Is this contamintion? - (Jun/06/2007 )

Hi,

I grow MDA-MB-468 cells in DMEM plus 10% FBS.
First, the cells look OK, but after one passage,
there are floating cells and debris in the medium.
It looks like cells dont grow well and eventually
all die in several days.
First I thought there were just cell debris in the
medium, but they are small, round, almost transparent,
and look like particles. They dont move at all.
They are distributed in the medium evenly, but there
is no turbidity.
I changed media and serum, and cleaned up the
incubator, but same thing persists with another frozen
stock.
I still havent even decided if they are organisms or just
inpurities in serum.
I think the cells were just fine at my previous lab several
months ago.
Do you have some ideas?

sometimes it is normal to have some debris or dead cells or debris of dead cells.
everytime we passage (by trypsinization or scraper) not all the cells will survive (maybe due to the process of detaching cells or centrifugation), so some cells will not attach the next day.
second possibility is your serum. serums always has some particles/debris in it. it is normal and do not need to worry about it. it scares me the first time when i saw something floating in media after adding FBS.
third, your cells start to divide and the small round shape cells are the ''children''.

to check if it is really contamination, take few ml of your culture/culture media and plate them on a agar plate. any agar for bacteria is OK. normally i used LB agar as it is available in my lab. if something grow (bacteria) the next day... discard your cells. if not, congrats!!! the debris might not harmful to your cells.
if you suspect your media/FBS contaminated, use the same method to check if any bact colony grow.
for safety purpose, carry out this contamination check once in a while.
you can also buy a mycoplasma contamination check kit to check your culture...

good luck!!!

actually, this may be contamination; sources could be the stock of cells, trypsin/EDTA solution, medium, FCS, culture dishes, pipettes, flow, incubator etc.

so you have to re-start with definite exclusion of any contamination;

to varify contamination, try a culture with penicillin and streptomycin added to the cell culture medium

It also could be that your cells need a few passages (2-3) to recover. could in your case this be the reason of this phenomenon?!

Take a sample from the medium and put it in an Agarplate... Incubate for a while and see if you have colonies.

D

I spread 100 ul of media on LB plate w/o antibiotics and put it at 37C.
The plate was clear the next day.
The cells continues to die. The number of debris (?) is increasing, but
the medium is not turbid.
I checked the incubator again. The temperature, humidity, and CO2
concentration (I checked with Fyrite) seem OK. MCF-7 cells in the same
incubator are doing OK.
So, I am not conclusive at the moment. It might be mycoplasma (I havent
checked it yet). It can be media as Almasy says (some guys in my previous
lab grow the cells in DMEM in CO2 incubator. Nobody in the present lab use
this cell line) or the lot of serum.
Now I am also trying other breast cell lines. Thanks guys, Ill let you know if
I find something.

slab

Your cells may be too old to work with too... As I understand, they never recover. So you may want to check passage number at witch cells have been frozen.

The story after the story.
On June 11, there were very few living cells.
I passaged the cells 1:1, this time in RPMI with
10% FBS.
To my surprise, the cells began to grow about 10
days later.
Now the cells look "normal" I checked expression
of some proteins and their expression pattern was
similar to that I checked before.
I don't know the reason.
Perhaps damage to the cells at freezing and thawing
might slow the growth, or RPMI was a better medium
than DMEM for the cells.
Other reasons suggested by you all are also possible.

slab

glad to hear that..... good luck!!