Oh my cell culture problems never cease!!! - (Jun/14/2011 )

I use CHO cells which are stably transfected with myoglobin gene

When i freeze these cells down, i used media w/ 10% FBS and 10% DMSO

When i thaw them again to re-use the cells, they take like forever to attach in growth media. I'm using a 150cm flask with 30ml media. My cells even otherwise take a long time to adhere, and generally are known to adhere overnight. It seems to be their characteristic property. But when i thaw out a fresh vial of cells and seed them, they take close to 36hrs... there are so many cells floating even after 24hrs. Its such a waste of time esp. with a PI who wants results overnight. Is this normal? Do freshly thawed cells take way long to adhere as opposed to cells that were split?

Another thing... what's the fastest easiest way to determine doubling time of cells? I think its ~14h but i don't know

sk

I have never used CHO so I can't say about the adhering thing.

However, I can comment that the easiest way to estimate doubling time is to seed a known number of cells in several wells and harvest at time points (say 24, 48, 72 hours) and count the number of cells you get (I would do live and dead counts for a bit of extra information), then plot a graph of cell count over time.

it is very normal that it takes longer time for cells which was frozen and thawed to adhere comparing to just split.

Frozen cells are like sleeping, so it takes a while for them to wake up.

Also, certain number of cell will die for freeze/thaw cycle no matter how careful you are,
so it is recommended for you to check the cell number and viability every-time you thaw a new vial
and culture them in flask with appropriate size depending on the viable cell number.

It might be a good idea to look for mycoplasma contamination since that could in some instances affect cell attachment.

Suhas on Tue Jun 14 13:06:14 2011 said:

Dear Suhas,

Just a couple of things:-

Parental CHO's should attach to the plastic within 10 minutes. However it maybe that by tranfecting them has changed this parameter.

Transfecting cells also can affect the "viability" of the cells....i.e. you might do a trypan exclusion on them and get 99% viable count....HOWEVER this is a very insensitive way of measuring viability and they may in fact be "suffering". The result of this suffering being a longer attachment time and also a longer population doubling time.

The freezing of the cells is VERY IMPORTANT. Try and use a controlled rate freezer that will take the cells down by 1degrees Celsuis/minute. If you cannot use one of them use a "Mr Frosty" from Nalgene (I think). You will always get cells that do not survive the freezing process....and this will generally increase when you transfect them.

If I read your post correctly you thaw your cells into a T150 cm flask.......This is very odd as when I freeze my cells into a 1.5ml cryovial, I have the cell suspension at 1 million cells/ml i.e. 1.5 million cells. Then I use a T25 for this initial seed. It is always better to have too many cells than too few when you first initiate your cells from frozen......they like "Neighbours".

Lastly in my experience PI/Group Leaders have no idea about growing times and the technical complexity of setting up and doing experiments.....generally because it has been decades since they did it last. They always want results yesterday and it is a skill you will learn....in educating them towards "reality".


Hope you find this useful.

Kindest regards.

Uncle Rhombus.