A method of preparing a liver organoid containing mesenchyme

Abstract

The application discloses a kind of hepatic organoids containing mesenchyme induction method, belong to organoid culture technical field.The method includes the following steps: (1) human embryonic stem cell WIBR3 pretreatment;(2) utilize human embryonic stem cell WIBR3 and induce liver organoids and mesenchymal cells;(3) mesenchymal cells and liver organoid cells are co-cultured, i.e. obtain the hepatic organoids containing mesenchyme.There is beneficial effect: the application changes the culture mode and method of the hepatic organoids containing mesenchyme.The hepatic organoids containing mesenchyme induction method proposed in the application is simple, and mesenchymal cells and other liver cells have the same genetic background source, suitable for the safety evaluation of drug and other chemical substances, liver fibrosis, non-alcoholic hepatitis modeling and pharmacodynamic evaluation.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of organoid culture, and particularly relates to a preparation method of a liver organoid containing mesenchymal cells. BACKGROUND

[0002] Due to the lack of suitable animal or cell models, the theoretical cognition of liver diseases, the discovery of drugs and treatment methods are hindered. Primary human hepatocytes retain many characteristics of hepatocytes, but can only survive for a few days in culture medium, so fresh tissues are constantly needed, and the complex structure of human liver is lacking. Cell lines from hepatocellular carcinoma and hepatocellular carcinoma can proliferate indefinitely, but they are cancerous and lack key features of normal hepatocytes. Therefore, there have been difficulties in drug discovery for diseases such as liver fibrosis and non-alcoholic liver disease, and organoids are expected to be a powerful tool for constructing liver fibrosis or non-alcoholic liver disease.

[0003] Since the occurrence and development of these diseases are closely related to the presence of liver mesenchymal cells (such as stellate cells), unlike hepatocytes and cholangiocytes that develop from endoderm, liver mesenchymal cells originate from mesodermal lineage differentiation. However, the source of mesenchymal cells is unstable, and the great difference in development from liver organoids hinders the co-culture, resulting in poor morphology and low culture efficiency of liver organoids containing mesenchymal cells.

[0004] Chinese patent application document with publication number CN117701492A discloses a construction method of liver organoids and its application. The liver organoids constructed by the 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 culture efficiency and stability of the liver organoids of the patent are poor, and therefore need to be further improved. SUMMARY

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

[0006] The present application solves the above technical problems by the following technical means:

[0007] The present application provides a preparation method of a liver organoid containing mesenchymal cells, comprising the following steps:

[0008] (1) Pretreatment of human embryonic stem cells WIBR3:

[0009] First, WIBR3 cells are cultured to be passaged, and then digested with Accutase digestion solution; after digestion is completed, DMEM / F12 medium is used to terminate digestion, the cell suspension is collected and centrifuged, the supernatant is discarded, and the cell precipitate is collected;

[0010] Then, the collected cell precipitate is resuspended with stem cell culture medium, and then plated in a well plate, and mixed after adding an apoptosis inhibitor.

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

[0012] A portion of the WIBR3 cells treated in (1) is cultured using a liver organoid culture medium to obtain liver organoid cells; another portion of the WIBR3 cells treated in (1) is induced to differentiate using a mesenchymal cell induction medium to obtain mesenchymal cells.

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

[0014] Further, in step (1), the WIBR3 cells are pre-cultured in a six-well plate of Matrigel gel (a basement membrane matrix extracted from mouse EHS sarcoma) with reduced growth factors, wherein the reduced growth factor Matrigel is diluted in DMEM / F12 at a ratio of 1:(50-200), uniformly plated at 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 as follows: the culture medium is stem cell culture medium mTeSR1, and the culture is carried out in a 37°C incubator for 1 day.

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

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

[0017] Further, in step (1), the centrifugation conditions are as follows: centrifugation is carried out at 1000-1200 rpm for 3-5 minutes.

[0018] Further, in step (1), after resuspension with the stem cell culture medium mTeSR1, the cells are plated in a 6-well plate at a cell density of (1-5) x 10 5 cells per well.

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

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

[0021] After 1-2 days of culture, the medium is changed and the liver organoid culture medium S1 is used for adherent culture;

[0022] After 2-4 days of culture, the medium is changed and the liver organoid culture medium S2 is used for adherent culture;

[0023] After 5-7 days of culture, the medium is changed and the liver organoid culture medium S3 is used for adherent culture.

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

[0025] Further, in step (2), the mesenchymal cell induction medium comprises 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, 100-300 ng / mL PDGF-BB.

