Main author: Berglind Eva Benediktsdóttir
Institution or Company: Lyfjafræðideild Háskóla Íslands, Lífvísindasetur Háskóla Íslands
Co-Authors, Institution or Company:
Erna María Jónsdóttir, Lyfjafræðideild Háskóla Íslands. Þórhildur Bergljót Jónasdóttir, Lyfjafræðideild Háskóla Íslands.
Introduction: EVs are nanosized vesicles released from human cells and participate in various physiological functions in health and disease states. The intrinsic ability of EVs to deliver proteins and miRNAs has sparked interest of their utility as a nanosized drug carriers. Methods for EV isolation need be scalable and reproducible to yield pure, bioactive EVs. The aim of this study was to refine the isolation and characterization methods for EVs to apply for future use as drug delivery nanocarriers.
Methods: The D492 and the MDA_MB_231 cell lines originate both from the breast epithelium, yet have very different origin (non-cancerous vs. cancerous). The cell lines were cultured in serum-free media, the conditioned medium was preclarified, concentrated followed by size-exclusion chromatography (SEC, Akta Start). Collected concentrated fractions (F0-F4) were analyzed with NanoSight and capillary western blotting (WB) to confirm the isolation of EVs.
Results: F4 of the MDA_MB_231 cell line was positive for EV markers (Alix, CD63) according to WB, with 3,2*108 ± 0,08*108 particles/ml, average size of 122,9 ± 2,2 nm. Repeated analysis of WB is needed for the isolation of fractions from the D492 cell line.
Conclusions: Current refined methods used for isolation and characterization of EVs show that Akta Start was well equipped to fractionate concentrated medium although the UV sensitivity was not optimal. The NanoSight was reliable in terms of particle size and quantity determination and capillary WB provided good resolution compared to traditional gel WB and isolated fractions could be characterized in terms of EV content.
