I am trying to induce angiogenesis in Adult Bovine aortic endothelial cells using Matrigel at a concentration of 37.5ug/ml. I wanted to confirm if the cells are actually showing lumen formation. I am attaching the pictures and would appreciate any suggestions or comments.
Would like to know how did you come up with matrigel 37.5 ug/ml? Usually people will mix matrigel with either culture medium or buffer in a ratio of 1:2 to 1:10. Tube length, area of tubular structure, or branch point counting are three commonly used method to quantify in vitro angiogenesis. The cells in your pictures look very unhappy and numerous intracytoplasmic vesicles can be ssen. To me they did not form appropriate tubular structure. The only way you can tell whether "lumen" is formed or not will be a cross-sectional cut and visulaize under electron microscope.
Dear Shambhavi,
The endothelial cells have not differentiated well into definitive tubes here clearly.You need to trouble shoot with the health of your cells and the appropriate passage for the endothelial cells you are using.The cytoplasmis vesicles are quite big here which clearly tells that the cells arent healthy at all.
I recommend that you use neat matrigel without any dilution or you can try a 1:1 dilution if required.BD biosciences recommends not to go below the 3mg/ml concentration.You are way below the limit.
All the best
Charan Thej
Stempeutics Research
Even though this assay is usually called a 'tube formation' assay, cells will only connect with eachother with the 'branches' that they form (which can be presented in the ways that Ru-Jeng mentioned), but will not form a lumen. Lumen formation requires (among other things) blood flow.
I agree with Ru-Jeng that these cells don't look all too happy to me, and would try and optimize your setup until you find that your cells really form a network structure.
Dear Shambhavi,
There are few references that could help to get the development of capillary tubes with lumen formation in vitro :
Folkman, J. & Haudenschild, C. (1980) Angiogenesis in vitro. Nature, 288, 551
Montesano, R. & Orci, L. (1985) Tumor-promoting phorbol esters induce angiogenesis in vitro. Cell, 42, 469
Montesano, R. & Orci, L. (1987) Phorbol esters induce angiogenesis in vitro from large-vessel endothelial cells. J Cell Physiol, 130, 284
Containing a troubleshooting table:
Arnaoutova, I. & Kleinman, H.K. (2010) In vitro angiogenesis: endothelial cell tube formation on gelled basement membrane extract. Nat Protoc, 5, 628
With my best wishes
Josette
While understanding the utility of the in vitro approach, it is clear that the process of lumen formation in culture is not the same as that seen in vivo, so you need to be careful not to over-interpret your data!
see: Egginton, S., Gerritsen, M. (2003). Lumen formation: in vivo vs. in vitro observations. Microcirculation 10: 45-61
Best wishes,
Stuart Egginton
University of Leeds
Thank you everyone for your suggestions. We bumped the concentration of matrigel to 8mg/ml and it appears that there might be angiogenesis taking place. However, my control cells still show some unnatural morphology. I am attaching the pictures and would appreciate any comments or suggestions. Thanks again.
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