CN111004770A

CN111004770A - Functional hepatocyte induction method and special three-dimensional induction culture medium and application thereof

Info

Publication number: CN111004770A
Application number: CN201910910755.8A
Authority: CN
Inventors: 王韫芳; 王术勇; 王璇; 谭作龙; 宿钰鑫; 胡健; 王勇
Original assignee: Institute of Pharmacology and Toxicology of AMMS
Current assignee: Institute of Pharmacology and Toxicology of AMMS
Priority date: 2019-09-25
Filing date: 2019-09-25
Publication date: 2020-04-14
Anticipated expiration: 2039-09-25
Also published as: CN111004770B

Abstract

The invention discloses a functional hepatocyte induction method, a special three-dimensional induction culture medium and application thereof, belonging to the technical field of bioengineering. The functional hepatocyte three-dimensional induction culture medium provided by the invention contains various micromolecules and cytokines, can be effectively used in a method for obtaining the functional hepatocyte by inducing a liver organoid with stem/progenitor cell characteristics, can be used for producing artificial liver and simulating in-vitro liver function, and can also be used as seed cells for establishing a liver disease treatment drug screening model and an in-vitro liver disease simulation model.

Description

Functional hepatocyte induction method and special three-dimensional induction culture medium and application thereof

Technical Field

The invention belongs to the technical field of bioengineering, relates to a functional hepatocyte induction method, a special three-dimensional induction culture medium and application thereof, and particularly relates to a three-dimensional culture medium for obtaining functional hepatocytes by using liver organoid with stem/progenitor cell characteristics and application of the functional hepatocytes obtained by induction in preparation of a medicament for treating liver diseases.

Background

The liver is the largest digestive organ of the human body and regulates many important physiological functions of the body, including metabolism of nutrients, synthesis of urea, synthesis of various important proteins, secretion of bile, detoxification of heterologous substances, and the like. Liver damage or disease caused by various reasons usually has serious consequences, such as viral hepatitis, hepatic fibrosis, hereditary metabolic liver disease, liver cirrhosis, biliary tract diseases and the like, which bring great harm to human bodies. According to relevant statistics, the number of newly added end-stage Liver disease (ESLD) cases is about 600 ten thousand every year worldwide, and about 100 ten thousand deaths occur. China is a country with The most extensive and largest harm degree of liver diseases, The morbidity and mortality of various liver diseases are in The top worldwide, The number of people died due to ELSD in China is nearly 40 thousands every year, and The number of people died due to The disease is 51% of The worldwide number (Wang FS, Fan JG, Zhang Z, etc.. The global burden of liver disease: The major impact of China [ J ]. Hepatology,2014,60: 2099. 2108.). Liver transplantation is the only effective treatment mode for end-stage liver diseases, but the serious shortage of donor livers is the most important factor for restricting the wide development of liver transplantation. In the united states, for example, the annual supply-to-demand ratio for organ transplantation is approximately 1: 4; in china, for example 2015, 2581 cases of liver transplants all over the country (data source: chinese liver transplantation registration system) and 30 or more ten thousand cases of patients waiting for liver transplantation fail to receive timely liver transplantation treatment, while patients listed on the transplantation waiting list are still rapidly increasing at a rate of 11% per year, and a large number of patients (about 17%) die unfortunately while waiting for liver transplantation. For many years, cell transplantation therapy using human hepatocytes as a source and medical adjuvant therapy using a bioartificial liver as a means have achieved good therapeutic effects in preclinical studies and small-scale clinical applications, however, obtaining and culturing human hepatocytes is still technically very difficult and difficult to apply in large scale, and artificial liver is also only a conservative adjuvant therapy mode in medical science and is difficult to fundamentally replace liver transplantation. In recent years, the development of stem cell technology provides a brand new cell source for acquiring functional liver cells, and opens up a new path for treating end-stage liver diseases caused by various reasons.

Developmentally, Liver Stem Cells have the potential for self-renewal and bidirectional differentiation, are seed Cells for the production of mature hepatocytes, are the major source of Liver epithelial system formation, and are therefore developmentally important (Miyajima A, tanakaM, Itoh T.Stem/Progenitor Cells in Liver Development, Homeostasis, Regeneration, and reproduction [ J ]. Stem Cell,2014,14: 561-. Because of this property of liver Stem cells, scientists have been obtaining liver Stem cells in vitro for many years by different approaches and techniques (Zhang RR, Zhang YW, Li B, etc. Heastic Stem cells with self-recycled and productive functional area harbored in CDCP 1-reactive subpartictions of human liver cells [ J ] Stem Cell research Ther,2018,9:29.) through their extensive expansion and differentiation to obtain sufficient functional hepatocytes. Obtaining hepatic stem cells from fetal liver and adult liver by cell isolation combined with cell surface specific markers and amplification in vitro is an important way, but the acquisition of human primary tissues is limited after all, which limits the application of this way in obtaining liver cells widely. With the establishment of human pluripotent Stem Cells (hPSCs), such as Embryonic Stem Cells (hESCs) and induced pluripotent Stem Cells (hipSCs), a completely new approach to the acquisition of functional hepatocytes is provided (Murry CE, Keller G. Differencention of Embryic Stem Cells clinical great positions: Lessons from Embryic Development [ J ] Cell,2008,132: 680. 661.). Pluripotent stem cells have the potential to self-renew indefinitely and to differentiate into any cell type in the body, and theoretically can provide a large number of liver stem cells, and then can provide sufficient functional hepatocytes.

Different laboratories at home and abroad have used different means to obtain the liver stem cells induced by hPSCs. For example, in 2014, the hans. clever team separated cells from the primary Liver tissue excised by surgery and screened the cells by surface labeling EPCAM, and the three-dimensional organoid Culture method combining cytokines and small molecule compounds using extracellular matrix gel matrix or BME succeeded in amplifying in vitro a large number of Liver stem cells with bidirectional differentiation potential (Huch M, Gehart H, van b extel R, etc.. Long-Term Culture of Genome-ble Stable stem cells from Adult Human Liver Cell [ J ]. 2015,160:299-312.), which established a good foundation for amplifying in vitro a large number of Liver stem cells and functional Liver cells, however, the primary Liver tissue has limitations in source and quantity, and is expected to be applied to large-scale clinical or pharmaceutical research. The inventors have also obtained liver organoids with bidirectional differentiation potential to functional hepatocytes and cholangiocytes by using sequential two-dimensional culture followed by three-dimensional culture with human embryonic stem cells (hESCs from commercial sources) as a source (joja, research on constructing liver organoids with bidirectional differentiation potential from human pluripotent stem cells, university of north and river medical science, master paper, 2018), and by using the liver organoids, sufficient functional hepatocytes can be obtained by three-dimensional induction culture, but the hepatocytes obtained by the method express a large amount of immature functional marker Alpha Fetoprotein (AFP), and have more limitations for their applications.

Disclosure of Invention

In view of one or more of the problems of the prior art, one aspect of the present invention provides a functional hepatocyte three-dimensional induction culture medium, comprising a first-stage three-dimensional culture medium and a second-stage three-dimensional culture medium; wherein:

the first-stage three-dimensional culture medium takes an HCM culture medium (Lonza)/HM culture medium (Sciencell) as a basic culture medium, and also comprises 0.01-1% BSA, 0.2-10% B-27 and one or more of the following components: antioxidants, L-ascorbate, ROCK signaling pathway inhibitors, hepatocyte growth factor, Oncostatin M, dexamethasone, penicillin and streptomycin;

the second-stage three-dimensional culture medium takes an HCM culture medium (Lonza)/HM culture medium (Sciencell) as a basic culture medium, and also comprises 0.01% -1% BSA, 0.2% -10% B-27 and one or more of the following components: antioxidants, L-ascorbate, ROCK signaling pathway inhibitors, dexamethasone, insulin-transferrin-selenium sodium, epidermal growth factor, adenylate cyclase agonists, vitamin K2, lithocholic acid, penicillin and streptomycin.

In the first-stage three-dimensional culture medium, the antioxidant is N-acetyl-L-cysteine, and the concentration is 0.1-10mM, preferably 1.25 mM; the concentration of the L-ascorbate is 0.05-2mM, preferably 0.2 mM; the ROCK signal pathway inhibitor is Y-27632(StemCell) with the concentration of 5-20 μ M, preferably 10 μ M; the hepatocyte cell growth factor is HGF, the concentration is 5-50ng/mL, and the optimal concentration is 20 ng/mL; the oncostatin M is OSM, the concentration is 5-50ng/mL, and the preferred concentration is 20 ng/mL; the dexamethasone is Dex, and the concentration is 0.1-1mM, preferably 0.5 mu M; the concentration of the penicillin is 10-1000U/mL, preferably 100U/mL; the concentration of streptomycin is 0.01-1mg/mL, preferably 0.1 mg/mL.

