I am trying to culture mouse embryonic stem cells in 3D chitosan hydrogel but can't harvest cells from hydrogel in order to do some assays like cell count or cell viability,etc. is there any effective method to detach cells from hydrogel without damaging them?
How about using dispase to incubate with the small blocks of reconstructed tissues? In my opinion, the isolation of cells from 3D engineered tissues shares the similar method with from in vivo tissues. Dispase or collagenase is a good choice to isolate from tissues. By the way, the cell viability could be determine by Alamar blue assay even in 3D tissues. Hopefully, it can help you.
Hello
Articles here will help you
Good luck
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3963751/
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0059482
If your hydrogel is porous, definitely the cells will penetrate deeply within the pore network. If you are not worried about the hydrogel structure, you can crash the hydrogel under ambient temperature to release the cells and quantify the cell number in the supernatant.
Another way is to trypsinize the cells, so that the cells are released rapidly into the medium.
Hope I was helpful. Thank you
Dear Dr. Gopal Shankar K, many thanks for your answer. how long should I keep my chitosan-based hydrogels in ambient temperature in order to release the cells efficiently?
As I told you, if you do not care about the hydrogel structure after the experiment, crush your hydrogel at 37 degree in order to release the cells and try also sonication under sterile conditions. After 5 minutes, I hope your cells will escape for the hydrogel due to mechanical disturbance and start to survive in the medium. Now you can quantify the cells in the media by MTT or alamar blue assay.
Remember to have respective controls to see the difference
Dear Dr. Gopal Shankar K, many thanks for your help. I won't need my hydrogel after cell detachment but I'm concerning about cell safety while crushing the hydrogel. how can I crush it without crushing the viable cells?
In case of cell viability assay, use the protocol of MTT assay. You can refer to the below link
https://www.ncbi.nlm.nih.gov/pubmed/27836656
You could to use a Lysozyme to biodegrade chitosan blocks without degrade yours cells.