Can anyone give me some suggestion about how to make HUVEC spheroid sprouting out? I can make HUVEC spheroid well. But after seeded into the collagen, there are only outgrowth of HUVEC cells but no sprouting. The rat tail collogen gel was solidified well. What is the problem?
Dear Chen,
Firstly do you have a positive control for angiogenesis using the sheroids?
Do you have any proangiogenic cytokine promoting endothelial tube formation representing neoangiogenesis?
An appropriate thickness of the collagen is required for efficient sprouting.
In the absence of a stimuli which promotes endothelial cell differentiation,and the presence of only a proliferation stimuli may give this sort of a result where the cells just proliferate but do not differentiate into tubes.You may want to relook into the angiogenic promoter you are looking at.
All the best!!
Thank you! I did have positive control by applying 30ng/mL VEGF. But it is just the same in the negative control and positive control. I added 500uL of collagen gel into each well of 24 well plate. I hope that the thickness is enough in this case.
Did you try this with other HUVEC lines?You could also try this using cytodex beads which can be loaded with endothelial cells and then perform a sprouting assay.Also try using another lot of collagen.VEGF is a very potent inducer of angiogenesis and i do not see the reason being the cytokine for the assay not working well.
You may want to try it using matrigel as well instead of collagen as collagen is a better ECM compared to collagen for in-vitro angiogenesis.
if you wish to send your test items to a commercial vendor providing an angiogenesis assay service based on the HUVEC sprouting assay you are interested in, please check this website: http://www.proqinase.com/content/view/62
(Cell-based angiogenesis assay offered by ProQinase)
I suppose I have solved this problem recently. The collagen concentration should be increased to least 1.5mg/mL.
Can you tell me according to which protocol you are preparing the spheroids?
Can you please provide me your protocol. I am also performing the same experiment.
Try our protocol (HUVEC sprouting)Article A cost-effective method of (atelo)collagen type 1/3 (COL1/3)...
I would not recommend to use Matrigel since it is a heterogenic mixture of various bioactive proteins from mouse tumors. To have more control over the system, I would suggest to use a collagen gel and VEGF.
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