In the second-stage three-dimensional culture medium, the antioxidant is N-acetyl-L-cysteine, and the concentration is 0.1-10mM, preferably 1.25 mM; the concentration of the L-ascorbate is 0.05-2mM, preferably 0.2 mM; the ROCK signal pathway inhibitor is Y-27632(StemCell) with the concentration of 5-20 μ M, preferably 10 μ M; the insulin-transferrin-selenium sodium is ITS, the concentration is 0.5% -10%, and the optimal concentration is 1%; the epidermal growth factor is EGF, and the concentration is 5-50ng/mL, preferably 10 ng/mL; the dexamethasone is Dex, and the concentration is 0.1-1mM, preferably 0.5 mu M; the adenylate cyclase agonist is 8-Br-cAMP (Selleck) and has the concentration of 50-200 mu M, preferably 100 mu M; the vitamin K2 is vitamin K2(Sigma) with the concentration of 5-20 μ M, preferably 10 μ M; the Lithocholic acid is Lithocholic acid (Sigma) with the concentration of 5-20 mu M, preferably 10 mu M; the dexamethasone is Dex, and the concentration is 0.1-1mM, preferably 0.5 mu M; the concentration of the penicillin is 10-1000U/mL, preferably 100U/mL; the concentration of streptomycin is 0.01-1mg/mL, preferably 0.1 mg/mL.

The formula of the three-dimensional culture medium in the first stage is selected from any one of the following formulas:

formula 1: HCM/HM medium as basic medium, and also contains 1% B-27, 0.1% BSA, 1.25mM N-acetyl-L-cysteine, 10 μ M Y-27632, 20ng/mL HGF, 100U/mL penicillin and 0.1mg/mL streptomycin;

and (2) formula: HCM/HM medium as basic medium, and further contains 1% B-27, 0.1% BSA, 1.25mM N-acetyl-L-cysteine, 10. mu. M Y-27632, 0.2mM L-ascorbate, 20ng/mL HGF, 20ng/mL OSM, 100U/mL penicillin and 0.1mg/mL streptomycin;

and (3) formula: HCM/HM medium was used as a basal medium, and further contained 1% B-27, 0.1% BSA, 1.25mM N-acetyl-L-cysteine, 0.2mM L-ascorbate, 10. mu. M Y-27632, 20ng/mL HGF, 20ng/mL OSM, 0.5. mu. MDex, 100U/mL penicillin and 0.1mg/mL streptomycin.

The formula of the second-stage three-dimensional culture medium is selected from any one of the following formulas:

formula 1: HCM/HM medium as basic medium, and further contains 1% B-27, 0.1% BSA, 1.25mM N-acetyl-L-cysteine, 10. mu. M Y-27632, 1% ITS, 10ng/mL EGF, 100U/mL penicillin and 0.1mg/mL streptomycin;

and (2) formula: HCM/HM medium as basic medium, and further contains 1% B-27, 0.1% BSA, 1.25mM N-acetyl-L-cysteine, 10. mu. M Y-27632, 1% ITS, 10ng/mL EGF, 0.2mM L-ascorbate, 0.5. mu.M Dex, 100U/mL penicillin and 0.1mg/mL streptomycin;

and (3) formula: HCM/HM medium was used as a basal medium, and further contained 1% B-27, 0.1% BSA, 1.25mM N-acetyl-L-cysteine, 10. mu. M Y-27632, 1% ITS, 10ng/mL EGF, 0.2mM L-ascorbate, 0.5. mu.M Dex, 100. mu.M 8-Br-cAMP, 10. mu.M vitamin K2, 10. mu.M Lithocholic acid, 100U/mL penicillin and 0.1mg/mL streptomycin.

In another aspect of the present invention, there is provided a method for inducing functional hepatocytes, comprising the steps of:

s1: directionally inducing and differentiating the pluripotent stem cells into liver stem/progenitor stage cells; wherein the pluripotent stem cells do not comprise human pluripotent stem cells or embryonic stem cells that are directly disaggregated from a human embryo or blastocyst;

s2: performing three-dimensional amplification culture on the liver stem/progenitor stage cells obtained in the step S1 by using a three-dimensional amplification culture medium to obtain liver organoids;

s3: after digesting the liver organoid obtained in S2 into single cells, the functional hepatocytes were induced to differentiate using the above-mentioned three-dimensional medium for induction of functional hepatocytes.

The method for inducing functional hepatocytes further includes, after step S2 and before step S3:

s2-1: pretreating the liver organoids obtained in the step S2 by using a pre-differentiation culture medium;

step S3 is to digest the liver organoids pretreated in step S2-1 into single cells, and then to induce differentiation into functional hepatocytes using the above-mentioned functional hepatocyte three-dimensional induction medium.

In the above method for inducing functional hepatocytes, the pretreatment conditions in step S2-1 are: 37 ℃ and 5% CO₂Culturing in an incubator for 4 days, wherein the pre-differentiation medium is replaced once every 2 days; the pre-differentiation culture medium takes Advanced DMEM/F-12 culture medium as a basal culture medium, and also comprises 0.01% -1% BSA, 0.2% -10% B-27, 0.2% -10% N2 additive, 1-50mM HEPES, 0.1% -10% glutamine additive, and one or more of the following components: antioxidants, gastrin, Sirt1 protein inhibitors, hepatocyte growth factor, bone morphogenetic protein, fibroblast growth factor, penicillin and streptomycin;

preferably, the antioxidant is N-acetyl-L-cysteine at a concentration of 0.1-10mM, preferably 1.25 mM; the Gastrin is Gastrin, and the concentration of the Gastrin is 1-100nM, preferably 10 nM; the Sirt1 protein inhibitor is nicotinamide with the concentration of 1-100mM, preferably 10 mM; the hepatocyte cell growth factor is HGF, the concentration is 5-100ng/mL, preferably 50 ng/mL; the bone morphogenetic protein is BMP7 or BMP4, the concentration is 5-100ng/mL, preferably 25ng/mL, the fibroblast growth factor is FGF2 or FGF4, the concentration is 5-100ng/mL, preferably 25 ng/mL; the concentration of the penicillin is 10-1000U/mL, preferably 100U/mL; the concentration of the streptomycin is 0.01-1mg/mL, preferably 0.1 mg/mL;

further preferably, the formula of the pre-differentiation medium is as follows: the culture medium is an Advanced DMEM/F12 culture medium and further comprises 0.1% BSA, 1% N2 additive, 1% B-27, 1% glutamine additive, 10mM HEPES, 1.25 mMN-acetyl-L-cysteine, 10mM nicotinamide, 10nM Gastrin, 25ng/mL BMP7, 25ng/mL FGF4, 50ng/mL HGF, 100U/mL penicillin and 0.1mg/mL streptomycin.

In the method for inducing functional hepatocytes, in step S1, the pluripotent stem cells are H9-based stem cells.

In the above method for inducing functional hepatocytes, the conditions of the three-dimensional expansion culture in step S2 are as follows: 37 ℃ and 5% CO₂Culturing in an incubator for 9-10 days, wherein the three-dimensional amplification culture medium is replaced once every 2 days; and/or

In the above functional hepatocyte induction method, the three-dimensional amplification medium described in step S2 comprises Advanced DMEM/F-12 medium or Advanced RPMI1640 medium as basic medium, and further comprises 0.01% -1% BSA, 0.2% -10% B-27, 0.2% -10% N2 additive, 1-50mM HEPES, 0.1% -10% glutamine additive, and one or more of antioxidant, Sirt1 protein inhibitor, gastrin, TGF- β signal pathway inhibitor, Wnt signal pathway agonist, epidermal growth factor, adenylate cyclase agonist, penicillin and streptomycin;

preferably, the antioxidant is N-acetyl-L-cysteine with the concentration of 0.1-10mM, preferably 1.25mM, the Sirt1 protein inhibitor is nicotinamide with the concentration of 1-100mM, preferably 10mM, the Gastrin is Gastrin with the concentration of 1-100nM, preferably 10nM, the Wnt signal pathway agonist is Wnt3a and R-spondin1 with the concentration of 10-1000ng/mL, preferably 100ng/mL, the concentration of R-spondin1 is 10-1000ng/mL, preferably 250ng/mL, the TGF β signal pathway inhibitor is A83-01 with the concentration of 0.1-10 μ M, preferably 5 μ M, the adenylate cyclase agonist is forskolin FSK with the concentration of 1-100 μ M, preferably 10 μ M, the epidermal cell growth factor is EGF with the concentration of 5-200ng/mL, preferably 50ng/mL, the concentration of penicillin is 10-100 μ M, preferably 0.01-1000 mg/mL, and the concentration of streptomycin is preferably 0.1-100 mg/mL;

further preferably, the formulation of the three-dimensional amplification medium is selected from any one of the following formulations:

formula 1: taking Advanced DMEM/F-12 medium as a basic medium, and further comprising 0.1% BSA, 1% B-27, 1% N2 additive, 10mM HEPES, 1% glutamine additive, 1.25mM N-acetyl-L-cysteine, 50ng/mLEGF, 250ng/mL R-Spondin1, 100U/mL penicillin and 0.1mg/mL streptomycin;

and (2) formula: advanced DMEM/F-12 medium as basal medium, further comprising 0.1% BSA, 1% B-27, 1% N2 additive, 10mM HEPES, 1% glutamine additive, 1.25mM N-acetyl-L-cysteine, 10mM nicotinamide, 5. mu. M A83-01, 50ng/mL EGF, 250ng/mL R-Spondin1, 100U/mL penicillin and 0.1mg/mL streptomycin;

and (3) formula: takes Advanced DMEM/F-12 culture medium as basic culture medium, and also comprises 0.1% BSA, 1% B-27, 1% N2 additive, 10mM HEPES, 1% glutamine additive, 1.25mM N-acetyl-L-cysteine, 10mM nicotinamide, 10nM Gastrin, 5 mu M A83-01, 10 mu M Forskin, 50ng/mL EGF, 100ng/mL Wnt3a, 250ng/mLR-Spondin 1, 100U/mL penicillin and 0.1mg/mL streptomycin.

