Method for culturing mesenchymal stem cells without serum

Abstract

The invention relates to a method for culturing mesenchymal stem cells without serum, and belongs to the field of stem cells. The method includes the steps that mononuclear cells are separated from marrows, and the mesenchymal stem cells with positive cell phenotypes CD73, CD90 and CD105 are obtained through multiplication culture. A serum-free medium is used during culturing, by optimizing components of the medium and the proportion of all the components, pollution to foreign proteins and exogenous microorganisms is avoided effectively, a stem cell form and stem cell features obtained throughculturing are good, and the proliferation rate of cells and positive expression rate of the mesencymal stem cells are increased remarkably. Through the method, new purposes are provided for large-scale preparation of the mesenchymal stem cells and cell transplantation treatment.

Description

technical field [0001] The invention belongs to the field of stem cells, and in particular relates to a serum-free method for culturing bone marrow mesenchymal stem cells. Background technique [0002] Bone marrow mesenchymal stem cells (BMSCs) are derived from undifferentiated mesenchymal cells of the mesoderm, and have the ability to differentiate into a variety of mesoderm tissue cells, such as osteoblasts, chondrocytes, and adipocytes Wait. In recent years, it has been found that BMSCs have the potential to differentiate across germ layers, such as cardiomyocytes derived from endoderm and nerve cells derived from ectoderm. The discovery of the ability to differentiate across germ layers creates broader prospects for the application of BMSCs. BMSCs can differentiate into osteoblasts, chondrocytes, adipocytes, muscle cells and even neuron-like cells under the induction of different physical and chemical environments and cytokines. [0003] In recent years, the rapid dev...

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Problems solved by technology

But it also contains some inhibitory factors or toxic substances that are not conducive to cell growth, and have potential cytotoxic effects

A large number of complex proteins in serum bring difficulties to the standardization of cell culture, and also bring great difficulties to the separation and purification of cell culture expression products

For example, Chinese patent CN105238749B discloses a method for resuscitating bone marrow mesenchymal stem cells, which provides DMEM medium containing 10% fetal bovine serum to expand and cultivate bone marrow mesenchymal stem cells, but animal serum such as fetal bovine serum may exist Known or unknown pathogens carried by animals pose a threat to possible clinical applications in the future and increase the difficulty of large-scale cultivation

[0004] For another example, Chinese patent CN101412985B discloses a serum-free medium for in vitro culture and expansion of bone marrow mesenchymal stem cells, by adding insulin, transferrin, ethanolamine, sodium selenite, growth factors, paste Wall factors, hormones, putrescine, inorganic salts, vitamins, albumin and antioxidants can enable bone marrow mesenchymal stem cells to attach to the culture medium under serum-free conditions, realize in vitro culture and expansion, and maintain multi-directional differentiation Potential, but at 0.5*10 4 cells / mL was used as the initial cell volume, and the number of cells after expansion and culture for 10 days was only 2*10 5 cells / mL, the cell proliferation ability is far from meeting the requirements of large-scale culture

[0005] It can be seen that the bone marrow mesenchymal stem cell culture method adopted in the prior art has problems such as unsatisfactory cell proliferation rate and complicated medium components.

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