Hello everyone,
We will start culturing HASMCs and HAECs soon in our lab and I am trying to get ready. However, I am confused regarding what type of medium to use. I have done a literature scan and it seems a variety of different media are in use by different researchers. Do you have any experience? What do you use to keep these cells happy?
What I have learned from what I read:
1. HASMCs--> DMEM low glucose (or DMEM/F12 3:1 ratio) + 10% FBS + ascorbic acid + nonessential aminoacids + pen/strep
-DMEM or DMEM/F12?
-What quality of FBS do you use? US origin?
2. HAECs--> Medium 199 (life technologies) + 10% FBS (life technologies) + pen/strep
-What difference would it make to have HEPES in Medium 199?
3. Trypsin (which concentration? and with or without EDTA?)
4. Use DPBS (instead of PBS which has Ca in it)
And also in some papers it is suggested to use collagen or gelatin coated plates to seed these cells on. What is your experience?
Sorry, I asked quite a bit of questions! Thanks very much for your time!
For the HAECs, if you make your own medium, you must add an ECGS (endothelial cell growth supplement) aliquot containing a mixture of growth factors, fresh to each batch of media. These are available from our core and sometimes can be purchased.
Alternatively, you can go with a commercial medium such as Lonza EGM-2 (which comes as a bullet kit) or Vasculife VEGF medium from Lifeline technologies. Both work extremely well in maintaining EC phenotype. I prefer to use accutase for dissociation since it is much gentler than trypsin. Also, I use 0.1% gelatin for coating.
Thanks very much Amogh. If I am to use ECGS, which medium.would you recommend? A chemically defined one or Medium 199 +FBS? Thanks very much again.
If you have a good budget, I will recommend to purchase media from Lonza: for EC - EGM-2 or EGM-2mv and for SMC - SmGM. Lonza usually has several similar media for the same type of cells. When you culture EC, coat the plates with rat tail collagen type I for 30 min at 37C. We use 0.05% Trypsin to harvest cells.
Thank you Dmitry. Unfortunately our budget here is very limited and this is why I am trying to make my own media and to decide the right ingredients. It seems Lonza is quite good, another researcher also recommended it and kindly provided the ingredients of the media supplement. Thanks again.
hello Godze. have you managed to work with Smooth muscle cells? i would like to ask you some informations about growing time from thawing to confluence and form subculturing to confluence for smooth muscle cells? can you give me some tips?
thanks in advance
Hi Luca, I wish I could but the cells we experienced problems with the cells we ordered (very few cells survived each time we thawed a vial even though we followed the exact protocol which was sent by the company and never managed to reach confluency). Now, we are in the process of ordering a new vial of HASMC. Sorry, couldn't be any help.
were they Lonza cells? I normally find myself comfortable with that brand
Actually they were from ATCC. I am also very surprised why such a thing happened.
Hello :) I have been growing HAoECs in PromoCell growth media that comes with a supplement pack - nice and easy and the cells have grown really well no problems! Good luck!
http://www.promocell.com/products/cell-culture-media/media-for-primary-cells/endothelial-cell-media/
Hi Sophie,
I know it is long shot but I want to ask if you use any coating on flask surface. I am having trouble on HAoSMCs. By fibronectin coating I did not had any problem with HAoECs.
Hi Derya, sorry only replying now -
I have never needed to coat my wells or flasks for HAECs or HASMCs which I get from Promocell!
Best wishes,
Sophie
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