Isolation and identification of exosomes from human adipose-derived mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.1715

Abstract

Abstract:

BACKGROUND: Adipose-derived mesenchymal stem cells as one of the important seed cells can be extracted in large quantities by simple methods. However, clinical application of the cells is limited by rigorous storage conditions and inconvenient transportation. Exosomes can be secreted by adipose-derived mesenchymal stem cells, whose structure is stable and difficult to decompose, providing new possibilities for clinical application of adipose-derived mesenchymal stem cells.
OBJECTIVE: To isolate and identify human adipose-derived mesenchymal stem cells and induce multidirectional differentiation of the cells as well as to isolate and identify the exosomes from adipose-derived mesenchymal stem cells.
METHODS: Adipose-derived mesenchymal stem cells were isolated from superficial abdominal subcutaneous adipose tissue of normal adult women after liposuction. The cells were purified by cell adherence method. The cells were digested and passaged by trypsin. The third generation cells were identified by flow cytometry, followed by adipocyte induction and identification as well as osteoblast induction and identification. Adipose-derived mesenchymal stem cells of the 3^rd to 6^th generations were collected. The exosomes were extracted from cell supernatant by differential centrifugation. The protein concentration was measured by BCA. The morphology of the exosomes was observed by transmission electron microscopy. The diameter distribution of the exosomes was measured by particle size analyzer. The exosomes were identified by immunomagnetic beads and flow cytometry.
RESULTS AND CONCLUSION: Adipose-derived mesenchymal stem cells were long spindle-shaped, fibroblasts-like, when arranged tightly. Flow cytometry showed that adipose-derived mesenchymal stem cells were positive for CD29, CD44, CD90 and CD105, but negative for CD34 and CD45. Alkaline phosphatase staining was blue-purple on the 9^thday after induction of osteoblasts. Alizarin red S staining showed red calcium nodules in the extracellular matrix on the 21^st day after induction. Oil red O staining was orange on the 14^th day after induction of adipocytes. Under the transmission electron microscope the exosome was cup-shaped with a diameter of (81.225±22.226) nm and had a membrane structure. The protein concentration was about 1.5 g/L and the diameter distribution ws concentrated in 70-100 nm. Flow cytometry showed that the exosomes were positive for CD9, CD29, CD44, CD63, CD90 and CD105, but negative for CD34 and CD45. To conclude, the exosomes derived from adipose-derived mesenchymal stem cells can be successfully extracted by ultracentrifugation and identified by transmission electron microscopy, immunomagnetic beads and flow cytometry.

Key words: adipose-derived mesenchymal stem cells, exosomes, adipogenic induction, osteogenic induction, cell identification, immunomagnetic beads, flow cytometry

CLC Number:

R459.9
R364

Wang Jing, Cai Xia, Wang Zhiguo, Xu Quanchen, Li Kun, Hua Cheng. Isolation and identification of exosomes from human adipose-derived mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(17): 2651-2658.

Figures/Tables 2

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