Abstract Number: PB1968
Meeting: ISTH 2020 Congress
Background: Smooth muscle cells and endothelial are often sites of pathology and drug action. The potential to grow these cells from blood (termed blood outgrowth endothelial and smooth muscle cells; BOECs and BO-SMCs respectively) without the need for surgery has revolutionised the vascular field. Unlike BOECs, for which there are >100 publications, there are only 12 papers on BOSMCs isolated from humans and no studies that have managed to isolate extracellular vesicles (EVs) from both cell types. EVs are involved in cell communication and can also serve as biomarkers in disease conditions.
Aims: Obtain fluorescently labelled EVs from pre-labelled BOECs and BOSMCs under inflammatory conditions.
Methods: BOECs and BOSMCs were isolated from healthy donors (Ahmetaj-Shala et al, FASEB J, 2020). Cells grown to confluence on a T25 were washed with PBS and gently scraped in fresh PBS. The suspension was centrifuged at 200 x g for 5 minutes. The pellet was resuspended with 0% serum EGM-2 media, labelled with fluorescent CellTracker Red CMTPX (10 µM) and incubated for 30 minutes in the dark. Cells were then seeded on a T25 and stimulated with LPS (1 µg/ml) and a control sample was collected. Cells were then incubated for 16 hours, at 37oC, 5% pCO2. The media was collected and, together with the control sample, centrifuged first at 2,000 g for 10 minutes to pellet cells and debris. The supernatant was recovered and centrifuged again at 15,000 g, for 1 hour. The EV-containing pellet was resuspended and analysed through flow cytometry (BD Fortessa).
Results: Both BOECs and BOSMCs were successfully labelled and generated EVs after stimulation with LPS. Consequently, EVs were also fluorescently labelled and tracked after flow cytometric analysis (Figure 1).
Conclusions: This novel and robust method will allow further exploratory research in order to understand the physiology of BOECs, BOSMCs and their EVs.
To cite this abstract in AMA style:Ferreira P, Kirkby N, Mitchell J, Ahmetaj-Shala B. Generation and Isolation of Extracellular Vesicles from Endothelial and Smooth Muscle Cells Grown out of Human Blood [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/generation-and-isolation-of-extracellular-vesicles-from-endothelial-and-smooth-muscle-cells-grown-out-of-human-blood/. Accessed December 3, 2021.
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