I recently bought in a new (very expensive) immortalised cell line, Human Brain Microvascular Endothelial Cells, or HBMECs. They were delivered in a vial on dry ice, I stored them overnight at -80oC and then seeded them into a pre-coated T75 the next day. They came with a very detailed protocol which I followed to the letter, including buying all the appropriate reagents etc, but it meant that all were new to me.

24hours after seeding, the cells look mostly dead. A few have adhered here and there but do not look at all like endothelial cells, if anything they look senescent. 48 hours and there's still no proliferation, they look very unhappy.

The rat tail Type I collagen I used to coat the flask (diluted in PBS, left to coat for ~1.5 hours as recommended) looks like it has started to peel off at the sides and small tendrils are now sort of floating in the media...

The same cell line from another seller has been reported to have 'wandered' from its original phenotype but the company selling these ones assured us they would still be okay...

I wondered if anyone had used either the cell line or the collagen and could give any tips. Also any thoughts on what might be happening and if it's retrievable would be very much appreciated!

Thanks