My lab has tried several variations of cell extraction buffers to remove cells and leave an Extracellular Matrix (ECM). We tried NH4OH with 3 pre-washes with PBS and 5 post washes with H2O. We tried PBS+ 0.5% Tritonx-100+ 20mMNH4OH, and every time the ECM comes off. We grow NIH 3T3 fibroblast in 12 well plates coated with 0.1% porcine gelatin. Even with the coated plates the ECMs still come off. We tried reducing the amount of days of ECM deposition to see if that would help but so far we are getting the same results. Any insight would help! thanks
Hi
Our lab uses the following formula for three days without problem (on Fibroblasts).
0.5% Triton-X 100, 20 mM NH4OH.
I recommend to add 50 ug/ml ascorbic acid to increase ECM synthesis and coat the culture plate with fibronectin before cell culture. Both improve the ECM adhesion to dish.
Good luck
Hello,
Some of my colleagues also work on decellularized ECM from fibroblasts here. They also use macromolecular crowding, that is the use of macromolecules in the media which prevent the diffusion of the ECM components, thus promoting the deposition and assembling. I leave you a good review on that here: Article Macromolecular Crowding Meets Tissue Engineering by Self-Ass...
Also, as indicated previously, the addition of coating (fibronectin, poly-L-lysine) and ascorbic acid or growth factors can enhance the deposition of ECM. As well the oxigen tension can promote this (i.e. 2% O2).
Regarding the decellularization itself, non-ionic or zwitterionic detergents together with hypotonic solutions are the best bet (as you are using).
Hope it helps,
Regards
Hi Omar, you may try to culture your cells in larger cell culture dishes. We use almost the same protocol than you in 60 mm and 100 mm Petri dishes and don't get your issue. Good luck
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