CN104694456B - Hepatic lineage after the method for in vitro culture hepatic lineage and the optimization of use this method culture - Google Patents

Abstract

The invention belongs to biological technical fields, and in particular to it is a kind of optimized after hepatic lineage extracorporeal culturing method and using this method obtain optimization after hepatic lineage.The extracorporeal culturing method is included using sandwich cell culture method, free serum culture and inducing culture.For hepatic lineage close to the 26S Proteasome Structure and Function of primary liver cell, the expression of drug transporters, bile acid transporter and/or cholic acid synzyme is significantly higher than traditional hepatic lineage after the optimization of acquisition；Its drug choleresis index (BEI) and inherent bile clearance rate (CL_b,int) it is significantly higher than traditional hepatic lineage；There is the polarization expression of drug transporters in it.

Description

Method for culturing hepatic-like cells in vitro and optimized hepatic-like cells cultured by this method cell

technical field

The invention belongs to the field of biotechnology, and in particular relates to a method for culturing hepatic cells in vitro and optimized hepatic cells cultured by the method.

Background technique

The liver is the main organ that determines drug metabolism and toxicity, and is also the target organ of various viral hepatitis and liver tumor diseases. At present, in the pharmaceutical industry, primary hepatocytes (including human and animal primary hepatocytes) are known as pharmacokinetics because of their good correlation with the expression of drug metabolizing enzymes and drug transporters, new drug development and clinical phenomena. The gold standard for medical research and an invaluable resource for liver transplantation and bioreactors (Sinz, Wallace et al. 2008). The current international market for primary hepatocytes is conservatively estimated at $2 billion. However, primary hepatocytes cannot be passaged, and the expression of various proteins decreases, coupled with high price and donor dependence, which limit its further use in scientific research and clinical practice. Therefore, there is an urgent need for a functional hepatocyte that functions similarly to hepatocytes, can be stably passaged, is reliable, and is readily available, both in scientific research and in the practice of the pharmaceutical industry.

The FDA white paper in 2010 pointed out for the first time that drug transporters have the same important position as drug metabolizing enzymes in the development of new drugs, and stipulated 7 drug transporters that must be investigated, of which 5 drug transporters are in the metabolism and detoxification of the liver play a significant role in (International Transporter, Giacomini et al.2010). This content was established in the 2012 FDA drug interaction guidance.

However, the currently available cell lines similar to liver cells have various shortcomings, especially the lack of suitable expression of drug transporters, and cannot be used for liver-related drug hepatobiliary distribution, metabolism, and toxicity studies. For example, the most common liver-derived cell line, HepG2, has almost no expression of important drug transporters, let alone bile duct-like structures. Currently recognized as the cell line HepaRG that is closest to primary hepatocytes, although the expression of drug metabolizing enzyme CYP3A4 is relatively abundant and can be induced, the expression of its drug transporter is only recognized by Pgp, and the immunohistochemical test shows that it cannot achieve polarized expression (Andersson, Kanebratt et al. 2012) (see Figure 4).

In recent years, many in vitro experiments have confirmed that embryonic stem cells or induced pluripotent stem cells can be differentiated into liver-like cells. However, these techniques are limited by the high cost and complexity of donor sources (ES cells from embryos) and induction processes (derived from iPS cells from human epithelial cells need to undergo three steps of transdifferentiation) and cannot provide suitable hepatic-like cells for drug development (Jensen, Hyllner et al. 2009). Although many literatures have reported that these cells have a certain expression of drug-metabolizing enzymes, there are few reports on the expression of drug transporters, and they are not satisfactory. At present, the latest direct transdifferentiation technology in the world can differentiate mouse fibroblasts into mouse-derived induced hepatocyte-like cells (iHep). Although iHep has many advantages compared with existing cell lines in terms of morphology, function and application, iHep does not significantly improve the expression of transporters (Fig. 1). It is immature in drug transporter and bile acid synthesis and secretion, and has no polarized expression of drug transporter and detectable ability of bile acid synthesis and secretion. In fact, bile acid synthesis and secretion and drug detoxification ability are important signs of mature hepatocytes. Experimental data show that in existing iHep cells, the expression levels of drug transporters and bile acid-related synthetases and transporters are extremely low compared to their expression levels in primary mouse liver cells, only 5-10 %(figure 1). Similarly, human induced hepatocyte-like cells (HiHep) can also be obtained from human non-liver cells by direct transdifferentiation technology, but the cells also have the above-mentioned defects.

Contents of the invention

technical problem

In 2011, a paper was published in Nature, and it was proposed for the first time in the world that mouse tail fibroblasts could be directly transdifferentiated into liver-like cells by means of epigenetics (Huang PY, He ZY, Ji SY, Sun HW, Xiang D, et al. (2011) Induction of functional hepatocyte-like cells from mouse fibroblasts by defined factors. Nature475:386-U142.). Compared with primary hepatocytes, the source of hepatic-like cells generated by the above-mentioned direct transdifferentiation technique is not limited by donor resources, and theoretically can be passed on indefinitely on the basis of maintaining genetic traits. They not only have multiple physiological characteristics and functions of liver cells, but also have a clear genetic background and the expression of certain drug-metabolizing enzymes. Therefore, it has the potential to replace primary hepatocytes and become a new cell model for drug development and clinical practice.

However, this cell line only has preliminary expression of drug transporters and bile acid metabolizing enzymes, and various related proteins are not polarized on the cell membrane. The drug interaction guidelines issued by the FDA in 2012 clearly pointed out that only when drug transporters are polarized on the liver cell membrane can cells have the ability to polarize bile salt uptake and secretion, which is an important characteristic for evaluating liver-like cells.

