Multi-lineage liver organ metabolic maturation method and application

Abstract

The invention provides a multi-lineage liver organ metabolic maturation method and application, and the method can significantly improve the expression level of metabolism-related genes on the premise of maintaining various cell types related to liver physiology. Comprise drug metabolism (phase I metabolic enzyme, phase II metabolic enzyme and phase III transporter), lipid metabolism, glycometabolism, bile acid metabolism related markers and the like; moreover, the overall expression profile is close to that of primary liver tissues, and the mature liver organs produced by the method are particularly suitable for the applications of preclinical drug hepatotoxicity screening, metabolism evaluation and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell culture, and in particular relates to a method for metabolically maturing multi-lineage liver organoids and its application. Background Art

[0002] Since the first successful induction of differentiation of human pluripotent stem cells (PSC) into hepatocytes in 2010, in the past decade, through the continuous efforts of scientists around the world, this technology has made great breakthroughs in both the purity of 2D hepatocytes and the complexity of 3D liver organoids, providing an important model for the basic research and medical application of liver diseases. On the one hand, these 2D / 3D liver derivatives can be continuously induced and differentiated from PSC in vitro, which once and for all solves the problem of limited sample sources. On the other hand, the advantage of humanization can not only produce results close to the real world in preclinical drug research, but also provides a new candidate solution for liver regeneration and repair.

[0003] As a substitute model for the largest metabolic organ in the human body, at present, the insufficient maturity of PSC liver derivatives, especially the problem of low metabolic capacity, is the key bottleneck hindering their further popularization and application: most of the maturity based on 2D hepatocyte induction methods is only approximate to the fetal level; although 3D liver organoids, relying on the advantage of multi-cell interaction, have improved the expression of mature 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 adult liver; therefore, it seriously affects the accuracy of disease simulation and hepatotoxicity screening research. Summary of the Invention

[0004] In view of this, the present invention aims to propose a method for metabolically maturing multi-lineage liver organoids and its application. 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, glucose metabolism, and bile acid metabolism, etc., 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 realized as follows: A method for metabolically maturing multi-lineage liver organoids, the method comprising the following steps: S1. Induce PSC to form multi-lineage liver organoids having at least hepatocytes, cholangiocytes, macrophages, and hepatic stellate cells; S2. Metabolic maturation culture: Continuously culture the multi-lineage hepatic organoids with a maturation-promoting medium containing maturation-promoting inducing factors until the organoids achieve maturation; wherein, the maturation-promoting inducing factors include HGF, OSM, COL-1, thyroid hormone T3, TGFβ inhibitor, and γ-secretase inhibitor, and the maturation-promoting medium further includes the basal medium StemPro34 and William's E.

[0006] In some embodiments, the concentration of each maturation-promoting 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; The volume ratio of StemPro34 is 5 - 30%, and the volume ratio of William's E is 50 - 95%.

[0007] 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; The γ-secretase 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.

[0008] In some embodiments, the maturation-promoting medium further includes 0.5 - 2.5 μM DEX.

[0009] In some embodiments, the method for inducing PSC to form multi-lineage hepatic organoids is: sequentially inducing the differentiation of PSC with a medium containing BMP4 and bFGF, a medium containing BMP4, bFGF, and a Wnt agonist, a medium containing COL-1 and LN-411, a medium containing VEGF, EGF, and bFGF, and a medium containing FSK and a cAMP agonist.

[0010] In some embodiments, the specific method for inducing PSC to form multi-lineage hepatic organoids is: S11. Induce PSC to simultaneously generate HAND1 + mesoderm and FOXA2 + endoderm; S12. Continuously induce the generation of HHEX + mesoderm progenitor cells containing KDR + and HNF4α+ Posterior endoderm of the foregut; S13. The derivative obtained in S12 is enzymatically digested, centrifuged and collected, and after resuspending the single cells with a medium containing VEGF, EGF, and bFGF, COL1 and LN411 are added for 3D repolymerization; After culturing until the medium is changed, continue to use the medium at this stage to induce the generation of CD31 + / CD34 + Intrahepatic endoderm of nascent endothelial cells, and COL1 and LN411 are no longer added thereafter; S14. Use a medium containing FSK and cAMP agonist to continue to induce the generation of multi-lineage liver organoids.

[0011] In some embodiments, the culture days of S1 are 24-30 days, and the culture days of S2 are 8-10 days; in S2, the medium is changed every 72 h.

