Optimization of Production and Application of Human Extracellular Vesicles
Objective:To optimize the production and application of extracellular vesicles(EVs)for protein delivery through the use of genetic engineering.Methods:qRT-PCR,flow cytometry and Western blot(WB)were applied to determine the expression of target genes in producer cells.EVs were isolated and purified from the conditioned medium derived from engineered cells by sequential ultracentrifugation.The particle concentration and size distribution of EVs were measured by nanoparticle tracking analysis.The representative EV markers were characterized by WB.The therapeutic effect of EVs was estimated in a human alveolar epithelial cell injury model.Cell viability was determined by CCK8 assay,cell apoptosis was assessed by Annexin V,and expression of Wnt/β-catenin downstream genes such as AXIN2,TCF4,and NKD1 was tested by qRT-PCR.Results:CD9 or CD81 overexpression was confirmed by flow cytometry in HEK293T and HEK293F.CD9 was superior to CD81 in promoting EV production.Moreover,CD9 tripled the yield of EVs in both adherent cells and suspension cells.We also investigated whether this platform could be used to deliver proteins such as Wnt3a.The results showed that Wnt3a was highly loaded on EVs and that CD9 Wnt3a EVs improved the cell viability and reduced the apoptosis after cell injury.Conclusions:We have developed a novel platform that can enhance the production of EVs based on CD9 overexpression.In addition,this approach is also compatible with protein delivery,providing a potential cell-free therapeutic option for clinical applications.
万方数据
维普