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

[0027] After 8-12 days of culture, the medium is changed and the liver organoid culture medium S4 is used for adherent culture;

[0028] After 4-6 days of culture, the medium is changed and the liver organoid culture medium S5 is used for adherent culture;

[0029] After 4-6 days of culture, the medium is changed and the liver organoid culture medium S6 is used for adherent culture;

[0030] After 4-6 days of culture, the medium is changed and the liver organoid culture medium S7 is used for adherent culture.

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

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

[0033] Further, the liver organoid culture medium S5 further comprises 0.1-0.5 % BSA, 25-100 ng / mL BMP7, 25-100 ng / mL FGF4, 25-100 ng / mL HGF.

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

[0035] Further, in step (3), the cell ratio of mesenchymal cells to liver organoid cells is 3:1, and the number of liver organoid cells in each 20 μL gel droplet is 3x10 4 .

[0036] The present application has the following beneficial effects:

[0037] 1. The present application induces iPS to differentiate into mesenchymal cells and liver organoids at the same time, and further co-cultures to form liver organoids with interstitium, which form a liver fibrosis or non-alcoholic liver disease disease model, providing a good research foundation for subsequent liver fibrosis and non-alcoholic liver disease drug discovery.

[0038] 2. The present application provides a preparation method of liver organoids with interstitium, which has simple preparation method, high culture efficiency, and the prepared liver organoids with interstitium can be used for chemical substance liver fibrosis and non-alcoholic liver disease drug discovery.

[0039] 3、The application changes the culture mode and method of the liver organoids containing mesenchyme. The liver organoids containing mesenchyme provided by the application have simple induction method, the mesenchymal cells and other liver cells have the same genetic background source, are suitable for safety evaluation of drugs and other chemical substances, liver fibrosis, non-alcoholic hepatitis modeling and pharmacodynamic evaluation, etc., and the prepared liver organoids containing mesenchyme can be used for chemical substance liver fibrosis and non-alcoholic liver disease drug discovery. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 is the liver organoid containing mesenchyme in Example 1 of the application;

[0041] Figure 2 is the metabolic enzyme activity of the liver organoid containing mesenchyme in Example 1 of the application;

[0042] Figure 3 is the result of liver fibrosis modeling of the liver organoid containing mesenchyme in Example 1 of the application by LPS;

[0043] Figure 4 is the result of non-alcoholic hepatitis modeling of the liver organoid containing mesenchyme in Example 1 of the application;

[0044] Figure 5 is the comparison chart of the morphology and yield of the organoids obtained in Example 1 and Comparative Example 1 of the application. DETAILED DESCRIPTION

[0045] To make the purpose, technical scheme and advantages of the embodiments of the application more clear, the technical scheme of the embodiments of the application will be described clearly and completely below in combination with the embodiments of the application. Obviously, the described embodiments are part of the embodiments of the application, rather than all the embodiments of the application. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the application.

[0046] The test materials and reagents used in the following examples, etc. can be obtained from commercial channels if not otherwise specified.

[0047] The specific techniques or conditions not specified in the examples can be carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0048] Example 1:

[0049] The preparation method of the liver organoid containing mesenchyme derived from embryonic stem cell WIBR3 comprises the following steps:

[0050] (1) Pretreatment of human embryonic stem cell WIBR3:

[0051] ①WIBR3 cells were cultured in a six-well plate pre-coated with Matrigel gel, and the culture medium was stem cell culture medium mTeSR1. The cells were cultured in a 37°C incubator for 4 days to be passaged. When the cells were digested, 500 μL of Accutase digestion solution was added to each well of the six-well plate, and the cells were digested in a 37°C incubator for 3 minutes;

[0052] ②The digestion was terminated by adding DMEM / F12 with a volume of 3 times that of the digestion solution, and the cell suspension was centrifuged at 1000 rpm for 3 minutes. The supernatant was discarded, and the cell pellet was collected and counted;

[0053] ③The cells were resuspended in mTeSR1 and plated in a six-well plate at a cell density of 1×10 5 cells per well, and 10 μM of the apoptosis inhibitor Y-27632 was added, and the cells were mixed gently;

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

[0055] A portion of the WIBR3 cells treated in (1) were cultured using a liver organoid culture medium (37°C incubator). The specific steps were as follows: after 1 day of culture, the culture medium was replaced with liver organoid culture medium S1; after 2 days of culture, the culture medium was replaced with liver organoid culture medium S2; after 5 days of culture, the culture medium was replaced with liver organoid culture medium S3, and liver organoid cells were obtained;

[0056] Another portion of the WIBR3 cells treated in (1) were induced and differentiated using a mesenchymal cell induction medium, and mesenchymal cells were obtained;

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

[0058] After 10 days of culture using liver organoid culture medium S4, liver organoid culture medium S5 was used; after 4 days of culture, liver organoid culture medium S6 was used; after 4 days of culture, liver organoid culture medium S7 was used; and the culture was continued for another 4 days.

