The short answer is Yes. However, when you say these cells proliferate "very slow", how slow? Being the primary cells, these cells under optimal conditions proliferate once every 24-36 h. This is considered to be normal. Therefore, find out optimal conditions.

See link: http://www.lonza.com/products-services/bio-research/primary-cells/human-cells-and-media/endothelial-cells-and-media/huvec-human-umbilical-vein-endothelial-cells.aspx?gclid=CN-lqdTP9MgCFQkyaQodJHoC4g

Very slow compared to what? To previous aliquots thawed from the same cell bank, or to cells from a previous lot?

There is strong batch-to-batch variability with primary cells sources, especially when the cells are from a single donor. Once we bought new HUVECs that performed very poorly in our lab (doubling time slightly over 50h), so we sent a complaint and got another lot from the company with no added expense.

Thank you for your answer.

Normally, the cell that I used always double after 1-2 days. 

Recently, I have changed medium (I am still using the same cell but higher passage) and  the doubling time is more than 4-5 days. 

How many passages are we talking about?

We used to restrict our assays to passages 3 to 5. (liquid nitrogen cell bank was prepared at passage 2 from the purchased aliquot)

More than that and the risk of losing the phenotype (along with the proliferative properties) would be too great.

HUVEC cell line should be maitained in 10 passages, we usually use cells within 7 passages, VEGF adding can promote cell growth, and you can try FGF-2 too.

It depends on the passages. You should use them within 10 passages, but in my experience after 7 they are not good anymore. If your cells are less then 7 passages, you could give them time to recover after thawing (up to 2 weeks) or add more growth factors depending on the experiments you want to do with the cells. Otherwise try to thaw another vial and if it happens again maybe there are problems with freezing/thawing, medium preparation or contamination. 

Now, I am using passage 6.  I will try another vial and see what will happen.

Thank you for all the answers :)

You said you changed medium, do you mean you changed the formula of the medium? Please note that if the cells were used to the old medium (presumably it was the more nutritious one such as EGM2), they would not like the different medium that they are now cultured in, especially if they are the less nutritious one like M199-based formula 

Proliferation is not a "normal" state for endothelial cells that usually form a confluent monolayer and have a very low turn over in vivo (see reviews from William Aird). Nevertheless, for research purpose we need to expand primary endothelial cells for experiments. HUVEC are commonly used and commercialy available. HUVEC are not a cell line but are primary cells and optimal culture requires specific culture medium supplemented with growth factors (VEGF, cortisone...etc) such as Endothelial growth basal medium (EGBM) from Promocell or the equivalent from Lonza or other providers. Using this culture medium will help you to amplify your cells up to the 7th passage with no changes in morphology and function. This "amplification" phase is required to reach cell confluency where endothelial cells will acquire their specific cobblestone morphology. Then, you should remove the growth medium and incubate your cell  for 8-12h minimum in a "deprivation" medium (such as the ECBM, endothelial cell basal medium with no supplement) before to use/treat your cell monolayers.

I am working with primary cultured HUVEC for more than 15 years. I have isolated the cells from umbilical cord and grow in M199. I think the following steps definitely increase the growth of the HUVECs:

1. Add at least 1x 10 5 (ten to the power five cells) in each 100 mm petri plate. If you add less cells HUVEC may grow slowly or may not grow at all !!!!

2. Subculture the cells when they are 70-80% confluent, not 100% confluent. 100% confluent inhibit the growth of HUVEC.

3.  Add 10 % serum, instead of 5% serum

3. Add freshly prepared L-glutamine (L-glutamine degrade easily, so do not use a prepared solution.

4. Add VEGF from Sigma.

Please remember, after 5-7 passage primary cultured HUVEC may start loosing some of its properties and will die after 50 passage..

Hi Ratima,

For optimal proliferation of endothelial cells, they should always be used until p5 and cultured as recommended by clonetics, in greater amounts of media as cells become more than 50% confluent. Media should be changed every 48h. Also, HUVECs should always be divided when about 70-80% confluent, not more; otherwise their ability to proliferate, migrate or invade in response to angiogenic growth factors will start to decrease.

We have worked with several endothelial cell-specific culture media over the years. Although several are of good quality, our conclusion is that the EGM2 bullet kit from Clonetics is the most efficient to sustain faster endothelial cell proliferation. 

However, from your message, your problerm may not necessarily come from the media you use, but from the fact that your cells' passage is too high. As someone points out in their answer, we also freeze stocks of HUVECs at p1 and use them from p2-p5 only. This way, all of the biological assays performed with these cells will be more reproducible, and cells will maintain their ability to proliferate or respond in any other way to growth factor stimulation. This is critical when you work with endothelial cells. Although HUVECs may still look OK after p5, they are definitively different than early passage cells. 

Hope this helps - good luck,

Isabelle

Hoi Ratima,

this problem we also had once in our lab, but that was because passage of the cell were at P8. 

The information that Isabelle wrote is very true. I think you should check the passage of  your cell. You will also see that the cell aren't that beautiful in your flask. The look very long and thin. From that reason you can also conclude that the cells are overdone. If you freeze your cells it is better to do that in an early passage, eg: P1 or P2. In my lab we use vascular cell basal medium from ATCC and that already works well.

In our lab we changed from using EGM2 to EGM2 with additional FCS, final concentration of 18%. This improved growth. (Since FCS for us is cheaper than EGM2, it also safes some money)

Hi,

I am agree with Isabelle Royal's suggestions.

In our lab we use Human Endothelial Cell Growth Medium: All-in-one ready-to-use from cell-application (cat. No. 211-500) and it works really well.

Thank you,

Swati

Dear Tapan kumar Mukherjee , What happens if you split/subculture the cells after being 100% confluent? Do they recover at all? Or should they be dumped?

Greetings,

Kimberly

The most important factors are plating density and passage number. We plate at 10000 cells per cm2, and keep the passage below 5. We also routinely coat the flasks with gelatin to aid cell adhesion, and we change the media every 2 days. Naturally, a good method of freezing down the cells will maximize the number of cells that are viable when they are thawed for use. We use a rate freezer for this and use cell freezing medium, and when we thaw the cells we warm them very rapidly in a water bath. Ensure that you use appropriate media. We have found that MesoEndo by Cell Applications is very good, with an extra 5% of FBS to make a total of 10% FBS. Growth factors may help but probably only minimally. The optimal time to passage the cells is at 90% confluence. Trypsin can be used for this, although we found that Accutase was more gentle and hence is better for cell survival but more expensive. I hope that is somewhat helpful.