I'm going to start culturing these cerebral endothelial cells:
http://www.cell-systems.com/cert/22/137/Products/Products/ACBRI-Site-Specific-Cells/Human-Brain-Microvascular-Endothelial-Cells
Eventually I want to perform tube formation and migration assays with them. In literature I could not find tube formation assays with these cells, as compared to HUVECs where you find the most info. I wonder if you have observed tube formation capacity. The company could not tell.
I also need to understand better what coating would be best, and what growth conditions. I need to find the "minimum" good conditions, preferably in defined medium without serum, as I will try treatments to see enhanced proliferation and angiogenic capacity, but this might be modest.
Please reply here if you have experience with these cells or with a particular aspect of it. If you don't have them from Cell Systems, but isolate them yourself, please reply anyways, your experience would be very helpful and appreciated.
Dear Virginia, I once carried out apoptosis assay with hMECS,I cultured them in MV2 media from Promo-Cell and detached them from small culture flask with gentle pipeting. you can also try CSC complete serum free medium from Cell Systems. I know that the teams from Howard Fines from NIH, Prof DeClerck from Leuven and Shalom Avraham's group from Harvard did tube formation assays successfully from hbMECs. May be you could search their articles and see how you could optimize their protocols from your purpose.
Good luck!
Dear Virginia,
I have used the Matrigel (BD) and EGM media for tube-formation assays of hBMECS.
My paper may help for your study (2009, Hayashi S et.al, PMID: 19815818 )
Best to you!
Dear Syeda and Shin-ichiro,
thanks a lot for your helpful references!
CULTURING:
I want to culture hBMECs in defined medium. Getting rid of serum would be great, but probably not possible. I was thinking of doing so just 24 hours prior to treatment and during treatment. But still I would like to have a defined set of nutrients and growth factors.
CSC complete serum from Cell Systems requires the addition of Rocket Fuel. The content is proprietary, except for there being EGF and bFGF and iron supplemented serum. I read on some papers that these cells like to have also insulin, transferrin and epidermal and endothelial growth factors. Some also use BSA in the serum-free pre-treatment option to help attachment i suppose, but not sure at all.
Any clue on what is best to feed these cells?
ATTACHMENT:
Fibronectin seems to be a good coating.
Do you know if it´s best to buy fibronectin coated flasks or fibronectin solution to coat surfaces myself ?
Do you observe spontaneous tube-formation on fibronectin?
Thanks for your time and help
Hi Virginia
we have used an immortalized rat brain microvascular endothelial cell line which resulted in nice tube formation in matrigel. We used to starve the cells overnight in serum/growth factors free DMEM/F12. Matrigel experiments were done in EBM +0.5% FBS. Best results are obtained after 48hrs. You can find more info in our paper:
http://www.ncbi.nlm.nih.gov/pubmed/24755675
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