I have porcine chondrocytes and they are in short term culture in 24 well plates. Normally we would use accutase and collagenase to detach this type of cell. However, in 24 well plates the cells just do not want to come of the plastic. This happens regardless of how long they are in the incubator for. Any suggestions of how I can detach them with out completely destroying them, as I need to use them for cell counting, are welcome.
Depending from which tissue you are isollating the chondrocytes, you might need to change your enzymes solution to target the specific collagens and/or glycans produced by them. I used to work also with porcine chondrocytes (isolated from articular cartilage), but used trypsin/EDTA solution to detach the cells with high yield.
Hi
I am also working with human knee meniscus primary cells and encounters same problem. So what I used to do is, Initially for few minutes (not more than 2 minutes) the cells are treated with trypsin/EDTA and wash out the cells and again repeat the same process. Twice or thrice same procedure is followed.
The chondrocytes adherence are depend on the many factors, one among them is the serum concentration. In our lab we use the low serum media (3 % FBS). Additionally, for the cell detachment we have been using the trypsin/EDTA 0.25 % for 3 Mins. That works fine.
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