[0059] During the co-culture, the mesenchymal cells gradually differentiated into liver mesenchymal cells, i.e., mature liver organoids containing mesenchymal cells were obtained. Figure 1

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

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

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

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

[0064] The liver organoid culture medium S6 includes Hepatocyte medium, 100x of B27 without vitamin A, 100x of N2, 8 mM of HEPES, 3 mM of Nicotinamide, 20 μM of Gastrin.

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

[0066] The metabolic enzyme activity test of the mature mesenchymal-containing liver organoid is carried out by immunofluorescence, and the results are shown in Figure 2 The liver fibrosis and non-alcoholic hepatitis modeling are further carried out, the fibrosis markers are characterized, and the results are shown inFigure 3 , as shown in FIG. 4.

[0067] Stability characterization:

[0068] After the liver organoids cells, mesenchymal cells of step (2) were frozen and then recovered, the co-culture was performed again:

[0069] Using WIBR3-derived mesenchymal cells passaged for 5 times or more to co-culture with liver organoids, due to the limited activity of liver organoids after recovery from cryopreservation, the ratio of mesenchymal cells to liver organoid cells was changed from 3:1 to 1:1, and the number of liver organoid cells in each 20 μL gel droplet was 3 x 10 4 ), and the subsequent steps were the same as above, i.e., 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; continuing to culture for 4 days to obtain mature liver organoids containing mesenchyme, which still had good morphology. Therefore, the repeatability and stability of the liver organoids containing mesenchyme of this embodiment were good.

[0070] Example 2:

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

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

[0073] The mesenchymal cell induction medium includes the 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] The liver organoid culture medium S4 includes Hepatocyte medium, 100x B27 without vitamin A, 100x N2, 5 mM HEPES, 1.25 mM Nicotinamide, 10 μM Gastrin, 1 μM A83-01, 2 μM Forskolin 1, 50 ng / mL Wnt-3a, and 50 ng / mL EGF.

[0075] Liver organoid media S5 includes Advanced DMEM / F12, 100x B27 without Vitamin A, 100x N2, 10 mM HEPES, 5 mM Nicotinamide, 50 μΜ Gastrin, 0.5% BSA, 100 ng / mL BMP7, 100 ng / mL FGF4, 100 ng / mL HGF.

[0076] Liver organoid media S6 includes Hepatocyte media, 100x B27 without Vitamin A, 100x N2, 5 mM HEPES, 1.25 mM Nicotinamide, 10 μΜ Gastrin.

[0077] Liver organoid media S7 includes Hepatocyte media, 0.1% BSA, 100x B27 without Vitamin A, 0.1 mM Ascorbic acid, 1.25 mM Nicotinamide, 2 μΜ Y-27632, 10 ng / mL OSM, 0.5 μΜ Dexamethasone.

[0078] Example 3:

[0079] This example differs from Example 1 in that there are slight differences in the specific parameters in the media components (as shown below), and otherwise is the same as Example 1.

[0080] Liver organoid media S1-S3 includes the base media Hepatocyte media, B27 without Vitamin A, 200 ng / mL Activin A, 100 ng / mL Wnt-3a, 50 ng / mL FGF2, 50 ng / mL BMP4, wherein 100x B27 without Vitamin A is used in liver organoid media S1, and 50x B27 without Vitamin A is used in liver organoid media S2.

[0081] Mesenchymal cell induction media includes the base media high glucose DMEM, 15% KSR, 200 mM Glutamine, 100x NEAA, 0.1 μΜ beta-mercaptoethanol, 100 ng / mL FGF2, 100 ng / mL PDGF-BB.

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

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

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

[0085] Liver organoid media S7 includes Hepatocyte media, 0.5% BSA, 100x B27 without vitamin A, 0.2 mM Ascorbic acid, 5 mM Nicotinamide, 10 mM Y-27632, 20 ng / mL OSM, 5 mM Dexamethasone.

[0086] The mesenchyme-containing liver organoids produced in Example 2 and Example 3 were similar in morphology to those of Example 1.

[0087] Comparative Example 1:

[0088] This comparative example differs from Example 1 in that the co-culture of step (3) was delayed for 5 days after step (2), otherwise the same as Example 1.

