We thawed and seeded a vial of bEnd.3 cells (ATCC) last week, but after 10 days they do not look like endothelial cells. The suggested media is DMEM + 10% FBS; we have been successfully growing human endothelial cell line HBEC-5i (also from ATCC) with DMEM/F12 (1:1), 10% FBS, 1X endothelial growth supplement, and 25 ug/mL gentamicin, on flasks coated with 0.1% gelatin, so we kept the same formulation for bEnd.3. After the first few days, the cells appeared stringy and a lot were non-adherent, but checking the product sheet and website FAQ's this seemed normal. We changed the media every 3 days, centrifuging the old media to recover floating cells so they were not lost. But now, after 10 days, the cells still do not possess any endothelial characteristics - as shown in the attached images (5x, scale bar = 200 um), they look more like astrocytes or microglia! We are wondering if anyone has had similar issues with bEnd.3 cells upon initial seeding, and whether they were resolved?
We used to grow bovine aortic endothelial cells and I must say, when I saw your images, they reminded me of some of our cultures. We sometimes observed different morphologies in our cultures that were never fully understood. Maybe when your cells become confluent, they'll appear more endothelial-like? We also observed that endothelial cells appear fibroblast-like when grown on conditioned medium from smooth muscle cells.
If you look at the photos at https://www.atcc.org/products/crl-2299, yours resembles that on the left.
We reached out to ATCC and they are sending us a new vial. The problem with these were that, indeed as Thomas J Raub mentioned, they look perhaps like what ATCC suggests for low density, but they never adopted the endothelial morphology even after several days in culture and media changes (retaining floating cells). We have switched out all our media, tips, gloves etc to rule out possible contamination - when we try with the new batch I will provide an update!
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