The invasive properties of WI-38 and MRC-5 fibroblasts were investigated using a spheroid invasion assay performed and quantified as previously described [48 (link)]. Briefly, 2500 cells per well were seeded in 100 µl of DMEM containing 10% FBS into a cell-repellent 96-well microplate (650790, Greiner Bio-one, Frickenhausen, Germany). After overnight incubation at 37 °C, the cells formed spheroids, 70 μL of medium was removed from each well and the remaining medium with spheroid was overlaid with 2.5% bovine collagen I (5005, Advanced BioMatrix, Carlsbad, CA, USA). Following the polymerization of collagen, fresh serum-free DMEM supplemented with PBS, 20 µg/mL 2D EVs or 20 ng/mL of PDGF-B (100-14B, Peprotech, London, UK) was added. The cells were allowed to invade the collagen matrix for 48 h, after which they were stained with Hoechst (62249, Thermo Fischer Scientific, Waltham, MA, USA). Images were acquired on an Axio Observer 2 fluorescence microscope (Zeiss) using a 5× objective. Cell invasion is determined as the average of the distance invaded by the cells from the center of the spheroid as determined by the cell dissemination counter software aSDIcs. Three to five spheroids per condition were analyzed and three independent experiments were performed.
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