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Efficient and reliable preparation method of hESC-MSC

A technology for culture medium and embryonic stem cells, applied in the field of biomedicine, can solve problems such as affecting differentiation efficiency and reducing cell viability, and achieve the effects of short time consumption, fast adherence, and strong proliferation and differentiation ability.

Active Publication Date: 2020-03-10
SHANXI UNIV
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AI Technical Summary

Problems solved by technology

Since hESCs are very sensitive to changes in the culture system when cultured in vitro, this scheme has a fundamental disadvantage, that is, changing all culture systems of hESCs (including coating materials and culture medium) at one time will greatly reduce the viability of the cells. thereby affecting its differentiation efficiency

Method used

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  • Efficient and reliable preparation method of hESC-MSC
  • Efficient and reliable preparation method of hESC-MSC
  • Efficient and reliable preparation method of hESC-MSC

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Embodiment Construction

[0036] 1. Cell culture

[0037] 1. Embryonic stem cell culture: hESCs cell line: RC9 (UK), cultured in a feeder-free culture system coated with vitronectin, passaged once every 4-6 days. When subcultured, digest with 0.5mM EDTA, place at room temperature for 4min, discard EDTA when the edge of the colony becomes clear, wash 2 times with PBS without Ca2+ and Mg2+, add mTeSR medium, and blow the colony into small colonies in a "Z" shape. Colonies were inoculated into vitronectin-coated culture dishes, and the medium was changed every day.

[0038] 2. Umbilical cord-derived mesenchymal stem cell (UC-MSC) culture: the umbilical cord tissue used for cell isolation was obtained from newborns delivered by caesarean section in the First Hospital of Shanxi Medical University, and was donated by the parturient mother and signed a consent form. Cut the amniotic membrane tissue at the umbilical cord-placenta junction into small pieces of about 2 mm, and digest it with collagenase type II...

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Abstract

The invention belongs to the technical field of biological medicines, and provides a preparation method of hESC-MSC. The method comprises that a supporting / coating material is changed and a culture solution system is changed. Embryonic stem cells (hESCs) start a differentiation process by a method based on mechanical partitioning and planking. The hESC gradually enters the differentiation mode, and the high survival rate is kept in the differentiation process, so that the better differentiation efficiency is achieved. Meanwhile, screening is performed by adjusting a passage mode, adopting a partitioning method and then adopting a digestion method according to the growth characteristics of hESC and MSC, so that the operation is simple and convenient, and the effect is obvious. The obtainedhESC-MSC can be used for inhibiting CD4+T-cell proliferation and promoting the increasing of CD4+Foxp3+(Treg). The hESC-MSC has an immunomodulatory capacity equivalent to or higher than that of umbilical cord MSCs and has powerful proliferative capacity and a simple, convenient and efficient differentiation effect in a short time, and derived cells have typical MSC cytological characteristics.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to an efficient and reliable hESC-MSC preparation method, in particular to a high-purity, high-potential embryonic stem cell-derived mesenchymal-like stem cell, method and application thereof. Background technique [0002] Mesenchymal stem cells (MSCs) can be isolated from adults and widely exist in various organs and tissues of adult animals. Under certain conditions, they can differentiate into various functional cells according to specific programs, such as osteoblasts, chondrocytes Cells and fat cells, so that the corresponding tissues and organs maintain a dynamic balance of growth and decline. It was first discovered in bone marrow by Friedenstein in 1968. It is a fibroblast adherent cell and has the ability to differentiate into osteoblast, chondrocyte and adipocyte both in vivo and in vitro. In 1991, Caplan named this type of cells with common characteristics...

Claims

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Application Information

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IPC IPC(8): C12N5/0775C12N5/0735C12N5/077A61K35/28A61P37/02
CPCC12N5/0662C12N5/0654C12N5/0653C12N5/0655A61K35/28A61P37/02C12N2506/02C12N2533/52C12N2533/54C12N2533/80
Inventor 叶进培
Owner SHANXI UNIV
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