In the above method for inducing functional hepatocytes, the specific method for directionally inducing and differentiating pluripotent stem cells into hepatocytes/progenitor stage cells in step S1 comprises:

1) digesting the multifunctional liver cells into single cells by using Dispase, and then inoculating, wherein a culture medium is TeSR-E8 culture medium added with 10 mu M Y-27632(Stem Cell) to promote Cell adhesion;

2) after 24h cells were attached, the cell culture medium was changed to RPMI1640 medium (Gibco), and 1% B-27(Gibco), 100ng/mL Activin A (R & D), 50ng/mL Wnt3a (R & D) were added to induce differentiation to the qualitative endoderm stage;

3) discarding waste liquid every other day, replacing cell culture medium with RPMI1640 culture medium, adding 1% B-27, and 100ng/ml of actin A for one day;

4) repeating the step 3) once, and finishing the induction of the qualitative endoderm stage;

5) discarding waste liquid every other day, replacing cell culture medium with HCM culture medium (Lonza), adding 10ng/mL FGF-2(R & D) and 20ng/mL BMP-4(R & D), acting for 5 days, changing culture medium every 2 days, and inducing to obtain liver stem/progenitor stage cells.

The functional liver cells induced by the above-mentioned method for inducing functional liver cells also belong to the present invention.

The application of the functional liver cell as seed cell for manufacturing artificial liver, establishing liver disease treatment drug screening model, in vitro liver function simulation model and in vitro liver disease simulation model also belongs to the content of the invention.

The three-dimensional induction culture medium for the hepatocytes, which is provided based on the technical scheme, can be effectively used for liver organoids which are obtained by sequential two-dimensional culture and three-dimensional culture and have bidirectional differentiation potential towards functional hepatocytes and cholangiocytes. In the method for obtaining the functional liver cells by induction, the three-dimensional induction culture medium is serum-free and non-nourishing layer cells, the functions of the functional liver cells obtained by induction are more mature, a large number of markers for expressing the maturation of the liver cells, such as ALB, CK18, AAT, TF, CYP3A4, CYP2C9, CPS1, ASS1 and the like, the expression quantity of Alpha Fetoprotein (AFP) which is an immature function marker is greatly reduced, in addition, the functions of albumin secretion, urea synthesis, drug metabolism, glycogen synthesis, indigo green uptake and the like of the liver cells generated by induction are greatly enhanced, and the three-dimensional induction culture medium can be better used for manufacturing artificial livers and can be used as ideal seed cells for establishing liver disease treatment drug screening models, in-vitro liver function simulation models and in-vitro liver disease simulation models. In the induction method of the liver cells, the used pluripotent stem cells can be obtained commercially, can be obtained easily, can be used for establishing a stable cell line, and has the potential of differentiating to liver stem cells, so that liver organoids can be obtained simply and efficiently, and a large amount of functional liver cells can be obtained.

Drawings

FIG. 1 is a technical roadmap for the present invention;

FIG. 2 is a phase contrast image (A) of pluripotent stem cells at different stages of induction; phase contrast images (B) of hEHOs continuous growth; phase contrast images of hEHOs at passage 1, passage 20 and after resuscitation cryopreserved (C);

FIG. 3 is a graph of hEHOs growth curves (A) at generations 1-3 and 13-15; karyotyping of hEHOs at passage 1 and passage 15, with a chromosome number of 46 (B);

FIG. 4 shows principal component analysis (A) of genome-wide RNA-Seq of

generations

1 and 3 of PHH, hAHos, FSCs, HS, HB, and hAHos; cluster analysis (B); a thermal map (C);

FIG. 5 is an immunofluorescence image of hEHOs specific liver stem/progenitor-related protein;

FIG. 6 is a phase contrast image of hEHOs induced differentiation to mature hepatocytes at day 1 and day 15;

FIG. 7 is a bar graph of gene level expression of hEHOs induced differentiation to produce mature hepatocytes versus starting cells hEHOs and primary hepatocytes;

FIG. 8 is an immunofluorescence staining image of hEHOs induced differentiation to mature hepatocytes in liver-associated proteins;

FIG. 9 is an AFP/ALB immunofluorescent staining image and quantification of hepatocytes generated by hEHos induced differentiation;

FIG. 10 is a flow assay of hEHOs induced differentiation to mature hepatocytes;

FIG. 11 is a graph showing the difference between the uptake and excretion of indocyanine green (A-B) in the functional characterization of hEHOs induced differentiation to produce mature hepatocytes; glycogen particle staining pattern (C); CDFDA staining pattern (D);

FIG. 12 is a histogram of the quantitative determination of mature hepatocyte function induced by hEHOs differentiation;

FIG. 13 is a transmission electron micrograph of mature hepatocytes generated by hEHos induced differentiation.

Detailed Description

According to the stage rule that the pluripotent stem cells are differentiated into the liver cells, the early stage of liver lineage specification (HS) is taken as the initial stage of liver organoid establishment, and the stage is considered as the stage of liver stem cell existence. Then, with reference to the method for culturing other organs (Sato T, Vries RG, snippet HJ et al. SingleLgr5 stem cells builded crypt-villus structures in vitro with a gene culture medium. Nature 2009; 459:262.Eiraku M, Takata N, Ishibashi H et al. Self-organizing nucleic-cup morphinesis in this-geographic culture medium. Nature 472:51.Lancaster MA, Renner M, Martin C-A et al. nucleic organization model human introduction and genome vector 2013; 373. Takasatto M, PX, Kinect et al. nucleic organization gene expression vector protein 2017. Bo. cell III. Biogene, Bo cell III, Bo et al. knot protein III, Bo cell protein, WO 8. Nature 2013; 373. Takasara M, Er, PX, Kinect. culture medium protein gene strain I.2017. Bo. knot protein, Bo. Japan strain culture medium strain, Bo. M20132. Japan strain, Bo. Biostrain culture medium strain, Bo strain, Na # 3. III strain, Na # 12. III. coli strain, Bo. III. M.7. coli strain, Bo. M. coli strain, Bo. M.7. coli strain, Bo. M. Biostrain, Bo. Bio strain, Bo. Na 3. Nature et al., the liver organoids are cultured in large quantities and efficiently at the HS stage. Secondly, the modification of the present invention on the basis of the prior art protocol for hepatocyte-induced differentiation (3D human tissue from complex cells in vitro and support complex functions in vivo. apparatuses of metabolism 2018; 92:3117-3129.Ogawa S, Suripathiat J, Virtan C et al. Three-dimensional culture and mapping protocol for the synthesis of liver cells in vitro. 12.8; 92: 20157-3129. acquisition S, modification 2013; 140:3285-3296.Gieseck RL,3, handle NRF, culture R et al. cell-derived metabolism. development 201372. medium cells of culture medium. 21. and 8683. 9. adapting to liver cells in vitro. 12. 9. 12. 3. 7. 9. 3. 12. 3. 7. medium cells of culture medium. expression of culture medium. 21. 3. 9. 12. 9. 7. 12. 7. 3. 7. 3. 12. 3. 12. 3. Establishes a special three-dimensional induced differentiation method and a three-dimensional induced culture medium suitable for hEHOs to differentiate into hepatocytes. After corresponding induction, the morphology, gene expression, protein expression and important in vitro functions of hepatocytes obtained from hEHOs, including the albumin secretion function, drug metabolism function, urea synthesis function, glycogen synthesis function, etc., of hepatocytes were examined correspondingly.

The present invention will be described in detail below by way of specific embodiments.

The methods used in the following examples are conventional unless otherwise specified, and specific procedures can be found in: molecular Cloning: A Laboratory Manual (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold spring harbor).

The percentage concentration is a mass/mass (W/W, unit g/100g) percentage concentration, a mass/volume (W/V, unit g/100mL) percentage concentration, or a volume/volume (V/V, unit mL/100mL) percentage concentration, unless otherwise specified. The "%" in the medium means the percentage concentration relative to the total amount of the medium, in mL/100 mL.

The various biological materials described in the examples are obtained by way of experimental acquisition for the purposes of this disclosure and should not be construed as limiting the source of the biological material of the invention. In fact, the sources of the biological materials used are wide and any biological material that can be obtained without violating the law and ethics can be used instead as suggested in the examples.

The embodiments are implemented on the premise of the technical scheme of the invention, and detailed implementation modes and specific operation processes are given, which are helpful for understanding the invention, but should not be taken as limiting the content of the invention.

Example 1 Induction culture of hepatocytes

In this example, pluripotent stem Cells (cell line H9, human embryonic stem Cells (human embryonic stem Cells) purchased from the american research center for wicel) were used as starting Cells, and then subjected to a qualitative endoderm stage, a hepatic stem/progenitor stage, and a liver organoid stage in this order as shown in fig. 1, to thereby induce hepatocytes. The method specifically comprises the following steps:

1.1 culture of cells of the cell line H9

1) The Matrigel (Corning) was taken out of the freezer at-80 ℃ one day in advance, thawed in the freezer at 4 ℃ and, after thawing, diluted with pre-cooled DMEM/F12 medium (Gibco) at a ratio of 1: 100.

