Metabolic maturation methods and applications of multi-lineage liver organoids

Abstract

The present invention provides a method and application for metabolic maturation of multi-lineage liver organoids. This method can significantly increase the expression levels of metabolism-related genes, including drug metabolism (phase I metabolic enzymes, phase II metabolic enzymes and phase III transporters), lipid metabolism, sugar metabolism, and bile acid metabolism-related markers, while maintaining multiple cell types related to liver physiology; and the overall expression profile is close to that of primary liver tissue. The mature liver organoids produced by this method are particularly suitable for applications such as hepatotoxicity screening and metabolic evaluation of preclinical drugs.

Description

Technical Field

[0001] The present invention belongs to the field of cell culture technology, and in particular relates to a multi-lineage liver organoid metabolic maturation method and application. Background Art

[0002] Since the first successful induction of hepatocyte differentiation from human pluripotent stem cells (PSCs) in 2010, the sustained efforts of scientists worldwide over the past decade have yielded significant breakthroughs in the purity of 2D hepatocytes and the complexity of 3D liver organoids, providing important models for basic research and medical applications in liver disease. On the one hand, these 2D / 3D liver derivatives can be continuously induced to differentiate in vitro from PSCs, addressing the issue of limited sample sources. On the other hand, the advantages of humanization not only enable near-real-world results in preclinical drug research but also provide new candidate options for liver regeneration and repair.

[0003] As a surrogate model of the largest metabolic organ in the human body, the current lack of maturity of PSC liver derivatives, especially the low metabolic capacity, is a key obstacle to their further promotion and application: the maturity of most 2D hepatocyte induction methods is only close to the fetal level; and although 3D liver organoids have the advantage of multicellular interactions and have improved the expression of maturity markers to a certain extent, key metabolic genes such as phase I metabolic enzyme CYP3A4 and phase II drug metabolizing enzyme UGT1A1 are still at low expression levels, far from that of adult livers; therefore, it seriously affects the accuracy of disease simulation and hepatotoxicity screening studies. Summary of the Invention

[0004] In view of this, the present invention aims to propose a multi-lineage liver organoid metabolic maturation method and application, which can significantly increase the expression levels of metabolism-related genes, including drug metabolism (phase I metabolic enzymes, phase II metabolic enzymes and phase III transporters), lipid metabolism, sugar metabolism, and bile acid metabolism, while maintaining multiple cell types related to liver physiology; and the overall expression profile is close to that of primary liver tissue, which is suitable for toxicity screening and metabolic evaluation of candidate drugs.

[0005] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0006] A method for metabolic maturation of multi-lineage liver organoids, comprising the following steps:

[0007] S1. Induce PSCs to form multi-lineage liver organoids with at least hepatocytes, cholangiocytes, macrophages, and hepatic stellate cells;

[0008] S2. Metabolic maturation culture: Use maturation-inducing medium containing maturation-inducing factors to continue culturing multi-lineage liver organoids until the organoids achieve maturity; among them, maturation-inducing factors include HGF, OSM, COL-1, thyroid hormone T3, TGFβ inhibitor and γ-secretin inhibitor. Maturation-inducing medium also includes basal medium StemPro34 and William's E.

[0009] In some embodiments, the concentration of each maturation-inducing factor is: 10-75 ng / ml HGF, 10-75 ng / ml OSM, 20-80 ng / ml COL-1, 2-8 μM thyroid hormone T3;

[0010] The volume ratio of StemPro34 is 5-30%, and the volume ratio of William's E is 50-95%.

[0011] In some embodiments, the TGFβ inhibitor is any one of A83-01, SB431542, and RepSox, the concentration of A83-01 is 1-7.5 μM, the concentration of SB431542 is 10-50 μM, and the concentration of RepSox is 1-7.5 μM;

[0012] The γ-secretin inhibitor is any one of DBZ, RO4929097, and Compound E. The concentration of DBZ is 0.5-3 μM, the concentration of RO4929097 is 10-30 μM, and the concentration of Compound E is 2-5 μM.

[0013] In some embodiments, the maturation-enhancing medium further comprises 0.5-2.5 μM DEX.

