5 replies to this topic

[Prateek]

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

[Irene G]

Hi!
I don't culture keratinocytes but I think you might ask Lonza for the optimal culture conditions for this cells.
Good luck
Irene

[bob1]

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.

[Prateek]

Irene G, on 27 May 2011 - 01:31 AM, 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.

[Prateek]

bob1, on 27 May 2011 - 04:05 PM, 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.

[bob1]

It all depends on the concentration of the trypsin and the exact protocol that is used as to whether 6 ml is too much.