In-vitro amplification culture method and application of hepatocytes

A technology of in vitro expansion and hepatocyte application in cell culture active agents, hepatocytes, artificial cell constructs, etc.

Active Publication Date: 2020-04-10
CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is not yet possible to realize the continuous proliferation of human hepatocytes and the efficient repopulation ability in vivo without gene editing.

Method used

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  • In-vitro amplification culture method and application of hepatocytes
  • In-vitro amplification culture method and application of hepatocytes
  • In-vitro amplification culture method and application of hepatocytes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0180] Example 1, HM medium combined with hypoxic culture can promote long-term expansion of human hepatocytes in vitro

[0181] Through the screening and optimization of the medium formula, the inventors obtained a medium that can promote the expansion of human hepatocytes in vitro, and named it as HM medium. Human hepatocytes were divided into 1×10 5 Cells / well, spread in 6-well plate, and add HM medium for culture. Real-time observation in the live cell workstation, it can be found that human hepatocytes undergo epithelial-mesenchymal transition in cell morphology on the second day, followed by a large number of cells proliferating on the second day to the third day ( figure 1 A and B). Ki67 staining at day 3 revealed that 80% of the cells were proliferating, and 3 hours of Brdu incubation further showed that 20% of the hepatocytes were replicating ( figure 1 C). However, human hepatocytes rarely proliferated and replicated in YAC, a medium that could induce the prolife...

Embodiment 2

[0184] Example 2, Wnt3a is necessary for the promotion of hepatocyte proliferation in HM medium components

[0185] HM medium contains multiple growth factors and small molecular compounds, and the inventors found that when the Wnt3a conditioned medium was removed, human hepatocytes could not be expanded ( figure 2 A and B).

[0186] When using 50ng / ml Wnt3a purified protein or Wnt signaling pathway activator CHIR (0.1uM) to replace Wnt3a conditioned medium, human hepatocytes can also be expanded, and both are similar in cell morphology and expansion fold ( figure 2 A and B).

[0187] The above results prove that the activator of Wnt signaling pathway in HM medium is necessary for the proliferation of hepatocytes, and the activator of Wnt signaling pathway can be Wnt3a conditioned medium, activating protein or small molecule of Wnt signaling pathway.

Embodiment 3

[0188] Example 3. Proliferated human hepatocytes maintain part of the functions of mature hepatocytes

[0189] After cultured in HM medium, human hepatocytes maintain the expression of mature hepatocyte genes, and the expression level is different from that of primary human hepatocytes (PHH; freshly recovered uncultured primary human hepatocytes), such as ALB, HNF4A , TTR and CYP3A4 expression levels were lower than PHH, while CYP1A2, CAR, C3 and UGT1A1 expression levels were close to or higher than PHH ( image 3 A). Immunofluorescence staining further showed that more than 97% of proliferating human hepatocytes (ProliHH) expressed mature hepatocyte marker proteins ALB and AAT ( image 3 B). In order to analyze the level of hepatocyte gene maintenance of ProliHH, the present inventors compared PHH, ProliHH, and hepatocyte-like cells (HLC) derived from embryonic stem cell differentiation and fibroblast transdifferentiation (HLC; hepatocytes derived from embryonic stem cell d...

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Abstract

The invention relates to an in-vitro amplification culture method and application of hepatocytes. According to the invention, a culture system for reprogramming human hepatocytes into proliferative intermediate state cells between mature hepatocytes and liver precursor cells is established, and the liver reproductive capacity of the human hepatocytes is verified in animal bodies. According to themethod, exogenous genes do not need to be introduced into the hepatocytes; amplification of the hepatocytes can be achieved through conventional culture; the obtained hepatocytes can be subjected to passage; and maturation culture can be further carried out to obtain functional human liver maturation cells.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically, the invention relates to a method for expanding and culturing hepatocytes in vitro and the application of using the expanded hepatocytes for metabolic analysis and cell transplantation. Background technique [0002] The liver is one of the most functional and complex real organs in the body. Its main functions include regulating the balance of carbohydrates and lipids, participating in the metabolism and biotransformation of exogenous substances, the generation and excretion of bile, the storage of vitamins, and the secretion of proteins. Synthesis, generation and elimination of coagulation substances, and plays an important role in specific and nonspecific immunity. As the center of human substance metabolism, the liver plays an important role in various physiological and pathological processes, and many diseases can also affect the liver. At present, the most effective treat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12Q1/02A61K35/407A61P1/16
CPCC12N5/067G01N33/5067A61K35/407A61P1/16C12N2500/32C12N2500/38C12N2501/11C12N2501/119C12N2501/12C12N2500/02C12N2501/415C12N2501/39C12N2501/345C12N2501/237C12N2501/01C12N2503/02C12N5/0671C12N5/0672C12N2506/14C12N2501/15C12N2501/727
Inventor 惠利健张鲁狄张坤
Owner CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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