Help needed! - HUVEC - C cells - RGD targeting using Human Umbilical Endothelial Cell Line - C (Jan/22/2007 )
Hello everyone,
I am doing some research on RGD targeting using Human Umbilical Endothelial Cell Line - C (HUVEC-C).
The growth medium I am is DMEM with 20 % FCS, 0.1 mg/ml heparin, 0.05 mg/ml mitogen (endothelial cell growth supplement) and 1 % Penicillin-streptomycin. The cells grow quite slowly. I am wondering if anyone can give me some tips or suggestions on culturing HUVEC cells.
Also, I heard HUVEC can only be used within 10 passages? I don’t know if this is true? As the HUVEC I got are passage 9 and 14 already.
Thank you for replying to both questions in advance!
JMC2
Dear jmc2,
Primary HUVECS once obtained can be used from P1-P10. However they do change significantly over that period of time, i.e. Prostenoid profile. You need to do 1:2 splits, anything higher than that and the cells will suffer. Make sure also that you split than before full confluency i.e at approx 60-70%. Optimise which Tissue culture plastic is best, they all have different properties.
Your specific HUVEC cell line may have a longer life if they have been virally transformed. Whatever you do you must check that your markers are present at each passage number. Make " Master cell stocks" of your lowest passaged cells.
Good luck.
I am doing some research on RGD targeting using Human Umbilical Endothelial Cell Line - C (HUVEC-C).
The growth medium I am is DMEM with 20 % FCS, 0.1 mg/ml heparin, 0.05 mg/ml mitogen (endothelial cell growth supplement) and 1 % Penicillin-streptomycin. The cells grow quite slowly. I am wondering if anyone can give me some tips or suggestions on culturing HUVEC cells.
Also, I heard HUVEC can only be used within 10 passages? I don’t know if this is true? As the HUVEC I got are passage 9 and 14 already.
Thank you for replying to both questions in advance!
JMC2
jmc2,
The media formulation I use when culturing HUVECCs is:
500 ml F-12k media
50ml (10%) heat inactivated FBS
5ml (1%) NEAA
10ml (2%) PenStrep
15mg endothelial growth factor
They do grow slowly, I usually split once every 6-7 days (1:2) and change the media once after 3-4 days. I usually grow them in a polystyrene flask but I have used collagen I type flasks as well. I find that they're stubborn to get off the flask so I remove the supernatant and wash for ~30 with 1 mM K3 EDTA/PBS, remove, and than trypsinize for no longer than 10mins.
I've had good luck thus far using this media formulation. I've passaged them up to 30 passages before, I don't personally use them for experiements so I can't tell you if they change after a certain number of passages. You will be able to tell when they start to die.