Comparison of different extracellular vesicles populations derived from adipose tissue derived stromal cells depending on conditioning period

Introduction: Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSC) became an important player in regenerative medicine over past years. The most common protocol to isolate EVs from cell culture, includes a period of fetal calf serum (FCS)-free conditioning. Different conditioning periods are common ranging from 12h up to 96h. Recent guidelines(MISEV 2018) only paid little attention to this variable without any recommendations. The aim of this study was to investigate EVs released from adipose tissue stromal cells (ADSCs) in different periods. Materials and Methods: For EV preparation, condition medium from primary ADSC culture was collected each 24h by replacing it with fresh FCS-free GM for 72h. Thus, we obtained EVs released within first 24h (24h), 48h-72h (48h), and 48h-72h (72h). The groups were compared by their size, number, phenotype, and amount of protein. The proliferation potential was assessed by a coculture with primary Schwann cells. Results: EVs from all the groups (24h, 48h, and 72h) fulfilled the characterization criteria according to MISEV 2018. However, the properties of EVs differed significantly by size, number, EV to protein ratio and the expression profiles CD9, CD63, CD81. Further, Schwann cells treated with “24h” EVs exhibited a significant increase in proliferation. The proliferative effect of EVs was diminished with longer conditioning period. Conclusion: For groups working with EVs it might be enticing to prefer longer FCS-free conditions for a higher yield. More, as EVs from all groups fulfilled the minimal criteria, which generates an erroneous belief of a coherent population. With our approach, we identified distinct populations of EVs with significantly different proliferative potential. For the sake of reproducibility, our data demonstrate the urgent need for a regulation of the FCS-free period for at least EVs from primary ADSCs. Future experiments are needed to validate our results for EVs from immortalized cells, or for further cell lines.