Therefore, in the prior art, for example, no matter whether embryonic stem cells or induced pluripotent stem cells are differentiated into liver-like cells or the liver-like cells obtained by direct transdifferentiation technology do not form polarized expression of drug transporters and/or The ability to synthesize and secrete bile acids can be detected. In this application, liver-like cells obtained from animal-derived non-liver cells or human-derived non-liver cells will be defined as traditional liver-like cells by using existing technologies (including direct transdifferentiation technology and embryonic stem cell or pluripotent stem cell induced differentiation technology) , which lack polarized expression of drug transporters and/or detectable bile acid synthesis and secretion capacity, are functionally and morphologically distinct from primary hepatocytes.

In order to improve the expression and function of bile acid synthase, bile acid transporter and drug transporter in traditional hepatic-like cells produced by existing technologies (including direct transdifferentiation technology and induced differentiation technology of embryonic stem cells or pluripotent stem cells), this application Through multiple tests, the inventor has produced a special in vitro culture method suitable for traditional hepatic-like cells produced by existing technologies (including direct transdifferentiation technology and embryonic stem cell or pluripotent stem cell induced differentiation technology). This approach effectively stimulates further differentiation, expression of bile acid synthase and polarized distribution of related drug transporters in conventional hepatic-like cells generated by existing techniques, including direct transdifferentiation techniques and embryonic stem cell or pluripotent stem cell-induced differentiation techniques, thereby The traditional hepatoid cells have similar bile uptake and secretion capabilities as the primary hepatocytes, and the polarized expression of drug transporters can be used in related research work.

Therefore, an object of the present invention is to provide a method for in vitro culturing of traditional hepatic-like cells obtained by the prior art. Specifically, the method can further promote the growth and differentiation of traditional liver-like cells and increase the expression level and polarized expression of drug transporters, bile acid transporters and/or bile acid synthases.

Another object of the present invention is to provide an optimized liver-like cell, the expression of bile acid synthase, bile acid transporter and drug transporter in the optimized liver-like cell is greatly increased, and the function is close to that of primary liver cells .

Technical solutions

The present invention provides a method for culturing traditional hepatic cells in vitro, which comprises:

Step 1. Cultivate traditional hepatic-like cells by sandwich cell culture method, that is, inoculate traditional hepatic-like cells on a 24-well plate containing rat tail collagen I, add ordinary medium, and culture cells for 24-48 hours , spread Matrigel to form a sandwich culture structure, and continue to culture for 24-48 hours;

Step 2. Remove the original normal medium and replace it with induction medium to continue culturing for 3-5 days to obtain optimized hepatic cells;

Wherein, the induction medium is supplemented with an inducer on the basis of a serum-free medium, that is, the common medium removes 1% fetal bovine serum, and supplements cAMP (cyclic adenosine monophosphate), 3MC (3-methylcholesterol Anthracene), PB (phenobarbital), TCA (taurocholic acid) and VPA (valproic acid) at least one nuclear receptor agonist, the above-mentioned each nuclear receptor agonist in the induction medium The concentration ranges are cAMP1-10μM, 3MC1-10μM, PB10-200μM, TCA5-50μM and VPA0.2-4μM;

Preferably, the induction medium contains two or three selected from cAMP, 3MC, PB and TCA, further preferably, the induction medium contains cAMP with a concentration of 2 μM and PB with a concentration of 50 μM, or contains cAMP at a concentration of 2 μM, 3MC at a concentration of 2 μM, and PB at a concentration of 50 μM, or containing cAMP at a concentration of 2 μM, 3MC at a concentration of 2 μM, PB at a concentration of 50 μM, and TCA at a concentration of 20 μM.

More preferably, the induction medium contains cAMP at a concentration of 2 μM, PB at a concentration of 50 μM and TCA at a concentration of 20 μM.

Wherein, the conventional hepatoid cells do not have polarized expression of drug transporters and/or detectable ability to synthesize and secrete bile acid.

Preferably, the traditional hepatoid cells are transfected with hepatocyte nuclear factor.

Preferably, the traditional liver-like cells are liver-like cells differentiated from embryonic stem cells or induced pluripotent stem cells; or the traditional liver-like cells are liver-like cells obtained from non-liver cells of animal origin by direct transdifferentiation technology, For example, mouse-derived liver-like cells obtained from mouse tail fibroblast (TFF) cells or mouse embryonic fibroblast (MEF) cells; or, the traditional liver-like cells are human-derived non-liver cells using direct transdifferentiation technology, preferably Human liver-like cells obtained from human embryonic fibroblasts; (for example, refer to Huang PY, He ZY, Ji SY, Sun HW, Xiang D, et al. (2011) Induction of functional hepatocyte-like cells from mouse fibroblasts by defined factors. Nature 475:386-U142. Formed by the method described).

More preferably, the mouse-derived hepatoid cells are transfected with hepatocyte nuclear factor 4α (Hnf4α); and the human-derived hepatoid cells are transfected with hepatocyte nuclear factor 4A (HNF4A).

In another aspect, the present invention provides an optimized hepatic-like cell, wherein the optimized hepatic-like cell is obtained by the above in vitro culture method.

