Hello eveyone,
I am trying to establish a primary myoblast culture from zebrafish but my cells does not attach after 1st passage. I am trying to establish tissue explant culture since I do not have enough tissue to start with the enzymatic way. Cells proliferate from the tissue but they do not survive after 1st passage. I am using matrigel as coating material and I have tried trypsinization, cell scraper and accutase in order to detach the cells but they did not efficiently attached after all. What can I change other than these?
A few suggestions that might resolve the issue,
Increase the matrigel concentration to 50ug/cm2
Decrease the concentration of Trypsin to 0.05% or use EDTA instead of trypsin.
Make sure your tissue culture dishes are Tissue Culture Treated.
Your media should contain 20% serum, Chick Embryo Extract, and bFGF for these cells to do well.
Lowering trypsin concentration to 0,05% or using trpLE could help if the problem is the passage (cells should be round and clear after trypsin, if not they are stressed). You could also try saving and re-using some of the old medium or a few explants in order not to loose media conditioning all of a sudden.
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