[0014] In some embodiments, the method of inducing PSC to form multi-lineage liver organoids is: inducing differentiation of PSC using a culture medium containing BMP4 and bFGF, a culture medium containing BMP4, bFGF and a Wnt agonist, a culture medium containing COL-1 and LN-411, a culture medium containing VEGF, EGF and bFGF, and a culture medium containing FSK and a cAMP agonist in sequence.

[0015] In some embodiments, the specific method of inducing PSCs to form multi-lineage liver organoids is:

[0016] S11. Use medium containing BMP4 and bFGF to induce PSCs and produce HAND1 + Mesoderm and FOXA2 + Endoderm;

[0017] S12, use culture medium containing BMP4, bFGF, and Wnt agonist to continue inducing KDR + HHEX of mesodermal progenitor cells + 、HNF4α + posterior foregut endoderm;

[0018] S13, enzymatically digesting the derivatives obtained in S12, collecting them by centrifugation, and resuspending the single cells in a medium containing VEGF, EGF, and bFGF, and then adding COL1 and LN411 for 3D repolymerization;

[0019] After the culture medium was replaced, the culture medium was used to induce the production of CD31 + / CD34 + Hepatic endoderm of primary endothelial cells, and no COL1 and LN411 were added thereafter;

[0020] S14. Continue to induce the generation of multi-lineage liver organoids using culture medium containing FSK and cAMP agonist.

[0021] In some embodiments, the culture duration of S1 is 24-30 days, and the culture duration of S2 is 8-10 days; in S2, the culture medium is replaced every 72 hours.

[0022] The present invention also provides a maturation-promoting culture medium, which is used for the above-mentioned multi-lineage liver organoid metabolic maturation method, including a basal culture medium and maturation-promoting induction factors, the basal culture medium includes StemPro34 and William's E, and the maturation-promoting induction factors include HGF, OSM, COL-1, thyroid hormone T3, TGFβ inhibitor and γ-secretin inhibitor.

[0023] The present invention also provides a metabolically mature multi-lineage liver organoid constructed by the above-mentioned multi-lineage liver organoid metabolic maturation method, in which the adult hepatocyte marker ALB is upregulated to an expression level close to that of adult primary liver, and the expression of drug metabolism-related genes phase I metabolic enzymes, phase II metabolic enzymes, phase III transporter pumps, and related markers of sugar metabolism genes, lipid metabolism genes, and bile acid metabolism genes are all upregulated to a level close to that of primary liver tissue.

[0024] The present invention also provides an application of the metabolically mature multi-lineage liver organoids described above in drug toxicity screening and drug metabolism evaluation.

[0025] Compared with the existing technology, the multi-lineage liver organoid metabolic maturation method and application described in the present invention have the following advantages:

[0026] The multi-lineage liver organoid metabolic maturation method described in the present invention can significantly increase the expression levels of metabolism-related genes, including drug metabolism (phase I metabolic enzymes, phase II metabolic enzymes and phase III transporters), lipid metabolism, sugar metabolism, and bile acid metabolism, while maintaining multiple cell types related to liver physiology; and the overall expression profile is close to that of primary liver tissue, which is suitable for toxicity screening and metabolic evaluation of candidate drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The accompanying drawings, which constitute part of the present invention, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0028] Figure 1 Analysis of organoid cellularity before and after maturation; pre-maturation refers to liver organoids induced to differentiate to differentiation day 24; post-maturation refers to liver organoids cultured to differentiation day 32; biological replicates, n = 4; *, P < 0.05;

[0029] Figure 2 Figure 2: Expression analysis of liver maturation genes before and after maturation culture. A: AFP expression analysis before and after maturation. B: ALB expression analysis before and after maturation. C: Expression analysis of liver metabolism-related genes before and after maturation. The expression level before maturation (differentiation day 24) was set as 1, and the values ​​in each group are relative expression levels compared with that before maturation. Primary liver tissue was used as the positive control. Biological replicates were n = 4. *, P < 0.05; **, P < 0.01; P***, P < 0.001.

