Failure in cell detachment after trypsinization - (Nov/12/2008 )
Dear all,
I am routinely using trypsin to harvest my cells, but it happens that a few cell lines are unable to detached after trypsin incubation and tapping of the flasks, and situation didn't turn better even i prolonged the trypsin incubation time. Typically i will add 1ml of trypsin per 25cm2 flasks, and incubate them for at least 8 mins. It's problematic as i can only retrieve approximately less than 10% of cells from the flask, it minimized the amount of substances to be extracted out. Is there anyone having similar experience and kindly give me some advices?
Thanks in advance
I know that with some cell lines- primary keratinocytes for example, if the cells are more than 80% confluent then the trypsinization will not detach all cells. It could be a similar problem you're having. Make sure your cells are not too confluent before trypsinizing.
Or you might switch to a more rude approach and scratch the cells off.
It's really pleased to have such fast responses.
Actually i forgot to mention what's happening after what i encountered. The subcultured cells tensed to have more difficulties to detach from the bottom of the flask, no matter how many times i passaged it out later.
Will it be that once they became undetachable, the situation will be last forever?
I've heard of passaging the cell line too many times will cause the cell reluctant to detach, is it true?
I haven't experienced this myself, but I have heard of it.
Do you have earlier passaged cells frozen down?
What cell line are you culturing?
They are tongue epithelial cell line culture from tumoric tissue cells. Due to an early accident, we've only got hihger passage number cell lines left, and they all behave like this.
What brand of flask are you using?
You may wish to culture this cell line using another brand of flask.
I am routinely using trypsin to harvest my cells, but it happens that a few cell lines are unable to detached after trypsin incubation and tapping of the flasks, and situation didn't turn better even i prolonged the trypsin incubation time. Typically i will add 1ml of trypsin per 25cm2 flasks, and incubate them for at least 8 mins. It's problematic as i can only retrieve approximately less than 10% of cells from the flask, it minimized the amount of substances to be extracted out. Is there anyone having similar experience and kindly give me some advices?
Thanks in advance
WOOOOOOOOOOOOOOOw, 8 minutes for trypsinisation.........that's far too long. Things to try:
As suggested by another poster, try a different flask manufacturer.....all of them produce different plastics with different adherence capabilities for different cells....this is usually done when optising your cell maintenance and growth conditions.
Try washing the cells first with PBS (w/o divalent cations) maybe 3/4 times....THEN wash again with an EDTA solution.
Definetely INCREASE the volume of Trypsin solution you put onto the cells.....BUT DO NOT INCREASE THE CONCENTRATION.
Try incubating the cells at 37oC when trypsinising the cells.
As stated by someone else .......ALWAY passage your cells when they are SUB-CONFLUENT....many cells when they reach confluence are far more difficult to trypsinise.
Are the cells able to grow in SUSPENSION.....this then means that no washing/trypsinisation is required.
Hope one of these suggestions helps you out
Kindest regards
Rhombus
Thanks for all the suggestions. I will try my best to solve the problem and report the how things going later.