Keratinocyte Primary Culture - Cells not growing after 3rd passaging (May/25/2011 )
I am new to cell culture and have just started my PhD. I am working on Keratinocyte and am facing problem in expanding the primary cells. I ordered cells from Lonza Biosciences and they claim the cells have the minimum capacity of 15 doubling populations. But just after second passaging (third if you count the one done by the company) my cells are not proliferating. Can anyone please help me in finding the reason behind it?
Prateek
Hi!
I don't culture keratinocytes but I think you might ask Lonza for the optimal culture conditions for this cells.
Good luck
Irene
You are probably letting them get too confluent, try splitting them when they are 70-80% or less confluent. Make sure that you don't split them too hard either, primary cells do not like to be at too low a density either.
Irene G on Fri May 27 09:31:58 2011 said:
Hi!
I don't culture keratinocytes but I think you might ask Lonza for the optimal culture conditions for this cells.
Good luck
Irene
I am using the Lonza KGM media. I guess the problem lies somewhere else.
bob1 on Sat May 28 00:05:45 2011 said:
You are probably letting them get too confluent, try splitting them when they are 70-80% or less confluent. Make sure that you don't split them too hard either, primary cells do not like to be at too low a density either.
Hi Bob,
I split the cells at 70-80% confluent as suggested by the company protocol. But They advise to use 6 ml of trypsin for T-75 flask. Is this amount too much or is this okay for keratinocyte? For fibroblast I use 1ml for T-75.
It all depends on the concentration of the trypsin and the exact protocol that is used as to whether 6 ml is too much.