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Method for separating and culturing macaque adult hepatic precursor cells

A technology of liver precursor cells and culture method, which is applied in the field of separation and culture medium of adult liver precursor cells in rhesus monkeys, can solve the problems of not fully confirming the characteristics of liver precursor cells, and achieve the effect of easy promotion and simple method

Inactive Publication Date: 2010-10-20
昆明亚灵生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current reports have not fully confirmed the characteristics of liver precursor cells, such as the bidirectional (hepatic and biliary) differentiation ability of cells

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Take 5 grams of rhesus monkey adult liver tissue, soak and wash with PBS for 3 times, cut into small pieces, digest the tissue piece with DMEM containing 5 mg / ml type IV collagenase for 30 minutes, collect the cells in the supernatant, and wash with DMEM Three times and centrifuge at 1200 rpm for 5 min. The collected cells were suspended in DMEM with 10% fetal bovine serum, and placed in a sealed centrifuge tube for suspension culture to form cell clusters. Individual cell clumps were picked out under a microscope and seeded on culture plates plated with rat tail collagen. After the cells were overgrown on the culture plate, they were digested with trypsin-EDTA at 37°C for 10-30 minutes, and the digested cells were sorted by flow cytometry to E-cad+ cells to obtain adult liver precursor cells of rhesus monkeys.

[0024] Among the above methods, see (Malhi, 2002) for the preparation method of rat tail collagen.

[0025] Cell culture medium contains 10mmol / L nicotinamid...

Embodiment 2

[0027] Take about 8 grams of rhesus monkey adult liver tissue, soak and wash 3 times with PBS, cut into small pieces, digest the tissue piece with DMEM containing 5mg / ml type IV collagenase in a shaker at 37°C for 50 minutes, and collect Cells in supernatant were washed three times with DMEM and centrifuged at 1200 rpm for 5 min. The collected cells were suspended in DMEM with 10% fetal bovine serum, placed in a sealed centrifuge tube and cultured in a shaker at 37° C. to form a cell mass. Individual cell clumps were picked out under a microscope and seeded on culture plates plated with rat tail collagen. Cell culture conditions are 37°C, 5% CO 2 , the culture medium was changed every two days. After the cells were overgrown on the culture plate, they were digested with trypsin-EDTA at 37°C for 10-30 minutes, and the digested cells were sorted by flow cytometry to E-cad+ cells to obtain adult liver precursor cells of rhesus monkeys.

[0028] The preparation method of rat ta...

Embodiment 3

[0030] The isolated rhesus monkey adult liver precursor cells were cultured with the cell culture solution of Example 1 on the culture plate plated with rat tail collagen, and could be passaged multiple times at a ratio of 1:2 or 1:3 to grow and expand.

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PUM

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Abstract

The invention relates to a method for separating and culturing macaque adult hepatic precursor cells, in particular to a separation and culture solution of the macaque adult hepatic precursor cells, belonging to a separation and culture solution of animal precursor cells. The method comprises the following steps: digesting a small quantity of hepatic tissues obtained by a macaque liver removal surgery or other ways by utilizing IV-type collagenase; collecting and suspending the cells in 10% of a fetal calf serum DMEM for culture; selecting and then inoculating a single cell block in the culture plate of a rat tail collagen bed board, wherein the culture solution is the special one; and finally sorting E-cad+cells from the cultured cells by the flow cytometry to obtain the target cells. The cells separated by the method of the invention can be applied to cell differentiation mechanism study and can become ideal master cells for treating late-period hepatic diseases.

Description

technical field [0001] The invention belongs to the separation and culture solution of animal precursor cells, in particular to the separation and culture solution of rhesus monkey adult liver precursor cells. Background technique [0002] There are currently 30 million chronic hepatitis patients in my country, some of whom gradually develop into liver fibrosis. If the liver fibrosis worsens further, it will lead to liver cirrhosis, liver failure, and portal hypertension. Generally speaking, early liver fibrosis is reversible, while advanced liver fibrosis (cirrhosis) is irreversible. Organ transplantation is currently the only feasible solution for the treatment of advanced liver diseases. However, the severe shortage of donor organs limits its application. [0003] More and more studies have shown that cell replacement therapy based on liver stem (precursor) cells has become a new hope for the treatment of liver diseases. Adult liver precursor cells (Hepatic Progenitor ce...

Claims

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Application Information

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IPC IPC(8): C12N5/071
Inventor 纪少珲金立方季维智
Owner 昆明亚灵生物科技有限公司
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