM beta-mercaptoethanol, 100-300 ng / mL FGF2, and 100-300 ng / mL PDGF-BB.

[0026] Furthermore, in step (3), the step of co-culturing mesenchymal cells and liver organoid cells comprises:

[0027] After 8-12 days of culture, the medium was changed and adherent culture was performed using liver organoid medium S4;

[0028] After 4-6 days of culture, the medium was changed and adherent culture was performed using liver organoid medium S5;

[0029] After 4-6 days of culture, the medium was replaced and adherent culture was performed using liver organoid medium S6;

[0030] After 4-6 days of culture, the culture medium was replaced and the hepatic organoid culture medium S7 was used for adherent culture.

[0031] Furthermore, the liver organoid culture medium S4-S6 includes Hepatocyte culture medium or Advanced DMEM / F12, 100x B27 without vitamin A, 100x N2, 5-10mM HEPES, 1.25-5mM Nicotinamide, and 10-50μM Gastrin.

[0032] Furthermore, the liver organoid culture medium S4 also includes 1-5 μM A83-01, 2-10 μM Forskolin1, 50-100 ng / mL Wnt-3a, and 50-100 ng / mL EGF.

[0033] Furthermore, the liver organoid culture medium S5 also includes 0.1-0.5% BSA, 25-100 ng / mL BMP7, 25-100 ng / mL FGF4, and 25-100 ng / mL HGF.

[0034] Furthermore, the liver organoid culture medium S7 includes Hepatocyte culture medium, 0.1-0.5% BSA, 100x B27 without vitamin A, 0.1-0.2mM ascorbic acid, 1.25-5mM Nicotinamide, 2-10μM Y-27632, 10-20ng / mL OSM, and 0.5-5μM Dexamethasone.

[0035] Furthermore, in step (3), the cell ratio of mesenchymal cells to liver organoid cells was 3:1, and the number of liver organoid cells in each 20 μL gel droplet was 3×10 4 .

[0036] The beneficial effects of the present invention are:

[0037] 1. The present invention induces iPS cells to differentiate into mesenchymal cells and liver organoids at the same time, and further co-cultures them to form liver organoids with mesenchyme to form a liver fibrosis or non-alcoholic liver disease model, providing a good research basis for the subsequent discovery of drugs for liver fibrosis and non-alcoholic liver disease.

[0038] 2. The present invention proposes a method for preparing liver organoids containing stroma. The preparation method is simple and has high culture efficiency. The prepared liver organoids containing stroma can be used for drug discovery for chemical liver fibrosis and non-alcoholic liver disease.

[0039] 3. The present invention changes the culture mode and method of liver organoids containing stroma. The induction method of liver organoids containing stroma proposed in the present invention is simple, and stroma cells and other liver cells have the same genetic background source, which is suitable for safety assessment of chemical substances such as drugs, liver fibrosis, non-alcoholic hepatitis modeling and efficacy evaluation, etc. The prepared liver organoids containing stroma can be used for drug discovery of chemical substances for liver fibrosis and non-alcoholic liver disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a diagram of the induction of a liver organoid containing stroma in Example 1 of the present invention;

[0041] Figure 2 It is the metabolic enzyme activity of the liver organoid containing stroma in Example 1 of the present invention;

[0042] Figure 3 This is the result of liver fibrosis modeling using LPS in the interstitial liver organoids in Example 1 of the present invention;

[0043] Figure 4 This is the result of modeling non-alcoholic hepatitis using the mesenchymal-containing liver organoids in Example 1 of the present invention;

[0044] Figure 5 The figure is a comparison chart of the morphology and yield of organoids obtained in Example 1 of the present invention and Comparative Example 1. DETAILED DESCRIPTION

[0045] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0046] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0047] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0048] Embodiment 1:

[0049] The method for preparing mesenchymal-containing liver organoids derived from embryonic stem cells WIBR3 comprises the following steps:

[0050] (1) Human embryonic stem cell WIBR3 pretreatment:

[0051] ① Use six-well plates pre-coated with Matrigel to culture WIBR3 cells. The culture medium is stem cell culture medium mTeSR1. Culture in a 37°C incubator for 4 days until the cells can be passaged. When digesting, add 500 μL of Cliputase digestion solution to one well of the six-well plate and digest in a 37°C incubator for 3 minutes.