[0012] The present invention also provides a maturation-promoting medium, which is used for the method for metabolically maturing the above-mentioned multi-lineage liver organoids, and includes a basal medium and a maturation-promoting inducer. The basal medium includes StemPro34 and William's E, and the maturation-promoting inducer includes HGF, OSM, COL-1, thyroid hormone T3, TGFβ inhibitor, and γ-secretase inhibitor.

[0013] The present invention also provides a metabolically mature multi-lineage liver organoid constructed by the method for metabolically maturing the above-mentioned multi-lineage liver organoids. The adult hepatocyte marker ALB in this liver organoid is up-regulated to be close to the expression level of adult primary liver, and the related markers of phase I metabolic enzymes, phase II metabolic enzymes, phase III transport pumps, and sugar metabolism genes, lipid metabolism genes, and bile acid metabolism genes related to drug metabolism are all up-regulated to be close to the primary liver tissue.

[0014] The present invention also provides an application of the above-mentioned metabolically mature multi-lineage liver organoid in drug toxicity screening and drug metabolism evaluation.

[0015] Compared with the prior art, the method for metabolically maturing the multi-lineage liver organoids and the application thereof according to the present invention have the following advantages: The method for metabolically maturing the multi-lineage liver organoids according to 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, etc., on the premise of 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 metabolism evaluation of candidate drugs. Description of the Drawings

[0016] The accompanying drawings, which form a part of the present invention, are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings: Figure 1 Analysis of the composition of organoid cells before and after maturation culture; before maturation refers to hepatic organoids induced to differentiate until differentiation day 24; after maturation refers to hepatic organoids after maturation culture until differentiation day 32; biological replicates n = 4, *, P < 0.05; Figure 2 Analysis of the expression of liver maturation genes before and after maturation culture; A is the analysis of AFP expression levels before and after maturation; B is the analysis of ALB expression levels before and after maturation; C is the analysis of the expression levels of liver metabolism-related genes before and after maturation; the expression level before maturation (differentiation day 24) is set to a value of 1, and the values of each group are relative expression levels compared thereto; primary liver tissue is set as the positive control; biological replicates are all n = 4, *, P < 0.05; **, P < 0.01; P***, P < 0.001; Figure 3 Principal component analysis of gene expression before and after maturation culture, principal component analysis after transcriptome sequencing; adult liver tissue, fetal liver tissue, PSC-derived hepatocytes, and PSCs are set as controls, biological replicates n = 3, and the data of PSC-derived hepatocytes are from DOI: 10.1038 / s41422-019-0196-x; Figure 4 For the promoting maturation effects of each inducer component, the expression level value of the group with all maturation-promoting factors added is set to 1, and the values of each factor-removed group are relative expression levels compared thereto; biological replicates n = 4; *, P < 0.05; **, P < 0.01; P***, P < 0.001; ALB is a marker of adult hepatocytes; CYP3A4 is a phase I drug-metabolizing enzyme of hepatocytes; UGT1A1 is a phase II drug-metabolizing enzyme; N.D., below the limit of detection; Figure 5 For the effects of different TGFβ inhibitors or γ-secretase inhibitors on the promoting maturation effect; the expression level before maturation (differentiation day 24) is set to a value of 1, and the values of each group are relative expression levels compared thereto; biological replicates n = 3; ALB is a marker of adult hepatocytes; CYP3A4 is a phase I drug-metabolizing enzyme; UGT1A1 is a phase II drug-metabolizing enzyme; Figure 6Promoting maturation effect of basal medium components; A, influence of retention / removal of basal medium on expression of maturation markers; the expression level before maturation (day 24 of differentiation) was set as 1, and the values of each group were relative expression levels compared with it; biological replicates of this method, n = 4; biological replicates of StemPro34 removal group and William's E removal group, n = 3; ALB is a marker of adult hepatocytes; CYP3A4 is a phase I drug-metabolizing enzyme of hepatocytes; UGT1A1 is a phase II drug-metabolizing enzyme; **, P < 0.01; P***, P < 0.001; B, influence of retention / removal of basal medium on the cell composition of organoids, biological replicates, n = 3. Detailed implementation manners

[0017] It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments may be combined with each other.

[0018] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0019] The reagents used in the examples and comparative examples are shown in Table 2-3.