2) The diluted Matrigel was uniformly spread in a six-well plate at 1mL per well, and incubated in an incubator at 37 ℃ for 1 hour for use.

3) H9 cells to be passaged were removed from the 37 ℃ incubator, aspirated, and washed once with DMEM/F12 medium.

4) 1mL of 0.1mg/mL Dispase (Sigma) digested cells was added to each well and placed in an incubator at 37 ℃ for 8-10 min.

5) Discarding Dispase, gently washing twice with DMEM/F12 medium, adding 1mL of DMEM/F12 medium into each well, lightly scraping the cell clones attached to the bottom of the well with a tip, transferring the scraped cells to a 15mL centrifuge tube, washing the original well with DMEM/F-12 medium, and putting the original wells together into a 15mL centrifuge tube.

6) Centrifuge at 1000rpm for 3min at room temperature.

7) The supernatant was discarded and the cells were gently resuspended in TeSR-E8 medium (StemCell).

8) The cells were plated in six-well plates containing previously coated Matrigel, and the cells were changed daily.

1.2 Induction of differentiation of the H9 cell line into hepatic Stem/progenitor cells

In the step, the induced differentiation of the H9 cells cultured in the step 1.1 is continued, and the method specifically comprises the following steps:

1) when the fusion degree of the H9 cells cultured in the step 1.1 in the hole reaches about 70-80%, the cells can start to induce.

2) Matrigel, lamin (Gibco), Collagen IV (Gibco) were applied uniformly in a ratio of 3:1:1 to 24-well plates 3 hours in advance and dried in a room temperature ultra clean bench (Beijing Touhal technologies, Ltd.).

3) Following the procedure for passaging the H9 cell line in step 1.1 above, H9 cells were digested into single cells using Dispase at 1X 10⁵The cells were seeded at density in the 24-well plates of step 2) in TeSR-E8 medium plus 10. mu. M Y-27632(Stem Cell) to promote Cell attachment.

4) After 24h of cell attachment, the cell culture medium was changed to RPMI1640 medium (Gibco), and 1% B-27(Gibco), 100ng/mL Activin A (R & D), 50ng/mL Wnt3a (R & D) were added to induce differentiation to the qualitative endoderm stage.

5) The waste liquid is discarded every other day, the cell culture medium is replaced by RPMI1640 medium, and 1% B-27 and 100ng/ml of actin A are added for one day.

6) The waste liquid was discarded every other day, the cell culture medium was replaced with RPMI1640 medium, and 1% B-27 and 100ng/ml of actin A were added thereto for one day. Qualitative endoderm stage induction was terminated and induction was directed to the hepatic stem/progenitor stage.

7) The waste liquid is discarded every other day, the cell culture medium is replaced by HCM medium (Lonza), 10ng/mL FGF-2(R & D) and 20ng/mL BMP-4(R & D) are added for 5 days, the liquid is changed every 2 days, and the induction of the hepatic stem/progenitor stage cells is finished.

The cell morphology at different induction stages in the above steps was observed, and the results are shown in fig. 2A, which shows that: cells (pluripotent stem cells-qualitative endoderm stage cells-hepatic stem/progenitor cells) exhibit different cell morphologies at different induction stages.

1.3 three-dimensional amplification culture of cells in the liver Stem/progenitor stage

In the step, the three-dimensional amplification culture is continuously carried out on the liver stem/progenitor stage cells obtained in the step 1.2, and the method specifically comprises the following steps:

1) after the end of the liver stem/progenitor stage at step 1.2 above, 1 XPBS (Ca-free) was used²⁺、Mg²⁺Preparation of PBS: mixing 8g NaCl, 0.2g KCl, 0.24g KH₂PO₄，1.44g Na₂HPO₄Dissolved in 1L of deionized water, filtered through a 0.22 μm filter membrane after sufficient dissolution and put into an autoclave for autoclaving for use, as follows), washed twice, then digested into single cells with 0.25% Trypsin-EDTA (Gibco), and the digestion is stopped with fetal bovine serum (Gibco).

2) Centrifuge at 1000rpm for 5min at room temperature.

3) BME-2(R & D) and/or Matrigel were placed on ice in advance for use.

4) The centrifuged cells were resuspended in BME-2 or Matrigel, and 30-50. mu.L droplets (3,000-10,000 cell density) per well were seeded into low-adsorption 24-well plates and incubated in a 37 ℃ incubator for 7-8 min.

5) After the gel is solidified, adding a three-dimensional amplification culture medium (formula: advanced DMEM/F12 medium + 0.1% BSA (Gibco) + 1% N2 additive + 1% B-27+ 1% glutamine additive (Glutamax, Gibco) + N-Acetyl-L-cysteine (N-Acetyl-L-cysteine, 1.25mM, Sigma) + Gastrin (10nM, Sigma) + Nicotinamide (Nicotinamide, 10mM, Sigma) + HEPES (10mM, Gibco) + A83-01 (5. mu.M, Selleck) + Forskolin (10. mu.M, Selleck) + R-spondin1(250ng/mL, R & D EGF) + (50ng/mL, R & D) + Wnt3a (100/mL, R & D) + penicillin +0.1mg/mL streptomycin).

6) The three-dimensional amplification medium was changed every two days. Obtaining liver organoids (hEHOs) after three-dimensional expansion culture of liver stem/progenitor cells.

The liver organoids (hEHOs) are stem cell groups with self-renewal capacity, can be bidirectionally differentiated into functional liver cells and bile duct cells, have similar spatial tissues with the liver organs after the three-dimensional induced differentiation is carried out on mature liver cells, and can reproduce partial functions of the liver organs, thereby being capable of copying and simulating the three-dimensional environment of the liver tissues in vivo.

Wherein the formula of the three-dimensional amplification culture medium in the step 5) can be any one of the following formulas:

and (2) formula: takes Advanced DMEM/F-12 culture medium as basic culture medium, and also comprises 0.1% BSA, 1% B-27, 1% N2 additive, 10mM HEPES, 1% glutamine additive, 1.25mM N-acetyl-L-cysteine, 10mM nicotinamide, 5 mu M A83-01, 10 mu M Forskolin, 50ng/mL EGF, 250ng/mL R-Spondin1, 100ng/mLWnt3a, 100U/mL penicillin and 0.1mg/mL streptomycin.

The liver organoids obtained in step 1.3 above were subjected to the following treatments and functional verification.

1.3.1 passage of liver organoids

1) And (3) carrying out continuous subculture after the cells in the step 1.3 are subjected to amplification culture for about 7-9 days, carrying out subculture according to the ratio of 1:6-8, blowing the cells in original holes by a mechanical method, transferring the cells to a 15mL centrifuge tube, and precooling for 30min on ice.

2) Centrifuge at 800rpm for 3min at room temperature.

3) The supernatant was discarded and the cells were digested into small pieces with additional Accutase (Sigma).

4) Centrifuge at 1000rpm for 5min at room temperature.

5) Resuspend cells with the three-dimensional amplification medium of step 1.3 above, which is also subculture medium, and inoculate as described above.

6) The culture medium was changed every two days.

1.3.2 cryopreservation of liver organoids

1) After the cells in the step 1.3.1 are subcultured for about 7-10 days, the cells are digested according to the method in the step 1.3.1, and then centrifuged.

2) And (4) discarding waste liquid after centrifugation, performing cell cryopreservation by adopting serum-free rapid cell cryopreservation liquid (StemCell), and placing in liquid nitrogen for long-term storage.

1.3.3 Resuscitation of liver organoids

1) The water bath (Beijing Changfeng instruments and meters) was preheated to 42 ℃ in advance.

2) The cells frozen in step 1.5 above were taken out from liquid nitrogen and rapidly thawed in a 42 ℃ water bath.

3) After the cells had thawed, they were transferred to a 15mL centrifuge tube.

4) Centrifuge at 1000rpm for 5min at room temperature.

5) The supernatant was discarded and the recovery medium was the same as the expansion medium described above in step 1.3, but 10. mu. M Y-27632(Stem Cell) was added to the first two days of culture to promote Cell aggregation.

6) After two days, the waste liquid is discarded and replaced by a three-dimensional amplification culture medium without Y-27632.

7) After the cells are cultured for about 7-10 days, subculture is carried out according to the method.

The morphology of the liver organoids that continued to grow was observed, and the results are shown in fig. 2B, which shows the morphology from day 0 to day 10, indicating that the liver organoids grew well; the results of observing the passage of the liver organoids and the morphology after the resuscitation and the cryopreservation are shown in fig. 2C, which shows the morphology diagrams of the liver organoids of the 1 st generation and the 20 th generation and the morphology after the resuscitation and the cryopreservation, and it can be seen that the liver organoids still maintain the vitality and the higher viability after the passage to the 20 th generation, and the morphology of the cells after the resuscitation and the cryopreservation is clear.

1.3.4, multiplication Curve

1) The cells after the passage were cultured at 1X 10 per well⁴Inoculating the cells in the three-dimensional amplification culture medium at a density and culturing for 8-10 days;

2) digesting the obtained liver organs into single cells by using Accutase enzyme, and counting trypan blue by using a blood counting plate at each time point;

3) the proliferation curve was determined according to the number of cells at the final time point, y (t) 0 × e (growth rate × t); y 0-number of cells per time point; t ═ time) were calculated quantitatively;

4) doubling time Each time point was calculated as ln (2)/growth rate.