[0030] Figure 3 Principal component analysis of gene expression before and after maturation culture and principal component analysis after transcript sequencing; adult liver tissue, fetal liver tissue, PSC-derived hepatocytes, and PSCs were used as controls, with biological replicates n = 3. Data for PSC-derived hepatocytes are from DOI: 10.1038 / s41422-019-0196-x.

[0031] Figure 4 The expression level of the group with all the maturation-promoting factors added was set as 1 to represent the maturation-promoting effect of each inducing factor component, and the values ​​of the group with each factor removed were the relative expression levels compared with the group without the maturation-promoting factors; biological replicates, n=4; *, P<0.05; **, P<0.01; P***, P<0.001; ALB is a marker for adult hepatocytes; CYP3A4 is a phase I drug-metabolizing enzyme in hepatocytes; UGT1A1 is a phase II drug-metabolizing enzyme; ND, not reached the limit of detection;

[0032] Figure 5The effect of different TGFβ inhibitors or γ-secretin inhibitors on maturation promotion was shown. The expression level before maturation (differentiation day 24) was set as 1, and the values ​​of each group are relative expression levels compared with it. Biological replicates were performed in n = 3. ALB is a marker for adult hepatocytes. CYP3A4 is a phase I drug metabolizing enzyme. UGT1A1 is a phase II drug metabolizing enzyme.

[0033] Figure 6 Maturation-promoting effect of basal culture medium components; A: Effect of retaining / discarding basal culture medium on the expression of maturation markers; the expression level before maturation (differentiation day 24) was set as 1, and the values ​​in each group are relative expression levels compared with it; n=4 biological replicates for this method; n=3 biological replicates for the StemPro34 withdrawal group and the William's E withdrawal group; ALB is a marker for adult hepatocytes; CYP3A4 is a phase I drug-metabolizing enzyme in hepatocytes; UGT1A1 is a phase II drug-metabolizing enzyme; **, P<0.01; P***, P<0.001; B: Effect of retaining / discarding basal culture medium on the cell composition of organoids, with n=3 biological replicates. DETAILED DESCRIPTION

[0034] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0035] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only 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 making creative efforts are within the scope of protection of the present invention.

[0036] The reagents used in the examples and comparative examples are shown in Table 2-3.

[0037] The present invention provides a method for metabolic maturation of multi-lineage liver organoids, comprising the following steps:

[0038] S1. Induce PSCs to form multi-lineage liver organoids with at least hepatocytes, cholangiocytes, macrophages, and hepatic stellate cells;

[0039] Specifically, PSCs were induced to differentiate using a culture medium containing BMP4 and bFGF, a culture medium containing BMP4, bFGF and a Wnt agonist, a culture medium containing COL1 and LN411, a culture medium containing VEGF, EGF and bFGF, and a culture medium containing FSK and a cAMP agonist in sequence.

[0040] More specifically, the following steps are included:

[0041] S11. Use medium containing BMP4 and bFGF to induce PSCs and produce HAND1 + Mesoderm and FOXA2 + Endoderm;

[0042] S111. Culture in conventional culture medium for 1 day. The conventional culture medium includes a basal culture medium and additives. The basal culture medium is RPMI-1640 containing 2% B27 by volume. The additives include 10-75 ng / ml BMP4 and 100-150 ng / ml Activin A.

[0043] S112. Culture the cells for 2-3 days in a first culture medium comprising a basal medium (RPMI-1640 containing 2% B27 by volume) and additives (100-150 ng / ml Activin A, 1-10 ng / ml BMP4, and 50-200 ng / ml bFGF). Change the culture medium every 24 hours.

[0044] S12, use culture medium containing BMP4, bFGF, and Wnt agonist to continue inducing KDR + HHEX of mesodermal progenitor cells + HNF4α + posterior foregut endoderm;

[0045] The culture medium used in this stage is the second culture medium, which includes a basal culture medium and additives. The basal culture medium includes 2% B27 by volume, 23% StemPro34 by volume, and 75% IMDM by volume; the additives include 10-50 ng / ml BMP4, 200-500 ng / ml bFGF, and Wnt agonists. The Wnt agonists include CHIR99021, Wnt3a, and Wnt7a. When CHIR99021 is selected as the Wnt agonist, its concentration is 1-5 μM. The culture period is 3-4 days, and the culture medium is replaced daily.

