My lab has some mice that we cannot afford to euthanize. We would like to use ear punch biopsies and tail clips for fibroblasts, but I am not sure if there will be enough cells in each to set up a culture. If anyone has had success using ear biopsies and 2-3 mm tail clips for primary cells, could you please share your experience and/or protocols? Thank you!
Hi Swetha
Culturing ear fibroblasts is quite straight forward. Cut off a piece of the ear and wash it for 2-3 minutes with 70% ethanol. Dry and cut the piece of the ear in small pieces and incubate with collagenase (1000U/ml,) in media (DMEM high glucose, 15% DMEM, 1%Glutamax, 1% Pen/Strep) for 30min @37 C. Spin @ 300 g for 5min and discard the supernatant. Resuspend the cell pellet with media and bring it into culture. Split them 1:3 when they reach confluency. After several passages they might immortalize (visible as cell colonies). Good luck
Hi Swetha,
I agree with Philip. In my experience, I've obtained primary cultures from marsupials and monotremes using similar methods, but I didn't even need to use collagenase as the cells just grew out. I'd follow Philip's protocol, adding that we found Amniomax to be an excellent medium for generating explant cultures.
Antibiotics is a must. Once you have enough cells, split off a small well and culture without antibiotics and see when you are infection-free.
Kind regards,
Jason