I'm working with HUVEC isolated from normal donors but I have some problems. I found a lot of variability in their responsiveness to my stimuli (e.g. TNF-alpha), is it possible to speak about responder and "not-responder" HUVEC according to the different donor? Someone has my same problems? Do you know some principles to discriminate against responder and not responder donors?
Actually, I'm using M199 (Sigma) supplemented with endothelial cell grow supplement and 20%FBS, but I need to eliminate serum for my experiments. Is there someone who has experience in HUVEC culture without serum?
Finally, do you know any continuous HUVEC cell line?
There certainly could be differences in response to TNF-alpha when using HUVECs from single donors. For this reason, some people prefer to use HUVECs isolated from multiple donors and the cells pooled (see this link
. This tends to balance out the response variability.
Other causes of variability in the response to cytokines are inconsistent degree of confluency, serum-starvation times, and passage number. Standardizing the experimental parameters can do wonders for improving the consistency of experiments.
HUVECs can survive for limited time periods in the absence of serum. Here's a quote from an article available at this link
: HUVECs can survive in serum-free medium (M199 without FCS) for up to 12 h without losing their phenotypic features. However, for longer experiments, in particular those lasting 24 h, the absence of growth factors triggers apoptosis, an irreversible mechanism. This process can be circumvented by the use of an enriched medium but with a low concentration of proteins such as the commercial medium Ultroser G (Biosepra, cat. no. 259515) (see refs. 6,8,23) at 1% in M199 without FCS; in this medium, HUVECs can survive for 48 h. Some other low-serum media that support HUVECs viability can also be used.
Cell Applications, Inc. sells HUVECs and also has a good page of FAQs. It can be found at this link
I do not know of an immortalized cell line equivalent to HUVECs.
If you have trouble accessing the article, post your email address and I will the send the pdf.