Hi Aleks,

Just to be clear, you are starting with frozen cells in the ampules that were shipped from Gibco.   The cells have never been thawed previously nor grown in anyone's lab locally and then refrozen.  You thaw, plate in M200 with LSGS, place into 5% CO2 incubator and 2 days later they are dead.  Is that correct or is there more to it?

Hi Robert, 

That's exactly what I have done yes - can you help? 

Aleks,

 It's possible that you have a fairly fundamental problem with your cell culture set up or with your thawing procedure.  

If you or others in your lab are growing other cell types in the same incubator and those cells are growing well, then the incubator set up is probably fine.  

If the HUVEC arrive from Gibco still frozen and you either thaw immediately or put them into liquid nitrogen immediately, then that's best.    

If you thaw other cell types and they survive well, then you're thawing procedure is probably fine. 

 If your M200 and LSGS are fresh, that should not be the problem.  Of course, you are not adding any FBS or other serum to the media!

 If you are using the same type of tissue culture flasks for these cells and other adherent cells and the other cell types grow well in the flasks, the flasks are not the problem. 

If you are following the rather detailed instructions from Gibco for handling these cells, then that should avoid most problems. 

We found it helpful to remove the DMSO containing media 24 hours after plating. This improved cell survival for us. 

Also, we avoided centrifugation when DMSO was present, as this as this tended to damage the cells. 

 There are additional precautions that we take with HUVEC, but these apply only when doing trypsinization and transfers. But it sounds like your problems occur before you do trypsinization.

Hello Robert,

Thank you very much for the detailed response it is much appreciated! I will revisit the points you have suggested and give it another try. 

For culturing HUVECs we always coat the flasks with 1% gelatin (or collagen can be used), and add EGM2 bulletkit from Lonza to culture them. 24h after seeding, we change the medium. You can try to increase the FCS in your M200 medium to see if they survive. Do they form a confluent layer within those 48 hours? Or do they not even attache when you seed them?  

 Hi Dafna, 

Thank you for your reply. They don't become confluent at all within 48 hours which was strange. It did say on protocol that it will take a few days to reach 80% confluency but got nowhere near there as they detached. When I seed them they do attach pretty quickly I should say so it must be something within the 48 hours of culture that leads to the problem. I wasn't aware of the collagen coating and a few people have mentioned it now so will give that a try too. 

I would summarize the following suggestion:

1. Coat your plate/flasks with 0.1% gelatin and keep them in PBS at 4 0C until use.

For preparing 0.1% gelation weight 1 gm gelatin (Sigma), add into 1 liter of MQ water and autoclave. Allow it to cool down and in the laminar flow hold add 3-5 ml of gelatin to a 100 mm petri plate to cover the entire plate. Close the lid and put into 37 0C for 30 minutes. Remove gelatin after incubation and add PBS and keep it into 4 0C until use.

2. Thaw the cells immediately after receiving and put into plate/flasks without disturbing.

3. Please check the pH of the medium (7.4 ) before use. Add 5-10% serum and VEGF from Sigma/other company into the cell culture medium.

4. Next day change the medium to remove DMSO.

The EGM-2 media from Lonza works well for HUVECs. Gelatine or collagen coating of flasks will be fine. If you are following the supplier's protocol exactly, but are unable to get the cells to grow, it's worth going back to them and ask them for a replacement. 

Thanks for all the suggestions. From the recommendations here and talking to someone that worked with these cells within the University, I believe what I was missing is coating of the flasks. This wasn't suggested or recommended on the supplier protocol, so I will be having another go with collagen coating of the flasks to grow these cells. Hopefully there will be no problems.

I've gone through the answers and they're all very good, and even though you seem to have succeeded, I'd like to share our experience with HUVEC (ATCC in our case, not sure how much it matters...).

We have been growing HUVEC fairly successfully on routine basis without coating. Sometimes that might be useful, depending on your experimental protocols. We use standard cell-culture plastic, use F12 medium from Gibco supplemented with 20% FBS (10% works also), and add 0,1mg/ml heparin and 0,05mg/ml Endothelial Cell Growth Supplement (ECGS), both from sigma. We add this directly to the culture flask rather than the medium bottle, somehow that seems better... Medium change every 2-3days.

Note though that HUVEC dont really grow much past 80%, be ready to use them around then.

Good suggestion. My only concern is 20% serum although very good for cell culture they may interfere with many drugs including growth factors, steroids etc. In our case when we grow HUVEC in highly enriched medium and then shifted to low/no serum medium for our treatment with various agents cells may not behave normally, particularly if you keep the cells longer (more than over night) in low serum medium. However, you can use charcoal-dextran treated serum for culture also.

Very correct Tapan Mukherjee. We have noticed the same. we solved the issue by reducing FBS to 1%  ON upon aliquoting the HUVEC. then they respond great to 1-5% BSA (depending on growth factors) up to 72h or up to 2 weeks if the protocol allows to change medium every 3 days 

Endogrow culture media obtained from Millipore work very well for HUVEC. Since I use this culture medium, I am not obliged to coat my flasks with gelatin. It costs a little expensive but the cells look like better.

There are no tricks:

A protocol can be found here: Circ Res. 2009;104(5):589-99 or in: Biol Chem. 2008;389(10):1333-8 Media were purchased by PromoCell, but other brands should also work (e.g. LONZA). Standard T175 flasks (from Saarstedt) were used (Falcon, TPP etc. should also work), no coating was done!

My experience with HUVEC is something I'd want to share (From ATCC).

Without coating, I've been culturing HUVEC on a regular basis with reasonable results. I utilize a corning cell-culture flask, Gibco's F12K nutrient mixture basal media supplemented with 10% FBS, and Sigma's 0.1 mg/mL heparin sodium salt and 0.03 mg/mL Endothelial Cell Growth Supplement (ECGS). This time, I'm using the complete media by mixing all the ingredients mentioned above and changing it up every two days. I think this way will help you. For growing HUVEC cell lines, the passage number is crucial. This cell line's number does not increase to your satisfaction after passage-20.

use TC treated vessels. Corning for example, lonza media works great