[0089] This delayed co-culture mode resulted in mesenchyme-containing liver organoids that were smaller in morphology and had a lower yield, as shown in Figure 5

[0090] ​The above examples are only used to illustrate the technical solutions of the present application, and are not intended to limit the present application; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that the technical solutions recorded in the foregoing examples can be modified, or some technical features can be replaced by equivalent features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for preparing a liver organoid containing mesenchyme, characterized by, The method comprises the following steps: (1) Pre-treatment of human embryonic stem cells WIBR3: Firstly, WIBR3 cells are cultured to the time of passaging, and then the cells are digested with Accutase digestion solution; after the digestion is completed, the digestion is terminated by using DMEM / F12 culture medium, the cell suspension is collected and centrifuged, the supernatant is discarded, and the cell precipitate is collected; Then, the collected cell precipitate is resuspended by using a stem cell culture medium, and then is plated in a well plate and mixed after adding an apoptosis inhibitor; (2) Induction of liver organoids and mesenchymal cells by using human embryonic stem cells WIBR3: Part of the WIBR3 cells treated in (1) are taken and cultured by using a liver organoid culture medium to obtain liver organoid cells; and another part of the WIBR3 cells treated in (1) are taken and induced and differentiated by using a mesenchymal cell induction culture medium to obtain mesenchymal cells; The step of culturing by using the liver organoid culture medium comprises: After 1-2 days of culture, the culture medium is replaced, and the liver organoid culture medium S1 is used for adherent culture; After 2-4 days of culture, the culture medium is replaced, and the liver organoid culture medium S2 is used for adherent culture; After 5-7 days of culture, the culture medium is replaced, and the liver organoid culture medium S3 is used for adherent culture; The liver organoid culture media S1-S3 comprise a basic medium RPMI 1640 or Hepatocyte medium, 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 100x of B27 without vitamin A is used in the liver organoid culture medium S1, and 50x of B27 without vitamin A is used in the liver organoid culture medium S2; The mesenchymal cell induction culture medium comprises 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; (3) Co-culture of mesenchymal cells and liver organoid cells, to obtain liver organoids containing mesenchymal cells; The step of co-culturing mesenchymal cells and liver organoid cells comprises: After 8-12 days of culture, the culture medium is replaced, and the liver organoid culture medium S4 is used for adherent culture; After 4-6 days of culture, the culture medium is replaced, and the liver organoid culture medium S5 is used for adherent culture; After 4-6 days of culture, the culture medium is replaced, and the liver organoid culture medium S6 is used for adherent culture; After 4-6 days of culture, the culture medium is replaced, and the liver organoid culture medium S7 is used for adherent culture; The liver organoid culture media S4-S6 comprise Hepatocyte medium or Advanced DMEM / F12, 100x B27 without vitamin A, 100x N2, 5-10 mM HEPES, 1.25-5 mM Nicotinamide, and 10-50 μM Gastrin; The liver organoid culture medium S7 comprises Hepatocyte medium, 0.1-0.5% BSA, 100x B27 without vitamin A, 0.1-0.2 mM ascorbic acid, 1.25-5 mM Nicotinamide, 2-10 μM Y-27632, 10-20 ng / mL OSM, 0.5-5 μM Dexamethasone.

2. The production method according to claim 1, characterized by, In step (1), the WIBR3 cells are pre-cultured in a six-well plate of Matrigel gel with reduced growth factors, wherein the reduced growth factor Matrigel is diluted in DMEM / F12 at a ratio of 1: (50-200), uniformly laid on the bottom of the well, and added at 1 mL for a six-well plate, 0.8 mL for a twelve-well plate, or 0.5 mL for a twenty-four-well plate; the culture condition is that the medium is selected to be stem cell culture medium mTeSR1, and the culture is carried out in a 37°C incubator for 1 day.

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

4. The production method according to claim 1, characterized by, In step (1), the centrifugation conditions are as follows: centrifuge at 1000-1200 rpm for 3-5 minutes; after resuspending in stem cell culture medium mTeSR1, centrifuge at (1-5) × 10⁻⁶. 5 Cells were seeded at a density of 2-10 μM in 6-well plates; the apoptosis inhibitor Y-27632 was used.

5. The preparation method according to claim 1, characterized in that, The liver organoid culture media S1-S3 comprise the basic medium RPMI 1640, B27 without vitamin A, 150 ng / mL Activin A, 80 ng / mL Wnt-3a, 35 ng / mL FGF2, and 40 ng / mL 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.

6. The method of claim 1, wherein, The mesenchymal cell induction medium comprises the 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.

7. The preparation method according to claim 1, characterized in that, The liver organoid culture medium S7 comprises 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.

8. The method of claim 1, wherein, In step (3), the cell ratio of mesenchymal cells to liver organoid cells was 3:1, and the number of liver organoid cells per 20 μL of gel droplet was 3 x 10 4 .

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