[0046] S13. The derivative obtained in S12 was enzymatically digested and collected by centrifugation. The single cells were resuspended in a medium containing VEGF, EGF, and bFGF, and COL-1 and LN-411 were added. After mixing, the cells were seeded into a 96-well U-shaped culture plate at a seeding volume of 10,000 cells / well for 3D repolymerization.

[0047] The culture medium used is the third culture medium, which includes a basal culture medium and additives. The basal culture medium includes 2% B27 by volume, 23% StemPro34 by volume, and 75% IMDM by volume; the additives include 50-150ng / ml VEGF, 5-25ng / ml EGF, 10-50ng / ml bFGF, Wnt agonist, TGFβ inhibitor, 0.25-1.25mg / ml COL-1, 1-5μg / ml LN-411, Wnt agonists include CHIR99021, Wnt3a, and Wnt7a. When CHIR99021 is selected as the Wnt agonist, its concentration is 1-10μM. The TGFβ inhibitors include A83-01 and SB431542. When A83-01 is selected as the TGFβ inhibitor, its concentration is 1-10μM.

[0048] After the culture medium was changed, the fourth culture medium without COL-1 and LN-411 was used to induce the production of CD31 + / CD34 + Primary endothelial cells of the hepatic endoderm.

[0049] Specifically, the culture medium used was Medium IV, which includes a basal medium and additives. The basal medium includes 2% B27 by volume, 23% StemPro34 by volume, and 75% IMDM by volume. The additives include 50-150 ng / ml VEGF, 5-25 ng / ml EGF, 10-50 ng / ml bFGF, a Wnt agonist, and a TGFβ inhibitor. The Wnt agonists include CHIR99021, Wnt3a, and Wnt7a, and the TGFβ inhibitors include A83-01 and SB431542. When CHIR99021 is selected as the Wnt agonist, its concentration is 1-10 μM, and when A83-01 is selected as the TGFβ inhibitor, its concentration is 1-10 μM.

[0050] The culture time is 4-5 days, and the culture medium is replaced every 48 hours.

[0051] S14. Continue to induce the generation of multi-lineage liver organoids using culture medium containing FSK and cAMP agonist;

[0052] The culture medium used in this stage is the fifth culture medium, which includes basal culture medium and additives. The basal culture medium includes 7.5% FBS by volume, 17.5% StemPro34 by volume, and 75% HCM by volume; the additives include 5-25ng / ml HGF, 10-25ng / ml OSM, 1-10μM FSK, 0.25-1mM cAMP agonist, and 0.1-0.5μM DEX. The cAMP agonist is 8-Br-cAMP. The culture period is 14-20 days, and the culture medium is replaced every 72h.

[0053] S2. Metabolic maturation culture: Use maturation-inducing medium containing maturation-inducing factors to continue culturing multi-lineage liver organoids until the organoids achieve maturity; among them, maturation-inducing factors include HGF, OSM, COL-1, thyroid hormone T3, TGFβ inhibitor and γ-secretin inhibitor. Maturation-inducing medium also includes basal medium StemPro34 and William's E and DEX. The culture period is 8-10 days, and the medium is replaced every 72 hours.

[0054] The concentrations of various maturation-inducing factors are: 10-75 ng / ml HGF, 10-75 ng / ml OSM, 20-80 ng / ml COL-1, and 2-8 μM thyroid hormone T3.

[0055] The volume ratio of StemPro34 is 5-30%, and the volume ratio of Williams' E is 50-95%. Specifically, the volume ratio of StemPro34 can be 10% and the volume ratio of Williams' E can be 88%.

[0056] The concentration of DEX is 0.5-2.5 μM.

[0057] The TGFβ inhibitor was any one of A83-01, SB431542, and RepSox, with the concentration of A83-01 being 1-7.5 μM, the concentration of SB431542 being 10-50 μM, and the concentration of RepSox being 1-7.5 μM;

[0058] The γ-secretin inhibitor is any one of DBZ, RO4929097, and Compound E. The concentration of DBZ is 0.5-3 μM, the concentration of RO4929097 is 10-30 μM, and the concentration of Compound E is 2-5 μM.