The expression level of the drug transporter of the optimized hepatic-like cells is more than double that of the traditional hepatic-like cells;

The expression level of the bile acid transporter of the optimized hepatic-like cells is significantly higher than that of the traditional hepatic-like cells;

The expression level of bile acid synthase of the optimized liver-like cells is significantly higher than that of the traditional liver-like cells;

The drug bile secretion index (BEI) and intrinsic bile clearance (CL _b,int ) of the optimized hepatic-like cells are significantly higher than those of the traditional hepatic-like cells;

After the optimization, the hepatic-like cells showed polarized expression of drug transporters.

Wherein, the bile acid transporter is Bsep, Mrp2 and/or Ntcp; the drug transporter is P-gp, Bcrp, Oatp1b2, Oatp1a1 and/or Oatp1a4; the bile acid synthase is Cyp7a1.

Beneficial effect

In order to improve the expression and function of bile acid synthase, bile acid transporter and drug transporter in traditional hepatoid cells produced by direct transdifferentiation, this application adopts an in vitro culture method including the following characteristics: after transfection of Hnf4α/HNF4A into traditional After the hepatic-like cells were expressed, they were cultured by the sandwich culture method, and then the traditional hepatic-like cells were activated and optimized by using serum-free medium and adding nuclear receptor inducing factors.

The optimized liver-like cells obtained by the in vitro culture method described in this application have the following advantages: (1) the mRNA levels of drug transporters and endogenous bile transporters and their related nuclear receptors are similar to their mRNA levels in primary hepatocytes The levels are basically close; (2) bile canaliculi can be formed in vitro and have secretory function; (3) the bile secretion rate of the optimized hepatic cells and primary hepatocytes is very well correlated.

Hnf4α/HNF4A significantly increased the mRNA expression of Bsep, Mrp2, Ntcp, and Cyp7a1 in mouse/human hepatoid cells.

Different from the traditional culture method of liver-like cells, the in vitro culture method described in this application adopts serum-free culture, so that the mRNA expression level of liver drug transporters in liver-like cells is significantly improved, especially the bile acid uptake transporter Ntcp and the expression levels of the efflux transporter Bcrp were significantly increased.

The in vitro culture method described in the application adopts a three-dimensional cell culture method (sandwich cell culture method), so that the expression level of the mRNA of the liver drug transporter in the hepatoid cells is significantly increased.

The addition of cAMP, PB and/or TCA etc. to the serum-free medium greatly increased the mRNA levels of the main liver bile acid transporters Bsep, Mrp2 and Ntcp. In addition, cAMP, PB and/or TCA etc. have a great effect on increasing the mRNA levels of drug transporters (P-gp, Bcrp, Oatp1a1/4 and Oatp1b2) in the liver. The mRNA level of bile acid synthase Cyp7a1 can be increased by 4 times under the induction of cAMP. The increased expression of these drug transporters, bile acid transporter and bile acid synthase is an important indicator of the enhanced ability of hepatic cells to metabolize and distribute drugs and endogenous substances.

This work will lay the foundation for the practical application of epigenetics and direct transdifferentiation technology, making it possible for new liver-like cells to be used in the development of new drugs.

Description of drawings

Figure 1A-Figure 1F show the effects of different experimental conditions and different induction factors on the gene expression levels of bile acid synthase (Cyp7a1) and various bile acid transporters in mouse-derived traditional liver-like cells.

Figures 2A-2G show the effects of different experimental conditions and different induction factors on the gene expression levels of various drug transporters in mouse-derived traditional liver-like cells or on the BEI (%).

Figures 3A-3B show the effects on gene expression levels of drug efflux transporters and uptake transporters (3A) and related nuclear receptors (3B) in mouse-derived optimized liver-like cells obtained in Example 1. Among them, rat tail fibroblasts and mouse liver cells were used as controls.

Fig. 4 is an image obtained under an electron microscope after immunohistochemical staining of HepaRG cells. HepaRG cells are currently the only liver-like cell line approved by the FDA, which can be used for research on the induction of liver drug-metabolizing enzymes. However, it can be seen from Figure 4 that the expression of drug-metabolizing enzymes (CYP3A4, Figure 4C) in this cell line is acceptable, but the expression of important drug and bile acid transporters (Figure 4A: P-gp, Figure 4B, MRP2) is very low. Sufficient, only expressed in some cells, and uneven.

Fig. 5 is an image of mouse liver cells, traditional hepatoid cells and the optimized hepatoid cells obtained in Example 1 under a fluorescence microscope to observe whether CDF is secreted and pooled in the formed bile duct area. It can be seen that both the mouse liver cells and the optimized hepatic-like cells obtained in Example 1 have CDFs converging in the area of the formed bile duct, while this phenomenon is not observed in traditional hepatic-like cells.

Figure 6 is a fluorescent confocal microscope photo. The first and third rows are photos of mouse liver cells, in which the important bile acid and drug transporters Bsep, Mrp2, Pgp and Bcrp are polarized along the bile duct structure; the second and fourth rows are Example 1 The obtained photo of optimized liver-like cells, in which Bsep, Mrp2, Pgp and Bcrp also showed polarized expression. It can be seen that the morphology of the mouse liver cells is very similar to that of the optimized liver-like cells obtained in Example 1.

Figure 7 shows the inner bile of hepatic cells and primary mouse hepatocytes before and after optimization of DPDPE (enkephalin), d8-TCA (isotope-labeled taurocholic acid), rosuvastatin and methotrexate Correlation of clearance.

Fig. 8 is the gene expression level (Fig. 8a) and BEI (%) (Fig. 8b) of the drug transporter of human-derived optimized hepatic-like cells obtained in Example 2.

