Could someone share, in your own opinion, the simpliest method to isolate and characterize mouse brain microvascular endothelial cells?
After treatment of brain tumor tissue with collagenase and hyaluronidase, microvascular endothelial cells can be obtained by flow cytometry using the antidody for CD31 (PECAM-1), CD133 (AC133), Flk-1. (VEGFR-2 ), Tie-2, or VE-cadherin. As comapred with normal vessel endothelial cells, tumor endothelial cells strongly express Dickkopf-homolog 3 (Dkk-3), CD13, Collagen2a，integrin αvβ3. That is why these markers are also useful to for the enrichment of endothelial cells specific to tumor angiogenesis.
Hi Saimonas,
I recently published an optimised protocol for easily isolating, culturing and characterising robust preps of brain microvascular cells from rat. I didn't try it on mice but given the general translatability of brain endothelial cell preps across species I'm confident it should work. It's a very reproducible protocol in our hands and you get a good number of cells to use.
Good luck!
Marc
Article Modelling the endothelial blood-CNS barriers: A method for t...
Thank you very much!
Marc, nice protocol, a bit of shame that there's a lack of some reagents in our lab, maybe we'll sort something out..
Hi Salmonas
My protocol is similar to Marc's protocol.
Briefly, the cerebrum was excised from a 16-week- rat, dissected into 2-mm
pieces, and homogenized with a Potter-Elvehjem homogenizer. To the homogenate, a similar volume of 32% dextran was added to give a 16% dextran solution, followed by centrifugation at 4500g for 15 min at 4ºC. The pellet was resuspended and digested in 0.066% collagenase/dispase and 0.033% bovine serum albumin in 1x PBS (-) for 3 h at 37 ºC. The homogenate was then centrifuged (700g, 2 min), and the pellet was re-suspended in DMEM, supplemented with 20% FBS, 100 units/ml PC and 100 ug/ml SM and 2.5 ug/ml amphotericin B (Gibco Laboratories) and transferred to culture dishes.
Then use specific culture medium to select endothelial cells.
See this paper
Asashima T et al. Eur J Cell Biol 81:145-152, 2002.
Hisashi
Thank you, Hisashi!
Could you tell me the composition of the specific culture medium you use to select endothelial cells? And is it necessary to use dispase, maybe collagenase would be enough?
The culture medium was Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 1.5 mg/mL sodium bicarbonate, 15 ug/mL bovine endothelial cell growth factor (bECGF), antibiotics, and 10% fetal bovine serum (FBS).
However, this is very old protocol. I think that there are several endothelial specific medium (commercially available)
And you can use MACS or cell sorting combined with this method.
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