The results are shown in FIG. 3A, which shows the doubling curves of liver organoids, and it can be seen that the cell doubling time of liver organoids from passage 1 to passage 3 is 62.24 + -1.09 hours, and the cell doubling time from passage 13 to passage 15 is 68.69 + -1.84 hours, and it can be seen that the liver organoids still maintain the stable increase of cell doubling time during the long-term subculture.

1.3.5 karyotyping

1) The liver organoid medium of passage 1 and passage 15 cultured in 1.3.4 above was supplemented with 40ng/mL colchicine and incubated for 4 hours at 37 ℃ in an incubator.

2) After 4 hours, wash twice with 1 × PBS.

3) Individual cells were digested with Accutase and manipulated according to standard karyotyping procedures.

As a result of karyotyping of liver organoids, as shown in fig. 3B, it was found that the karyotype of liver organoids at both passage 1 and passage 15 was normal, and the number of chromosomes n was 46.

1.3.6 determining the developmental stage of hEHOs

1.3.6.1 obtaining of Foregut Stem Cells (FSCs), adult liver-derived liver organoids (hAHOs), hepatic progenitors (HB), Primary Human Hepatocytes (PHH)

(1) Obtaining Foregut Stem Cells (FSCs)

1) Qualitative endoderm cells derived from human pluripotent stem cells (commercially available pluripotent stem cell lines ESC or iPSC such as H1, H7, H9, H13, H14, etc.) were cultured in RPMI1640 medium supplemented with 2% B27 and 50ng/mLActivin a for 3-4 days;

2) the qualitative endoderm cells obtained above were digested with 0.25% pancreatin and re-seeded in Matrigel-coated well plates;

3) the foregut stem cell culture medium is based on an RPMI1640 culture medium, and is added with 1% B27, 1% NEAA (Gibco), 10ng/mL Activin A, 20ng/mL bFGF, 10ng/mL BMP4, 20ng/mL HGF and 50ng/mL EGF for acting for 3 days, so that the obtained foregut stem cells are obtained.

(2) Hepatic progenitor cell (HB)

Human pluripotent stem cell (commercially available pluripotent stem cell lines ESC or iPSC, e.g., H1, H7, H9, H13, H14, etc.) derived liver lineage-specific early stage (HS) 1% B27, 0.2mM L-ascorbate, 4.5X 10 based on HM medium was added^-4MTG (Sigma), 20ng/mL HGF, 0.5 MuM Dex and 20ng/mL OSM, and the obtained hepatic progenitor cells are obtained after 9 days of action.

(3) Obtaining of Primary Human Hepatocytes (PHH)

1) Methods for isolation of primary human hepatocytes reference published literature (leculus EL, alexandree. isolation and culture of primary hepatocytes from recovered human liver tissue. methods Mol Biol 2010; 640:57-82.) wherein primary human liver tissue used is obtained from a donor in liver transplantation surgery, with informed consent from a patient in the Beijing friendship hospital. The method is approved by medical ethics committees of Beijing friendship hospitals and military medical research institutes;

2) primary human liver tissue was washed with HBSS solution containing 0.5mM EGTA (Sigma) and 0.5% BSA for 15-30 min and perfused with preheated 1mg/mL collagenase type IV (Sigma) for 20-40 min;

3) passing the liver cell suspension obtained by digestion through a 100-micron filter screen, and centrifuging for 3 minutes at 75 g;

4) the cell pellet was resuspended in Percoll (GE healthcare, density 1.130g/mL) solution and centrifuged at 100-150g for 10min at room temperature;

5) the cell pellet was washed three times with DMEM/F12 (Gibco);

6) the primary human hepatocyte inoculation culture medium is Williams' E culture medium (Gibco), added with 1% glutamine and 5% fetal bovine serum (Gibco), and inoculated with cells in a pore plate coated by type I collagen (Corning);

7) after the cells are attached for 4-10 hours, the culture medium is changed to HM medium.

(4) Obtaining of liver organoids (hAHos) derived from adult liver

1) Adult liver-derived liver organoids (hAHOs) are described in published literature (Huch M, Gehart H, van Boxtel R et al Long-Term Culture of Genome-Stable Bipotent Stem Cells from AdultHuman liver 2015; 299-312.Broutier L, Andersson-Rolf A, Hindley CJ, etc. Culture and estelliment of self-renewing human and mouse adult slides and fingerprints 3D oligonucleotides and the human genetic management. Nature Protocols 2016; 11:1724.) method wherein the primary human liver tissue used was obtained from a donor in liver transplantation surgery, informed consent was obtained from patients in the Beijing friendship hospital. The method is approved by medical ethics committees of Beijing friendship hospitals and military medical research institutes;

2) flow sorting EpCAM⁺(BD biosciences, PE-CD326) cells were resuspended in Matrigel or BME2 and seeded on low adsorption 24-well plates as 3000-10000 cells;

3) hAFOs medium based on Advanced DMEM/F12 supplemented with 1% N2, 1% B27, 1.25mM N-acetyl-L-cysteine, 10nM gastrin, 10mM nicotinamide, 1% glutamine, 5. mu. M A83-01, 10. mu. MForskolin, 50ng/mL EGF, 250ng/mL R-Spondin1, 50ng/mL EGF, 100ng/mL FGF10, 25 ng/mGF, 100U/mL penicillin and 0.1mg/mL streptomycin for 10-14 days;

4) passage of hAHos, cryopreservation recovery as described in 1.3.1-1.3.3

1.3.6.2, extracting RNA samples of hEHOs 1 st generation and 3rd generation cells by a Trizol method; for reference, RNA samples of Foregut Stem Cells (FSCs), adult liver-derived liver organoids (hAHOs), hepatic progenitors (HB), HS cells (obtained by the pluripotent stem cell-induced differentiation process in step 1.2 above), Primary Human Hepatocytes (PHH) were extracted in the same manner:

1) according to 10cm²Adding Trizol cell lysate (Gibco) into the cell culture solution at a ratio of/mL, standing at room temperature for 5min to fully lyse the cells, and fully dissolving out RNA;

2) centrifuging at 12000rpm for 5min at 4 deg.C, and removing precipitate;

3) adding chloroform solution into 200 μ L chloroform/ml Trizol, brushing for 15s, shaking, mixing, and standing at room temperature for 10 min;

4) centrifuging at 12000rpm for 5min at 4 deg.C;

5) layering the solution into three layers, wherein RNA is positioned in an upper aqueous phase, DNA and protein are positioned in a middle layer and lower phenol layer, and only the upper aqueous phase is sucked into another 1.5ml EP tube;

6) adding 500 mul of isopropanol/ml of Trizol into the precooled isopropanol solution, turning upside down and uniformly mixing, and standing for 10min at room temperature;

7)12000rpm, 10min, 4 ℃ centrifugation, supernatant fluid discarding, RNA depositing in the bottom of 1.5ml EP tube;

8) 1.5ml of an EP tube was gently shaken by adding 1ml of 75% ethanol/ml Trizol to a 75% ethanol solution;

9) centrifuging at 12000rpm for 5min at 4 deg.C, and removing supernatant;

10) drying in a super clean bench for 5-10 min;

11) adding 15-20 μ L of RNA enzyme-free water solution RNA, standing at room temperature for 3-5min, and mixing well;

12) the RNA concentration was determined by Nanodrop 2000 (Thermo).

1.3.6.3 Gene sequencing and analysis (cooperative completion of Beijing Annuoda Co., Ltd.)

Using Illumina based on the RNA samples of FSCs, hAHos, HB,

hAHos generation

1 and 3, HS, PHH extracted in step 1.3.6.1

Ultra^TMThe RNA Library Prep kit (E7530L, NEB, USA) was used to generate RNA sequencing libraries. Sequencing was performed by Annuodda (China) and RNA sequencing was performed using Noves 6000. Differentially Expressed Genes (DEG) were analyzed by DESeq2, with genes with q ≦ 0.05 and | log2_ ratio | ≧ 1 identified as DEG. Heatmap generation and principal component analysis were performed using the R-package. The raw RNA data of the above 6 cells were uploaded to GeneExpression Omnibus database (access number GSE 128717).

To determine the developmental stage of hEHOs, transcriptomes were established from hEHOs including passage 1 and passage 3 cells, hESC (human pluripotent stem cells, e.g., the H9 cell line) derived FSC cells, HS cells, HB cells, hAHOs cells and PHH cells. As shown in panel a in fig. 4, principal component analysis showed clear clustering for each analysis group, and hEHO (generation 1 and generation 3) stably showed more similarity to HS and HB, but remained different from them. Hierarchical clustering analysis, as shown in panel B of fig. 4, showed that hEHOs is intermediate between FSC and the two sets of immature liver lines (HS and HB), closely related to HS and HB, far from PHH. As shown in panel C of fig. 4, significant expression of mature hepatocyte markers (including CYP3a4, CYP1a2, CYP2E1, CYP2a6, CYP2C8, CYP2C9 and CYP2D6) was in PHH, while foregut specific transcription factors including SOX17, SOX2 and CER1 were expressed in FSC. hEHOs showed activation of several transcription factor (PROX1, ONECUT2, SOX9, FGFR4, FOXA1, FOXA3 and ONECUT1) genes of early liver development, while upregulating components of Wnt signaling (LGR5, ZNRF3, RNF43, AXIN2, CD44, DKK1, Wnt7A and Wnt10A) were expressed in hAHOs, suggesting that hEHOs showed similarities to HS and HB in the whole genome gene expression profile.

