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Method for carrying out isolated culture on animal embryo liver stem cells

A liver stem cell, separation and culture technology, applied in the field of cell separation, can solve the problems of complicated operation and high cost of separation, purification and culture of embryonic liver stem cells, and achieve the effects of ensuring digestion efficiency, controlling digestion speed and good activity.

Inactive Publication Date: 2016-09-07
吉林省中科生物工程股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the method of the present invention, the liver stem cells with high purity can be obtained simply and conveniently by taking out the liver of the animal embryo, then cultivating and centrifuging and purifying, further adding enzyme

Method used

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  • Method for carrying out isolated culture on animal embryo liver stem cells
  • Method for carrying out isolated culture on animal embryo liver stem cells
  • Method for carrying out isolated culture on animal embryo liver stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] 1) Take out the liver of the human embryo under aseptic conditions, place the embryonic liver tissue in a petri dish and wash it thoroughly with cold PBS at 4°C, then use ophthalmic scissors to cut the liver into 1mm×1mm×1mm tissue pieces, and again with Wash with cold PBS at 4°C to remove blood cells, and obtain liver tissue pieces after washing;

[0062] 2) Add 0.05% type IV collagenase solution dropwise to the cleaned liver tissue, and digest it in a 37°C water bath for 15 minutes; then, add an equal amount of DMEM / F12 complete culture solution to stop the digestion, and blow the mixture repeatedly with a pipette , and filtered with a 200-mesh sieve to obtain liver stem single cell suspension;

[0063] 3) Centrifuge the hepatic stem single-cell solution obtained in step 2) for 5 min under a centrifugal force of 55×g, and transfer the supernatant obtained after centrifugation to another container, and place it on ice.

[0064] 4) Add PBS solution again to the precipi...

experiment example 1

[0071] 1) The purified hepatic stem cells of Example 1 were inoculated on coverslips pre-treated with 0.2% gelatin, then placed in 24-well culture plates, and incubated at 37° C., 5% CO 2 Cultivate in an incubator under conditions for 24 hours, until the hepatic stem cells grow into a single layer; then, discard the culture medium, wash the cultured cells with PBS solution 3 times (5min / time), and then add 4% paraformaldehyde to fix for 25min;

[0072] 2) Wash the fixed cells with PBS, and then permeabilize the cells with 0.3% TritonX-100 for 10 min;

[0073] 3) Wash the permeabilized cells with PBS, and block with goat serum for 50 min;

[0074] 4) Add anti-AFP, CK18, AAT, CK19 antibodies (1:100) to the blocked cells respectively, place them in a wet box, and culture overnight at 4°C;

[0075] 5) Immunofluorescence cytochemical detection: wash part of the overnight cultured cells in step 4) with PBS solution, then add Alexa Fluor 488 Conjugate secondary antibody (1:200), and...

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Abstract

The invention provides a method for carrying out isolated culture on animal embryo liver stem cells. The method comprises the following steps of: taking out animal embryo liver, carrying out culture and centrifugal purification, then adding enzyme for removing fibroblasts, and carrying out purification and culture again, so that liver stem cells with high purity can be simply and conveniently obtained, and further the technical problems that operations in separation, purification and culture of the embryo liver stem cells are complex and cost is high in the prior art are solved. The method provided by the invention has the advantages of simple operation, high efficiency, high purity of the liver stem cells obtained through culture, good activity, high multiplication rate and stable characters; meanwhile, massive cells can be provided for fundamental research and clinical application on liver diseases.

Description

technical field [0001] The invention relates to the technical field of cell separation, in particular to a method for isolating animal embryonic liver stem cells. Background technique [0002] The liver is an important organ to maintain the normal metabolism of the human body. In my country, liver disease is a common disease that endangers people's health. At present, for acute and chronic liver failure, advanced liver cirrhosis and liver cancer caused by various factors, the therapeutic effect of existing drugs is not obvious. Liver transplantation is currently the most common and effective method, but due to the shortage of transplanted donor livers, immune rejection after transplantation, and high costs during the transplantation and anti-rejection process, liver transplantation is largely limited. The widespread development and application of transplantation in clinic makes it difficult for liver transplantation to become a routine treatment. [0003] In recent years,...

Claims

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Application Information

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IPC IPC(8): C12N5/0735
CPCC12N5/0606C12N2509/00
Inventor 石毅董欣熊丽东
Owner 吉林省中科生物工程股份有限公司
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