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Method for producing liver progenitor cells of mice

A liver precursor cell and production method technology, applied in the field of mouse liver precursor cell production, can solve the problems affecting the purity of isolated cells and complicated operation steps

Inactive Publication Date: 2014-02-19
CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The equipment required by MACS is simple and can be operated in an ultra-clean bench. However, multi-marker sorting needs to be carried out step by step. The operation steps are relatively complicated. At the same time, there is a certain proportion of non-specific binding, which affects the purity of the separated cells.

Method used

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  • Method for producing liver progenitor cells of mice
  • Method for producing liver progenitor cells of mice
  • Method for producing liver progenitor cells of mice

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Isolation of Liver from Mouse Embryo

[0053] 1. Pregnant mice (C57BL / 6) of different gestational ages (gestational age E9.5-E16.5) were sacrificed by cervical dislocation after deep anesthesia. Open the abdomen with surgical scissors, take out the uterus containing the fetal mouse, and put it in PBS pre-cooled on ice.

[0054] 2. Separate the fetal mouse from the uterus with ophthalmic forceps and place it in cold PBS.

[0055] 3. Using a stereo microscope, separate the embryonic livers of different stages from the fetal mice, and remove the surrounding visceral tissues. The embryonic liver is pink compared to the surrounding tissue and is easier to distinguish from other surrounding visceral tissue (white).

Embodiment 2

[0056] Example 2: Preparation of single cell suspension of liver

[0057] The embryonic liver isolated in Example 1 was cut into pieces with scissors, and then digested with liver digestion solution (EDTA solution containing 0.25g / 100ml trypsin, prepared by adding 0.25g trypsin and 0.025g EDTA to 100ml PBS), Make it dispersed into a single cell suspension.

Embodiment 3

[0058] Embodiment 3: the culture of cell

[0059] 1. Inoculate the dispersed single-cell suspension (the cells in which are called P0 generation cells) to the γ-ray-treated (sterilized) mouse fibroblast feeder layer (mouse embryo fibroblast, MEF, catalog number MUIEF-01002) and cultured on liver precursor cell culture medium. The formula of liver precursor cell culture medium is supplemented with 10% (v / v) fetal bovine serum, 2mmol / L L-glutamine, 100μM non-essential amino acids, 100U / ml penicillin and 100U / ml streptomycin, 28.6 μM beta-mercaptoethanol in DMEM medium. cells in 5% CO 2 Cultured in a 37°C incubator with 95% air, and the medium was changed every two days. After 7 days of culture, a single clone of many regular cells (called P1 generation) can grow on it.

[0060] The monoclonal cells are picked and inoculated on the new feeder layer and liver precursor cell culture medium to continue to expand the culture, and a large number of cell clones can be grown on the ...

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Abstract

The invention relates to a method for producing liver progenitor cells of mice. The method comprises the following steps: (1) separating the liver from a mouse embryo with the embryonic day being 13-15 days; (2) obtaining cell suspension of the liver; (3) sub-culturing cells on a mouse fibroblast feeding layer to obtain a monoclonal antibody; (4) sub-culturing the monoclonal antibody on gelatin; (5) obtaining the liver progenitor cells. The method is simple and easy to operate, is not limited by instruments, and has large yield and high purity. The obtained liver progenitor cells do not have tumorigenicity and have a potency of two-way differentiation to parenchymal liver cells and biliary epithelial cells.

Description

technical field [0001] The invention relates to the field of cell biology, in particular to a method for producing mouse liver precursor cells. Background technique [0002] Liver precursor cells play a very important role in liver regeneration and development. Liver precursor cells (hepatoblast) have a strong self-renewal ability, and at the same time have the ability to bidirectionally differentiate into hepatic parenchymal cells and bile duct epithelial cells. It is an ideal cell source for studying liver development and hepatocyte differentiation. [0003] This cell first appeared on the 8.5th day of mouse embryonic liver development and was induced by cardiac mesoderm. During this period, it mainly expressed alpha-fetoprotein (AFP) and albumin (ALB). On the 13.5th day of embryonic development, this bidirectionally differentiated cell begins to differentiate into hepatic parenchymal cells and bile duct epithelial cells [Jung J et al., 1999; Zhao R et al., 2005; Lemaigr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
Inventor 安威孙广永董凌月
Owner CAPITAL UNIVERSITY OF MEDICAL SCIENCES