3T3 Swiss albino fibroblasts - Help needed (May/31/2006 )

Dear All,
I am working with the 3T3 swiss albino fibroblasts. I have noticed a problem with my culture. Since the first seeding, I have noticed that at every seeding stage there are lot of dead cells in my flasks. I know these cells are contact inhibitant. But still after each trypsinisation i find lot of dead cells. This has been a problem from the time I had these cells from ATCC. Can any body tell me whether this is a common things with these cell lines and how does it grow after seeding? I mean they become confluent easily or the growth rate is not that great. Because I found them growing slow and more over I find many dead cells. Am I doing something wrong? or its is a cell behavior problem. I asked with the ATCC tech dept they said the dead cells could be because of trypsin. I will change it and see if it helps. Meanwhile if any one can give me some tips or suggestions, it would be helpful.

Thanks,
Naveen

Hi Naveen

I have never worked with these cell lines but generally, a lot of cells die when there is contamination. Generally, there is change in cell morphology, slow growth rate and cell death associated with contamination. The original culture might be contaminated. The literature regarding growth rate etc. comes with the culture or you can look in the web.

Wish you good luck

There are always some dead cells in the culture flasks. And with trypsinisation, u are physically manipulating the cells so u will see more dead cells.

Some cells grow slowly so check with ATCC abt their growth rate. Also check if you are using the right medium and if has all the components (FBS, L-Gln, etc)

Dear Maashu and scolix,
Thanks for your replies....Infact if the cells were contaminant, it should grow and all the cells should die..right? But that was not the case..with me...the first time when I received them from ATCC and after seeding them in 25 cm2 flask I had lot of dead cells the next day. I changed the media and put in fresh and cells grew well. I passaged it twice until i seeded them on to 150 cm2 flask. But at each trypnisation step I had plently of dead cells again, but they used to grow well...later....when i added fresh media. And I am using the media recommended by ATCC. I have just taken a stocked sample out and grown it...it grows well, but the same problem of dead cells in each growth step persits. This actually reduces my cell count and more over they grow slowly as more time is required to attain confluency. I dont know what the other reason could be. They are contact inhibitant. But so many dead cells, i have no idea. Its not contamination i guess, because then the cells should not grow only....but they do grow...can you guys have any other insight about this problem?...anyways thanks for all your helpful suggestions...

hi naveenACT,

i never use trypsin with these cells, but instead scrap in medium, gently dissociate by pipeting and pass them 10-20 dil.
i use DMEM w/ genta and 10% FCS, nothing else
i have checked for mycoplasma contamination when cells grown lesser and in a doubt, treated them with Plasmocin for 2w
i always be carefull the cells not reach 100% confluence