[0059] Specifically, 10 ng / ml HGF, 20 ng / ml OSM, 20 ng / ml COL-1, 1 μM A83-01, 2.5 μM T3, 0.5 μM DBZ, and 0.5 μM DEX can be selected. Furthermore, the concentrations of each factor are not limited to these and can be achieved within the stated ranges. TGFβ inhibitors are not limited to the listed A83-01, SB431542, and RepSox, and γ-secretin inhibitors are not limited to the listed DBZ, RO4929097, and Compound E. Any inhibitor that can achieve the objectives of the present invention is contemplated and is not specifically listed here.

[0060] Furthermore, the multi-lineage hepatic organoids used for metabolic maturation are not limited to those obtained using the aforementioned method. For example, multi-lineage hepatic organoids obtained with application numbers CN202210119937.5 and CN202211314796.9 can also be cultured using this maturation-enhancing medium. Only one such multi-lineage hepatic organoid is used as an example here. This method is suitable for maturation of hepatic organoids derived from PSCs.

[0061] Example 1 Construction of mature multi-lineage liver organoids

[0062] S1. Induction of differentiation of multi-lineage liver organoids (differentiation days 1-24):

[0063] When the confluence of PSCs reaches 80-95%, differentiation is initiated.

[0064] S11 (differentiation days 1-3): Induce PSCs and produce HAND1 + Mesoderm and FOXA2 + Endoderm.

[0065] Differentiation day 1: The culture medium used was RPMI-1640 containing 2% B27 by volume plus 50 ng / ml BMP4 and 100 ng / ml Activin A.

[0066] Differentiation Days 2-3: Culture for 2 days in the first culture medium, which consists of RPMI-1640 containing 2% B27 by volume supplemented with 100 ng / ml Activin A, 5 ng / ml BMP4, and 50 ng / ml bFGF. Change the culture medium every 24 hours.

[0067] S12 (differentiation day 4-6): Produce KDR + HHEX of mesodermal progenitor cells + HNF4α + Endoderm posterior to the foregut.

[0068] The cells were cultured for 3 days in a second culture medium containing 2% by volume of B27, 23% by volume of StemPro34, and 75% by volume of IMDM supplemented with 10 ng / ml BMP4, 500 ng / ml bFGF, and 3 μM CHIR99021. The culture medium was changed daily.

[0069] S13 (differentiation day 7-10): generate CD31 + / CD34 + Primary endothelial cells of the hepatic endoderm.

[0070] S131, 3D repolymerization based on extracellular matrix (ECM) components - COL-1 and LN-411:

[0071] Differentiation day 6 derivatives were enzymatically digested and collected by centrifugation. Single cells were resuspended in a third culture medium supplemented with COL-1 at a final concentration of 0.25 mg / ml and LN-411 at 1 μg / ml. After mixing, the cells were seeded into a 96-well U-shaped culture plate at a seeding size of 10,000 cells / well for 3D repolymerization.

[0072] The third culture medium used was: 2% by volume of B27, 23% by volume of StemPro34, and 75% by volume of IMDM supplemented with 100 ng / ml VEGF, 10 ng / ml EGF, 10 ng / ml bFGF, 3 μM CHIR99021, 2.5 μM A83-01, 0.25 mg / ml COL-1, and 1 μg / ml LN-411.

[0073] S132, after the first culture medium change, continue to use the fourth culture medium to induce the production of CD31 + / CD34 + The fourth culture medium does not contain COL-1 and LN-411.

[0074] The fourth culture medium used was: 2% by volume of B27, 23% by volume of StemPro34, and 75% by volume of IMDM, supplemented with 100 ng / ml VEGF, 10 ng / ml EGF, 10 ng / ml bFGF, 3 μM CHIR99021, and 2.5 μM A83-01.

[0075] In S13, the culture medium was changed every 48 h.

[0076] S14 (Differentiation Days 11-24): Generation of multi-lineage liver organoids.

[0077] The culture medium used was 7.5% FBS, 17.5% StemPro34, and 75% HCM (volume ratio) supplemented with 10 ng / ml HGF, 20 ng / ml OSM, 1 μM FSK, 500 μM 8-Br-cAMP, and 0.5 μM DEX. The culture medium was changed every 72 h.

