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Serum-free medium for culturing placental 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 increased cost and risk, lack of culture medium, etc., and achieves improved in vitro separation and preparation efficiency, high growth and proliferation rate, and increased number of Effect

Active Publication Date: 2015-08-05
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 addition, after the mesenchymal stem cells are extracted and obtained from the tissue, the culture of the mesenchymal cells still needs to be cultured with the addition of fetal bovine serum, and there is no suitable medium for industrial application. Added costs and risks, need to be further explored

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0031] 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 concentrated solution The liquid is sterilized by filtration through a sterile 0.22μm water phase membrane or organic phase membrane (this step does not need to be carried out if a sterile concentrate is purchased), and refrigerated or frozen for later use.

[0032] Design different concentrations for different growth factors (see Table 1) and add them 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 (see Table 3) were investigated to obtain the optimal growth factor combinati...

Embodiment 2

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

[0043] 1. Cells are cultured adherently in a petri dish, and the culture medium and suspended cells are discarded after 48 hours. Change the medium every 2-3 days. When the cell fusion rate reaches 90%, tryspin-EDTA digests and harvests the cells. After the harvested cells are washed once with PBS, they are re-inoculated and added with fresh medium for subculture.

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

Embodiment 3

[0046] For the determination of the surface markers of cells cultured in serum and serum-free media, flow cytometry was used to determine the ratio of different labeled 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 chorionic cells + , CD73 + And CD105 + The average cell numbers were 99.06%, 99.98%, and 99.10%, respectively, indicating that the primary adherent spindle cells have significant mesenchymal cell characteristics. After 2 passages, adherent culture, in a serum medium, CD90 + And CD105 + The cells are significantly reduced, and CD90 in serum-free medium + And CD105 + Cells can still exist in 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 chorion on the fetal side of the placenta have been passaged in serum-free mediu...

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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 culture placental mesenchymal stem cells in a serum-free manner. Background technique [0002] Mesenchymal Stem Cells (MSCs) are a type of stem cells that have two important characteristics of stem cells: strong self-proliferation ability and multi-differentiation potential. MSCs originated from the mesoderm, and theoretically it can differentiate into mesoderm tissue. Human mesenchymal stem cells were first isolated from bone marrow, and under specific conditions in vivo and in vitro, they have a variety of adult cells such as osteoblasts, chondrocytes, adipocytes, endothelial cells, nerve cells, muscle cells, and liver cells. The 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 hematopoietic support, and are eas...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0775
Inventor 章毅陆瑶洪艳霍思维
Owner 章毅
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