I have done all the above assays you have mentioned.I culture HUVEC in M199 with 20%FBS and penstrep.I'll be happy to help if you have specific questions on the migration,proliferartion and tube formation.

Regards..

The biggest challenge with HUVECs is fibroblast or smooth muscle cell contaminants. Over time, these contaminating cells can overtake the population, especially in co-culture settings. You should source your HUVECs carefully (commercial vs. lab-made stocks tend to behave quite differently).

Proliferation assays are reasonably straightforward (I recommend S-phase entry studies), as are motility assays. Tube formation is a bit more complex. Neither tubulogenesis in 2D culture nor bead-based sprouting assays are good models of angiogenesis, so you may want to add a bit more detail to your question to get feedback on better approaches to looking at vessel formation.

I agree with Dr.Cohen regarding the taking over of smooth muscle cells over time.Seeding at high density of 10000 cells/cm.sq is one of the ways to overcome this problem.

For the migration,proliferation and tube formation assays,optimizing the cell number for each assay is very important with HUVEC.

I purchased HUVEC (pool) from Lonza, and used their culture medium. Those cells can be split, in my hand I used the cells between passage 3-7, the results are consistent. I have done the proliferation,migration, and tube formation assay. If you are interested, you might check our paper: Dimethylarginine dimethylaminohydrolase 1 modulates endothelial cell growth through nitric oxide and Akt. Arterioscler Thromb Vasc Biol. 2011 Apr;31(4):890-7.

It is better to use HUVECs of the same passage for direct comparisons (say, to study the effects of a particular compound). If not possible, at least try to use your cells withing passages 2-4 (at most 5). If someone in your institution is isolating HUVECs from umbilical cords, you may want to get some cells from them (freshly isolated ones are always better).

As for culturing, M199 supplemented with 15-20% FBS (heat inactivated- put in a water bath for 60 minutes at 55-60C) and pen-strep isfine. Addition of commercially available growth supplements can stimulate cell proliferation, but not a good idea when you are actually trying to measure cell division/angiogenesis etc.

Hope this helps.

You can seed the cells on BD matrigel matrix plus collagen 1 in corning Transwell 24 well plate as 3D culture for tube formation study.

How better is the tube formation efficiency of endothelial cells with addition of type 1 collagen in Matrigel?

How can the stability of the tubes be maintained for more than 16 hours in an in vitro 3D system?

Thank you for all of your replies - I am going to write some experimental protocols shortly and will take your comments into consideration.