Comparison and characterization of extracellular vesicles isolated from endometrial cell lines by different methodologies

Abstract

Extracellular vesicles (EVs) are membraneous vesicles released by a variety of cells into the extracellular microenvironment. They act as media to transfer proteins, small RNAs and mRNAs to distant sites. EVs may take important roles in the embryoendometrial dialog within the uterine microenvironment for successful implantation. There are different methologies for purifying EVs, however, there is not much information on comparison of these metholdogies. In this study, we compared four different EVs isolation methods using endometrial cell line as source of EVs. Four different methods were applied to isolate EVs from endometrial Ishikawa cell conditioned media; ultracentrifugation (UC), polymer-based precipitation (PBP), size based ultra-filtration (SF) and polymer-based precipitation with size based ultra-filtration (PBP+SF). The efficiency of these isolation methods were assessed by quantitation of the EVs isolated, expression of EVs enriched proteins, expression of specific microRNAs, ability to transfer EVs’ small RNAs and internalization of EVs by recipient trophoblasts and blastocysts. The numbers of EVs isolated were assessed by counting under electronic microscopy while expression of exosome-enriched proteins CD63 and TSG101 were examined by Western blotting, microRNAs expressions were determined by RT-QPCR. Bioactivity was estimated by the ability to transfer EVs contents; either fluorescence-labeled EVs or miRNAenriched EVs were added to recipient trophoblast cell line Jeg-3. Successful entries of the labeled EVs were assessed by confocal fluorescence microscopy. EVs were isolated from miRNA let-7a enriched Ishikawa cells and cocultured with Jeg-3 recipient cells. The expression levels of let-7a in recipient cells were measured. Western blotting demonstrated that EVs isolated by all the 4 methods expressed exosome-enriched proteins CD63 and TSG101. Electronic microscopy revealed that the diameters of the isolated EVs are of no significant difference between different methods (UC: 39.97±12.42 nm, PBP:34.56±4.98nm, PBP+SF: 33.35±4.63 nm and SF: 30.22±2.92nm). Electronic microscopy revealed that PBP could recovery the highest number of EVs while UC is the least efficient (PBP:207.4±68.5, PBP+SF: 163.2±91.5, SF: 127.2±89.7 and UC: 44.9±35.1,). For bioactivity analysis, confocal fluorescence microscopy showed that nearly all the recipient cells cocultured with EVs isolated by PBP or PBP+SF were EV-fluorescence positive (PBP+SF: 95%±9% and PBP 100%±0%) while only 51.4%±34% of the cells were fluorescence positive by EVs from UC. Moreover, the EVs isolated from the PBP could transfer more miRNA to the recipient cells (PBP+SF: 2.3±0.98-fold and PBP 2.4±1.1-fold) when compared to the UC (0.8±1.2-fold). In summary, the PBP and PBP+SF methods were much more efficient than UC and SF in isolating bioactive EVs. The SF column helps to reduce small size contaminant and thus has the advantage of getting a EV preparation with higher purity.