Detailed ways

The present invention is further illustrated below through specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

Example 1 Obtainment of liver-like cells after optimization of mouse source

Step 1. Using direct transdifferentiation technology to generate traditional mouse-derived liver-like cells

1) Molecular cloning and lentivirus production

First construct the vector of the target gene: the multiple cloning site is inserted into the PmeI restriction enzyme site of the lentiviral vector pWPI with the reporter gene GFP, and the cDNAs of the three transcription factors of the target genes Gata, Hnf1α, and Foxa3 are respectively connected to this On the modified vector, three constructed pWPI vectors containing transcription factors and two packaging plasmids psPAX2 and pMD2.G were added to 293T cells. After 48 hours, the supernatant containing the lentivirus was collected and filtered through a 0.45 μm filter, aliquoted into EP tubes, and frozen at -80°C.

2) Culture of mouse tail fibroblast (TFF) cells or mouse embryonic fibroblast (MEF) cells:

Rat tail fibroblasts were first cut off a 5cm tail from a 2-month-old mouse, removed the dermis, chopped it to about 1cm, and placed it on a 6cm flat plate covered with collagen I. The medium was 10% FBS DMEM in a volume of 5ml. After 5 days, the fibroblasts were transferred to a new 6 cm plate coated with collagen I. Generally, the 7th to 9th passages of mouse tail fibroblasts are used for lentivirus transfection.

Mouse embryonic fibroblasts were first obtained from 13.5-day-old mouse embryos, the head and viscera were removed, the remaining tissues were minced, digested with 0.25% trypsin at 37°C for 15 minutes, and then the cells were transferred to a 6cm flat plate covered with collagen I for growth , the medium is still DMEM containing 10% FBS, the volume is 5ml. Generally, the third passage of mouse embryonic fibroblasts is used for lentivirus transfection.

3) Transfection of lentivirus and acquisition of mature traditional liver-like cells

On the first day, 1.5× ¹⁰⁵ mouse tail fibroblasts or mouse embryonic fibroblasts were plated on a 6 cm plate covered with collagen I, and three transcription factors Gata, Gata, and Hnf1α, Foxa3, and polybrene (polybrene, the final concentration reaches 8 μg/ml or 4 μg/ml) was added at the same time, and the fresh medium was changed on the third day, and the medium was still DMEM containing 10% FBS, and fresh culture was changed on the fourth day Base, the medium is common medium, after two days to change the common medium, transfection 14-21 days, to obtain mature traditional liver-like cells. Note: Common medium formula: DMEM/F121:1 basal medium (Hyclone) containing 0.1 μM dexamethasone, 20 μg/LTGF-α, 10 μg/L EGF, 4.2 mg/L insulin, 3.8 mg/L human transferrin , 5μg/L selenite and 1% fetal bovine serum.

Step 2, transfection of hepatocyte nuclear factor 4α (Hnf4α)

The cDNA of the target gene hepatocyte nuclear factor 4α (Hnf4α) was connected to the lentiviral vector pWPI with the reporter gene GFP, and the constructed pWPI vector containing Hnf4α and packaging plasmids psPAX2 and pMD2.G were used to infect 293T cells. After 48 hours, the supernatant containing lentivirus was collected and filtered with a 0.45 μm filter, and distributed into EP tubes. Use a flow cytometer to detect the virus titer, and finally transfect the traditional liver-like cells obtained in step 1.

Step 3. The conventional hepatic-like cells transfected with Hnf4α obtained in step 2 are cultured by a sandwich cell culture method.

Firstly, digest and separate the formed traditional hepatic-like cells, then inoculate the traditional hepatic-like cells on a 24-well plate containing rat tail collagen I at a density of 1×10 ⁶ cells/cm ² , add 500 μl ordinary medium, and culture the cells After 24 hours, Matrigel was spread to form a sandwich culture structure, and culture was continued for 24 hours. Common medium formula: DMEM/F121:1 basal medium containing 0.1μM dexamethasone, 20μg/L TGF-α, 10μg/L EGF, 4.2mg/L insulin, 3.8mg/L human transferrin, 5μg/L L selenite and 1% fetal bovine serum.

Step 4. Optimizing culture to obtain optimized hepatic-like cells (mouse source)

After step 3, remove the original ordinary medium and replace it with induction medium, that is, add nuclear receptor agonists: cAMP (2 μM), PB (50 μM) and TCA (20μM), continue to culture for 3-5 days to obtain optimized hepatic cells.

Example 2 Obtainment of hepatic-like cells after optimization of human source

In addition to replacing mouse tail fibroblast (TFF) cells or mouse embryonic fibroblast (MEF) cells with human embryonic fibroblasts (derived from maternal placenta samples), human embryonic fibroblasts (derived from Pregnant women's placenta samples), using a method similar to Example 1 to obtain optimized human-derived hepatic cells.

Example 3 An experimental study was conducted on combinations of nuclear receptor agonists of different concentrations and types.

Firstly, hepatic-like cells transfected with Hnf4α were obtained on the basis of traditional hepatic-like cells (the steps are the same as steps 1-3 in Example 1). Then switch to the induction medium containing different concentrations and different combinations of nuclear receptor agonists for optimal selection, try a single nuclear receptor and its concentration, multiple combinations of nuclear receptor agonists and their concentrations, and finally obtain Most preferred combinations and concentrations of nuclear receptor agonists. The specific results are shown in Tables 1, 2, and 3 below.