[0020] The present invention provides a method for metabolically maturing multi-lineage liver organoids, which includes the following steps: S1. Induce PSCs to form multi-lineage liver organoids having at least hepatocytes, cholangiocytes, macrophages, and hepatic stellate cells; Specifically, use a medium containing BMP4 and bFGF, a medium containing BMP4, bFGF, and a Wnt agonist, a medium containing COL1 and LN411, a medium containing VEGF, EGF, and bFGF, and a medium containing FSK and a cAMP agonist to induce the differentiation of PSCs in sequence.

[0021] More specifically, it includes the following steps: S11. Use a medium containing BMP4 and bFGF to induce PSCs to simultaneously produce HAND1 + mesoderm and FOXA2 + endoderm; S111. Culture for 1 day in a conventional medium, which includes a basal medium and additives. The basal medium is RPMI-1640 containing 2% B27 by volume, and the additives include 10 - 75 ng / ml BMP4 and 100 - 150 ng / ml Activin A.

[0022] S112. Culture for 2 - 3 days in the first medium, which includes a basal medium and additives. The basal medium is RPMI - 1640 containing 2% B27 by volume, and the additives include 100 - 150 ng / ml Activin A, 1 - 10 ng / ml BMP4, and 50 - 200 ng / ml bFGF. The medium is changed every 24 h.

[0023] S12. Continue to induce the generation of HHEX + mesoderm progenitor cells containing KDR + HNF4α + posterior - lateral endoderm of the foregut; The medium used in this stage is the second medium, 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 10 - 50 ng / ml BMP4, 200 - 500 ng / ml bFGF, and Wnt agonists. The Wnt agonists include CHIR99021, Wnt3a, and Wnt7a. When the Wnt agonist is CHIR99021, its concentration is 1 - 5 μM, and the culture duration is 3 - 4 days. The medium is changed daily.

[0024] S13. Digest the derivative obtained in S12 by enzymatic digestion, collect by centrifugation, resuspend the single - cells with a medium containing VEGF, EGF, and bFGF, add COL - 1 and LN - 411, mix well, and inoculate into a 96 - well U - shaped culture plate at an inoculation density of 10000 cells / well for 3D repolymerization; The medium used is the third medium, 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, Wnt agonists, TGFβ inhibitors, 0.25 - 1.25 mg / ml COL - 1, and 1 - 5 μg / ml LN - 411. The Wnt agonists include CHIR99021, Wnt3a, and Wnt7a. When the Wnt agonist is CHIR99021, its concentration is 1 - 10 μM. The TGFβ inhibitors include A83 - 01 and SB431542. When the TGFβ inhibitor is A83 - 01, its concentration is 1 - 10 μM.

[0025] After culturing until the medium is changed, continue to use the above-mentioned fourth medium without COL-1 and LN-411 to induce the generation of CD31 + / CD34 + intrahepatic endoderm of nascent endothelial cells.

[0026] Specifically, the medium used is the fourth medium, 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, Wnt agonists, and TGFβ inhibitors. The Wnt agonists include CHIR99021, Wnt3a, and Wnt7a, and the TGFβ inhibitors include A83-01 and SB431542. When the Wnt agonist is CHIR99021, its concentration is 1 - 10 μM, and when the TGFβ inhibitor is A83-01, its concentration is 1 - 10 μM.

[0027] The culture time is 4 - 5 days, and the medium is changed every 48 h.

[0028] S14. Use the medium containing FSK and cAMP agonist to continue to induce the generation of multi-lineage liver organoids; The medium used at this stage is the fifth medium, which includes a basal medium and additives. The basal medium includes 7.5% FBS by volume, 17.5% StemPro34 by volume, and 75% HCM by volume; the additives include 5 - 25 ng / ml HGF, 10 - 25 ng / ml OSM, 1 - 10 μM FSK, 0.25 - 1 mM cAMP agonist, 0.1 - 0.5 μM DEX. The cAMP agonist is 8-Br-cAMP, and the culture days are 14 - 20 days, and the medium is changed every 72 h.

[0029] S2. Metabolic maturation culture: Use the maturation medium containing maturation induction factors to continue culturing multi-lineage liver organoids until the organoids are mature; among them, the maturation induction factors include HGF, OSM, COL-1, thyroid hormone T3, TGFβ inhibitor, and γ-secretase inhibitor. The maturation medium also includes the basal medium StemPro34, William's E, and DEX. The culture days are 8 - 10 days, and the medium is changed every 72 h.

