Hi, I'm wondering if anyone can help?
I work with carp hepatocyte primary cell cultures: I isolate the hepatocytes using a protocol well established in this lab, which basically involves in situ perfusion of the liver via the aerteria coelica, and a 2-step perfusion method with collagenase D (Roche).
I have been doing this for around a year, and has worked perfectly 95% of the time, getting between 3 and 9 plates of viable cells each fish...
HOWEVER, recently, the last 12-13 fish, everything appears to be working fine, until I come to the low-speed centrifugation of the cell suspension - which should result in a nice pellet of hepatocytes, but recently I am getting a VERY low yield - in some cases hardly any pellet - and as the pellet is so small, it is not easily seperated from all the other debris, so not really useable at all. THis is getting really frustrating and just wondered if anyone has any advice?? Or experienced similar problems?
I haven't changed any part of the protcol so am really baffled! The one thing I thought it might be is that I started to use a new batch of collagenase D just before this started happening - could this be the problem? Perhaps it is a 'dodgy batch'... although I have to stress that the liver appears visually as it should do after the correct digestion time - I can monitor the digestion by eye, and so surely would be able to tell if it had been overdigested.... ?