difficulty in passaging HK-2 cells - (Nov/14/2011 )
I am using HK-2 cells (human kidney proximal tubule immortalized cells) and I am finding it extremely hard to detach them from the surface of the flask. I am using low glucose DMEM, 10% FBS, 1 Penn strep for their maintenance as mentioned in several papers. I have tried washing with 1 ml trypsin prior to washing with PBS, incubation at 37C for 10 minutes, pipetting vigorously with trypsin. But nothing really works. Does anyone have an experience of handling these cells or any other suggestions?
When you say you wash with trypsin prior to PBS, is that a mistake? Do you mean you wash with PBS prior to adding 1ml trypsin?
What concentration of trypsin are you using? The 1ml, what size flask are you adding that too?
Are you letting them grow to 100% before splitting (in my experience, using a different cell type to you though, cells are much harder to lift if they are confluent or over confluent).
According to the atcc entry for these cells, you should not let them reach 100% confluent (sub at 80%) and trypsin should work just fine though.
Hi leelee, thanks for reply. I am washing with trypsin prior to PBS just to neutralize residual media because then it inactivates a fraction of trypsin. I am using 0.05% trypsin and using 3.5 ml for T75 flask for trypsinization.
Confluency can be a possible reason. I will give a try using little less confluent cells. Thanks again!
Ah ok, I'm with you- you wash with trypsin, then PBS, then add your trypsin to lift the cells.
Hope the less confluent cells are more co-operative for you
Just a couple of tips.
Try different plastics........they all have surfaces with individual characteristics. The cells will adhere differently to each surface, hopefully one will be less adherent than another.
Passage the cells at a low density.
Try increasing the concentration of Trypsin
Increase the number of PBS washes.
Hope one of these suggestions helps.
Thanks a lot uncle rhombus. I will try different plastics. Increasing PBS washes has definitely helped. Also I started smacking the flask on fist and trypsinizing immediately. Altogether it's working now.