Primary culture and serum-free multidimensional induced differentiation method of adipose-derived stem cells

Abstract

The invention belongs to the technical field of biology tissue engineering, and discloses a primary culture and serum-free multidimensional induced differentiation method of adipose-derived stem cells. A collagenase dissociation method is adopted for obtaining the adipose-derived stem cells from subcutaneous and omentum tissue, primary culture is performed until 80% is merged, subculturing is performed, and identification of the fat mesenchyme stem cells is performed. Third generation cells in favorable growth state are selected, a serum-free induced culture medium is added for forming fat andosteogenesis induced differentiation, and finally, oil red O dying and alizarin red dying are performed for identifying the differentiation effect and Real-time RCR is used for detecting the situation of fat formation and osteogenesis gene expression. The adipose-derived cells are easy to obtain, and through primary culture, the fat mesenchyme stem cells are adequate in survival volume and survival period, and ripe lipoblast and osteoblast can be formed through induced differentiation. The primary culture and serum-free multidimensional induced differentiation method provides ideal seed cellsfor cell treatment of metabolic diseases of diabetes, obesity, osteoporosis diseases and the like. A new way is provided for organizational project biolization bones and biolization fat, and the method has wide clinical application prospects.

Description

technical field [0001] The invention belongs to the technical field of biological tissue engineering, in particular to a method for primary culture of human adipose stem cells and serum-free multi-directional induction differentiation. Background technique [0002] At present, the existing technologies commonly used in the industry are as follows: [0003] The induction methods of human adipose-derived stem cells (ASCs) mainly include in vitro induction, gene modification, protein transduction and tissue microenvironment induction, in which in vitro induction uses a combination of different stimulating factors to induce differentiation of stem cells cell. There are a lot of research reports on the use of mesenchymal stem cells to induce differentiation into adipogenic and osteogenic cells in vitro, mainly including: 1) adipogenic and osteogenic differentiation induction cells prepared with 10% FBS DMEM as the basal medium. liquid. 2) The differentiation time is relativel...

AI Technical Summary

Problems solved by technology

2) The differentiation time is longer, mostly around 28 days

Its main disadvantages are: 1) Stem cells differentiate into adipocytes in serum-containing medium, which is subject to many interference factors and a long differentiation cycle; 2) Most of the traditional chemical reagents are toxic, which is prohibitive and affects subsequent clinical applications; 3) The adipogenic differentiation cells after induction are low and in poor condition; 4) The induction differentiation conditions are different in each laboratory, the induction differentiation mechanism is not clear, and the induction differentiation efficiency is low; 5) Serum is prone to contamination of cultured primary cells Mycoplasma pollution, etc., and will affect the differentiation process of stem cells

Chinese patent application 201210197360.6 discloses a serum-free culture medium for adipose-derived mesenchymal stem cells. The modified medium uses high-glucose DMEM as the base medium and adds taurine, reduced glutathione, and ceruloplasmin. However, reduced glutathione can protect osteoblasts in high glucose environment and promote osteogenic differentiation of adipose-derived mesenchymal stem cells, but has little effect on adipogenic differentiation. Poor cell adhesion, slow proliferation, and complex components

Chinese patent application 201310134502.9 discloses a serum-free culture medium for adipose-derived mesenchymal stem cells, which uses low-sugar DMEM as the base medium, although components such as basic fibroblast growth factor, heparin and glutamine are added, However, the effect of growth and proliferation of adipose-derived mesenchymal cells is not ideal due to the influence of materials and other aspects.

Chinese patent application CN104762260A discloses a preparation method and application of adipose-derived mesenchymal stem cells and their preparations. The purity of adipose-derived mesenchymal stem cells obtained by this method is as high as 90%. To achieve the purpose of anti-aging, the effect of inducing differentiation has not been studied

[0006] (1) The cell proliferation rate is not ideal, and the cell components are complex. The primary cells are derived from adipose tissue. In addition to the mesenchymal stem cells we need, they also contain fibroblasts, preadipocytes and mature adipocytes. Complex, and slow proliferation, less than the amount of cells required for the experiment

[0007] (2) The time of induction and differentiation is long, the aging state of cells is not good, and the differentiation fluid is toxic to cells, etc.

Traditional differentiation takes 28 days. With the aging of cells and the toxicity of differentiation fluid to cells, the state of cells is not good, which will affect the experimental results.

[0008] (3) The traditional differentiation solution contains serum, and the growth factors and cytokines in the serum affect the detection of cytokines in metabolic diseases, affect the differentiation process of stem cells, and lead to large research errors

Images