Passage/freezing T-cell line calculation problems - (Jan/10/2010 )

Hi,
I have a specific questions on how to do the calculations and procedure for passage and freezing of non-adherent immortalized T cells. I have them in 25-ml flasks and I need to do the following:
Passage at concentration of 10e5 cells/ml in 10-ml flasks
Freeze back at concentration of 50x10e5 cells/ml (1ml cells in 1ml media).
I have the cell count for two of them as an example: A = 30 x 10e5 c/ml and B = 5 x 10e5 c/ml

I keep getting confused as to how to get the final concentrations. I know the formula Vol final x concentration final = vol initial x concentration initial. And I can solve for the initial volume I need to add, but I don't really understand what that volume means. I don't know if I am supposed to spin down the cells and at what volume to resuspend them in. I know this is a basic concept for technique, but I can't figure out all of the steps accurately, so I would appreciate if someone could explain how to do this in a step-by-step manner.

Thank you for your time!

T-cells are suspension cells right? (I don't use them myself)

Note you may find it easier to use units you are familiar with like 50 x10⁵ = 5x10⁶ or 5 million.
Also note that in the Ci x Vi= Cf x Vf formula each side is the same as working out "n" in n=c x v. The former formula can be substituted into my examples below, but I have broken it down for ease of comprehension.

If so - you have a culture of cells growing in a volume of liquid.

Collect the liquid and cells into a tube, take an aliquot for counting (20-100 ul). Remember the volume of cells that you have (e.g. 20 ml)

Count the aliquot. (you gave 30 x10⁵ cells per ml in your example; also make sure your numbers are LIVE cells, don't count the dead as they play no part in your culture)

so you have:
n=c x v
n=30 x10⁵ cells/ml x 20 ml
n=6x10⁷ cells total (60 million)

For passage:
Dilute all the cells to 500,000 cells/ml and aliquot out into new flasks, which will give you a ton of flasks and use a ton of medium too. Using the Ci x Vi etc formula, the Vf number you got is the number of ml you will have finally. Subtract the initial volume you have (20 ml in my example) and the resulting number is the amount of medium you will need to use.

OR

work out how many cells you need for a flask (e.g. 500,000 cells/ml x 20 ml = 10,000,000 cells) and from that work out the volume required from the original culture:
v=n/c
v=10,000,000 cells/3,000,000 cells/ml
v=3.333 ml
Take 16.667 ml of medium, add it to a new flask and then add 3.333 ml of cell suspension.


For freezing:
Spin the cells down and resuspend at 5 million cells per ml in your freezing mix using the total cell number. This will give lots of frozen tubes (60 million cells/5million cells per ml = 12 tubes), assuming 1 ml/tube.

OR

Work out how many ml of frozen cells you want (e.g. 4) and work out how many cells you need from the original cell suspension concentration:
n=v x c
n=4 ml x 5 million cells/ml
n=20 million cells.

you have 3 million cells/ml:
v=n/c
v=20 million cells/3 million cells/ml
v= 6.667 ml
Take 6.667 ml of your original culture into a tube, spin down and then add 4 ml of freezing mix.

Thanks, that's really helpful. I just have a follow-up about freezing the cells. If I have a lot of samples (like 14), then it seems difficult to spin down different volumes to get each at the appropriate concentration for freezing. Since they are all at a different cell count, they wouldn't balance out and therefore couldn't all spin at the same time (it seems trivial, but it takes time when dealing with many samples). Would there be a calculation if I were to spin down 10ml of each sample (for example) to determine how much media to resuspend in to have the correct final concentration? I would need to find the amount to resuspend in to have the correct concentration of 5x10e6 cells/ml and add 1ml of resuspended cells to 1ml of freezing media (since media is at 2x concentration). I hope that made sense.

Thanks again!

You need to count your cells....

Use "number of cells =volume x concentration" (i.e. 10 ml x the number you get from counting in cells/ml)

spin down

Resuspend in appropriate volume to give 5 x 10⁶ cells/ml using v= n/c (i.e. volume = number of cells from first calulation/5 x 10⁶ cells/ml)