HUVEC

HUVEC cells are primary cells, hence they survive for a limited number of population doublings. HUVECs can be ordered from ATCC. The cells here have a life expectancy of 50 to 60 population doublings. Remember, however, that population doublings is not the same as passage number. Depending on how much the cells are cut back prior to passage, a cell line may go through multiple population doublings between each passage.

The recommendation of 2-6 passages for your experiments refers to the passage number starting from the primary culture. This could be equivalent to 8-24 population doublings, if we assume 4 doublings per passage. This number is variable and depends on the fraction of cells from one flask that is used to seed a new flask. It could be that this range of passage number was chosen to be well short of the life expectancy of the cells.

hi i work on HUVEC to assay their angiogenic response to a combination of specific growth factors for my theses. i have read that i need to perform my assays at 2-6 passages of HUVEC, my question is that what does 2-6 passage means, is it the number of passages after the defrizzing of cryopreserved cell lines i order from a cell bank or it means 2-6 passage after the primary cultures of HUVEC? in that case i have to isolate primary HUVEC cells!

good information and i learned a lot from it

Hi Blue Devil,
thank you very much for your helpful reply. Unfortunately I don't access to this article and I'll be grateful if you could send me the pdf version. My mail adrees is: cinzia.cordazzo@gmail.com.
Thanks a lot,
Cinzia

I just emailed the article. Post again if you don't get it.

Hi Blue Devil,

thank you very much for your helpful reply. Unfortunately I don't access to this article and I'll be grateful if you could send me the pdf version. My mail adrees is: cinzia.cordazzo@gmail.com.

Thanks a lot,

Cinzia

Hi all,
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?
Thank you
Cinzia

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.

Hi all,

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?

Thank you

Cinzia

Hi, I'm a student and I have a big trouble with HUVEC and I hope someone give an idea. I use HUVEC isolated directly to the umbilical cord and use the M199 medium suplemented with bovine fetal serum and human fetal serum to 20%, and endothelial growth factor for the culture, the cells grow well and has normal morphology. But when I test the fluorescent response to nicotine, acetylcholine and choline using the Eclipse E600 microscope and Fluo-3, HUVEC don't respond. The cells have a good response to ATP. I don’t know why the cells hasn't cholinergic response; I dont know if is a problem with the nicotinic receptor alpha-7 or another reason. If someone has an idea to solve this problem please send me a e-mail to hilda.andrea@gmail.com.
Thanks
Hilda.

HUVECs should definitely respond to nicotine and ACh. The article at this link has a fairly detailed protocol for detecting calcium influx following nicotine treatment of cells. They use Fura-2 instead of Fluo-3. The article gives appropriate nicotine concentrations (60-100 microM) and a time course of calcium influx. I would first check to see if your protocol is reasonable based on these guidelines. Notice that the HUVECs are washed in serum-free buffer prior to and throughout the assay. Concentrate first on getting nicotine to work before tackling the others.
If you have trouble accessing the article, post your email address and I will the send the pdf.

Hi Blue Devil!, thanks for answer my question, if you can send me the article in pdf will be grateful, my e-mail is hilda.andrea@gmail.com. Thanks a lot and I'm going to check my protocol carefully. I hope this works .

It's on the way. Post again if you don't get it.

Hi, I'm a student and I have a big trouble with HUVEC and I hope someone give an idea. I use HUVEC isolated directly to the umbilical cord and use the M199 medium suplemented with bovine fetal serum and human fetal serum to 20%, and endothelial growth factor for the culture, the cells grow well and has normal morphology. But when I test the fluorescent response to nicotine, acetylcholine and choline using the Eclipse E600 microscope and Fluo-3, HUVEC don't respond. The cells have a good response to ATP. I don’t know why the cells hasn't cholinergic response; I dont know if is a problem with the nicotinic receptor alpha-7 or another reason. If someone has an idea to solve this problem please send me a e-mail to hilda.andrea@gmail.com.
Thanks
Hilda.

HUVECs should definitely respond to nicotine and ACh. The article at this link has a fairly detailed protocol for detecting calcium influx following nicotine treatment of cells. They use Fura-2 instead of Fluo-3. The article gives appropriate nicotine concentrations (60-100 microM) and a time course of calcium influx. I would first check to see if your protocol is reasonable based on these guidelines. Notice that the HUVECs are washed in serum-free buffer prior to and throughout the assay. Concentrate first on getting nicotine to work before tackling the others.
If you have trouble accessing the article, post your email address and I will the send the pdf.

Hi Blue Devil!, thanks for answer my question, if you can send me the article in pdf will be grateful, my e-mail is hilda.andrea@gmail.com. Thanks a lot and I'm going to check my protocol carefully. I hope this works .

Hi Blue Devil!, thanks for answer my question, if you can send me the article in pdf will be grateful, my e-mail is hilda.andrea@gmail.com. Thanks a lot and I'm going to check my protocol carefully. I hope this works .

Hi, I'm a student and I have a big trouble with HUVEC and I hope someone give an idea. I use HUVEC isolated directly to the umbilical cord and use the M199 medium suplemented with bovine fetal serum and human fetal serum to 20%, and endothelial growth factor for the culture, the cells grow well and has normal morphology. But when I test the fluorescent response to nicotine, acetylcholine and choline using the Eclipse E600 microscope and Fluo-3, HUVEC don't respond. The cells have a good response to ATP. I don’t know why the cells hasn't cholinergic response; I dont know if is a problem with the nicotinic receptor alpha-7 or another reason. If someone has an idea to solve this problem please send me a e-mail to hilda.andrea@gmail.com.
Thanks
Hilda.

HUVECs should definitely respond to nicotine and ACh. The article at this link has a fairly detailed protocol for detecting calcium influx following nicotine treatment of cells. They use Fura-2 instead of Fluo-3. The article gives appropriate nicotine concentrations (60-100 microM) and a time course of calcium influx. I would first check to see if your protocol is reasonable based on these guidelines. Notice that the HUVECs are washed in serum-free buffer prior to and throughout the assay. Concentrate first on getting nicotine to work before tackling the others.

If you have trouble accessing the article, post your email address and I will the send the pdf.

Hi, I'm a student and I have a big trouble with HUVEC and I hope someone give an idea. I use HUVEC isolated directly to the umbilical cord and use the M199 medium suplemented with bovine fetal serum and human fetal serum to 20%, and endothelial growth factor for the culture, the cells grow well and has normal morphology. But when I test the fluorescent response to nicotine, acetylcholine and choline using the Eclipse E600 microscope and Fluo-3, HUVEC don't respond. The cells have a good response to ATP. I don’t know why the cells hasn't cholinergic response; I dont know if is a problem with the nicotinic receptor alpha-7 or another reason. If someone has an idea to solve this problem please send me a e-mail to hilda.andrea@gmail.com.

Thanks

Hilda.

Ladies and gentleman,

due to miscalculation we have produced a full set of HUVECs treated with continuous shear stress of 0,15 dyn/cm2. The shear stress was apllied in a cone plate system for 2 - 6 - 12 hours (n=6 in each time group). Samples are first passages of self isolated HUVECs, every shear treated group has static control pair. Trough the whole work HUVECs were incubated with Promo Cell Endothelial Cell MV medium.

The cells and supernatant stored in liquid nitrogen and -80 freezer respectively are ready for further analysis.
The samples could complete an experiment set with higher shear stress values.

Anyone interested in these samples?

Wish you a nice day