[0078] By differentiation day 24, multilineage hepatic organoids were formed, with a sinusoidal endothelial network running throughout the hepatic clusters. These cells contained five cell types: sinusoidal endothelial cells, hepatocytes, cholangiocytes, macrophages, and hepatic stellate cells.

[0079] S2, metabolic maturation culture (differentiation day 25-32):

[0080] After differentiation day 24, switch to the maturation medium for hepatic multilineage organoids and change the medium every 72 hours.

[0081] The maturation-promoting medium contains 2% B27 by volume, 10% StemPro34 by volume, and 88% William's E, supplemented with 10 ng / ml HGF, 20 ng / ml OSM, 20 ng / ml COL-1, 1 μM A83-01, 2.5 μM T3, 0.5 μM DBZ, and 0.5 μM DEX.

[0082] By differentiation day 32, organoids had reached maturity and highly expressed drug-metabolizing enzymes.

[0083] After generating liver organoids according to the multi-lineage liver organoid differentiation protocol, they were cultured for 8 days to mature. The cell composition of the organoids before and after maturation was analyzed. Figure 1 As shown, flow cytometry analysis showed that compared with the mature culture before ALB + The hepatocyte content increased by approximately 7.4%, while the other four cell types were present and their relative abundance remained stable. This result indicates that the maturation-promoting medium can preserve the various cell types of multi-lineage liver organoids and promote the further differentiation of some immature hepatic progenitor cells into hepatocytes.

[0084] Correspondingly, mature culture significantly upregulated the adult hepatocyte marker ALB, which was close to the expression level of adult primary liver ( Figure 2 AFP, a marker of fetal liver cells, was significantly downregulated, although it was still higher than that of primary liver ( Figure 2Importantly, the results of liver metabolism-related marker expression analysis showed that: pharmacokinetic-related genes (phase I metabolizing enzymes such as CYP3A4, CYP2C9; phase II metabolizing enzymes such as UGT1A1, UGT1A7; phase III transporter pumps such as MDR1, MRP2), glucose metabolism genes (such as GBE1), lipid metabolism genes (such as APGL), and bile acid metabolism (such as BSEP) were all significantly upregulated ( Figure 2 Middle C). Compared with primary liver tissue, except for APGL, the expression levels of other markers showed no significant differences.

[0085] The results of principal component analysis based on transcript sequencing showed that the gene expression profile of mature PSC-derived multi-lineage liver organoids was closer to adult liver tissue, while that of pre-mature PSC-derived multi-lineage liver organoids was closer to fetal liver tissue ( Figure 3 ).

[0086] In summary, this maturation culture method achieved metabolic maturation of liver organoids.

[0087] Comparative Example 1 Effect of Maturation-Promoting Induction Factor

[0088] In order to study the effects of various maturation-inducing factors, the following comparative experiment was designed.

[0089] Table 1 Combination of different induction factors

[0090]

[0091] Note: “-” means not added, “+” means added.

[0092] Figure 4 The results of different induction factor combinations are compared. As can be seen from the figure, in the maturation-promoting culture medium of multi-lineage liver organoids, the best effect is achieved when the maturation-promoting induction factors simultaneously include HGF, OSM, COL-1, thyroid hormone T3, TGFβ inhibitor A83-01, and γ-secretin inhibitor DBZ. Removing any one of them will significantly affect the expression of key metabolic genes.

[0093] Comparative Example 2 Effects of Different TGFβ Inhibitors or γ-Secretin Inhibitors

[0094] In order to verify that other TGFβ inhibitors and γ-secretin inhibitors also have the maturation effect, verification experiments were conducted on other TGFβ inhibitors and γ-secretin inhibitors.

[0095] The results are as follows Figure 5As shown in the figure, similar maturation-promoting effects can be achieved at appropriate concentrations by replacing the TGFβ inhibitor type with 10μM SB431542 and 1μM RepSox, and replacing the γ-secretin inhibitor with 10μM RO4929097 and 2μM Compound E.