1.3.7 cellular immunofluorescence

1) HS cells and liver organoids were treated with 4% paraformaldehyde (formula: 3.56g of NaOH, 42.7g of PFA, 13.6gKH₂PO₄Dissolving in 1L PBS, heating and stirring with magnetic stirrer at 65 deg.C until clear and bright, and packaging for use) fixing at room temperature for 15-20min, and washing with PBS twice.

2) 0.25% Triton X-100(Sigma) for 10-15min, and washed twice with PBS.

3) Blocking was performed with sera from the same species as the secondary antibody (as shown in table 2 below) for 1 hour at room temperature.

4) Primary antibody (as shown in table 1 below) was diluted with the same serum as the secondary antibody species source, and the concentration of the primary antibody was diluted as described, incubated overnight at 4 ℃ in a refrigerator, and washed three times with PBS.

5) The secondary antibody was diluted with serum from the same species as the secondary antibody, incubated for 1 hour at room temperature in the dark, and washed three times with PBS.

6) Nuclei were stained with dapi (sigma) lining, incubated 10min at room temperature in the dark and washed three times with PBS.

7) And (5) sealing by using a water-soluble sealing agent.

8) Observed under a laser confocal microscope and photographed (Nikon Tie-A1).

The immunofluorescence results are shown in fig. 5, and it can be seen that liver organoids express specific proteins of liver stem/progenitor cells, such as SOX9, EPCAM, CK19, HNF4A and TBX3, protein AFP expressing immature liver cells, protein ALB not expressing liver cell maturation, dry markers OCT4 and SOX2, cells maintain the proliferation property, and Ki67 protein is positively expressed.

Table 1: primary antibodies for immunofluorescence detection of liver organoids (hEHOs)

A primary antibody	Company source	Goods number	An antibody species	Dilution ratio
					AFP (alpha-fetoprotein)	Abcam	Ab169552	Rabbit		200
AFP (alpha-fetoprotein)	Sigma	A8452	Mouse immunoglobulin 2a	200
					EPCAM (epithelial cell adhesion molecule)	Neomarker	MS-181	Mouse immunoglobulin	1	200
EPCAM (epithelial cell adhesion molecule)	CST	2929	Mouse immunoglobulin 1	800
					ALB(Albumin)	Bethyl	AF2400	Goat immunoglobulin		200
ALB(Albumin)	R&D	MAB1455	Mouse immunoglobulin 2a	200
					SOX9 (sex determination region Y Gene 9)	Abcam	Ab185966	Rabbit		100
CK19 (cytokeratin 19)	Abcam	ab7754	Mouse immunoglobulin 2a	200
					CK19 (cytokeratin 19)	Abcam	Ab52625	Rabbit		100
TBX3 (T-box protein 3)	Abcam	Ab99302	Rabbit								100
					OCT4 (octamer binding transcription factor 4)	Abcam	Ab19857	Rabbit		100
SOX2 (sex determination region Y-box protein 2)	R&D	245610	Mouse immunoglobulin 2a	100
					HNF4A (hepatocyte nuclear factor 4)	Santa Cruz	sc-6556	Goat immunoglobulin	100
Ki67	Thermo	14-5698-80	Rat immunoglobulin 2a	200

Table 2: secondary antibodies for immunofluorescence detection of liver organoids (hEHOs)

1.4 Induction of differentiation of hEHOs into hepatocytes

In this example, the inventors established a three-dimensional suspension induction protocol and a three-dimensional induction medium suitable for hEHOs to hepatocyte differentiation, aiming at the phenotypic characteristics of hEHOs, and specifically included the following steps:

1) hEHOs were pretreated in BME or Matrigel for 4 days in advance, with pretreatment medium Advanced DMEM/F12, 0.1% BSA, 1% N2 additive, 1% B-27, 1% glutamine additive, 10mM HEPES, 1.25mM N-acetyl-L-cysteine, 10mM Nicotinamide (Nicotinamide), 10nM Gastrin (Sigma), 25ng/mL BMP7 (bone morphogenetic protein 7, which can also be bone morphogenetic protein 4), 25ng/mL FGF4 (fibroblast growth factor 4, which can also be fibroblast growth factor 2), 50ng/mL HGF, 100U/mL penicillin and 0.1mg/mL streptomycin.

2) hEHos cells were digested into single cells by adding 0.25% Trypsin-EDTA.

2) Centrifuge at 1000rpm for 5min at room temperature.

3) Discarding supernatant, adding first-stage hepatocyte three-dimensional induction culture medium to resuspend cells, and performing 1-1.5 × 10⁵The density of the culture medium is inoculated into each well of a low-adsorption 24-well plate, and the first-stage three-dimensional induction culture medium is HM (ScienCell)/HCM (Lonza) + 0.1% BSA + 1% B-27+0.2mM L-ascorbate +1.25mM N-acetyl-L-cysteine +20ng/mL HGF +20ng/mL OSM (R)&D) + 10. mu. M Y-27632+ 0.5. mu.M Dex +100U/mL penicillin +0.1mg/mL streptomycin.

4) The medium was changed every three days for 3 days of induction.

5) After 3 days, the cells were replaced with second-stage three-dimensional induction medium of HM/HCM + 0.1% BSA + 1% B-27+0.2mM L-ascorbate + 1% insulin-transferrin-selenium sodium (ITS) + 10. mu.MY-27632 + 0.5. mu.M Dex +10ng/mL EGF + 100. mu.M 8-Br-cAMP (Selleck) + 10. mu.M VK2(vitamin K2, Sigma) + 10. mu.M LCA (Lithocholic acid, Sigma) +100U/mL penicillin and 0.1mg/mL streptomycin.

6) The medium was changed every two days, and the total induction period of the first stage induction and the second stage induction was 15 days.

As shown in FIG. 6, the hEHOs were digested into single cells on day 1, and induced by addition of induction culture, and then they were able to grow into smooth-edged three-dimensional cell balls with a diameter of 100-150 μm, i.e., induced hepatocytes, approximately 15 days or so.

Wherein the formula of the first-stage three-dimensional induction culture medium can also be as follows:

formula 1: HCM/HM medium as basic medium, and also contains 1% B-27, 0.1% BSA, 1.25mM N-acetyl-L-cysteine, 10 μ M Y-27-632, 20ng/mL HGF, 100U/mL penicillin and 0.1mg/mL streptomycin;

and (2) formula: HCM/HM medium was used as a basal medium, and further contained 1% B-27, 0.1% BSA, 1.25mM N-acetyl-L-cysteine, 10. mu. M Y27-632, 0.2 mML-ascorbate, 20ng/mL HGF, 20ng/mL OSM, 0.5. mu.MDex, 100U/mL penicillin and 0.1mg/mL streptomycin.

The formula of the second-stage three-dimensional induction culture medium can also be as follows:

and (2) formula: HCM/HM medium was used as a basal medium, and further contained 1% B-27, 0.1% BSA, 1.25mM N-acetyl-L-cysteine, 10. mu. M Y27-632, 1% ITS, 10ng/mL EGF, 0.2mM L-ascorbate, 0.5. mu.M Dex, 100. mu.M 8-Br-cAMP, 10. mu.M VK2, 10. mu.M LCA, 100U/mL penicillin, and 0.1mg/mL streptomycin.

Example 2: hepatocyte morphology and function verification

2.1 RNA extraction, reverse transcription and real-time quantitative PCR

In this step, RNA extraction was performed using an RNA extraction kit (Qiagen), reverse transcription was performed using a reverse transcription kit (TOYOBO), and real-time quantitative PCR was performed using a real-time fluorescent quantitative PCR instrument (Bio-Rad).

1) The hepatocytes (three-dimensional cell balls) induced in example 1 above were collected, and a suitable amount of lysate Buffer RLT (Qiagen) was added to the collected cells to sufficiently lyse the cells.

2) An equal volume of 70% ethanol was added to the lysate, mixed well with a pipette, and transferred to spinocolumn, 12000rpm at room temperature, and centrifuged for 30 s.

3) The waste liquid was discarded, 700. mu.L of Buffer RW1 to spin column was added, and the mixture was centrifuged at 12000rpm at room temperature for 30 seconds.

4) The waste solution was discarded, 500. mu.L of Buffer RPE was added to spin column, and centrifuged at 12000rpm at room temperature for 30 seconds.

5) The waste liquid was discarded, 500. mu.L of Buffer RPE was added to spin column, and centrifuged at 12000rpm at room temperature for 2 min.

6) The waste liquid is discarded, and the mixture is subjected to room temperature 12000rpm idle for 1 min.

7) Spin column was transferred to a new 1.5mL collection tube, 30. mu.L of RNeasy-free water was added, and the mixture was centrifuged at 12000rpm at room temperature for 1 min. And collecting the liquid in the tube to obtain the RNA extracting solution.

8) The concentration of RNA in the extract was determined using NanoDrop 2000.

9) The RNA was inverted based on the total amount of 1. mu.g RNA, 16. mu.L total volume of RNA and RNeasy-free water, denatured by heating at 65 ℃ for 5min, and then quickly transferred to ice for 2min precooling.

