Method for culturing liver stem cells

A technology for liver stem cells and cells, applied in the field of extraction and expansion of liver stem cells, can solve the problems of long time, many processing steps, and many red blood cells, and achieve the effect of shortening processing time and steps, reducing pollution opportunities, and improving cell viability.

Inactive Publication Date: 2012-04-04
JILIN TUO HUA BIOTECH
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  • Abstract
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  • Claims
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AI Technical Summary

Problems solved by technology

This method takes a long time to process liver tissue and has many processing steps, which is easy to cause pollution and damage to cells
Differential centrifugation does not use digestive enzymes but cuts the tissue and removes the tissue fragments at different speeds to leave cells behind. However, this method contains more red blood cells, which will affect the attachment and growth of liver stem cells.

Method used

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  • Method for culturing liver stem cells
  • Method for culturing liver stem cells
  • Method for culturing liver stem cells

Examples

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Embodiment Construction

[0029] Below in conjunction with accompanying drawing, further illustrate the present invention through the embodiment, but not as limitation to the present invention.

[0030] Main instruments and reagents:

[0031] Instrument: 371 type high temperature sterilization CO 2 The incubator was purchased from Thermo Company of the United States, the programmed cooling box was purchased from NALGENE Company of the United States, the large-capacity low-speed centrifuge was purchased from Thermo Company of the United States, and the flow cytometer was purchased from BD Company of the United States. The CKX41 inverted phase contrast microscope is a product of Olympus, Japan. The biological safety cabinet is a product of Beijing Hadonglian Company.

[0032] Reagents: α-MEM basal medium and superior fetal bovine serum (FBS) were purchased from Hyclone Company of the United States, trypsin was purchased from Gibco Company of the United States, FITC-labeled mouse anti-human CD34, CD44, ...

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Abstract

The invention relates to a method for culturing liver stem cells. The method comprises the following steps of: a, flushing a liver tissue with PBS (Phosphate Buffered Saline) containing double antibodies for three times under the sterile condition; b, shearing the liver tissue, and digesting with a 0.25 percent g/mL trypsinase solution for 30 minutes; C, neutralizing digestive juice with a complete medium; d, beating and uniformly mixing the digested and neutralized liver tissue in a mixed liquid of the digestive juice and a neutralizing solution, centrifuging at the speed of 500 revolutions/minute for five minutes, extracting a supernatant liquid, and centrifuging the supernatant liquid at the speed of 1,500 revolutions/minute for five minutes; e, discarding the supernatant liquid, suspending precipitates with the complete medium, inoculating into a culture bottle, and culturing at the temperature of 37 DEG C in the environment of 5 percent CO2; f, culturing for four days, washing non-adherent cells, and changing the liquid with the complete medium; and g, changing the liquid with the complete medium once every four days, and performing passage in the ratio of 1:2 when the cells grow and expand to over 80 percent of the bottom area of the culture bottle.

Description

technical field [0001] The invention belongs to the technical field of cell culture, and specifically relates to a method for extracting and expanding liver stem cells. Background technique [0002] In the past, the terminal treatment of liver cancer and advanced liver cirrhosis was liver transplantation, but the source of donors is scarce and there are many adverse reactions after transplantation. After the stem cell transplantation technology is applied in the clinic, it brings the hope of survival to the majority of patients. Initially, autologous bone marrow stem cells were used clinically, but the quality of bone marrow is affected by the patient's age, physical condition and other conditions, which often weakens the number and activity of stem cells. [0003] At present, the use of early (aborted) fetal tissue stem cells to treat related diseases has entered clinical practice. In 2003, the Ministry of Science and Technology and the Ministry of Health issued the "Guid...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0735
Inventor 毕薇薇
Owner JILIN TUO HUA BIOTECH
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