Serum-free medium for culturing placenta mesenchymal stem cells

A serum-free culture medium and stem cell technology, applied in the field of cell culture compositions, can solve the problems of lack of culture medium, increased costs and risks, achieve high growth and proliferation rate, improve in vitro separation and preparation efficiency, and increase the number of Effect

Active Publication Date: 2014-05-21
章毅 +10
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Most of these existing technical solutions isolate stem cells from the placenta to establish a placental stem cell bank. There are still many shortcomings, mainly reflected in the purity and/or the number of cells obtained, so that it is still difficult to meet people's expectations
[0009] In a

Method used

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  • Serum-free medium for culturing placenta mesenchymal stem cells
  • Serum-free medium for culturing placenta mesenchymal stem cells
  • Serum-free medium for culturing placenta mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Combination experiment of cytokines in the serum-free medium of embodiment 1

[0031]The selected growth factors: recombinant human fibroblast growth factor 2 (hFGF), human recombinant insulin (I), human recombinant growth hormone (hGH), human recombinant transferrin (TF) and BMP-4 concentrates were prepared to concentrate Liquid, sterilized by filtration through a sterile 0.22 μm aqueous phase membrane or organic phase membrane (this step is not necessary if you purchase a sterile concentrated solution), refrigerated or frozen for later use.

[0032] For different growth factors, design different concentrations (see Table 1) to add to the culture medium and find the optimal effect concentration of the growth factor according to the growth curve. After concentration screening experiments, different growth factors with optimal concentrations were combined (see Table 2), and their effects on the growth of stem cells were investigated (see Table 3), and the optimal growth ...

Embodiment 2

[0042] Take the chorion from the fetal side of the placenta, cut it into pieces, collect the cells after digestion with 0.13v / v% collagenase type II, and use cell culture medium (containing DMEM, 10v / v% fetal bovine serum, 2mML-glutamine) to adhere to the wall to cultivate.

[0043] 1. Cells were adhered to the culture dish, and the medium and suspended cells were discarded after 48 hours. The medium was changed every 2-3 days. When the cell fusion rate reached 90%, the cells were digested with Tryspin-EDTA and the cells were harvested. After the harvested cells were washed once with PBS, they were re-seeded, and fresh medium was added for subculture.

[0044] 2. Subculture the harvested cells, the medium used is DMEM as the base solution, and also contains 2mML-glutamine, 55uMb-mercaptoethanol, 1mM sodium pyruvate, 1v / v% non-essential amino acids (Gibco company), 10 μg / ml insulin, 0.1 μg / ml transferrin, 200 μg / ml glutathione, 0.1 ng / ml BMP-4, 10 ng / ml FGF-2 and 3 ng / ml hGH) ...

Embodiment 3

[0046] For the determination of the surface markers of cells cultured in serum-containing and serum-free media, flow cytometry was used to determine the ratio of different marker cell surface antigens in mesenchymal stem cells cultured and harvested in different culture systems (see Table 3).

[0047] table 3

[0048]

[0049] CD90 in primary placental fetal side chorionic cells + , CD73 + and CD105 + Cells averaged 99.06%, 99.98%, and 99.10%, respectively, indicating that primary adherent spindle cells have prominent mesenchymal properties. After 2 passages of adherent culture, in serum medium, CD90 + and CD105 + cells had a significant reduction, while CD90 in serum-free medium + and CD105 + Cells can still exist at a relatively high ratio, maintaining the characteristics of mesenchymal stem cells. According to the above comparison, it can be concluded that the cells isolated from the fetal side chorion of the placenta have the characteristics of surface antigens o...

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Abstract

The invention discloses a serum-free medium for culturing placenta mesenchymal stem cells. The serum-free medium takes a DMEM (Dulbecco Modified Eagle Medium) culture solution as a basis and also contains a fibroblast growth factor receptor 2, growth hormone, insulin, transferrin, glutathione, BMP-4, L-glutamine, sodium pyruvate, non-essential amino acids and beta-mercaptoethanol. According to various serum-free media provided by the invention, growth and proliferation of the placenta mesenchymal stem cells in a serum-free medium system can be effectively promoted, the placenta mesenchymal stem cells have higher growth and proliferation rate in the serum-free medium system compared with a serum cell culture medium, the characteristics of the stem cells are preserved, the serum-free medium has multiple differential potentials, and the stem cells can be directionally induced into fat cells and osteoblasts.

Description

technical field [0001] The invention relates to a composition for cell culture, in particular to a composition for culturing stem cells, which is used for culturing placental mesenchymal stem cells in a serum-free manner. Background technique [0002] Mesenchymal Stem Cells (MSCs) are a type of stem cells with two important characteristics of stem cells: strong self-proliferation ability and multi-differentiation potential. MSCs originate from mesoderm, and theoretically it can differentiate into mesoderm tissue. Human mesenchymal stem cells were first isolated from bone marrow. Under specific conditions in vivo and in vitro, they have the ability to develop into various adult cells such as osteoblasts, chondrocytes, adipocytes, endothelial cells, nerve cells, muscle cells, and liver cells. Ability to differentiate (J. Orthop. Res., 1991, 641-650; Science, 1999, 28, 143-147). [0003] The latest research shows that mesenchymal stem cells have immune regulation and hematopo...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
Inventor 章毅陆瑶洪艳霍思维
Owner 章毅
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