10) Adding 5 × RT.MM in 4 μ L reverse transcription reagent to reach total volume of 20 μ L, setting reverse transcription program of common PCR instrument at 37 deg.C for 15min, 50 deg.C for 5min, 98 deg.C for 5min, and terminating at 4 deg.C.

11) Triple distilled water was prepared, and the cDNA obtained by reverse transcription was mixed with SYBR Green (TOYOBO) to prepare a Q-PCR mixture, and primers (shown in Table 3 below, the amount of each primer pair was 2OD, and the mixture of primers shown in Table 3 was added to each well) in advance of a 96-well plate, and the total amount of each well was 20. mu.L (system including: primers, SYBR GREEN, cDNA and Mix), sealing a 96-well plate (Bio-Rad) by a sealing membrane, putting the plate into a real-time quantitative PCR instrument, and carrying out detection procedures as follows: cycling was performed 40 times in total at 95 ℃ for 3min, 95 ℃ for 10s, 60 ℃ for 35s, 65 ℃ for 5s, and 95 ℃ for 5 s. The real-time quantitative PCR amplification results are shown in FIG. 7.

Table 3: PCR primer sequences

As shown in fig. 7, which shows real-time quantitative PCR results of hepatocytes induced by hEHOs, it can be seen that whether hepatocytes are induced by hEHOs of generation 1 (generation 1) or by hEHOs of generation 15 (generation 15), the induced hepatocytes are significantly up-regulated in expression of genes related to liver maturation (HNF4A, AAT, CK18, TAT, TF, G6PC, ALB), genes related to urea metabolism (OTC, CPS1, ASS1, ASL, ARG1), genes related to drug metabolism (CYP3a4, CYP2C9, CYP2E1), nuclear receptor genes (pxc, pr, FXR) compared to hEHOs before induction, wherein some genes are close to the gene expression level of primary hepatocytes, such as the hepa, altf genes, etc., indicating differentiation of hEHOs into hepatocytes.

2.2 immunofluorescence analysis of hEHOs-derived hepatocytes

1) The hepatocytes (three-dimensional cell spheres) obtained by induction in example 1 were fixed with 4% paraformaldehyde (same as in example 1) at room temperature for 15-20min and washed twice with PBS.

2) 0.25% Triton X-100(Sigma) for 10-15min, and washed twice with PBS.

3) Blocking was performed with serum from the same species as the secondary antibody (as shown in table 2 above) for 1 hour at room temperature.

4) Primary antibody (shown below in table 4) was diluted with the same serum from the same species as the secondary antibody, and the concentration of the primary antibody was diluted as described, incubated overnight in a refrigerator at 4 ℃, and washed three times with PBS.

7) And (5) sealing by using a water-soluble sealing agent.

8) Observed under a laser confocal microscope and photographed (Nikon Tie-A1).

9) Image J software calculates immunofluorescence quantitation results.

10) The AFP/ALB in hepatocytes induced from liver organoids (control group) obtained in example 1 (inventive group) and before the present inventors (jongzi, study of construction of liver organoids with bidirectional differentiation potential from human pluripotent stem cells, hebei medical university, master paper, 2018) were subjected to immunofluorescence quantitative staining according to the methods of steps 1) -9) described above.

The immunofluorescence results of the hepatocytes obtained by induction in example 1 are shown in fig. 8, and it can be seen that the proteins related to liver maturation (ALB, AAT, TF, HNF4A, CK18, CYP3a4, CPS1) are all significantly expressed, and the expression of the immature related protein AFP is weak, so that the hepatocytes obtained by hEHOs induced differentiation are closer to the mature hepatocytes.

The immunofluorescence staining results of AFP/ALB in hepatocytes of the present invention group and the control group are shown in fig. 9, where panel a in fig. 9 shows the staining results of the control group, and panel B in fig. 9 shows the staining results of the present invention group, and it can be seen that the expression level of protein ALB related to liver maturation in hepatocytes obtained by induction of the present invention group is significantly increased and the expression level of protein AFP related to immature liver in hepatocytes obtained by induction of the control group is significantly decreased, and it can be seen that hepatocytes obtained by hEHOs induced differentiation in example 1 are closer to mature hepatocytes than hepatocytes obtained by previous induction of the present inventors.

Table 4: primary antibody for immunofluorescence detection of hepatocytes

A primary antibody	Company source	Goods number	An antibody species	Dilution ratio
					AAT (aspartate aminotransferase)	Abcam	ab166610	Rabbit	100
AFP (alpha-fetoprotein)	Abcam	ab169552	Rabbit							200
					ALB(Albumin)	Bethyl	AF2400	Goat immunoglobulin	200
ALB(Albumin)	R&D	MAB1455	Mouse immunoglobulin 2a	200
					TF (transferrin)	Abcam	ab82411	Rabbit	200
CPS1 (carbamoyl phosphate synthetase)	Santa cruz	sc-30060	Rabbit	200
					CK18 (cytokeratin 18)	Abcam	ab181597	Rabbit	200
CYP3A4 (cytochrome P4503A4 enzyme)	Santa Cruz	sc-53850	Mouse immunoglobulin 1	100
					MRP2 (multidrug resistance associated protein-2)	Abcam	ab172630	Rabbit	200
HNF4A	Santa Cruz	sc-6556	Rabbit	100

2.3 flow cytometry analysis of hEHOs-derived hepatocytes

1) Using Accutase enzyme to digest the liver cells induced and differentiated by hEHOs into single cells;

2) cells were fixed in 4% paraformaldehyde (same step 2.2) for 15min at room temperature and washed twice with 1 × PBS;

3) blocking with serum from the same species as the secondary antibody for 30min at room temperature;

4) the primary antibody (shown in table 4 above) was incubated at room temperature for 1 hour and washed three times with 1 × PBS;

5) the secondary antibody (shown in table 2 above) was incubated for 40 minutes at room temperature in the dark and washed three times with 1 × PBS;

6) the cells were resuspended in1 × PBS, filtered through a 70 μm sieve before loading, and analyzed by flow cytometry.

The hepatocyte flow results are shown in fig. 10, and it can be seen that about 93% of the hepatocytes positive to ALB and 92.7% of the cells positive to both ALB and AAT indicate that the efficiency of hepatocyte induction differentiation is high; also 20% of hepatocytes were ALB positive and AFP double positive, and this population of cells was close to immature fetal hepatocytes, accounting for less. It can be seen that the method according to the invention induces the obtained hepatocytes to become more mature.

2.4 liver function testing

2.4.1 glycogen particle staining (PAS)

1) hEHOs-induced hepatocytes were fixed with 4% PFA (Sigma) in 1.5mLEP tubes at room temperature for 15-20min and washed twice with PBS after fixation.

2) Periodic Acid Solution (Periodic Acid Solution) was added and incubated at room temperature for 5 min.

3) Centrifuging at 1000rpm for 4min at room temperature, discarding the waste liquid, and washing twice with PBS.

4) Schiff's Reagent (Sigma) was added and incubated at room temperature for 15 min.

5) Cells were collected by centrifugation and washed three times with PBS for 5min each time.

6) Hematoxylin (Vector laboratories) solution was added to line the nuclei for 90 s.

7) The cells were washed three times with PBS for 5min each, observed under an inverted phase contrast microscope (Leica) and the images were taken.

2.4.2 Indocyanine Green (ICG, Sigma) uptake and excretion

1) hEHos-induced hepatocytes were collected and washed once with PBS.

2) 1mg/mL of ICG (Sigma) reagent was added and incubated at 37 ℃ in an incubator for 30 min.

3) After completion, the cells were washed three times with PBS and PBS was added.

4) The images of the ICG uptake by the cells were observed under an inverted phase contrast microscope (Leica) and photographed.

5) After the end of the image taking, PBS was removed, and fresh second-stage hepatocyte induction medium (same as above) was added and placed in a 37 ℃ incubator for further culture and observation.

6) The cells were observed to secrete ICG every 1h under the mirror until they completely excreted ICG and photographed.

7) The second stage hepatocyte induction culture medium was replaced with fresh one and then placed in a 37 ℃ incubator for further culture.

2.4.3 CDFDA (Sigma) staining

1) The hEHOs-induced hepatocytes were collected and washed once with PBS.

2) Add 2. mu.M CDFDA reagent and incubate for 30min at 37 ℃ in an incubator.

3) The waste liquid is discarded by centrifugation, and precooled PBS is added for washing for three times.

4) Staining of intracellular microcapillaries was observed under a laser confocal microscope (ZEISS) and images were taken.

The results of the above 2.4.1 to 2.4.3 are shown in FIG. 10, in which the A-D frames in FIG. 11 represent: indocyanine green uptake, indocyanine green excretion, glycogen particle staining and CDFDA staining results show that hepatocytes induced by hEHOs possess the specific functions of hepatocytes: such as the uptake and excretion of indocyanine green (ICG), the storage of glycogen Particles (PAS), the excretion function of the micro bile duct (CDFDA).

2.4.4 ELISA detection of Albumin

In the step, a human BSA detection kit (Bethy Laboratories) is adopted to carry out ELISA detection on Albumin, and the method specifically comprises the following steps:

1) the second-stage hepatocyte three-dimensional induction culture medium of hEHOs is replaced by HM culture medium without phenol red 24h in advance, two blank control holes are made simultaneously, and the culture medium is placed in an incubator at 37 ℃ for 24 h.

2) Cell supernatants were collected by centrifugation after 24 h.