[0030] The concentration of each maturation induction factor is: 10 - 75 ng / ml HGF, 10 - 75 ng / ml OSM, 20 - 80 ng / ml COL-1, 2 - 8 μM thyroid hormone T3.

[0031] The volume ratio of StemPro34 is 5 - 30%, and the volume ratio of William's E is 50 - 95%. Specifically, the volume ratio of StemPro34 can be selected as 10%, and the volume ratio of William's E is 88%.

[0032] The concentration of DEX is 0.5 - 2.5 μM.

[0033] 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 - secretase 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.

[0034] 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. Moreover, the concentration of each factor is not limited to this, and it can be achieved within the above - mentioned range. The TGFβ inhibitor is not limited to the listed A83 - 01, SB431542, and RepSox, and the γ - secretase inhibitor is not limited to the listed DBZ, RO4929097, and Compound E. As long as the purpose of the present invention can be achieved, they are not listed one by one here.

[0035] In addition, the multi - lineage liver organoids for metabolic maturation culture are not limited to the multi - lineage liver organoids obtained by the above - mentioned method. For example, the multi - lineage liver organoids obtained by the application numbers CN202210119937.5 and CN202211314796.9 can also use this maturation - promoting culture medium. Here, only one kind of multi - lineage liver organoid is taken as an example. This method is suitable for the maturation of liver organoids developed from PSCs.

[0036] Example 1 Construction of mature multi - lineage liver organoids S1. Inductive differentiation of multi - lineage liver organoids (differentiation days 1 - 24): When the confluence of PSCs reaches 80 - 95%, initiate differentiation.

[0037] S11 (differentiation days 1 - 3): Induce PSCs to simultaneously produce HAND1 + mesoderm and FOXA2 + endoderm.

[0038] Differentiation day 1: The medium used was RPMI-1640 containing 2% B27 by volume supplemented with 50 ng / ml BMP4 and 100 ng / ml Activin A.

[0039] Differentiation days 2-3: Cultured in the first medium for 2 days. The first medium used was RPMI-1640 containing 2% B27 by volume supplemented with 100 ng / ml Activin A, 5 ng / ml BMP4, and 50 ng / ml bFGF, and the medium was changed every 24 h.

[0040] S12 (differentiation days 4-6): Generate HHEX + mesoderm progenitor cells + HNF4α + posterior foregut endoderm.

[0041] Cultured in the second medium for 3 days. The second medium used was IMDM containing 2% B27 by volume, 23% StemPro34 by volume, and 75% IMDM supplemented with 10 ng / ml BMP4, 500 ng / ml bFGF, and 3 μM CHIR99021, and the medium was changed daily.

[0042] S13 (differentiation days 7-10): Generate intrahepatic endoderm containing CD31 + / CD34 + nascent endothelial cells.

[0043] S131, 3D repolymerization based on extracellular matrix (ECM) components - COL-1 and LN-411: The derivatives on differentiation day 6 were enzymatically digested, centrifuged and collected, and the single cells were resuspended using the third medium, and the third medium was supplemented with COL-1 at a final concentration of 0.25 mg / ml and LN-411 at 1 μg / ml; after mixing, they were seeded into a 96-well U-bottom culture plate at an inoculation density of 10,000 cells / well for 3D repolymerization.

[0044] The third medium used was: IMDM containing 2% B27 by volume, 23% StemPro34 by volume, and 75% 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.

[0045] S132. After culturing until the first medium change, continue to use the fourth medium to induce the generation of intrahepatic endoderm containing CD31 + / CD34 + nascent endothelial cells, and the fourth medium does not contain COL-1 and LN-411.

[0046] The fourth medium used is: adding 100 ng / ml VEGF, 10 ng / ml EGF, 10 ng / ml bFGF, 3 μM CHIR99021, and 2.5 μM A83-01 to IMDM containing 2% (v / v) B27, 23% (v / v) StemPro34, and 75% (v / v) IMDM.

[0047] In S13, change the medium every 48 h.

[0048] S14 (differentiation days 11 - 24): Generate multi-lineage liver organoids.

[0049] The medium used is: adding 10 ng / ml HGF, 20 ng / ml OSM, 1 μM FSK, 500 μM 8-Br-cAMP, and 0.5 μM DEX to HCM containing 7.5% (v / v) FBS, 17.5% (v / v) StemPro34, and 75% (v / v) HCM, and change the medium every 72 h.