Table 1: Single nuclear receptor agonists and their concentration ranges

Table 2: Various combinations of nuclear receptor agonists and their application concentrations

Table 3 The most preferred nuclear receptor agonist combination and its application concentration

EXPERIMENTAL EXAMPLES Functionality and gene expression testing to confirm successful cell optimization

After the cell optimization is completed (usually takes 3-5 days), verify whether the optimization process is successful from the perspective of gene expression and function:

1. Determination of the expression of drug transporters, bile acid transporters and bile acid synthase by quantitative PCR (QPCR)

First, the total RNA of the cells was extracted with Trizol, and the RNA was extracted according to the RNA extraction kit. After diluting the extracted total RNA to a certain multiple, measure the light absorption values at two wavelengths of 260nm and 280nm with a UV spectrophotometer, and then calculate the concentration of the extracted total RNA according to the following formula: [RNA concentration (μg/ml )]=A260×40×dilution factor, the ratio of the two readings at 260nm and 280nm (A260/A280), which can reflect the purity of nucleic acid, generally A260/A280 is between 1.8-2.0. 2 μg of RNA was reacted by reverse transcription system to generate cDNA. The reverse transcription reaction conditions were: 37°C for 15 minutes, 85°C for 5s, and then kept at 10°C. Quantitative PCR operation was performed by SYBR Premix Ex Taq kit, and primers such as Pgp, Bcrp, Mrp2, Ntcp, and Bsep were designed and synthesized according to requirements, and then entered into the instrument ABI7500 quantitative PCR system for determination.

Determine whether the expression of important drug transporters, bile acid transporters, etc. has significantly increased compared with traditional cells, and the expression of 2/3 of the measured objects has increased by more than 50% as qualified.

I. Referring to Fig. 1A-Fig. 1F, in each coordinate diagram, the first group of histograms (the three leftmost histograms) represent rat tail fibroblasts (TTF), primary hepatocytes (PriHep) and adopt the embodiment 1 The gene expression levels of bile acid synthase (Cyp7a1) and various bile acid transporters of mouse-derived traditional liver-like cells obtained in step 1; Mouse-derived traditional liver-like cells were subjected to sandwich culture (i.e., only step 1 and step 3 of Example 1), serum-free culture (i.e., after step 1 of Example 1, 1% fetal Serum-free medium with bovine serum, cultured for 48 hours) and long-term normal culture for 6 weeks after the maturation of traditional hepatoid cells (that is, after step 1 of Example 1, normal culture medium was used for another 6 weeks). Then measure the gene expression levels of bile acid synthase (Cyp7a1) and various bile acid transporters under these three kinds of culture conditions respectively; Mouse-derived traditional liver-like cells were cultured for 3-5 days in induction medium (adding 2 μM cAMP, 2 μM 3MC, 50 μM PB, 20 μM TCA or 1 mM VPA of each nuclear receptor agonist in serum-free medium) or transfected with Hnf4α The gene expression levels of bile acid synthase (Cyp7a1) and various bile acid transporters were measured over a period of 1 day.

It can be seen from the results in Figure 1 that:

(1) For traditional hepatic-like cells, culture conditions such as sandwich culture, serum-free culture, or 6-week long-term culture can all increase the levels of hepatic bile acid transporters (Bsep, Mrp2, Ntcp) and bile acid synthase (Cyp7a1) mRNA expression (Fig. 1A-Fig. 1D);

(2) When nuclear receptor activators such as cAMP (2 μM), 3MC (2 μM), PB (50 μM), TCA (20 μM) or VPA (1 mM) were added to the induction medium, bile acid transporters Bsep, Mrp2 , Ntcp expression were all increased (Fig. 1A-Fig. 1C), while bile acid synthase (Cyp7a1) mRNA expression was increased by about 4 times under the induction of cAMP, but the induction effect of PB or TCA alone was not obvious (Fig. 1D).

(3) In BEI experiments (Fig. 1E and Fig. 1F), culture conditions such as sandwich culture, serum-free culture or 6-week long-term culture, induction of cAMP or TCA, and transfection expression of transcription factor Hnf4α can significantly Increasing the BEI (%) value of CLF (Bsep substrate) and D8-TCA (Bsep and Mrp2 substrate) indicates that the efflux of CLF and d8-TCA increases, thus further explaining the effect of different culture conditions and different induction factors on traditional liver samples. Effects on gene expression levels of various bile acid transporters in cells.

II. Referring to Fig. 2A-Fig. 2E, similar to Fig. 1, in each coordinate diagram of Fig. 2, the first group of histograms (three histograms on the far left) represent rat tail fibroblasts (TTF), primary liver Cells (PriHep) and the gene expression levels of the drug transporter of mouse-derived traditional liver-like cells obtained in Step 1 of Example 1; The liver-like cells were subjected to sandwich culture (i.e., only step 1 and step 3 of Example 1), serum-free culture (i.e., after step 1 of Example 1, the common culture medium was used to remove 1% of fetal bovine serum) Serum-free medium, cultured for 48 hours) or traditional hepatic-like cells matured and then long-term common culture for 6 weeks (that is, after step 1 of Example 1, cultured with normal medium for another 6 weeks), and then assayed for drug transporters The third group (the 6 histograms on the far right) represents the mouse-derived traditional liver-like cells obtained in step 1 of Example 1 in the induction medium (common medium with 2 μM of each nuclear receptor agonist added respectively). cAMP, 2 μM 3MC, 50 μM PB, 20 μM TCA or 1 mM VPA) or transfected with Hnf4α were cultured for 3-5 days, and the gene expression levels of drug transporters were determined.