3) Preparing working solution in advance, diluting 20 multiplied by Buffer C into 1 multiplied by Buffer C according to the instruction of the kit, and diluting 20 multiplied by Wash Buffer into 1 multiplied by Wash Buffer.

4) 100 μ L of the standard, the sample to be tested and the blank were added to the 96-well plate coated with the ALB antibody, and three duplicate wells were set for each sample.

5) Incubate at room temperature for 1 h.

6) The wells were emptied of liquid and 200. mu.L of 1 × Wash Buffer was added to each well for four washes.

7) mu.L of Anti-albumin Detection Antibody was added to each well.

8) Incubate at room temperature for 1 h.

9) The wells were emptied of liquid and 200. mu.L of 1 × Wash Buffer was added to each well for four washes.

10) 100 μ L of HRP Solution A was added to each well.

11) The wells were emptied of liquid and 200. mu.L of 1 × Wash Buffer was added to each well for four washes.

12) 100 μ L of TMB Substrate was added to each well.

13) Incubate for 30min at room temperature in the dark.

14) Add 100. mu.L of stop solution per well

15) Detecting absorbance value with wavelength of 450nm with enzyme labeling instrument within 30 min.

16) And drawing a standard curve and calculating a result according to the curve.

2.4.5 Urea detection

The step of detecting urea by using a urea detection kit (Bioassay Systems) specifically comprises the following steps:

1) the second-stage three-dimensional induction medium of hepatocytes of hEHOs was replaced with phenol red-free HM medium containing 10mM ammonium chloride 24h in advance, and the medium was incubated at 37 ℃ in an incubator for 24 h.

2) Cell supernatants were collected by centrifugation after 24 h.

3) Preparing a working solution for urea detection in advance, namely adding an equal volume of solution A and an equal volume of solution B.

4) 50 μ L of the standard, the sample to be tested and ultrapure water were added to each well.

5) Add 200. mu.L of urea detection working solution into each well, tap the plate and mix well.

6) And (5) incubating at room temperature for 20min, and incubating at room temperature for 50min if the urea content of the sample to be detected is low.

7) Detecting absorbance value with wavelength of 520nm with enzyme labeling instrument within 20 min.

8) And calculating the result according to a formula.

2.4.6 detection of CYP3A4 enzyme Activity

The step of adopting CYP3A4 detection kit (Promega) to carry out CYP3A4 enzyme activity detection specifically comprises the following steps:

1) hEHOs-induced hepatocytes were collected, phenol red-free HM medium containing 3. mu.M Luciferin-IPA was added to prepare a sample to be tested, and 3. mu.M Luciferin-IPA blank control wells were set.

2) Incubate at 37 ℃ for 1 h.

3) Centrifuging at 1000rpm at room temperature for 5min, collecting supernatant, washing cells with PBS for three times, replacing with fresh second-stage hepatocyte three-dimensional induction culture medium, and culturing in 37 deg.C incubator.

4) 50 μ L of test sample and blank were added to each well of an opaque 96-well plate, with three replicate wells per sample.

5) Add 50. mu.L of Luciferin deletion Reagent and gently tap the plate and mix well.

6) Incubate at room temperature for 20 min.

7) And detecting the fluorescence value of each hole by using a microplate reader within 20 min.

8) And calculating a result.

The results of 2.4.4-2.4.6 are shown in FIG. 12, which shows that hEHOs induced hepatocytes have typical liver functions, such as albumin secretion, urea synthesis and CYP3A4 liver pharmazyme metabolism.

2.5 preparation and Observation of samples for Transmission Electron microscopy

The hepatocytes obtained by hEHO induction to be tested were fixed with 2.5% glutaraldehyde overnight at 4 ℃ one day in advance, and the fixed cells were sent to the electron microscopy center of Beijing university medical department for sampling, sectioning, staining, and photography, with the results shown in FIG. 13.

According to fig. 13, it can be seen that the induced hepatocytes have ultrastructures associated with mature hepatocytes, such as a large number of mitochondria, a rough endoplasmic reticulum, lipid droplets, glycogen particles, and bile capillary ducts composed of tight junctions at the junction of cells and cells.

From the results of the above example 2, it can be seen that the method according to the present invention induces the obtained functional hepatocytes to be more mature, which greatly expresses markers of hepatocyte maturation such as ALB, CK18, AAT, TF, CYP3a4, CYP2C9, CPS1, ASS1, etc., while the expression level of the immature functional markers alpha-fetoprotein (AFP) is greatly reduced, and in addition, the induced hepatocytes have greatly enhanced functions of albumin secretion, urea synthesis, drug metabolism, glycogen synthesis, indocyanine green uptake, etc., which can be better used for production and simulation of liver functions in vitro, and can provide ideal seed cells in terms of liver disease treatment, drug development and establishment of disease models.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Beijing Dabowei Yingyao pharmaceutical technology Co., Ltd

Military medical research institute of military science institute of Chinese people's liberation army

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Claims

1. A functional hepatocyte three-dimensional induction culture medium is characterized by comprising a first-stage three-dimensional culture medium and a second-stage three-dimensional culture medium; wherein:

2. The three-dimensional induction medium for functional hepatocytes according to claim 1, wherein the antioxidant in the first-stage three-dimensional medium is N-acetyl-L-cysteine at a concentration of 0.1 to 10mM, preferably 1.25 mM; the concentration of the L-ascorbate is 0.05-2mM, preferably 0.2 mM; the ROCK signal pathway inhibitor is Y-27632(StemCell) with the concentration of 5-20 μ M, preferably 10 μ M; the hepatocyte cell growth factor is HGF, the concentration is 5-50ng/mL, and the optimal concentration is 20 ng/mL; the oncostatin M is OSM, the concentration is 5-50ng/mL, and the preferred concentration is 20 ng/mL; the dexamethasone is Dex, and the concentration is 0.1-1mM, preferably 0.5 mu M; the concentration of the penicillin is 10-1000U/mL, preferably 100U/mL; the concentration of the streptomycin is 0.01-1mg/mL, preferably 0.1 mg/mL;

3. The functional hepatocyte induction three-dimensional culture medium according to claim 1 or 2, wherein the formulation of the first-stage three-dimensional culture medium is selected from any one of the following formulations:

and (3) formula: HCM/HM medium was used as a basal medium, and further contained 1% B-27, 0.1% BSA, 1.25mM N-acetyl-L-cysteine, 0.2mM L-ascorbate, 10. mu. M Y-27632, 20ng/mL HGF, 20ng/mL OSM, 0.5. mu.M Dex, 100U/mL penicillin and 0.1mg/mL streptomycin.

4. The functional hepatocyte three-dimensional induction culture medium according to any one of claims 1 to 3, wherein the formulation of the second-stage three-dimensional culture medium is selected from any one of the following formulations:

and (3) formula: HCM/HM medium was used as a basal medium, and further contained 1% B-27, 0.1% BSA, 1.25mM N-acetyl-L-cysteine, 10. mu. M Y-27632, 1% ITS, 10ng/mLEGF, 0.2mM L-ascorbate, 0.5. mu.M Dex, 100. mu.M 8-Br-cAMP, 10. mu.M vitamin K2, 10. mu.M Lithocholic acid, 100U/mL penicillin and 0.1mg/mL streptomycin.

5. A method of inducing functional hepatocytes, comprising the steps of:

s3: after digesting the liver organoid obtained in S2 into single cells, inducing differentiation into functional hepatocytes using the functional hepatocyte three-dimensional induction medium according to any one of claims 1 to 4.

6. The method of claim 5, further comprising, after step S2 and before step S3:

step S3 is to digest liver organoids after pretreatment in step S2-1 into single cells, and then to induce differentiation into functional hepatocytes using the three-dimensional induction medium for functional hepatocytes of any one of claims 1 to 4.

7. The method according to claim 6, wherein the preprocessing conditions in step S2-1 are: 37 ℃ and 5% CO₂Culturing in an incubator for 4 days, wherein the pre-differentiation medium is replaced once every 2 days; the pre-differentiation culture medium takes Advanced DMEM/F-12 culture medium as basic culture medium, and also comprises 0.01% -1% BSA, 0.2% -10% B-27, 0.2% -10% N2 additive, 1-50mM HEPES, 0.1% -10% glutamine additive and one or more of the following componentsThe method comprises the following steps: antioxidants, gastrin, Sirt1 protein inhibitors, hepatocyte growth factor, bone morphogenetic protein, fibroblast growth factor, penicillin and streptomycin;

further preferably, the formula of the pre-differentiation medium is as follows: the culture medium takes Advanced DMEM/F12 as a basic culture medium, and further comprises 0.1% BSA, 1% N2 additive, 1% B-27, 1% glutamine additive, 10mM HEPES, 1.25mM N-acetyl-L-cysteine, 10mM nicotinamide, 10nM Gastrin, 25ng/mL BMP7, 25ng/mL FGF4, 50ng/mL LHGF, 100U/mL penicillin and 0.1mg/mL streptomycin.

8. The method according to any one of claims 5 to 7, wherein in step S1, the pluripotent stem cells are H9-lineage stem cells.

9. A functional hepatocyte induced by the method of any one of claims 5 to 8.

10. Use of the functional liver cell of claim 9 as a seed cell for the manufacture of an artificial liver, the establishment of a liver disease treatment drug screening model, an in vitro model for simulating liver function, and an in vitro model for simulating liver disease.