I am getting HUVECs from someone that does not know where they come from nor the media they were grown before.
I have never worked with HUVECs and I have no idea which media could be used. Can I use 10% FBS RPMI with glucose and L-Glut? Is it better to use DMEM instead of RPMI or should I buy an alternative media?
Dear Daniel,
Based upon our previous work, we found the following formula works very well:
- M199
- 20% Fetal bovine serum
- Pencilin/Streptomycin 2 mmol/L.
- Amphotericin B 2 mmol/L
- Glutamine 2mmol/L
- HEPES 10 mmol/L
- Endothelial cell growth supplement (ECGs) 30-50 µg/ml
- Heparin 100 µg/ml
Best
This topic has been recently discussed:
https://www.researchgate.net/post/What_is_the_most_cost-effective_media_for_culturing_primary_HUVECs
In general, much of the original work with HUVECs was done in M199 supplemented with 20% FBS and 2 mM glutamine plus penn/strep. This was the original formulation of Jaffe in the seminal paper linked below:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC302542/pdf/jcinvest00186-0087.pdf
Since then a number of improved formulations have been used, adding various growth factors or reducing serum content.
If cost is your main driver, then the above media is going to be most cost effective and has a long track record.
Depending on what you are trying to study, the high serum concentration may swamp whatever you are interested in, so you may end up needing to use a modern, low serum commercial formulation, which will be 2-3x more expensive then the simple one desribed above. Personally, I had very good luck with Lifeline Cell Technology cells and media:
http://www.lifelinecelltech.biz/category_s/161.htm
Tech support was very good and the sales reps made life very easy. Prices were very good, but I'm not sure about european distribution. You may want to look up EGM or EGM-2 from Lonza which should be more readily available.
If you need to act fast and go with what you got, I'd lean to DMEM 20% FBS plus L-Glut.
Dear Daniel
I have used HUVEc's taken directly from patients at my hospital from their umbilical cord. They work very well and grow very very fast with DMEM media.
I prepare 50 ml complete media (DMEM) by adding 5 ml of FBS in to 50 ml tube, then 1 ml of Pen/strep solution (Penicilin 10,000 units per ml and Streptomycin 10,000 ug/ml) and then make the volume to 50 ml by adding DMEM.
I got confluency with in 24 to 48 hours after i start culturing in T25/T75 flasks respectively.
Thanks & Best Regards
Thanks very much Sibtain,
I will try your formula and see if they survive. Again, thank you for your answer
Thanks very much for your answer Peter. I need to get them going quickly so I will go for DMEM
I have grown HUVEC for two years using EGM-2 from Lonza. I didn't realize how good this media was until I gave some cells to my colleague in another lab. He tried to use DMEM with 20% FBS yielding very slow growth and bad looking cells. It improved a little after he reduced the FBS conc.n to 10%, i don't know why or how.
Dear Daniel,
Based upon our previous work, we found the following formula works very well:
- M199
- 20% Fetal bovine serum
- Pencilin/Streptomycin 2 mmol/L.
- Amphotericin B 2 mmol/L
- Glutamine 2mmol/L
- HEPES 10 mmol/L
- Endothelial cell growth supplement (ECGs) 30-50 µg/ml
- Heparin 100 µg/ml
Best
Hi
We have been using M199 supplemented with 20% fbs, ecgs and heparin, and it works like a charm!
Hi Daniel,
I am going to use very similar medium for HUVECs. Could you please let me know, if the protocol is suitable for signalling pathways elucidation by detection of protein phosphorylation (in case you have try it with your cells)? Thank you in advance.
Best regards,
Stoyan
Hi Daniel,
Could you please give me details about the media you use, i wil use HUVEC, which M199 you use? I found many different formula, it is red phenol free? Or with earl's salt or without, i will order one so, could you please give me cat #?
Thanks in advance
protocol: Circ Res. 2009;104(5):589-99 Media were purchased by PromoCell, but other brands should also work (e.g. LONZA).
I can only support the answer from Irfan. With the EGM-2 from Lonza you have the best growth. Although it is more expensive than DMEM or others, you get a more specific endothelial population and you can get 2-3 more population doublings, at the end you can profit with this investment in more and better cells.
You can use DMEM or M199 based medium for only several passages, afterward, the cells would change their morphology and tend to stop growing. By the way, HUVEC is very sensitive to FBS concentration. Too low or too high are both harmful.
For HUVEC growth EGM-2 media with supplement kit + 5%FBS are essential, if You want to harvest it is better to use EBM-2 as working media.
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