Improvement Of Isolation Methodology Of Exosomes Derived From Adipose-Derived Stem Cells And Its Effect On Osteogenesis

Objective: Most exosomes have a diameter of 30-150 nm and are difficult to purify.There are a variety of known extraction methods,of which overspeed centrifugation and solvent precipitation are widely used.The aim of centrifugation and solvent precipitation are widely used.The aim of this study is to find the most efficient method of extracting exosomes from adipose-derived stem cells by the improved ExoQuick kit method,ultracentrifugation method and ExoQuick kit method.Exosomes possesses bioactive substances similar to their source cells,which can transmit information between cells and exert various biological functions.On the basis of determination of exosomes through transmission electron microscopy and Western Blot,it is discussed whether the addition of exosomes on ADSCs with induction direction of osteogenic differentiation enhances their osteogenic efficiency.Methods: ADSCs were extracted and cultured,subcultured for three generations,and observed under microscope.Lipogenic and chondrogenic induction were carried out for the third generation of ADSCs,and oil red O staining was carried out on 14 days and Alican staining was carried out on 28 days,respectively,to observe the induction results under the microscope.Flow cytometry analysis was performed on the third-generation ADSCs to detect the expressions of CD10,CD31 and CD34.Exosomes from the third generation of ADSCs were extracted with the improved ExoQuick kit method,ultracentrifugation method and ExoQuick kit method,and morphology was observed with its.Identification of the expression of marker protein CD9 and CD63 proteins.exosomes-extracted from the three methods were added to the osteoblast induction process of adipose-derived stem cells for morphological observation,The above four groups were respectively used in the induction process of osteogenic differentiation of ADSCs,and the morphological changes of four groups of ADSCs and the expressions of Runx2 and OCN proteins were detected through a control experiment.Results: 1.Under electron microscope,the morphology of the third generation ADSCs cells was uniform and showed obvious direction.2.After adipogenic induction of ADSCs,the intracytoplasmic lipid droplets were stained red by oil red O staining.After thechondrogenic induction of ADSCs,the cytoplasm and extracellular matrix were stained blue by Alican staining.3.After flow detection,CD10 expression was positive,CD31 expression was negative,and CD34 expression was negative,which was consistent with the phenotype of ADSCs,indicating that the cultured cells were ADSCs.4.Three methods were able to observe the circular and quasi-circular membranous vesicle structure,and all methods expressed the surface markers CD9 and CD63,and the improved ExoQuick kit method had the highest expression.5.Compared with the group without exosomes,the expression of Runx2 and OCN proteins in the group with exosomes was significantly higher and the improved ExoQuick kit method has more expression of Runx2 and OCN proteins than the other two methods.Conclusion: the improved ExoQuick kit method is the optimal method for extraction of ADSCs-Exos.Exosomes extracted from them are able to enter ADSCs and promote osteogenic differentiation of ADSCs.Moreover,exosomes extracted with the improved ExoQuick kit method has the best induction effect.