Umbilical cord mesenchymal stem cell serum-free cultivation method and application

Abstract

The invention discloses an umbilical cord mesenchymal stem cell serum-free cultivation method, comprising the following steps: (1) preparing coating buffer for fibronectin, a recombinant human epidermal growth factor and a recombinant human fibroblast growth factor, and coating a plastic culture bottle; (2) taking human umbilical cord tissue Whartonps jelly, cutting into pieces, and adding into the culture bottle in the step (1); (3) for a product obtained in the step (2), adding into a serum-free culture medium; (4) putting into a 5-percent carbon dioxide incubator at 37 DEG C for culture, wherein mesenchymal stem cells overgrow in 10-12 days; (5) washing by a phosphate buffer solution, and performing Triple enzymic digestion; (6) adding the phosphate buffer solution, and stopping the digestion; (7) collecting mesenchymal stem cell supernate; (8) centrifuging; (9) collecting sediments to obtain the needed mesenchymal stem cells. By adopting the method, the mesenchymal stem cells can go down to the 20th generation, the characteristics of the mesenchymal stem cells are kept, diseases can be treated, and application prospect is achieved.

Description

technical field [0001] The invention relates to a culture method and application of umbilical cord tissue mesenchymal stem cells, in particular to a serum-free mesenchymal stem cell culture method and application. technical background [0002] Mesenchymal stem cells come from the mesoderm and have multidirectional differentiation. They can differentiate into bone cells, chondrocytes, fat cells, muscle cells, nerve cells, etc., and are used in the treatment of ischemic heart disease, osteoarthritis, and senile dementia. and other diseases have a good outlook. [0003] Mesenchymal stem cells usually come from bone marrow, but as the bone marrow donor ages, the proliferative ability of bone marrow-derived mesenchymal stem cells gradually decreases, and the application is limited. Therefore, it is necessary to find other sources of mesenchymal stem cells. [0004] In 2003, American scientist Kathy published in stemcells journal (stemcells 2003; 21:50-60) that mesenchymal stem c...

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Problems solved by technology

[0008] In order to overcome the shortcoming that the passage number of mesenchymal stem cells cannot exceed 8-10 passages in the serum-free medium, the purpose of the present invention is to provide a kind of fibronectin combined with recombinant human epidermal growth factor (rhEGF) and recombinant human adult Fiber growth factor (rhbFGF) is coated on plastic culture bottles, so that fibronectin and recombinant human epidermal growth factor, recombinant human fiber growth factor and plastic culture bottle materials are fully combined. Plastic culture bottles are generally made of polystyrene materials, which are harmful to proteins. With a certain adsorption force, when the umbilical cord tissue and serum-free medium are inoculated into the coated culture flask, fibronectin, recombinant human epidermal growth factor and recombinant human fiber growth factor will have a synergistic effect to jointly promote the adhesion of mesenchymal stem cells. It promotes the proliferation of mesenchymal stem cells and maintains the characteristics of mesenchymal stem cells. Its function far exceeds that of using fibronectin-coated plastic culture flasks alone. The mesenchymal stem cells prepared by this method can be passaged for 20 generations and still maintain mesenchymal stem cells stem cell characteristics

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