It can be seen from the results in Figure 2 that:

(1) For traditional liver-like cells, sandwich culture and serum-free culture can significantly increase the mRNA expression of efflux transporters Pgp and Bcrp and uptake transporters Oatp1b2 and Oatp1a1/4. Inducers cAMP, PB, and TCA could increase the mRNA expression of Pgp, Bcrp, Oatp1b2, and Oatp1a1/4, but VPA and 3-MC had no obvious effect on the gene expression levels of drug transporters.

(2) The transcription factor Hnf4α can also greatly increase the mRNA expression of efflux and uptake transporters. In addition, the results of BEI experiments (Fig. 2F and Fig. 2G) showed that sandwich culture and serum-free culture, as well as inducers cAMP, PB, TCA and Hnf4α, could increase the levels of rosuvastatin (Bcrp substrate) and methotrexate (Bcrp substrate) The BEI(5) value of .

III. Referring to the results in Figure 3A, it can be seen that from the perspective of gene expression levels, in the optimized hepatic cells obtained in Example 1, the expression levels of various important drug transporters and bile acid transporters measured in the liver are greatly improved, Comparable to primary hepatocytes. Referring to the results in Figure 3B, it can be seen that the mRNA expression of the measured nuclear receptors and efflux and uptake transporters was also significantly increased.

2. Co-localization of drug transporters and bile acid transporters

The experimental object is the optimized liver-like cells obtained in Example 1.

Experimental procedure: at room temperature, fix the cells with 4% formaldehyde for 1 hour; wash with PBS three times, each time for 5 minutes; ; Block with 3% BSA-PBS, room temperature for 1 hour; absorb the blocking solution, add primary antibody P-19 (mouse anti-Bsep, 1:100) and M2-Ⅲ-6 (mouse anti-Mrp2, 1:40), incubate at 4°C 16 hours or overnight; wash with PBS three times, 5min each time; after sucking off the PBS, add secondary antibodies FITC-anti-mouse IgG (1:300) and TRITC-anti-mouse IgG (1:400), incubate at room temperature for 1h in the dark; PBS Wash three times, 5min each time; suck off the PBS, add 2.5% diazabicyclooctane (9:1 glycerol/PBS preparation) to block and mount, cover with a cover slip, and observe Bsep and Co-localization of Mrp2. For the colocalization of P-gp and Bcrp, the procedure is similar to the colocalization of Bsep and Mrp2, except that the primary antibodies are CD44 (mouse anti-P-gp, 1:200) and mouse anti-CD338 (1:100). The results showed that there was a certain co-localization of transporters in hepatic-like cells after optimization.

Specifically, referring to Fig. 6, the optimized hepatic-like cells obtained in Example 1 have for the first time the polarized expression of important drug transporters similar to primary hepatocytes (for example, the efflux-type drug transporter Bsep, Mrp2 is expressed in the bile tubule side), this feature was only reflected in primary hepatocytes. However, this feature was not found in HepaRG, the best hepatoid cell model at present (see Figure 4). It can be clearly seen from the comparison that the optimized hepatic cells obtained by the in vitro culture method described in this application are significantly better than the existing best hepatic cell model HepaRG, and are comparable to the primary hepatic cell in vitro culture model.

3. Functional determination of optimized hepatic-like cells

The experimental object is the optimized liver-like cells obtained in Example 1.

I. Detect whether the optimized hepatic cells form a functional bile duct structure.

Experimental procedure: After step 4 of Example 1, on the 6th day of culture with induction medium, cells ^{in triplicate were washed twice with 1 ml of warm HBSS with or without Ca 2+ per} well. Second-rate. After washing, 1 ml of HBSS with or without Ca ²⁺ was added and the cells were incubated at 37°C for 15 ^min . The medium was aspirated from each well and the cells were incubated at 37°C for 10 minutes with 0.5 ml medium containing substrate (5 μM CDF, 2.5 μM CLF and 2.5 μM d8TCA). After incubation, the medium was discarded and the cells were washed 3 times with ice-cold regular PBS. Cell plates were stored at -80°C. CDF (5-(and-6)-carboxy-2',7'-dichloro-fluorescein, fluorescein) is used to detect the formation of bile ducts. After the cell optimization is completed, the cell morphology and the accumulation of CDF in the bile ducts can be determined by Phase contrast microscopy and fluorescence microscopy were evaluated.

Figure 5 is an image of the presence or absence of CDF aggregation observed under a fluorescent microscope. Among them, mouse liver cells have obvious CDF aggregation, and traditional liver-like cells have no obvious CDF aggregation. After optimization, liver-like cells have obvious CDF aggregation. The results It shows that the optimized hepatic-like cells obtained by the in vitro culture method described in this application can form a bile duct structure similar to primary hepatocytes in vitro.

II. Determination of Intrinsic Bile Clearance (CL _b,int )

The experimental subjects were the traditional hepatic cells obtained in Step 1 of Example 1 and the optimized hepatic cells obtained after Step 4 of Example 1.

