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A culture medium and culture method of liver stem cells

A technology of hepatic stem cells and culture methods, applied in non-embryonic pluripotent stem cells, animal cells, vertebrate cells, etc., can solve the problems of long time of liver tissue, accompanied by spontaneous differentiation, stem cell pollution, etc., to accelerate cell proliferation and avoid culture. The effect of prolonging the cycle and reducing cell damage

Active Publication Date: 2020-02-04
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the existing methods for culturing liver stem cells take a long time to process liver tissue, and many processing steps are likely to cause pollution and damage to stem cells
Moreover, due to the inappropriate selection of culture medium or culture conditions in the prior art, when hepatic stem cells are cultured in vitro, the proliferation rate is slow and will be accompanied by spontaneous differentiation; when cultured in vitro for a period of time, the cells will all differentiate to form terminally differentiated cells

Method used

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  • A culture medium and culture method of liver stem cells
  • A culture medium and culture method of liver stem cells
  • A culture medium and culture method of liver stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] 1) Obtain liver tissue:

[0041] Pregnant rats were sacrificed by dislocation, sterilized by 75% ethanol and transferred to an ultra-clean workbench, and the fetal liver was aseptically removed.

[0042] 2) Separation and extraction of liver stem cells

[0043] Fetal liver was rinsed several times with PBS, shredded, digested with 0.02% type I collagenase and 0.02% type II collagenase at 4°C overnight, and the digested tissue was filtered with a 200-mesh sieve, and the obtained cell suspension was centrifuged at 200g After 5 min, the cell pellet was washed twice with PBS. The cells were separated by immunomagnetic beads, and the specific steps were as follows:

[0044] 1. Prepare a single cell suspension (filter with a 30um nylon mesh to avoid clogging the column) (wet the filter with buffer before filtering)

[0045] 2. Add buffer to wash the cells, and centrifuge (300g, 10min) (4-8°C) 3: Add buffer to reselect the cell single-cell suspension,

[0046] 3. Filter ag...

Embodiment 2

[0060] In the cell precipitation medium (the medium formula is shown in Table 1), the final concentration was prepared to be 1 × 10 5 ml of cell suspension. Add the cell suspension to the 25cm 2 culture flask at 37°C, 5% CO 2 cultured in an incubator. After 48 h, the cell growth reached the logarithmic phase, and the cell growth was observed under an inverted phase-contrast microscope and photographed ( Figure 1-a ). After the cells grew to more than 80% confluence, the microscopic observation was carried out again ( Figure 1-b ), and then added 0.25% trypsin and 0.02% EDTA to digest and carry out repeated subculture for 3 times to obtain hepatic stem cells. During the culture process, the number of cells was recorded every day, and the growth curves were drawn for the stem cells cultured in different media. The results are shown in figure 2 .

[0061] Table 1 medium formula

[0062]

[0063] Microscopic observation results showed that hepatic stem cells grew adhe...

Embodiment 3

[0066] The hepatic stem cells cultured in different media in Example 2 were tested by flow cytometry, specifically:

[0067] Take hepatic stem cells in the logarithmic growth phase and adjust the cell density to 1×10 6 Take 2.5 μL of monoclonal antibodies against human CD34, CD44, CD105, and HLA-ABC respectively, add 500 μL of cell suspension, incubate at room temperature in the dark for 20 minutes, set up a blank isotype control at the same time, and centrifuge at 1500 r / min for 5 minutes , discard the supernatant, wash 2 times with PBS containing 10% FBS, resuspend with 500 μL 1640 and test on the machine. The statistical results of the data are shown in Table 2:

[0068] Table 2: Flow Cytometry Results

[0069]

[0070] Among them, the detection results of the hepatic stem cells cultured in medium 3 are as follows: Figure 3-a~Figure 3-b As shown, liver stem cells are a kind of mesenchymal stem cells, and mesenchymal stem cells have low expression of HLA-ABC and CD34 ...

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Abstract

The invention relates to the field of stem cell culture, particularly a liver stem cell culture medium and culture method. The liver stem cell culture medium provided by the invention comprises a basal culture medium, HGF, SCF and LIF. The liver stem cell culture method provided by the invention comprises the following steps: separating the primary liver stem cells by immune magnetic beads, and culturing in the culture medium. The experiment indicates that the cell trauma is small in the primary cell obtaining process. The detection indicates that the activity is kept at 85% or so. The liver stem cells cultured by the method can enhance the propagation speed by 1.5-2.8 times. The flow cytometry detection indicates that the liver stem cells can keep favorable dryness.

Description

technical field [0001] The invention relates to the field of stem cell culture, in particular to a culture medium and a culture method of liver stem cells. Background technique [0002] The liver is a vital organ of the human body and performs many important functions such as: glucose homeostasis, detoxification of heterosexual biomass or synthesis of macromolecules. my country is a "big hepatitis country" with a high incidence of various types of hepatitis. Various viral hepatitis and related liver diseases seriously endanger the health of our people. Impaired liver function can have significant health consequences, with acute or chronic liver disease being the ninth or fifth leading cause of death worldwide. So far, the only cure for advanced liver disease is liver transplantation. Liver cell transplantation is a new method. In the prior art, there has been a precedent of using the liver tissue of aborted fetus to cultivate liver stem cells for the treatment of liver cir...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/074
Inventor 葛啸虎陈海佳王一飞卢瑞珊王小燕李平
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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