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Method for panning and multiplication culture of liver stem cells and application thereof

A liver stem cell and culture medium technology, applied in the field of bioengineering, can solve problems such as defects in the differentiation potential of liver stem cell populations, and achieve excellent effects and clear ingredients

Inactive Publication Date: 2020-08-07
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, some cells of the hepatic organoids obtained through this invention will express the marker genes of mature hepatic parenchymal cells, which suggests that the hepatic stem cells obtained through organoid culture may have defects in differentiation potential

Method used

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  • Method for panning and multiplication culture of liver stem cells and application thereof
  • Method for panning and multiplication culture of liver stem cells and application thereof
  • Method for panning and multiplication culture of liver stem cells and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0112] Example 1 Preparation of culture medium

[0113] The medium components include:

[0114] The liquid basic medium is RPMI1640, 1% N2, 1% B27, 1% insulin-transferrin-sodium selenite (ITS) mixture, 1% glutaminase substitute, 10mM nicotinamide, 50ng / mL epidermis Growth factors, 20ng / mL bone morphogenetic factor-4, 100ng / mL fibroblast growth factor-10, 100ng / mL RSPO1, 3μM CHIR99021, 5μM A83-01, 10μM Y-27632.

Embodiment 2

[0115] Example 2 Screening of liver stem cells

[0116] 1.1 Two-step perfusion of mouse liver

[0117] 1)Constant flow pump pretreatment: the pipes of the constant flow pump are first perfused with absolute ethanol, then cleaned with autoclaved deionized water, and finally cleaned with D-Hanks for use;

[0118] 2) Experimental mouse anatomy: After anesthetizing the mouse with 20μL of avertin per gram of body weight by intraperitoneal injection, the mouse is fixed on the dissecting board, the mouse's thoracic cavity is cut, the inferior vena cava is found, and the disposable indwelling needle Insert it and fix it with surgical sutures; 3mL of anticoagulant is injected into the liver of the mouse through the indwelling needle;

[0119] 3) Mouse liver perfusion: first adjust the speed of the constant flow pump to 5rpm / min, then start the perfusion with D-Hanks; cut off the portal vein, ligate the superior vena cava, time for 15 minutes and adjust the speed of the constant flow pump to 20...

Embodiment 3

[0140] Example 3 Establishment and expansion of liver stem cells

[0141] 1. Experimental method

[0142] 1.1 Establishment of liver stem cells

[0143] 1) Liver non-parenchymal cells are cultured in panning medium for 4-7 days;

[0144] 2) Use a self-made elbow glass needle to gently scrape the epithelioid clones with uniform cell morphology and cell number greater than 50 and transfer to a 48-well cell culture well plate coated with 1% Matrigel for 3 days;

[0145] 3) Replace the fresh panning medium containing 1x double antibody, after that, change the medium every two days;

[0146] 4) When the cells grow to 80% confluence, they are successively passaged to 24-well plates, 12-well plates and 6-well plates at a ratio of 1:2.

[0147] 1.2 Identification of the division mode of established liver stem cells (PHK26 staining)

[0148] 1) The cells are digested into single cells with Accutase (Sigma);

[0149] 2) Resuspend fine cells in appropriate medium, count on hemocytometer, take 5×10 5 c...

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Abstract

The invention provides a culture medium for elutriating and amplifying liver stem cells and the application of the culture medium. Based on the clone formation capability of stem cells, chemical smallmolecules and cytokines in an elutriation culture medium are optimally combined, flow sorting of cell surface antigen markers is skipped, and a culture medium with clear components is researched anddeveloped. The optimized culture medium can be used for directly elutriating, separating and amplifying adult liver stem cells with liver / gallbladder bidirectional differentiation potential in adult livers, and has wide application prospect and huge market value.

Description

Technical field [0001] The invention relates to the technical field of bioengineering, in particular to a culture medium for panning and amplifying liver stem cells and its application. Background technique [0002] my country is a major liver disease country, and various acute and chronic liver diseases pose a huge threat to the health of our population. Different physical, chemical and biological factors including viral infection, alcoholic and non-alcoholic overeating, poisoning, autoimmunity and cholestasis caused liver damage and repeated stimulation of inflammation, which not only lead to the death of liver parenchymal cells, but also induce Fibrosis occurs in the interstitial tissues of the liver, which then evolves into end-stage liver diseases such as cirrhosis, liver failure, and even liver cancer. [0003] At present, the only option for effective treatment of end-stage liver disease is liver organ transplantation, which is limited by the extreme shortage of donor liver...

Claims

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Application Information

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IPC IPC(8): C12N5/074
CPCC12N5/0672C12N2500/25C12N2500/32C12N2501/11C12N2501/119C12N2501/15C12N2501/155C12N2501/415C12N2501/70C12N2501/727A61K35/407
Inventor 李尹雄伍飞马陈彦谭圣林潘廷才
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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