Hello!
I am just starting research in the filed of vasculogenesis and angiogenesis.
I have isolated huvec cells from umbilical vein several times.
I learned that during isolation, serum free media(m199) without antibiotics
should be used.
I want to know reason why I should use 0% m199 without
antibiotics during extraction of huvec.
And at the last step of huvec isolation, I suck up the supertenant after centrifugation and resuspend the pellet with 20% m199 containing antibiotics
and then start culturing resuspended pellet in cell dish washed with 0% m199 containing antibiotics.
At this step, is it okay to use media(Both 0% and 20% m199) containing antibiotics?
Thank you!
Any answers?
Hello
see this links and attachment :
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2884313/
http://4designbiosciences.com/pubs/Nakatsu%20and%20Hughes%20(MIE).pdf
Hope help you , Good Luck
Dear Saleh Alkarim,
Thank you for your lilnks and attachment.
These will be great information for me.
But What I exactly asked was about the isolation of huvec. The details of my questions are described above.
If you can answer my questions, i will be deeply appreciated. Thank you and happy new year!
Hi Lee,
the reason why you should use serum free media during HUVEC isolation is related to the inhibition of collagenase. As you surly applied collagenase digestion by cord cannulation, during this step many inhibitors contained in the serum would be compromise the enzymatic digestion and consequently the recovery of HUVECs. Nonetheless after that, cell suspension should be cultured in serum free media containing VEGF. Serum free media means that insulin, transferrin and selenium as well as growth factors are added chemically, not by serum supplementation. We do not use m199, check it for the serum substitutes. Hope this would be useful. If you have further queries, please do not esitate.
good luck
Simone
Hello Simone
Thank you for your reply!
I learned a lot from your answer.
But I always use m199 with every work related with huvec.
So as I have mentioned above,
I use 0% m199 without antibiotics during huvec isolation
and after that,
I use 20% m199 with antibiotics for resuspending pellet
because I have to culture the resuspended pellet in cell dish.
You mean that are there any problems with my use of m199?
I will be deeply appreciated for your answer
and thank you again for your reply.
Dear Lee,
We routinely use EGM-2 for HUEC culture, this medium is a reduce serum medium (2%) rich in VEGF, with antibiotics, specifically formulate for these cells. we also get better results applying Attachment Factor (AF) from Lifetech instead of Gelatin as coating proteins. However, I don't think that you'll have problem culturing in M199 (20%). I found a paper comparing EGM-2 and M199 confirming a higher proliferation rates in EGM-2, but you could get HUVECs in M199 too, take a look to
doi: 10.1016/j.tice.2011.03.004 ( http://www.ncbi.nlm.nih.gov/pubmed/21511321 )
Write me back if you want more details on our protocol.
Best wishes
Simone
Dear Simone
Thank you very much!
It is kind of you not only to answer but also propose additional questions to me.
Thanks again.
If I have any questions, I will contact you.
I am just a young researcher so I don't have much knowledge and experience
but I also want to help you if you need.
Best wishes
Dear simone,
Can I ask you about huvec split?
When I split huvec, I use pipet aid and suction tip.
I think you know quite well why such tools are required.
I am always cautious about changing pipet aid before pouring new fresh 20% m199
because trypsin left on old pipet aid should not be mixed with fresh media.
But it seems that i forgot chaning suction tip(pipet tip anchored at the end of suction tool) during whole huvec split process.
I am concerned about inappropriate mix between materials(0% m199 for washing, 20% m199 and trypsin) because I did not change suction tip as mentioned above.
Can you give me advice about this?
I think that suctioning is just removing materials from cell dish so
no change of suction tip is not a problem but I want to make sure about it.
Thank you
Best wishes
Hey Lee,
I'm not sure if I understand your question, guess you're talcking about pepetting technique during media change and HUVEC harvesting. As you did, normally you should use new tips any times you have to drain fresh medium from its bottle. This is mainly to avoid bacteria or fungi contamination, to which cultures are expose during the culture time, and preserve sterile fresh media. (do not trust so much in the antibiotics). Working with highly proliferative cells, not changing the tip would expose also to cross-contamination, when living ad proliferating cells are transferred into different cultures. This happen usually working with cell lines. I think that trypsin contamination of fresh medium is not a real problem, first because it will be diluted many times, second because trypsin activity is strongly inhibited by serum.
Hope this could answer to your question.
Don't worry, troubleshooting is the best way to get experience.
all the best,
SImone
Dear simone,
You have been great helper to me.
I am very impressed for your kindness and knowledge.
But what I exactly asked was about not the draining medium from bottle,
but whether changing suction tip should be required during huvec works(huvec split, media change, etc)
It seems that I have bothered you too much although you wanted to help me.
So I will ask same question again not personally but publicly in this site.
Thank you again and I will be appreciated again if you answered my question agian.
But I want you to do your job first because I don't want to bother you with too many personal questions or requiremets.
Thank you again.
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