[0052] ② Add DMEM / F12 with a volume three times the volume of the digestion solution to terminate digestion, collect the cell suspension and centrifuge at 1000 rpm for 3 minutes, discard the supernatant, collect the cell pellet and count;

[0053] ③ Resuspend in mTeSR1 and press 1×10 5 The cells were plated at a density of 100 μM / well in a 6-well plate, and 10 μM apoptosis inhibitor Y-27632 was added and gently mixed;

[0054] (2) Inducing liver organoids and mesenchymal cells using human embryonic stem cell WIBR3:

[0055] A portion of the WIBR3 cells treated by (1) was taken and cultured using a liver organoid culture medium (incubator at 37°C), wherein the specific steps are as follows: after 1 day of culture, the culture medium was replaced and cultured using a liver organoid culture medium S1; after 2 days of culture, the culture medium was used for culture using a liver organoid culture medium S2; after 5 days of culture, the culture medium was used for culture using a liver organoid culture medium S3 to obtain liver organoid cells;

[0056] Another portion of the WIBR3 cells treated with (1) is taken and induced to differentiate using a mesenchymal cell induction medium to obtain mesenchymal cells;

[0057] (3) Co-culture: Mesenchymal cells that had been passaged for 5 times were co-cultured with liver organoid cells (the cell ratio was 3:1, and the number of liver organoid cells in each 20 μL gel droplet was 1×10 4 ), the specific steps are:

[0058] Use liver organoid medium S4, and after 10 days of culture, use liver organoid medium S5; after 4 days of culture, use liver organoid medium S6; after 4 days of culture, use liver organoid medium S7; continue to culture for 4 days.

[0059] During the co-culture process, mesenchymal cells gradually differentiate into hepatic stromal cells, and mature hepatic organoids containing mesenchyme are obtained. Figure 1 (shown)

[0060] Among them, the liver organoid culture medium S1-S3 includes the basic culture medium RPMI 1640, B27 without vitamin A, 150ng / mL Activin A, 80ng / mL Wnt-3a, 35ng / mL FGF2, and 40ng / mL BMP4. Among them, 100x B27 without vitamin A is used in the liver organoid culture medium S1, and 50x B27 without vitamin A is used in the liver organoid culture medium S2.

[0061] The mesenchymal cell induction medium includes a basic medium high-glucose DMEM, 18% KSR, 200 mM Glutamine, 100x NEAA, 0.4 μM beta-mercaptoethanol, 200 ng / mL FGF2, and 200 ng / mL PDGF-BB.

[0062] Hepatic organoid culture medium S4 includes Advanced DMEM / F12, 100x B27 without vitamin A, 100x N2, 8mM HEPES, 3mM Nicotinamide, 30μM Gastrin, 3μM A83-01, 5μM Forskolin 1, 75ng / mL Wnt-3a, and 60ng / mL EGF.

[0063] Hepatic organoid culture medium S5 includes Hepatocyte medium, 100x B27 without vitamin A, 100x N2, 8mM HEPES, 3mM Nicotinamide, 30μM Gastrin, 0.35% BSA, 60ng / mL BMP7, 50ng / mL FGF4, and 70ng / mL HGF.

[0064] Hepatic Organoid Medium S6 includes Hepatocyte Medium, 100x B27 without Vitamin A, 100x N2, 8mM HEPES, 3mM Nicotinamide, and 20μM Gastrin.

[0065] Hepatic organoid culture medium S7 includes Hepatocyte medium, 0.3% BSA, 100x B27 without vitamin A, 0.15 mM ascorbic acid, 2.5 mM Nicotinamide, 6 μM Y-27632, 15 ng / mL OSM, and 2.5 μM Dexamethasone.

[0066] The metabolic enzyme activity of mature mesenchymal liver organoids was tested by immunofluorescence. Figure 2 As shown; liver fibrosis and non-alcoholic hepatitis models were further established, and fibrosis markers were characterized. The results are shown Figure 3 , as shown in 4.

[0067] Stability Characterization:

[0068] The liver organoid cells and mesenchymal cells in step (2) are frozen and then revived, and co-cultured again:

[0069] WIBR3-derived mesenchymal cells that had been passaged for 5 or more times were co-cultured with liver organoids. Due to the limited activity of liver organoids after cryopreservation and thawing, the ratio of mesenchymal cells to liver organoids was changed from 3:1 to 1:1. The number of liver organoid cells in each 20 μL gel droplet was 3×10 4 ), the subsequent steps are the same as above, that is, using liver organoid culture medium S4; after 10 days of culture, using liver organoid culture medium S5; after 4 days of culture, using liver organoid culture medium S6; after 4 days of culture, using liver organoid culture medium S7; continue to culture for 4 days to obtain mature liver organoids containing stroma, and the morphology is still good. Therefore, the liver organoids containing stroma in this embodiment have good repeatability and stability.

[0070] Embodiment 2:

[0071] The difference between this embodiment and embodiment 1 is that the specific parameters in the culture medium components are slightly different (as shown below), and the rest are the same as embodiment 1.

[0072] Hepatic organoid culture medium S1-S3 includes basal culture medium RPMI 1640, B27 without vitamin A, 100 ng / mL Activin A, 50 ng / mL Wnt-3a, 10 ng / mL FGF2, and 20 ng / mL BMP4, wherein 100x B27 without vitamin A is used in hepatic organoid culture medium S1, and 50x B27 without vitamin A is used in hepatic organoid culture medium S2.