[0096] Comparative Example 3 Effects of basal culture medium components

[0097] In order to verify the effects of basal culture medium components, StemPro34 and William's E were withdrawn, i.e., StemPro34 withdrawal group (0% StemPro34, 98% William's E) and William's E withdrawal group (98% StemPro34, 0% William's E), and the results were compared. Figure 6 shown.

[0098] As can be seen from the figure, removing any one of them will significantly affect the expression of key drug metabolism genes ( Figure 6 The reason may be related to the significant change in the proportion of liver physiologically relevant cell types ( Figure 6 (B) This indicates that the multi-lineage hepatic organoid maturation medium must contain both the basal medium StemPro34 and William's E.

[0099] Table 2 Reagent list

[0100]

[0101] Table 3 Antibody list

[0102]

[0103] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for metabolic maturation of multi-lineage liver organoids, characterized by: The method comprises the following steps: S1. Induce PSCs to form multi-lineage liver organoids with at least hepatic sinusoidal endothelial cells, hepatocytes, cholangiocytes, macrophages, and hepatic stellate cells; S11, first culture in RPMI-1640 medium containing 50 ng / ml BMP4, 100 ng / ml Activin A, and 2% B27 by volume for 1 day, then culture in RPMI-1640 medium containing 100 ng / ml Activin A, 5 ng / ml BMP4, 50 ng / ml bFGF, and 2% B27 by volume for 2-3 days to produce HAND1 + Mesoderm and FOXA2 + Endoderm; S12, use the culture medium containing 2% B27, 23% StemPro34, 75% IMDM, 10 ng / ml BMP4, 500 ng / ml bFGF, 3 μM CHIR99021 to continue to induce KDR + HHEX of mesodermal progenitor cells + 、HNF4α + posterior foregut endoderm; S13. The derivative obtained in S2 was enzymatically digested and collected by centrifugation. The single cells were resuspended in a medium containing 2% by volume of B27, 23% by volume of StemPro34, 75% by volume of IMDM, 100 ng / ml VEGF, 10 ng / ml EGF, 10 ng / ml bFGF, 3 μM CHIR99021, and 2.5 μM A83-01. 0.25 mg / ml of COL-1 and 1 μg / ml of LN-411 were then added for 3D repolymerization. After the culture medium was replaced, the culture medium was used to induce the production of CD31 + / CD34 + Hepatic endoderm of primary endothelial cells, and no COL1 and LN411 were added thereafter; S14. Continue to induce the generation of sinusoidal network liver organoids using a culture medium containing 7.5% FBS (volume ratio), 17.5% StemPro34 (volume ratio), 75% HCM (volume ratio), 10 ng / ml HGF, 20 ng / ml OSM, 1 μM FSK, 500 μM 8-Br-cAMP, and 0.5 μM DEX; S2. Metabolic maturation culture: Multi-lineage liver organoids are cultured in a maturation-promoting medium containing maturation-promoting factors until the organoids achieve maturation. The maturation-promoting factors include 10-75 ng / ml HGF, 10-75 ng / ml OSM, 20-80 ng / ml COL-1, 2-8 μM thyroid hormone T3, a TGFβ inhibitor, a γ-secretin inhibitor, and 0.5-2.5 μM DEX. The maturation-promoting medium also includes 2% B27 by volume, 5-30% StemPro34 by volume, and 50-95% Williams's E by volume. The TGFβ inhibitor is any one of A83-01, SB431542, and RepSox. The concentration of A83-01 is 1-7.5 μM, the concentration of SB431542 is 10-50 μM, and the concentration of RepSox is 1-7.5 μM. The γ-secretin inhibitor is any one of DBZ, RO4929097, and Compound E. The concentration of DBZ is 0.5-3 μM, the concentration of RO4929097 is 10-30 μM, and the concentration of Compound E is 2-5 μM.

2. The method for metabolic maturation of multi-lineage liver organoids according to claim 1, wherein: The culture duration for S1 is 24-30 days, and the culture duration for S2 is 8-10 days; in S2, the culture medium is changed every 72 hours.

3. An application of a metabolically mature multi-lineage liver organoid constructed by the metabolic maturation method of a multi-lineage liver organoid according to any one of claims 1-2 in drug toxicity screening and drug metabolism evaluation.

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