I am working with the effect of MSCs on the degradation of ECM components especially collagens.I am wondering if the MMPs present in the conditioned medium of MSCs can degrade collagens in an in-vitro setting?
Does anyone have an experience with such an assay?
I was thinking of a fluorescent tagged collagen type 1 in a 96 well plate incubated with conditioned media of various concentration. Do you think the mmps are active? What do you think about TIMPs Co present in the media.
If you would like to detect MMP activities, you could perform a zymogram. Serum used for the culture might have MMPs and TIMPs. In addition, the albumin might decrease efficiency of your test. You need to condition culture medium without serum and use it for the test.
TIMP might be present in the culture medium.
You can do collagen degradation assay in 2d on coverslips. I I use this assay on cancer cells to see there degradation potential and then validate this by using zymography to quantify the MMPs.
Coat the coverslip with a preferred concentration of collagen (1-10 ug/cm2). And fluorescently label it by staining. You could also directly coat fluorescence tagged collagen. Just make sure it is not exposed to light or it would bleach even before you can perform the experiment. Seed the cells and incubate. Fix the cells, stain with phalloidin and then image them. While imaging, compute the area of cell and degraded area.The ratio would give a measure of the degradation of ECM.
http://www.omicsonline.org/extracellular-matrix-density-regulates-extracellular-proteolysis-via-modulation-of-cellular-contractility-2157-2518.S13-003.pdf
Thank you Vandhana. This is very informative. What do you think is the time that cells secreting MMPs would take to degrade collagen when directly plated onto fluorescent collagen?
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