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All-small molecular culture medium for culturing liver organoid and application of all-small molecular culture medium

A technology of small molecules and medium, applied in the direction of culture process, tissue culture, cell culture active agent, etc., can solve the problems of high price and unfavorable application of liver organoid culture system, and achieve the effect of low cost and reduced culture cost

Active Publication Date: 2019-03-26
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, growth factors such as EGF, Noggin, R-spondin, FGF10, HGF, and [Leu15]-Gastrin I are required to be added to the culture of liver organoids. These growth factors are expensive, It is not conducive to the application of liver organoid culture system, which greatly limits the large-scale expansion of liver organoids in vitro, and is used for the treatment of liver medicine

Method used

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  • All-small molecular culture medium for culturing liver organoid and application of all-small molecular culture medium
  • All-small molecular culture medium for culturing liver organoid and application of all-small molecular culture medium
  • All-small molecular culture medium for culturing liver organoid and application of all-small molecular culture medium

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Effect test

Embodiment １

[0033] Example 1: Cultivation of liver organoids based on CBF medium

[0034] According to the use of the CBF medium provided by the present invention in culturing liver organoids, the isolated mouse liver bile ducts prepared in this example were cultured. Using the technology of hepatic bile duct cultured in vitro as liver organoids, the role of small molecule CBF in the culture process was explored. According to the literature (Laura Broutier.et al.(2016) . "Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation." Nat Protoc. 2016 Sep;11(9):1724-43.) In the reported method, hepatic bile ducts were isolated and cultured from mice, and the culture status of CBF in vitro was observed. The specific methods are as follows:

[0035] (1) Isolate the liver from the mouse, cut it into 5mm pieces in the pre-cooled PBS buffer of pH7.4 3 of small pieces. And rinse three pieces with PBS to remove blood cells, fib...

Embodiment 2

[0038] Example 2: Mature differentiation culture of liver organoids based on CBF medium

[0039] According to the purpose of culturing liver organoids in the CBF medium provided by Benfang, the liver organoids in this example were further induced to differentiate into mature hepatic parenchymal cells. According to the literature (Laura Broutier.et al.(2016) . "Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation." Nat Protoc. 2016 Sep;11(9):1724-43. ) reported that the hepatic bile ducts were isolated and cultured from mice, cultured in CBF medium for a period of time, and OSM and dexamethasone were added to further differentiate the liver organs. The specific methods are as follows:

[0040] (1) Isolate the liver from the mouse, cut it into 5mm pieces in the pre-cooled PBS buffer of pH7.4 3 of small pieces. And rinse three pieces with PBS to remove blood cells, fibers and adipose tissue. Put the was...

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Abstract

The invention discloses an all-small molecular culture medium for culturing a liver organoid and an application of the all-small molecular culture medium. The culture medium comprises a basic culturemedium and a combination of small molecules, and the maintained combination of the small molecules is CBF, wherein C is CHIR-99021, B is Blebbistatin, C is Forskolin. The small molecules CHIR, Blebbistatin and Forskolin are added into the basic culture medium, so that the addition of a growth factor is replaced, a hpatic bile duct organoid can be cultured in vitro for a long time, and differentiation can be further induced to form a parenchymal hepatic cell. The method is beneficial to the in-vitro mass amplification of the liver organoid and provides the basis for researches and therapy of liver related diseases.

Description

technical field [0001] The present invention relates to the field of biotechnology. Specifically, the present invention relates to a whole small molecule culture medium for liver organoid culture. More specifically, the present invention relates to the medium, CHIR-99021, blebbistatin and forskolin in the preparation medium The use and method of culturing liver organoids. Background technique [0002] The liver is an important metabolic organ of the body. After the liver is damaged, it will quickly start to repair and regenerate, and its repair and regeneration is mainly driven by liver cholangiocytes. In recent years, the in vitro culture system of hepatic bile ducts has promoted the research in the field of liver, and the effective experiments and observations of hepatic bile ducts have been directly carried out in vitro. Liver organoids cultured in vitro can be transplanted into damaged livers and repair damaged livers. In addition, liver organoids cultured in vitro can ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0671C12N2501/999C12N2500/32
Inventor 林鑫华赵冰王鑫
Owner FUDAN UNIV
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