To break cell clumps - suspension culture related (Dec/11/2009 )

Hi all,

I'm currently working on Lu165 cell line (suspension cells). However, the cells form huge clumps which is very difficult to break even if I pipette up and down vigorously for about 10 times. This brings to the question of difficulty in cell counting and subculturing as single cell suspension is hard to achieve. And I noticed, those plates with lots of cell clumps enters quiescent soon and the cells die off after that.

Do I add any chemicals to break the clumps? If so, what's the concentration and for how long?

Also another silly question - how to remove cell debris efficiently with suspension culture? I tried to centrifuge and aspirate the media but I don't think that works really well. The cell debris gets more and more as the passage number increase. I could be wrong - but will that affect the cells? It gets pretty scaring when you thought the cells is contaminated when you mistaken the cell debris as some microbes! LOL.

Any suggestions would be useful. Thanks~

wia84 on Dec 11 2009, 07:37 PM said:

Hi all,

I'm currently working on Lu165 cell line (suspension cells). However, the cells form huge clumps which is very difficult to break even if I pipette up and down vigorously for about 10 times. This brings to the question of difficulty in cell counting and subculturing as single cell suspension is hard to achieve. And I noticed, those plates with lots of cell clumps enters quiescent soon and the cells die off after that.

Do I add any chemicals to break the clumps? If so, what's the concentration and for how long?

Also another silly question - how to remove cell debris efficiently with suspension culture? I tried to centrifuge and aspirate the media but I don't think that works really well. The cell debris gets more and more as the passage number increase. I could be wrong - but will that affect the cells? It gets pretty scaring when you thought the cells is contaminated when you mistaken the cell debris as some microbes! LOL.

Any suggestions would be useful. Thanks~

Dr Teeth on Dec 14 2009, 07:38 PM said:

wia84 on Dec 11 2009, 07:37 PM said:

Hi all,

I'm currently working on Lu165 cell line (suspension cells). However, the cells form huge clumps which is very difficult to break even if I pipette up and down vigorously for about 10 times. This brings to the question of difficulty in cell counting and subculturing as single cell suspension is hard to achieve. And I noticed, those plates with lots of cell clumps enters quiescent soon and the cells die off after that.

Do I add any chemicals to break the clumps? If so, what's the concentration and for how long?

Also another silly question - how to remove cell debris efficiently with suspension culture? I tried to centrifuge and aspirate the media but I don't think that works really well. The cell debris gets more and more as the passage number increase. I could be wrong - but will that affect the cells? It gets pretty scaring when you thought the cells is contaminated when you mistaken the cell debris as some microbes! LOL.

Any suggestions would be useful. Thanks~

Try adding EDTA to a final concentration of 5 mM, before triturating your cells. You can them run your cell suspension through a 40 uM nylon cell strainer (depending on your cell size). Falcon makes some (REF 352340) that fit on top of a 50 ml tube and will not allow cell clumps to pass.

rhombus on Dec 15 2009, 03:23 AM said:

ChrisHarris on Dec 16 2009, 10:57 AM said: