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Cornea Epithelial Cell culture - (Mar/30/2006 )


Does anyone has a working protocol on how to obtain/culture rabbit cornea epithelial cells from rabbit whole eyes?

I have tried to soak dissected rabbit corneas in Dispase II (0.5% in PBS) for 1hr at 37degC as described in some papers, (in which they also mention they could lift off the epithelial cells!), but i couldn't get any sheets of cells that I could 'easily' lift off!



I know that it has been quite sometime since you put up your post, but in case you have not solved your problem, I will give you a briefing of what I do.
2mg/ml Dispase II made up in 10% DMEM (with antibiotics). Cut out the cornea into several pieces and place in the Dispases II solution at 37C for 2 hours (10ml in a petri).
After 2 hours place each piece in, say a 6 well plate with about 3ml PBS. Hold the tissue with a forcep and scrap the epithelium off with another forcep. Once this is done, collect the PBS, spin down at 1000rpm for 5 mins, resuspend in 5ml of the culture media and transfer in a T25 along with i3T3s.
Just did it 2 weeks back for the first time. Worked fine!