Stable cell line stops growing :( - (Jan/16/2007 )

Dear all,

I have established a HepG2 stable cell line harboring a promoter luciferase construct. I used co-transfection, that is, I transfected HepG2 cell with the promoter luciferase vector and pcDNA3.1. Everything were going alright in the past four months. The cell grew nicely under G418 selection and I have performed several experiments using the cell line, but strange thing happened two week ago. The cell did not grow after my last passage!!!!!!! The cells were floating in the medium, only a few of them attached. I have thawed my stock and same thing happened. Now, I can't move further and probably have to start again. It is so frustrating that my four month work is ruined.

Has anyone of you experienced this? What are the reason of this? Is the cell contaminated by mycoplasma?

Please advice. Thanks.

Charles

Is it your gene inserted was so cytotoxic that the cells were killed?
Some cells may grow slower after transfection.
I don't think there is mycroplasma contamination in your case, mycroplasma doesn't kill cells suddenly but progressively.
If there is too many floating cells, it maybe the time of trypsinization is too long or they die during passaging.

Regarding to the problem of cell death after thawing new stock, it maybe due to the problem of freezing cells. The cells easily die with inappropriate freezing~~

I hope it may help^^

How many passages can a stable cell line survive usually? My stable cell line is at passage 10 counting from clone harveting. I wonder if my promoter+luciferase construct may make the cell suffer.

Regarding freezing, how much serum is needed when freezing stable cell line? 10%, 20% or more?

Please advice

Charles

I don't know for how many passages the cells can still survive~~
For me, the cells can be used for about a month, the cells still survive but the gene seems to be lost.
I don't think the promoter+luciferase construct make your cells die.

For the cell freezing process, I usually use the normal % of serum as I maintain the cell culture, e.g. 10%. Usually, 5%-10% DMSO is also added to the cells.

I dont think luciferase is leathal to HepG2 cells. I think you maybe late at one time in passing these cells. HepG 2 line is quite hardy and they dont just die for no reason. Sometimes, low seeding density also affect their growth.

Regarding freezing cells, I usually use normal complete medium plus 10% DMSO. Slowly freeze cells (-20C for 4-6 hrs, -80C overnight and then to LN2), thaw cells fast (in 37C bath). It works well for me.

I work with primary and stable cancer cell lines / For frozen I use 90% serum+ 10% DMSO. Freeze cells in foam plastic ( tight to your tube) at -80 for 24 hours then LN2. Work well