24 replies to this topic

[Stephan]

After lifting my cells in a culture flask with trypsin/EDTA there are always a number of stubborn cells which will not lift.  I remove all the cells which are in suspension but I feel its a waste to dispose of the flask with many viable cells.  I rinse it out with PBS and then media(with serum) and then I place fresh media in and allow to incubate.  The cells seem to be alright but I'm worried they may end up with something wrong with them at the end of it all.

Does anybody else do this on a regular basis and any other thoughts?

[Penguin]

Stephan, on Jun 1 2009, 11:46 AM, said:

After lifting my cells in a culture flask with trypsin/EDTA there are always a number of stubborn cells which will not lift.  I remove all the cells which are in suspension but I feel its a waste to dispose of the flask with many viable cells.  I rinse it out with PBS and then media(with serum) and then I place fresh media in and allow to incubate.  The cells seem to be alright but I'm worried they may end up with something wrong with them at the end of it all.

Does anybody else do this on a regular basis and any other thoughts?

A post-doc in our lab says he used to select for cells that detach easily by only keeping cells which lift off the plate within the first minute (i.e. those cells expressing fewer cell surface attachment proteins (adherins???).  I guess by keeping a flask going from cells that did not detach you are selecting for cells with more adherins

P

[bob1]

Quite right, you are selecting for cell populations that adhere strongly, meaning that expression of a wide range of genes is altered.  Your cells will be undergoing laboratory selection, and drifting from the parent population (assuming that these are commercial cell lines, or other cell lines used by multiple labs) meaning that other labs may not be able to replicate your results.

[anemone]

We are also re-using culture flasks, but we always wait until all the cells detach from the bottom to prevent unwanted cell selection. Than we resuspend the cells in fresh media, remove part of it and leave the rest to grow.

If you use lower concantration of trypsine you should not be afraid of leaving the cells in it for a longer time to achive complete cell detachment.

This way you can re-use the culture flasks up to 5-times.

A.

[Stephan]

Thanks for the replies - this has definitely shed some light on the issue.  It makes me feel that saving some time and money now may actually end up with using more later and with the risk of creating unreproducible results.

[Dominic]

are flasks THAT expensive you would mess up your results - just to recycle them? (taking into account the price of everthing else used leading to that point)

d

[rhombus]

Dominic, on Jun 12 2009, 01:49 PM, said:

are flasks THAT expensive you would mess up your results - just to recycle them? (taking into account the price of everthing else used leading to that point)

d

Dom,

Wonderful to have you back and talking common sense....your not my long lost brother?

Uncle Rhombus

[Stephan]

rhombus, on Jun 12 2009, 05:47 PM, said:

Dominic, on Jun 12 2009, 01:49 PM, said:

are flasks THAT expensive you would mess up your results - just to recycle them? (taking into account the price of everthing else used leading to that point)

d

Dom,

Wonderful to have you back and talking common sense....your not my long lost brother?

Uncle Rhombus

Well, that all depends on where your funding comes from.  I have to beg, borrow and steal just about everything because of a few expensive purchases in the beginning.
But yea, sure they are not THAT expensive to risk messing my results but I didnt know that until now.

[Dominic]

brother? uncle?

make your mind up man.

d

[Minnie Mouse]

Stephan, on Jun 17 2009, 09:57 AM, said:

rhombus, on Jun 12 2009, 05:47 PM, said:

Dominic, on Jun 12 2009, 01:49 PM, said:

are flasks THAT expensive you would mess up your results - just to recycle them? (taking into account the price of everthing else used leading to that point)

d

Dom,

Wonderful to have you back and talking common sense....your not my long lost brother?

Uncle Rhombus

Well, that all depends on where your funding comes from.  I have to beg, borrow and steal just about everything because of a few expensive purchases in the beginning.
But yea, sure they are not THAT expensive to risk messing my results but I didnt know that until now.

I got the same problem during my PhD because I didn't have a sponsor to pay for my expensive PhD training cost   .  
I had to re-use the flasks for 5-10 times...not a good idea...but I did not have enough money to do experiments.

I know Uncle Rhombus will laugh at me.  

[THE ROCK]

Hi dear, you can use culture flask 4-5 times unless you devoid trypsin from it. Either wash the flask using PBS or Culture medium, then add cell s.

[Dominic]

how do you devoid trysin?

pbs wont get rid of previous inhabitants, nor will culture media

not sure what your on about rock

[HMG]

Stephan, on Jun 1 2009, 05:16 PM, said:

After lifting my cells in a culture flask with trypsin/EDTA there are always a number of stubborn cells which will not lift.  I remove all the cells which are in suspension but I feel its a waste to dispose of the flask with many viable cells.  I rinse it out with PBS and then media(with serum) and then I place fresh media in and allow to incubate.  The cells seem to be alright but I'm worried they may end up with something wrong with them at the end of it all.

Does anybody else do this on a regular basis and any other thoughts?

ya,we regularly reuse culture dishes upto 3 times.....there is no problem....but rinse properly

[Buddy]

If I wash the plate well with PBS, can I reuse 6 well plates?

[bob1]

THE ROCK, on Jul 10 2009, 06:59 AM, said:

Hi dear, you can use culture flask 4-5 times unless you devoid trypsin from it. Either wash the flask using PBS or Culture medium, then add cell s.

HMG, on Sep 9 2009, 09:46 AM, said:

ya,we regularly reuse culture dishes upto 3 times.....there is no problem....but rinse properly

Buddy, on Dec 6 2009, 04:53 PM, said:

If I wash the plate well with PBS, can I reuse 6 well plates?

If you all read the above posts you will see that you are in error... re-using is a problem, if you do it you are jeopardizing you results through a number of factors including cellular damage, cell type drift (lab evolution), and general contamination issues.

Be better scientists!