[0050] By differentiation day 24, multi-lineage liver organoids are formed, with a sinusoidal endothelial network running through the liver clusters. It contains 5 cell types: hepatic sinusoidal endothelial cells, hepatocytes, cholangiocytes, macrophages, and hepatic stellate cells.

[0051] S2. Metabolic maturation culture (differentiation days 25 - 32): After differentiation day 24, switch to the maturation-promoting medium for liver multi-lineage organoids and change the medium every 72 h.

[0052] The maturation-promoting medium is: adding 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 to William's E containing 2% (v / v) B27, 10% (v / v) StemPro34, and 88% (v / v) William's E.

[0053] By differentiation day 32, the organoids are mature and highly express drug-metabolizing enzymes.

[0054] After generating liver organoids according to the multi-lineage liver organoid induction and differentiation protocol, perform 8 days of maturation culture. Analyze the cell composition of the organoids before and after maturation culture respectively, as Figure 1As shown, flow cytometry analysis indicated that: compared with before maturation culture, the content of ALB + hepatocytes increased by approximately 7.4%, while the other four cell types were all present and their relative contents remained stable. This result indicated that this maturation-promoting medium could maintain various cell types of multi-lineage liver organoids and promote the further differentiation of some immature hepatic progenitor cells into hepatocytes.

[0055] Correspondingly, maturation culture significantly upregulated the adult hepatocyte marker ALB, which was already close to the expression level of adult primary liver ( Figure 2 B in); while the fetal hepatocyte marker AFP was significantly downregulated, although it was still higher than that of primary liver ( Figure 2 A in). Importantly, the results of the expression analysis of liver metabolism-related markers showed that: drug metabolism-related genes (phase I metabolic enzymes such as CYP3A4, CYP2C9; phase II metabolic enzymes such as UGT1A1, UGT1A7; phase III transport 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 C in). Compared with primary liver tissue, except for APGL, the expression levels of other markers showed no significant differences.

[0056] Principal component analysis results based on transcriptome sequencing indicated that the gene expression profile of PSC-derived multi-lineage liver organoids after maturation was closer to that of adult liver tissue; while PSC-derived multi-lineage liver organoids before maturation were closer to fetal liver tissue ( Figure 3 ).

[0057] In summary, this maturation culture method achieved the metabolic maturation of liver organoids.

[0058] Comparative Example 1 Role of maturation-promoting induction factors To study the roles of various maturation-promoting induction factors, the following comparative experiments were designed.

[0059] Table 1 Different induction factor combination schemes

[0060] Note: "-" represents not added, and "+" represents added.

[0061] Figure 4 For the result comparison of different induction factor combination schemes, it can be seen from the figure that in the maturation-promoting medium of multi-lineage liver organoids, the maturation-promoting induction factors had the best effect when they simultaneously included HGF, OSM, COL-1, thyroid hormone T3, TGFβ inhibitor A83-01, and γ-secretase inhibitor DBZ. Removing any one of them would significantly affect the expression of key metabolic genes.

[0062] Comparative Example 2: Effects of Different TGFβ Inhibitors or γ-Secretase Inhibitors To verify that other TGFβ inhibitors and γ-secretase inhibitors also have a maturation effect, verification experiments were conducted on other TGFβ inhibitors and γ-secretase inhibitors.

[0063] The results are as Figure 5 shown. When the TGFβ inhibitor types were changed to 10 μM SB431542 and 1 μM RepSox respectively, and the γ-secretase inhibitor was changed to 10 μM RO4929097 and 2 μM Compound E respectively, approximate pro-maturation effects could also be achieved at appropriate concentrations.

[0064] Comparative Example 3: Roles of Basic Medium Components To verify the roles of basic medium components, StemPro34 and William's E were removed respectively, that is, the StemPro34-removed group (0% StemPro34, 98% William's E) and the William's E-removed group (98% StemPro34, 0% William's E), and the comparison results are as Figure 6 shown.

[0065] It can be seen from the figure that removing any one of them will significantly affect the expression of key drug-metabolizing genes ( Figure 6 A in it), and the reason may be related to the significant change in the proportion of liver physiology-related cell types ( Figure 6 B in it). It shows that in the multi-lineage liver organoid pro-maturation medium, the basic media StemPro34 and William's E must be contained simultaneously.

