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In-vitro hepatocyte-like cell culture method and optimized hepatocyte-like cell cultured by the method

A technology of liver-like cells and in vitro culture, applied in the biological field, can solve the problem of low expression level and achieve the effect of improving expression level and increasing expression level

Active Publication Date: 2015-06-10
SHANGHAI INST OF MATERIA MEDICA CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

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 )

Method used

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  • In-vitro hepatocyte-like cell culture method and optimized hepatocyte-like cell cultured by the method
  • In-vitro hepatocyte-like cell culture method and optimized hepatocyte-like cell cultured by the method
  • In-vitro hepatocyte-like cell culture method and optimized hepatocyte-like cell cultured by the method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Obtainment of liver-like cells after optimization of mouse source

[0049] Step 1. Using direct transdifferentiation technology to generate traditional mouse-derived liver-like cells

[0050] 1) Molecular cloning and lentivirus production

[0051] 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.

[0052] 2) Culture of mouse tail fibroblast (TFF) cells or mouse embryonic fibroblast (MEF) cells: ...

Embodiment 2

[0063] Example 2 Obtainment of hepatic-like cells after optimization of human source

[0064]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.

Embodiment 3

[0065] Example 3 An experimental study was conducted on combinations of nuclear receptor agonists of different concentrations and types.

[0066] 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.

[0067] Table 1: Single nuclear receptor agonists and their concentration ranges

[0068]

[0069] Table 2: Various combinations of nuclear receptor agonists and their application concentrations

[0070]

[0071]

[0072] Table 3 T...

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Abstract

Provided are an in vitro culturing method for obtaining optimized hepatocyte-like cells and optimized hepatocyte-like cells obtained by using this method. The in vitro culturing method comprises the use of a sandwich cell culture method, serum-free culturing and an induction medium. The optimized hepatocyte-like cells obtained above have a structure and function close to that of primary liver cells, and the expression levels of the drug transporter, bile acid transporter and / or bile acid synthase thereof are significantly higher than that of traditional hepatocyte-like cells; the drug bile secretion index (BEI) and intrinsic biliary clearance (CL b,int) thereof are significantly higher than that of traditional hepatocyte-like cells; and the polarized expression of the drug transporter appears therein.

Description

technical field [0001] 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 [0002] 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...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCA61P1/16C12N5/067C12N2501/01
Inventor 潘国宇
Owner SHANGHAI INST OF MATERIA MEDICA CHINESE ACAD OF SCI
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