NSC-34 Cells sloughing off at media change - (Jan/11/2010 )

Hey fellow lab rats,

I was wondering if I could get your input on a disquieting technical challenge we have been facing as of late!
Lately, our mouse motor neuron-like hybrid cell line has been exhibiting somewhat odd behaviour. After sub-culturing the cells they attach and grow fine until the first media change (2-3d after sub-culturing), at which point those cells in the middle of the well slough off and only those lining the perimeter of the wells remain.

To troubleshoot this issue, we have
1) ensured that media is NOT added directly onto the well, but added to one side, &,
2) attempted to use a different brand of plates ,
but this has not resulted in any progress!


My only other explanation for this aberrant growth is that perhaps these cells have reached the point where they can not be passaged for conducive research purposes anymore!

I would truly appreciate any input that you may have on the issue

Sincerest thanks,

Pierre Zwiegers

The cells aren't grown at too high a density are they?

Have you checked for mycoplasma contamination?

Are these immortalised cells?

bob1 on Jan 11 2010, 03:56 PM said:

Why in the world would you think of mycoplasma contamination? Seems like the default response on this forum these days.

My first thought would be that it is purely technical, i.e. how are you removing the media from the wells? I am assuming with suction but if they are loosely attached, you can literally tear them off of the surface with suction if your technique is not adjusted. For example, if you do not tilt the well GENTLY to one side and SLOWLY aspirate the media from there, you will lose many cells. Many people think it is the adding of the fresh media which is the issue but often times it is the removing of the old media that provides the most stress, particualrly in multi-well plates when suction is used.

Do you actually NEED to change media? If not just leave them alone. There is no hard and fast rules about media changes, sometimes it is counter productive and it can actually be harmful to replace the media too often. I work with many cells that only need a change once a week.

Your point about senescence does not make much sense since you said they "grow fine" until you change media.

Dukey on Feb 24 2010, 05:10 PM said:

Because of this...

zwiegers on Jan 11 2010, 12:20 PM said:

Changes in behaviour (assuming no change in technique) are often caused by contamination, mycoplasma being one of the more cryptic. I have usually found that people don't just jump into neural cultures without experience, so I was assuming some level of expertise. My other thought of primary lines reaching end of lifespan would also be valid.

bob1 on Feb 25 2010, 03:24 PM said:

Assuming expertise in the lab is a rather costly exercise. You have to consider the most likely explanation and to me, it is most likely a technique issue based on the information the OP provided. If the cells fell off on their own then of course something else is going on, but it happens when the OP messes with them. Thats revealing.