Experimental procedure: After step 4 of Example 1, on the 6th day of culture with induction medium, cells ^{in triplicate were washed twice with 1 ml of warm HBSS with or without Ca 2+ per} well. Second-rate. After washing, 1 ml of HBSS with or without Ca ²⁺ was added and the cells were incubated at 37°C for 15 ^min . Aspirate the medium from each well and incubate the cells with 0.5 ml of medium containing substrates (2.5 μM DPDPE, 2.5 μM d8-TCA, 2.5 μM rosuvastatin, and 2.5 μM methotrexate) at 37°C for 10 Minutes, the 0-minute and 10-minute culture fluids were collected and frozen in a -80°C refrigerator, and the cells were washed 3 times with ice-cold regular PBS immediately after incubation, and the cell plates were stored at -80°C.

Note: The formula for calculating intrinsic bile clearance (CL _b,int ) is:

CL _b,int = (A _{plus_Ca++} -A _{minus_Ca++} )/AUC _0-10min

Wherein A _{plus_Ca++} represents the accumulated amount of the tested compound in cells treated with Ca ²⁺ , A _{minus_Ca++} represents the accumulated amount of the tested compound in cells treated without Ca ²⁺ , AUC _0-10min =incubation time×( C _{m,0 min} +C _{m,10 min} )/2, C _{m,0 min} and C _{m,10 min} represent the concentration of the tested compound at 0 min (start of incubation) and 10 min (incubation time) respectively.

See Figure 7. The experimental results prove that optimized hepatic cells and primary hepatic cells have a good correlation with the intrinsic bile clearance rate (CL _b,int ) of the drug, indicating that optimized hepatic cells are more effective than unoptimized hepatic cells closer to primary hepatocytes.

III. Determination of bile secretion index BEI (%) and intrinsic bile clearance (CL _b,int ).

The inventor of the present application also selects an important tool drug, analyzes the drug accumulation situation by isotope method or liquid phase tandem mass spectrometry, and calculates the bile secretion index BEI (%) of the selected drug [the formula for calculating BEI is BEI (%)=(A _{plus_Ca++} -A _{minus_Ca++} )/A _{plus_Ca++} ×100%, where A _{plus_Ca++} represents the accumulation of the tested compound in cells treated with Ca ²⁺ , and A _{minus_Ca++} represents the accumulation of the tested compound in cells not treated with Ca ²⁺ volume] and intrinsic bile clearance (CL _b,int ). (results see table 4 below)

Table 4 Comparison of bile secretion ability of important tool drugs in different cell models

Wherein, the traditional hepatic-like cells are obtained after step 1 of Example 1, and the optimized hepatic-like cells are obtained after step 4 of Example 1;

The traditional liver-like cell group was used as the control group, and the experimental results were statistically analyzed (ANOVA, SPSS);

**Represents extremely significant difference (p<0.01)

4. Detection of optimized human-sourced hepatic-like cells

The optimized human liver-like cells obtained in Example 2 were used to determine the expression level of drug transporter genes and BEI (%) by using similar experimental operations as above.

It can be seen from the results in Figure 8 that:

(1) The human-derived liver-like cells obtained in Example 2 can express a variety of important drug transporters, such as P-gp, MRP2, MRP3, BSEP, NTCP, OATP, etc. The mRNA expression levels of these transporters and human primary liver cells (PHH) were similar (Fig. 8a, mRNA levels were around 80% of human hepatocytes).

(2) The human-derived hepatoid cells obtained in Example 2 have the ability to secrete bile. The BEI of compounds DPDPE, TCA and CLF in human-derived hepatoid cells reached more than 50% of that of primary human hepatocytes (Fig. 8b).

Therefore, whether in terms of gene expression or function, the human-derived hepatic-like cells obtained by optimizing the in vitro culture method described in this application are comparable to primary human hepatocytes, and are expected to become cell lines that can replace primary human hepatocytes .

Based on the above experimental results, it can be concluded that the optimized hepatic cells obtained by the in vitro culture method described in this application (including hepatic cells from a variety of genera) can be used for new drug research and development, artificial liver device seed cells , in vitro hepatitis cell model and other scientific research fields.

In addition, it should be noted that although traditional hepatic-like cells were obtained by direct transdifferentiation technology in the examples, activation and further induced culture were carried out on this basis, those skilled in the art can think of this method according to the above disclosure. The in vitro culture technology described in the application is also applicable to other traditional hepatic cells obtained by other methods in the prior art, such as ES and hepatic cells obtained from iPS sources.

Although this application uses the in vitro culture and detection of mouse fibroblasts as an exemplary embodiment, the methods described in this application are not limited to hepatic-like cells produced by mouse cells, but are also applicable to optimization of non-liver cells from various species. Hepatic-like cells derived from cells (eg, fibroblasts), such as non-hepatic cells of human origin or non-liver cells of rat origin. The application also provides that the liver-like cells produced by human-derived fibroblasts produce good drug bile secretion index (BEI) and gene expression levels (close to parameters such as primary human hepatocytes) after applying the optimized culture method described in the application. ) as evidence.

