I'm planning to use HUVEC cells for a tube formation assay in matrigel to measure the anti-angiogenic effects of some drugs. I have no experience with this assay and I found that there are different kinds of HUVEC cells and I'm wondering which are better for my experiments. Single donor or pooled cells? Also, what medium do you recommend me to use? Thank you in advance!
hi there,
i have used from the kit and it really works fantastically. Angiogenesis starter kit from invitrogen. Got very good results also.
Hi Laura,
If you want to reproducibly show some mechanism it's better to use pooled donors as these batches are quite consistent. If you're interested in donor-to-donor variation or donor characteristics (e.g. sex differences), then you can better use single donors. For 95% of the questions the pool is better.
Otherwise HUVECs are pretty robust and you can't do much wrong, don't worry. For culture EGM-2 is great, but it may be a little expensive.
I think Hendrik has pretty much addressed the question of the pooled vs single donor, so i will attempt on the medium used for the experiment.
If you want to look at the anti-angiogenic effect of a drug, it's important not to use high FBS in your medium. While EGM-2 is good to maintain the propagation of your HUVECs, I would not normally use EGM-2 during an experiment when I want to look at an effect of a cytokine or drug as the rich growth factors in EGM-2 might mask the effects of your drugs.
M-199 (a standard medium for culturing HUVECs) with your normal antibiotics/antimicrotics, with 1% FBS (you can do a dose-finding with the FBS level from 0.1% up to 5% to see which is the best in your system for tube formation), and adjust the number of cells seeded (5000 - 30000 cells/well of a 96-wells dish) to optimise the experimental conditions. I will normally acquire the result after 24 h. (Please note that if you also want to use M-199 for maintaining your HUVECs you need to add EC growth supplement kit, perhaps from Sigma, with 10% FBS. There are plenty of protocol online on recipe regarding M199-cultured HUVECs)
When you have validated your experimental results, you can also use EGM-2 with the drug just to double validate your findings if you wish, that will be even more convincing for your findings.
Thank you for your help!
Following your recommendations I'm planing to use polled HUVEC cells. I can not use EGM2 because it has VEGF that can interfere with my experiment but I think I will use the EGM bullet kit from Lonza, that includes the basal media plus the supplements.
I have one more question about the media. Do you recommend to use this same supplemented media during the angiogenesis experiment? Or just to mantein the propagation of the cells and then change to a basal media during the experiment?
- Badges
- Science method