[0073] The mesenchymal cell induction medium includes a basic medium high-glucose DMEM, 20% KSR, 200 mM Glutamine, 100x NEAA, 0.5 μM beta-mercaptoethanol, 300 ng / mL FGF2, and 300 ng / mL PDGF-BB.

[0074] Hepatic organoid culture medium S4 includes Hepatocyte medium, 100x B27 without vitamin A, 100x N2, 5mM HEPES, 1.25mM Nicotinamide, 10μM Gastrin, 1μM A83-01, 2μM Forskolin 1, 50ng / mL Wnt-3a, and 50ng / mL EGF.

[0075] Hepatic organoid culture medium S5 includes Advanced DMEM / F12, 100x B27 without vitamin A, 100x N2, 10 mM HEPES, 5 mM Nicotinamide, 50 μM Gastrin, 0.5% BSA, 100 ng / mL BMP7, 100 ng / mL FGF4, and 100 ng / mL HGF.

[0076] Hepatic Organoid Medium S6 includes Hepatocyte Medium, 100x B27 without Vitamin A, 100x N2, 5mM HEPES, 1.25mM Nicotinamide, and 10μM Gastrin.

[0077] Hepatic organoid culture medium S7 includes Hepatocyte medium, 0.1% BSA, 100x B27 without vitamin A, 0.1 mM ascorbic acid, 1.25 mM Nicotinamide, 2 μM Y-27632, 10 ng / mL OSM, and 0.5 μM Dexamethasone.

[0078] Embodiment 3:

[0079] The difference between this embodiment and embodiment 1 is that the specific parameters in the culture medium components are slightly different (as shown below), and the rest are the same as embodiment 1.

[0080] Hepatic organoid culture medium S1-S3 includes a basal culture medium Hepatocyte medium, B27 without vitamin A, 200 ng / mL Activin A, 100 ng / mL Wnt-3a, 50 ng / mL FGF2, and 50 ng / mL BMP4, wherein the hepatic organoid culture medium S1 uses 100x B27 without vitamin A, and the hepatic organoid culture medium S2 uses 50x B27 without vitamin A.

[0081] The mesenchymal cell induction medium includes a basic medium high-glucose DMEM, 15% KSR, 200 mM Glutamine, 100x NEAA, 0.1 μM beta-mercaptoethanol, 100 ng / mL FGF2, and 100 ng / mL PDGF-BB.

[0082] Hepatic organoid culture medium S4 includes Advanced DMEM / F12, 100x B27 without vitamin A, 100x N2, 10mM HEPES, 5mM Nicotinamide, 50μM Gastrin, 5μM A83-01, 10μM Forskolin 1, 100ng / mL Wnt-3a, and 100ng / mL EGF.

[0083] Hepatic organoid culture medium S5 includes Hepatocyte medium, 100x B27 without vitamin A, 100x N2, 5mM HEPES, 1.25mM Nicotinamide, 10μM Gastrin, 0.1% BSA, 25ng / mL BMP7, 25ng / mL FGF4, and 25ng / mL HGF.

[0084] Hepatic organoid culture medium S6 includes Hepatocyte medium or Advanced DMEM / F12, 100x B27 without vitamin A, 100x N2, 10mM HEPES, 5mM Nicotinamide, and 50μM Gastrin.

[0085] Hepatic organoid culture medium S7 includes Hepatocyte medium, 0.5% BSA, 100x B27 without vitamin A, 0.2 mM ascorbic acid, 5 mM Nicotinamide, 10 μM Y-27632, 20 ng / mL OSM, and 5 μM Dexamethasone.

[0086] The morphology of the mesenchyme-containing liver organoids prepared in Example 2 and Example 3 is similar to that in Example 1.

[0087] Comparative Example 1:

[0088] The difference between this comparative example and Example 1 is that the co-cultivation of step (3) is performed 5 days after step (2), and the rest is the same as Example 1.

[0089] This delayed co-culture mode will result in smaller hepatic organoids containing stroma and lower yields, such as Figure 5 shown.

[0090] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a liver organoid containing stroma, characterized in that: The following steps are involved: (1) Human embryonic stem cell WIBR3 pretreatment: First, the WIBR3 cells were cultured until they could be passaged, and then digested with Accutase digestion solution. After digestion, the digestion was terminated with DMEM / F12 medium, the cell suspension was collected and centrifuged, the supernatant was discarded, and the cell pellet was collected. The collected cell pellets were resuspended in stem cell culture medium, plated in a well plate, and apoptosis inhibitors were added and mixed; (2) Inducing liver organoids and mesenchymal cells using human embryonic stem cell WIBR3: Taking a portion of the WIBR3 cells treated by (1), and culturing them in a liver organoid culture medium to obtain liver organoid cells; taking another portion of the WIBR3 cells treated by (1), and inducing differentiation in a mesenchymal cell induction culture medium to obtain mesenchymal cells; (3) Co-culturing mesenchymal cells and liver organoid cells to obtain liver organoids containing mesenchyme.

