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Sorting method of hepatocytes of which embryonic hepatic cells are differentiated

An embryonic stem cell and cell differentiation technology, applied in the field of liver cell sorting, can solve the problems of affecting cell viability, cell growth state deterioration, and difficulty in completing cell transplantation experiments, so as to improve the differentiation rate of stem cells, reduce cell damage, The effect of promoting liver cell differentiation

Inactive Publication Date: 2016-03-30
THE FIRST AFFILIATED HOSPITAL OF SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This kind of sorting method undergoes digestion by trypsin and machine sorting operations such as FCM, which seriously affects the cell viability, and the cell growth state is obviously deteriorated, making it difficult to complete the subsequent cell transplantation experiment.

Method used

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  • Sorting method of hepatocytes of which embryonic hepatic cells are differentiated
  • Sorting method of hepatocytes of which embryonic hepatic cells are differentiated
  • Sorting method of hepatocytes of which embryonic hepatic cells are differentiated

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] (1) BALB / c mouse embryonic stem cells (purchased from the Experimental Animal Center of Sun Yat-Sen University) in 5% CO 2 Under the condition of 37℃ in the incubator, the shaking culture or suspension culture in the embryoid body liquid medium starts to differentiate. After about 10 hours, the embryonic stem cells can be seen to form a spherical embryoid body and suspend in the culture medium. The embryoid body can be cultured for 6 days. Increased continuously over time; after 6 days of culture, embryoid bodies were obtained;

[0043] (2) On the 7th day, move the embryoid body obtained in step (1) to a 24-well plate for adherent culture, and each embryoid body radiates and grows around the attachment point to form a cell differentiation community;

[0044] The medium for adherent culture consists of basal medium and supplements; the basal medium is DMEM medium; the supplements include the following components: a volume fraction of 20% fetal bovine serum, a final conce...

Embodiment 2

[0053] (1) BALB / c mouse embryonic stem cells (purchased from the Experimental Animal Center of Sun Yat-Sen University) in 5% CO 2 Under the condition of 37℃ in the incubator, the shaking culture or suspension culture in the embryoid body liquid medium starts to differentiate. After about 10 hours, the embryonic stem cells can be seen to form a spherical embryoid body and suspend in the culture medium. The embryoid body can be cultured for 6 days. Increased continuously over time; after 6 days of culture, embryoid bodies were obtained;

[0054] (2) On the 7th day, move the embryoid body obtained in step (1) to a 24-well plate for adherent culture, and each embryoid body radiates and grows around the attachment point to form a cell differentiation community; When the stem cells are differentiated to the 15th day, use a cell shovel to scrape the cells located in the cell differentiation community area 1 and / or the cell differentiation community area 2 (area 1 is located in the ce...

Embodiment 3

[0067] (1) with embodiment 2;

[0068] (2) When the embryonic stem cells are differentiated to the 15th day, use a cell shovel to scrape off the area located in the cell differentiation community area 1 and / or the cell differentiation community area 2 (area 1 is located in the center area of ​​the cell differentiation community and its edge to the center of the cell differentiation community) The distance does not exceed 1 / 4 of the radius of the cell differentiation community (the distance from the edge of the cell differentiation community to the center); area 2 is the area outside the area 3 of the cell differentiation community; area 3 is the area located in the center of the cell differentiation community and its edge to the cell differentiation community The distance from the center is 3 / 4 of the radius of the cell differentiation community (the distance from the edge of the cell differentiation community to the center) of the normal growth state cells, which are cultured ...

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Abstract

The invention belongs to the technical field of cell culture, and in particular relates to a sorting method of hepatocytes of which embryonic hepatic cells are differentiated. According to the sorting method, the embryonic hepatic cells are cultured into embryoid bodies in a shaking or suspension culture mode, then the embryoid bodies are cultured in an adherent culture mode, so as to gradually grow and differentiate into round differentiated cell communities, and cells in a specific area are selected to be sorted in a specific period and are cultured again in the adherent culture mode according to spatial distribution regularities of the differentiated hepatic cells in the round cell communities. In the process of adherent culture, according to a cell differentiation situation, EGFs (Epidermal Growth Factor), TGFs (Transforming Growth Factor), HGFs (Hepatocyte Growth Factor), OSMs and the like are selected as hepatic growth factors in the process that the embryoid bodies are differentiated into the hepatocytes, so as to promote the hepatocytes to differentiate, the whole sorting process has the advantages of simplicity in operation and low cost, cellular damage can be obviously reduced, compared with the prior art, the hepatic cell differentiation rate is greatly increased, the sorted cells grow with good conditions, and the cell purity and the cell functional requirements of hepatocyte transplantation can be satisfied.

Description

technical field [0001] The invention belongs to the technical field of cell culture, and in particular relates to a method for sorting liver cells differentiated from embryonic stem cells. Background technique [0002] In the past, the cell sorting in the differentiation of embryonic stem cells required the use of trypsin to digest the cells and centrifugal pipetting, etc., which caused serious damage to the cells, which was not conducive to the application of cell transplantation. Because when embryonic stem cells differentiate into hepatocytes, embryonic stem cells form embryonic bodies (embryonic bodies, EBs) after suspension culture, which is equivalent to blastocysts of embryos in vivo, and then the embryonic bodies grow adherently and differentiate toward hepatocytes. Each embryoid body contains a large number of cells, and the cells are closely connected with each other and with the bottom of the culture flask, and it is even difficult to distinguish the cell boundari...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/067C12N2500/25C12N2500/44C12N2500/84C12N2501/11C12N2501/12C12N2501/148C12N2501/15C12N2501/237C12N2506/02
Inventor 胡安斌
Owner THE FIRST AFFILIATED HOSPITAL OF SUN YAT SEN UNIV
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