Objective: Emerging evidence suggests that ECs generate tissue specific angiocrine factors, creating an instructive vascular niche. Here, we aim to isolate, purify, characterize and culture ovarian ECs in serum free conditions, creating a platform for the exploration of endothelial-follicular communication.
Design: Laboratory study
Materials and Methods: i. Primary ovarian ECs culture: Mouse ovarian ECs were isolated via magnetic cell sorting using CD-31 antibody. After a culture period of 5-7 days, activation of ECs was initiated via lentivirus transduction, of myr-AKT gene. ii. Primary fresh ovarian ECs at the follicular and luteal phases: Intra vital staining using VECAD and Isolectin were performed, allowing ovarian EC isolation using FACS. Expression of selected angiocrines and surface markers was done by Whole genome transcriptom and qPCR assays.
Results: 98% of cultured activated ovarian ECs were shown to be CD31+, VECAD+ and CD45- on flow cytometry. Relative expression of dll1, mmp10 and fgf1 was higher in the cultured ECs, while expression of Jagged-1, VECAD and PDGF was comparable in both groups. Expression of Jagged-2, notch1-4, dll4, VEGFR1, VEGFR2, mmp4, il6, and il8 was lower in activated, cultured ECs in comparison to fresh ECs but fairly high compared to its own house keeping gene.
Conclusion: Here, we report successful isolation and durable culture of ovarian ECs, with a matched angiocrine profile to fresh ovarian ECs thus providing a novel tool with which to study biology of ovarian endothelial cells and cell-cell communication within the ovary.
