After thawing; attached then detached - (Aug/13/2006 )

Hi,

Need some help!!

I'm working with a fishliver cell line (plhc-1) with epithelia morphology.
In culture they are very stable, survive 'everything'.
This summer I have thawing cells from our N2 tank over and over again..... tryed different serum, different percent serum, centrifuged to remove DMSO and not removed DMSO until they attached..

My problem is: They attach and everything looks fine but then they start to come lose and I'm losing cells every day.
I know that the Freezing and thawing is difficult steps...but they are alive after thawing and then starts to die..

Any suggestions?? Thanks in advance!

How are you thawing the cell lines? Do you think your cell lines could've got contaminated? You are removing the DMSO from the cell lines fairly quickly right? Do you think you might need something to help them attach? Cell lines do tend to be stressed after the thawing process ... so maybe you aren't giving them enough time to resettle and to begin to proliferate.

Just some thoughts I had ...

It's odd that the cells attach then start to lift later on. As you say, they must be viable if they're attaching and you seem confident you've removed the DMSO (thaw quickly and remove DMSO as soon as possible).

Are you sure the media you're using for culturing is OK (all additives, nothing missing?). Does the pH in your cultures look fine? I gather other cells are growing in your incubator so that's not the problem.

It's a long shot but you could try using a different type of culture vessel. I had a bad experience many years ago with a batch of flasks that were defective. Everything was OK if cells were plated at very high density but very little attachment was observed in primary cultures and cultures subcultured at low density.

I'm probably most suspicious of your media or the incubator environment (temp and pH). Either that or the freezing process has not been very efficient and your cells, although viable, are extremely stressed. Ensure the density of your cells is fairly high following plating - it might help get them established.

Thanks to both of you for good advices!
I've added antibiotics to the cells and now testing for mycoplasma with a PCR kit.
I realise I probably need new fresh cells anyway but I really want to know whats wrong.