2. The preparation method according to claim 1, characterized in that: In step (1), WIBR3 cells are cultured in a six-well plate with growth factor-reduced Matrigel gel in advance, wherein the growth factor-reduced Matrigel is diluted in DMEM / F12 at a ratio of 1: (50-200), evenly spread on the bottom of the well, and added at 1 mL for a 6-well plate, 0.8 mL for a 12-well plate, or 0.5 mL for a 24-well plate; the culture conditions are: the culture medium is the stem cell culture medium mTeSR1, and the cells are cultured in a 37°C incubator for 1 day.

3. The preparation method according to claim 1, characterized in that: In step (1), the digestion conditions are as follows: 500 μL of Accutase digestion solution is added to a well of a six-well plate, and digestion is carried out in a 37° C. incubator for 3 to 10 minutes; when terminating the digestion with DMEM-F12, the added volume is 3 to 4 times the volume of the Accutase digestion solution.

4. The preparation method according to claim 1, characterized in that: In step (1), the centrifugation conditions are: centrifugation at 1000-1200 rpm for 3-5 minutes; after resuspending in stem cell culture medium mTeSR1, the mixture is centrifuged at (1-5)×10 5 The cells were plated in a 6-well plate at a density of 100 μM / well. The apoptosis inhibitor Y-27632 was used at a concentration of 2 to 10 μM.

5. The preparation method according to claim 1, characterized in that: In step (2), the step of culturing using a liver organoid culture medium comprises: After 1 to 2 days of culture, the medium was changed and adherent culture was performed using liver organoid medium S1; After 2 to 4 days of culture, the medium was changed and adherent culture was performed using liver organoid medium S2; After 5 to 7 days of culture, the medium was changed and adherent culture was performed using liver organoid medium S3; The liver organoid culture medium S1-S3 includes a basic culture medium RPMI 1640 or a Hepatocyte culture medium, B27 without vitamin A, 100-200 ng / mL of Activin A, 50-100 ng / mL of Wnt-3a, 10-50 ng / mL of FGF2, and 20-50 ng / mL of BMP4, wherein the liver organoid culture medium S1 uses 100x B27 without vitamin A, and the liver organoid culture medium S2 uses 50x B27 without vitamin A.

6. The preparation method according to claim 1, characterized in that: In step (2), the mesenchymal cell induction medium includes a basic medium high-glucose DMEM, 15-20% KSR, 200 mM Glutamine, 100x NEAA, 0.1-0.5 μM beta-mercaptoethanol, 100-300 ng / mL FGF2, and 100-300 ng / mL PDGF-BB.

7. The preparation method according to claim 1, characterized in that: In step (3), the step of co-culturing mesenchymal cells and liver organoid cells comprises: After 8-12 days of culture, the medium was changed and adherent culture was performed using liver organoid medium S4; After 4-6 days of culture, the medium was changed and adherent culture was performed using liver organoid medium S5; After 4-6 days of culture, the medium was replaced and adherent culture was performed using liver organoid medium S6; After 4-6 days of culture, the culture medium was replaced and the hepatic organoid culture medium S7 was used for adherent culture.

8. The preparation method according to claim 7, characterized in that: The liver organoid culture medium S4-S6 includes Hepatocyte culture medium or Advanced DMEM / F12, 100x B27 without vitamin A, 100x N2, 5-10mM HEPES, 1.25-5mM Nicotinamide, and 10-50μM Gastrin; The liver organoid culture medium S7 includes Hepatocyte culture medium, 0.1-0.5% BSA, 100x B27 without vitamin A, 0.1-0.2mM ascorbic acid, 1.25-5mM Nicotinamide, 2-10μM Y-27632, 10-20ng / mL OSM, and 0.5-5μM Dexamethasone.

9. The preparation method according to claim 8, characterized in that: The liver organoid culture medium S4 also includes 1-5 μM A83-01, 2-10 μM Forskolin 1, 50-100 ng / mL Wnt-3a, and 50-100 ng / mL EGF; the liver organoid culture medium S5 also includes 0.1-0.5% BSA, 25-100 ng / mL BMP7, 25-100 ng / mL FGF4, and 25-100 ng / mL HGF.

10. The preparation method according to claim 1, characterized in that: In step (3), the cell ratio of mesenchymal cells to liver organoid cells was 3:1, and the number of liver organoid cells in each 20 μL gel droplet was 3×10 4 .

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