cell culture split ratios. - (Oct/22/2010 )

Hi all,
i was wondering if someone could explain me how to split cells accoring to split ratios for adeherent cells. it is pretty straight forward for suspension cells: count your cells and dilute them in fresh media acc. to ur split ratio but with adeherents can we do the same..?? because every body resuspends their cells in diff volumes.
hope i have asked my question clearly. and it would also be helpful know if there are different ways of doing this.

thank you all in advance
k.sagi

k.sagi on Fri Oct 22 12:25:59 2010 said:

Adherent cells Splitting:

Adherent cells can be split in many ways. Trypsinize the cells, dilute by adding more growth medium to the cells. Then:

1) count the cells and split by cell numbers – like 1 X10^6 cells total in a T75 flask or 0.5 X 10^5 cells total in T75 flask
2) number of cells / area (cm2) For example: 1 X 10^5 cells/cm2 = for a T75 it would be 75 X10^5 or 7.5 X10^6 cells total (we did this for human embryonic stem cells)
3) By Splitting ratios: This is the easiest of all. Just prepare the new T75 flasks by adding 12ml or 15 ml of fresh growth medium and label the flasks.
Re-suspend the cells after trypsinizing, into 10 ml – then:
1:4 split = add 2.5ml of cell suspension to the labeled T75 flask with medium
1:5 split = add 2ml of cell suspension
1:20 split = add 0.5ml of cell suspension
and so on. If you want to do 1:12, the re-suspended cell volume should be made to a total of 12 ml and then work out your ratio. If you want to save medium, then re-suspend in 5ml final volume and
1:4 split = add 1.25ml of cell suspension to the labeled T75 flask with medium
1:5 split = add 1ml of cell suspension
1:20 split = add 0.25ml of cell suspension (I guess the volume need not be very accurate for routine splitting / cell maintenance)

k.sagi on Fri Oct 22 12:25:59 2010 said:

Dear k.sagi,

Split ratios are very important in order to maintain optimum growth and viability of cultured cells. In general primary cells have a lower split ratio i.e. 1:2 / 1:4 , when compared to immortalised cell lines i.e. 1:10 / 1:300.

If you are growing HUVECS for example you would use a split ratio of 1:3. This means that if you have a 80% sub-confluent T25cm flask, this would be trypsinised and passaged to give rise to 3 x T25cm flask's. It is very easy as long as you know the split ratio in the first place. If you over split the cells then this can cause early senescence and death.

Hope you find this useful

Kindest regards

Uncle Rhombus