Cell Clumping Problem - Cells Stick to Eachother and Not the Flask (Nov/20/2008 )
Hi Everyone!
I was hoping someone would be able to offer me some suggestions to reduce cell clumping following trypsinization.
I'm working on a primary culture of cells from fish gills.
The cells grow quite well initially in flasks for several days and detach from flasks quite easily with trypsin (0.5% with 5.3mM EDTA; 2 rounds of trypsinization x 2 min each, washing with PBS in between). I trypsinate these flasks when they are about 80% confluent. I inactivate the trypsin by pouring the cells into PBS + 10% FBS, and then spin down the cells (500g for 8 min) and resuspend in media (L-15) with 10% FBS. When I re-seed these cells in flasks, sometimes they adhere to the flask and grow quite well, but most of the time they just clump together rather than attaching to the flasks.
Does anyone have any idea why the clumping happens?
Is my trypsination procedure damaging the cells?
I appreciate any suggestions!
Many Thanks! 
Why do you do the repeat trypsinisation thing? I would trypsinise them once.
You could try the ATCC method of trypsin treatment: Wash monolayer in PBS, add trypsin and coat bottom of flask by tilting, then wash off trypsin with a minimal volume of PBS and wait for cells to detach.
Usually clumping is caused by trypsin treating for too long. Could shorter treatments work?
You could try the ATCC method of trypsin treatment: Wash monolayer in PBS, add trypsin and coat bottom of flask by tilting, then wash off trypsin with a minimal volume of PBS and wait for cells to detach.
Usually clumping is caused by trypsin treating for too long. Could shorter treatments work?
Hi bob1,
I repeated the trypsinization because 2 min is not enough time for all of the cells to detach and I was taught that several short rounds of trypsinization is less damaging than one long round of trypsinization. I'm fairly new to cell culture, so based on experience, I have no idea if this is true.
Thank you so much for your suggestion. I will certainly give the ATCC protocol a try.
While checking the ATCC website, I also found this tip: "To avoid clumping do not agitate the cells by hitting or shaking the flask while
waiting for the cells to detach."
I must admit, sometimes I got impatient and vigorously tapped the bottom of the flasks. I will stop doing this now.
Thanks again!