Hi,

1. For effective decellularization of  your scaffold you may use a trypsin based approach (protocol in link 1, page 7) which is also commonly used. Alternatively you may consider papain based approaches since trypsin could be sometimes harsh on the cells. For further options, you may see a comparison of decellulization approaches (attached pdf file). However, please not that effective decellularization depends on enzyme type and may also have some influence on cell viability and complete decellulization might not be fully achieved and might not be the best way to estimate numbers (in my opinion).

2. You may do an LDH assay as an alternative to Cyquant, since cyquant might sometimes count in non-viable cell population (since these cells also contain DNA). LDH can be released only from viable cells. Comparison of the measured LDH signal from lysed cells with the control LDH data from known numbers of lysed cell (standard series) enables exact estimation of the cell number on scaffolds at different time points 

http://epubs.rcsi.ie/cgi/viewcontent.cgi?article=1054&context=anatart

Dear Shreyas,

Thanks for the answer! However, since I'm interested in cell harvest from the scaffolds, rather than in decellularization, I can only use reagents that don't kill cells, such as papain. So the question is for how long and with what concentration should I use it? I work woth 0.75-1 % w/v collagen scaffolds.

Thanks,

Dear Alexandra,

You can use DAPI staining instead or along with other methods for confirmation of other data. you choose 10 fields and count cells, if you want to compare cell viability of some groups together. please be aware and don not use TritonXE100. Decelularization techniques more time damage to cells.

Hi Sasha,

Have you tried protease digest for 1 hour followed by 8-10 hrs collagenase digest then cell strain the digest? we tend to soften our cartilage with protease then do an overnight collagenase digest obviously the scaffold will not need so long but it might well be worth using a protease 1 hr prior to collagenase?

Richard