[0066] Table 2: Reagent List

[0067] Table 3: Antibody List

[0068] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for metabolic maturation of multi-lineage liver organoids, characterized in that: The method comprises the following steps: S1. Inducing PSCs to form multi-lineage hepatic organoids with at least hepatocytes, cholangiocytes, macrophages and hepatic stellate cells; S2. Metabolic maturation culture: Continuously culturing the multi-lineage hepatic organoids with a maturation-promoting medium containing maturation-promoting inducing factors until the organoids are matured; wherein, the maturation-promoting inducing factors include HGF, OSM, COL-1, thyroid hormone T3, TGFβ inhibitor and γ-secretase inhibitor, and the maturation-promoting medium further includes a basal medium StemPro34 and William's E.

2. The method for metabolic maturation of multi-lineage liver organoids according to claim 1, characterized in that: The concentration of each maturation-promoting inducing factor is as follows: 10 - 75 ng / ml HGF, 10 - 75 ng / ml OSM, 20 - 80 ng / ml COL-1, 2 - 8 μM thyroid hormone T3; The volume ratio of StemPro34 is 5 - 30%, and the volume ratio of William's E is 50 - 95%.

3. The method for metabolically maturing multi-lineage hepatic organoids according to claim 2, wherein: The TGFβ inhibitor is any one of A83-01, SB431542, 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-secretase inhibitor is any one of DBZ, RO4929097, 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.

4. The method for metabolically maturing a multi-lineage hepatic organoid according to claim 1, wherein: The maturation-promoting medium further includes 0.5 - 2.5 μM DEX.

5. The method for metabolically maturing a multi-lineage hepatic organoid according to claim 1, characterized in that: The method for inducing PSCs to form multi-lineage hepatic organoids is: sequentially inducing the differentiation of PSCs with a medium containing BMP4 and bFGF, a medium containing BMP4, bFGF and a Wnt agonist, a medium containing COL-1 and LN-411, a medium containing VEGF, EGF and bFGF, and a medium containing FSK and a cAMP agonist.

6. The method for metabolically maturing multi-lineage hepatic organoids according to claim 5, characterized in that: The specific method for inducing PSCs to form multi-lineage hepatic organoids is: S11. Induce PSCs to simultaneously generate HAND1 + mesoderm and FOXA2 + endoderm; S12. Continue to induce the generation of HHEX-containing mesoderm progenitor cells with KDR using a culture medium containing BMP4, bFGF, and Wnt agonist + + and HNF4α + posterior lateral endoderm of the foregut;​ S13. Enzymatically digesting and centrifugally collecting the derivatives obtained in S12, resuspending the single cells with a medium containing VEGF, EGF, bFGF, and then adding COL1 and LN411 for 3D repolymerization; After culturing until the medium is changed, continue to use the medium at this stage to induce the generation of intrahepatic endoderm containing CD31 + / CD34 + primary endothelial cells, and then no longer add COL1 and LN411; S14. Continuously inducing the generation of multi-lineage hepatic organoids with a medium containing FSK and a cAMP agonist.

7. The method for metabolic maturation of multi-lineage hepatic organoids according to claim 1, characterized in that: The culture days of S1 are 24 - 30 days, and the culture days of S2 are 8 - 10 days; in S2, the medium is changed every 72 h.

8. A maturation-promoting medium, characterized in that: The maturation-promoting medium is used for the metabolic maturation method of the multi-lineage hepatic organoids according to any one of claims 1 - 7, and includes a basal medium and maturation-promoting inducing factors, the basal medium includes StemPro34 and William's E, and the maturation-promoting inducing factors include HGF, OSM, COL-1, thyroid hormone T3, TGFβ inhibitor and γ-secretase inhibitor.

9. A metabolically mature multi-lineage hepatic organoid constructed by the method for metabolically maturing multi-lineage hepatic organoids according to any one of claims 1-7, characterized in that: The adult hepatocyte marker ALB of the liver organoids is upregulated to near the expression level of adult primary liver, and the expression of related markers of phase I metabolic enzymes, phase II metabolic enzymes, phase III transport pumps, as well as sugar metabolism genes, lipid metabolism genes, and bile acid metabolism genes related to pharmacokinetics are all upregulated to near the primary liver tissue.

10. Use of the metabolically mature multi-lineage liver organoids as described in claim 9 in drug toxicity screening and drug metabolism evaluation.

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