I'd like to create a cell line from a tissue. Until now, I remove with trypsin the cells from the tissue and culture them. Also I keep the tissue pieces in petri dishes. In the culture I use RPMI 10% FBS + Pen/Strep. But unfortunately i need many weeks to have a cell line. If anyone has an alternative protocol, please send it to me.
In my preparations (Heart, Muscle & Brain), I used collagenase B and dispase combination for 30-45 min at 37oC. You can also try the same combination. May be trypsin was too strong for your preparation.
I use Collagenase type II for 30 minutes first and then collagenase dispase for 15 minutes (both in agitation at 37°) to have a primary cells culture from skeletal muscle. I pre-plate in non collagenated dishes for 45 minutes two times and then i pass the cells in collagenated dishes. With this protocol in few days i have enough cells to use...I don't know actually if this protocol can be used for other tissues!
I also think trypsin could be too strong, I use collagenase type 2 for my explant of vascular smooth muscle cells.
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