I am working on reprogramming fibroblasts to neural stem cells. After the NSCs induction, I am feeling it difficult to detach the cells from the surface. Even after incubating to 0.25% trypsin/Accutase for around 10 mins, the cells are not lifting off the surface. Only ~50% of the cells can be harvested and toxicity is seen because of the exposure time to the enzymes. I have also tried tapping the dish but it's not working. I can see some fibre-like structure around the cells which I think is ECM and that is inhibiting trypsin activity.
Hello Rishabh Deo Singh
You may try cell dissociation reagents that are specific for extracellular matrix (ECM) substrates such as ECM-specific collagenase, elastase and dispase.
Please refer to the links below for more information on these products.
https://www.sigmaaldrich.com/IN/en/products/cell-culture-and-analysis/cell-culture-supplements-and-reagents/cell-dissociation-reagents
https://www.sigmaaldrich.com/IN/en/technical-documents/technical-article/research-and-disease-areas/cell-signaling/collagenase-guide
Best.
Dear Malcolm Nobre,
Thanks for your suggestion. I will try using collagenase.
Hi,
Try using a cell lifter to lift off the adherent cells from the dish.
I agree with Malcolm Nobre's answer. Those reagents are what I use to work with cells that produce a lot of ECM. Sometimes, I add in DNAse as well.
I find that collagenase solution tends to deactivate after a day or so of use, so I tend to aliquot and freeze it when I make the solution from powder and use each aliquot once/dissociation. Include calcium ions in the solution you use to dissolve the collagenase.
And this may be silly/obvious, but are you washing your plate with PBS or a trypsin wash before adding the trypsin? The cell media can inactivate trypsin.
Hi Shravani! Thanks for your suggestion. Yes, I am washing the cells twice with PBS. I also read that for cells releasing high ECM, it is recommended to incubate in PBS for 10-15 mins before trypsinisation. I am yet to try that.
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