English and Chinese abbreviations in the manual:

iHep, induced hepatocyte-like cell, induced hepatocyte-like cell;

HiHep, human induced hepatocyte-like cell, human induced hepatocyte-like cell;

Hnf4α, hepatocyte nuclear factor4α, hepatocyte nuclear factor 4α;

CDF,5-(and-6)-carboxy-2',7'-dichloro-fluorescein, fluorescein substrate;

CLF, cholyl-lysyl-fluorescein, fluorescein substrate;

DPDPE, [D-Pen ^2,5 ] Enkephalin hydrate, enkephalin;

d8-TCA, d8-sodium taurocholate acid, isotope-labeled taurocholate;

TCA, taurocholic acid, taurocholic acid;

PB, phenobarbital, phenobarbital;

MTX, methotrexate, methotrexate;

cAMP, N6,2'-O-Dibutyryladenosine3',5'-cyclic monophosphate sodium salt, here refers specifically to dibutyryl cyclic adenosine sodium salt, referred to as cyclic adenosine;

3-MC, 3-methylcholanthrene, 3-methylcholanthrene;

VPA, valproic acid, valproic acid;

CL _bile,int ,intrinsic biliary clearance, intrinsic bile clearance;

BEI, biliary excretion index, bile secretion index;

BA, bile acid, bile acid;

SCMH, sandwich-culture mouse hepatocyte, sandwich mouse liver cell culture;

TTF, tail-tip fibroblasts, rat tail fibroblasts;

PriHep, primary hepatocyte, primary liver cells;

FBS, fetal bovine serum, fetal bovine serum;

Mrp2, multidrug resistance-associated protein2, multidrug resistance-associated protein 2;

Bsep, bile salt efflux protein, bile acid efflux protein;

Ntcp, sodium taurocholate cotransporting polypeptide, sodium ion - taurocholate cotransporter;

P-gp, p-glycoprotein, p-glycoprotein;

Cyp7a1, cholesterol7-alpha hydroxylase, cholesterol 7-hydroxylase;

PXR, pregnane X receptor, progesterone receptor;

FXR, farnesoid X receptor, farnesoid X receptor;

AhR, aryl hydrocarbon receptor, aromatic hydrocarbon receptor;

CAR, constitutive androstane receptor, constitutive androstane receptor;

PPARγ: peroxisome proliferator-activated receptor alpha, peroxisome proliferator-activated receptor;

Nrf2: NF-E2related factor-2, nuclear factor E2 related factor 2;

ES cell, embryonic stem cell, embryonic stem cell;

iPS cell, induced pluripotent stem cell, induced pluripotent stem cell.

Claims (9)

1. it is a kind of optimized after hepatic lineage extracorporeal culturing method, including：

Step 1, using sandwich cell culture method culture tradition hepatic lineage；

Step 2 removes original culture medium, uses inducing culture instead and continues culture 3-5 days, hepatic lineage after being optimized；

Wherein, the inducing culture be serum free medium, and the inducing culture contain selected from cAMP, 3MC, PB, A kind of nuclear receptors agonists in TCA and VPA either either containing cAMP, 3MC and PB or contain containing cAMP and PB CAMP, PB and TCA or containing cAMP, 3MC, PB and TCA, above-mentioned each nuclear receptors agonists are in the inducing culture Concentration range is respectively cAMP 1-10 μM, 3MC 1-10 μM, PB 10-100 μM, TCA 5-50 μM and VPA 0.2-4 μM；

Wherein, traditional hepatic lineage is direct transdifferentiation technology and embryonic stem cell or versatile stem cell induction differentiation skill The hepatic lineage of art induction；

Wherein, traditional hepatic lineage has transfected hepatocyte nuclear factor, and the hepatocyte nuclear factor is selected from Hepatocyte nuclear factor 4 α (Hnf4 α) and Hepatocyte nuclear factor 4 A (HNF4A).

2. extracorporeal culturing method according to claim 1, wherein,

The inducing culture contains the cAMP that concentration is 2 μM and the PB that concentration is 50 μM or is 2 μM containing concentration CAMP, concentration are 2 μM of 3MC and concentration is 50 μM PB or containing the cAMP that concentration is 2 μM, the PB that concentration is 50 μM and Concentration is 20 μM of TCA or containing the cAMP that concentration is 2 μM, the 3MC that concentration is 2 μM, the PB that concentration is 50 μM and concentration 20 μM of TCA.

3. extracorporeal culturing method according to claim 1, wherein,

The tradition hepatic lineage is mouse source, rat source or people source tradition hepatic lineage.

4. extracorporeal culturing method according to claim 3, wherein,

The mouse source tradition hepatic lineage is to utilize direct transdifferentiation technology, is obtained by the non-liver cell in mouse source；The people source Traditional hepatic lineage is to utilize direct transdifferentiation technology, is obtained by the non-liver cell in people source.

5. extracorporeal culturing method according to claim 4, wherein, the non-liver cell in mouse source is rat-tail fibroblast Or rat embryo fibroblast cell；The non-liver cell in people source is mankind's embryo fibroblast.

6. according to the extracorporeal culturing method any one of claim 3-5, wherein,

The mouse source tradition hepatic lineage has transfected Hepatocyte nuclear factor 4 α (Hnf4 α)；The people source tradition hepatic lineage transfection Hepatocyte nuclear factor 4 A (HNF4A).

7. hepatic lineage after a kind of optimization, the cell is obtained by extracorporeal culturing method according to any one of claims 1 to 6 .

8. hepatic lineage after optimization according to claim 7, the expression of drug transporters is traditional hepatic lineage One times or more；And/or the expression of its bile acid transporter is significantly higher than traditional hepatic lineage；And/or its cholic acid synzyme Expression be significantly higher than traditional hepatic lineage；And/or its drug choleresis index (BEI) and inherent bile clearance rate (CL_b,int) it is significantly higher than traditional hepatic lineage；And/or there is the polarization expression of drug transporters in it.

9. hepatic lineage after optimization according to claim 8, wherein, the bile acid transporter for Bsep, Mrp2 and/or Ntcp；The drug transporters are P-gp, Bcrp, Oatp1b2, Oatp1a1 and/or Oatp1a4；The cholic acid synzyme is Cyp7a1。