I'm trying to recellularize 0.5 cm2 of decellularized colon mucosa with colorectal cancer cell line HT-29 and HCT-15. These cell line survive after seeding 1 day and then die. Any suggestion? Any protocol?
Edoardo,
What have you done in terms of cleaning up the residual mucosa? There may still be some DNA contamination left over from the original decellularization which will require additional treatment with DNase. I also suggest using a Ca, Mg PBS to do your washing to stabilize the mucosa.
Hope this helps,
Jamie
Thanks Jamie!
Actually, when i stain the recellularized section with DAPI I observ a moderate amount of DNA debris remnant.
In your opinion, could this fact influence the ability of cells to adhere to the matrix or triggering a sort of cell death (due to DNA debris captation by the cell)?
Thanks again for your suggestion...
No problem :)
Yes I think that will definitely cause a problem for your HT-29 and HCT-15 cells. This may not be the reason you are experiencing such a dramatic loss of cells after seeding but it may be a good place to start. After ECM/ Mucosa extraction I use a solution of 10ug/ml DNase I in PBS containing calcium and magnesium to clear residual DNA. This can be done at 37C for 30min (I usually stick the plates back in the incubator). Then 2x wash with Ca/Mg PBS and the plates can be stored or used right away.
I can send you the full decellularization protocol if you are interested.
Jamie
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