Human gallbladder stem cell acquisition and long-term in-vitro culture method

An acquisition method and in vitro culture technology, which are applied in cell dissociation methods, artificial cell constructs, cell culture active agents, etc., to achieve the effect of reducing preparation costs

Pending Publication Date: 2020-12-25
上海拜羡生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some of these types of cells are obtained through genetic modification, and some have unknown risks such as immunogenicity, so clinical application of cell transplantation has not been attempted (Wang J, Sun M, Liu W, Li Y, Li M, StemCell-Base

Method used

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  • Human gallbladder stem cell acquisition and long-term in-vitro culture method
  • Human gallbladder stem cell acquisition and long-term in-vitro culture method
  • Human gallbladder stem cell acquisition and long-term in-vitro culture method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1. Rapid isolation of human gallbladder stem cells

[0052] A. Obtaining cells from the mucosal layer of the inner wall of the gallbladder

[0053] After the human gallbladder tissue is obtained (removed by hospital surgery), it is stored in liquid basal medium and transported to the laboratory; the tissue is taken out in a sterile environment, cut longitudinally along the tissue and fully expanded, and washed repeatedly with sterile PBS Tissue until the color of the solution is clear and bloodless; transfer the tissue to a 10cm culture dish containing pre-cooled basal medium, scrape off the mucosal layer cells of the inner wall of the gallbladder with a sterile disposable scalpel blade, wash the inner wall of the gallbladder with the basal medium, Gallbladder tissue was discarded.

[0054] B. Obtain single cells from the mucosal layer of the inner wall of the gallbladder

[0055] Pipette the culture medium containing the cells into a 50ml centrifuge tube, cen...

Embodiment 2

[0061] Example 2. Expansion and culture of human gallbladder stem cells

[0062] 1. Expansion of cells

[0063] When the cell clones are full (7-10 days), they can be digested and passaged. The specific steps are: (A) Take out the supernatant and discard it, add pre-cooled liquid basal medium, use a 1ml pipette gun to absorb the Matrigel containing cells, and blow repeatedly until the colloid is broken, so that the clumps are evenly distributed in the liquid . (B) Pipette the cell suspension in each three wells into a 15ml centrifuge tube, make up the liquid to 14ml, and use a 25ml pipette to mix the cell suspension evenly. Centrifuge at 400g for 5 minutes and discard the supernatant as much as possible. Add TrypLE digestion solution, place in a 37°C water bath for 10 minutes for digestion, and shake appropriately during the period. (C) After digestion, add liquid basal medium to 14ml, mix well, centrifuge at 400g for 5 minutes, discard the supernatant, repeat washing 2 ti...

Embodiment 3

[0074] Example 3. Inducing human gallbladder stem cells to differentiate into cells with mature hepatocyte function

[0075] When the cell growth density is about 50%, replace with hepatic differentiation medium. The differentiation medium includes N-acetylcysteine, R-spondin, nicotinamide, recombinant human epidermal growth factor, recombinant human hepatocyte growth factor, TGFβ inhibitor, dexamethasone and oncostatin M to induce mature hepatocytes required components.

[0076] After two weeks of culture, the differentiated cells can differentiate and have the ability to take up low-density lipoprotein, store glycogen and synthesize fat ( Figure 6 ).

[0077] The above results show that the hepatic differentiation system established in the present invention can induce the differentiation of human gallbladder stem cells into cells with the function of mature hepatocytes.

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Abstract

The invention relates to the technical field of biomedical engineering, and provides a human gallbladder stem cell acquisition and long-term in-vitro culture method. The method comprises a human gallbladder stem cell acquisition method and a human gallbladder stem cell long-term in-vitro culture method. A primary culture medium and a cell expansion culture medium with clear chemical components areprovided. By adopting the two culture media, selective amplification of the gallbladder stem cells in human gallbladder tissues is realized, the cells can be continuously cultured in vitro for more than 100 days under the condition, and a stable liver stem cell related molecular phenotype is maintained; after induced differentiation, the cells have partial liver functions, including ingestion oflow-density lipoprotein, synthesis of fat and storage of glycogen. Therefore, the gallbladder stem cells obtained by the method can be used as seed cells for cell therapy of liver failure diseases, and can be used for drug screening and preparation of tissue engineering liver and artificial liver.

Description

technical field [0001] The invention relates to the technical field of biomedical engineering, and relates to a long-term in vitro culture and expansion method of human gallbladder stem cells, including a primary culture medium with clear chemical composition, which is used for the primary in vitro culture of human gallbladder stem cells; and a Chemically defined expansion medium for long-term expansion of human gallbladder stem cells. Background technique [0002] Gallbladder stem cells refer to a group of cells with the ability to proliferate and differentiate located on the biliary system. A large number of studies have shown that gallbladder stem cells are distributed in the small bile duct (Hering's duct) in the liver and extrahepatic bile duct tissue, including gallbladder, common bile duct and hepatic duct (Cardinale V, Wang Y, Carpino G, Mendel G, Alpini G, Gaudio E , Reid LM, Alvaro D, The biliary tree—a reservoir of multipotent stem cells. Nat Rev Gastroenterol He...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/0625C12N2509/00C12N2500/32C12N2500/30C12N2501/11C12N2501/113C12N2501/115C12N2501/119C12N2501/12C12N2501/15C12N2501/727C12N2501/998C12N2501/999Y02A50/30
Inventor 王敏君胡骏凯陈费陈锐赵健金宜强
Owner 上海拜羡生物科技有限公司
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