The split ratio indicates which portion of cells should be sub-cultured (or transferred) to the new growth vessel. In routine subcloning, you can divide the cells by diluting the cell suspension to an appropriate split ratio without having to count them.
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splitting between two identical culture vessels
Splitting between two different culture vessels
How are split ratio, population doubling (PD) and time to subculture correlated?
At which split ratio is it best to pass the cells?
What do split ratio means?
The split ratio determines which fraction of cells should be subcultured or passed into the new growth vessel.
If the split ratio is 1: 2, then half of the cells in the initial culture must be moved to the new subculture vessel (with a surface equal to that of the starting trypzinized culture vessel); if the split ratio is 1: 3, only one third of the cells from the original culture must be moved to a new culture vessel; if the split ratio is 1: 4, then 1/4 of the cells and so on.
How to do in practice?
splitting between two identical culture vessels
if the culture vessels in which you want to subcultivate the cells have the same surface area of the starting vessel(e.g. you move the cells from a T75 to a T75), it is super easy.
After trypsinization and centrifugation to remove trypsin, the cells are resuspended in a specific X volume of medium.
If it is therefore necessary to divide the cells in a ratio of 1: 2, it will be necessary to move half of this volume X of medium (X / 2, containing half the cells of the original suspension) to the new growth vessel and add cell-free growth medium to reach the volume of medium in which you usually growth your cells.
If it is therefore necessary to divide the cells in a ratio of 1: 4, it will be necessary to move 1/4 of this volume X of cell suspension (X / 4, containing 1/4 of the cells of the original suspension) to the new growth vessel aand add cell-free growth medium to reach the volume of medium in which you usually growth your cells.
and so on…
the formula will be
X = V x S.r.
where it is:
X= volume of cell to be passed in the new vessel
V = volume in which you resuspended the trypzinized cells
S.r. = split ratio (let’s say 1/4 = 0.25, referring to the previous example)
A few examples
Suppose to trypsinize the cell culture growing on a T25 flask and, after centrifugation, resuspend the cells in 5 ml of medium.
To divide the cell suspension 1: 2, you can put half the amount of cell suspension (2.5 ml) in a new T25 and add 2.5 ml of new medium (if you usually put a total of 5 ml of medium in your T25).
What if you had to split your cell with a 1: 3 split ratio?
You can resuspend your cell in, let’s say, 3ml, then put 1 ml in a new T25 flask, adding 4ml of fresh medium to the flask OR you can resuspend your cells in 15ml, then put 5ml of cell suspension (1/3 of the original) in a new flask.
So let’s play a little more:
If you suspend your cells post-trypsinization in 5ml of medium you can split your cell at different ratio
- 1:10; Transfer 0.5 ml of the suspension to a new T25 flask and add the complete support
- 1: 5; Transfer 1 mL of the suspension to a new T25 flask and add the complete support
- 1: 2; Transfer 2.5 ml of suspension to a new T25 flask and add the complete support
Splitting between two different culture vessels
but if the growth support in which you want to pass your cells does NOT have the same area as the starting one, how to do and calculate the correct dilution of the cell suspension?
Let’s start with an example: you want to pass the cells from a T75 to a T 25 with a split ratio of 1: 4.
To do this, it is also necessary to take into account the surface variation which in a T25 is 1/3 of the starting one.
If you switched to a T75,you would simply take 1/4 of the initial volume X of cells, but as I switch to a T25,you also have to divide by the vessel volume reduction factor, i.e. 1/3.
In a nutshell: suppose you resuspend the trypsinized cells in 12 ml, if you wanted to pass them in a T75 with a split ratio of 1: 4, you would take 3ml and you would move them to a t75, instead wanting to pass them in a T25 (which has 1/ 3 of the volume of a T75), then you will only take 1ml of cell suspension and put it in the T25
the formula will be
X = V x S.r. x V.r
where it is:
X= volume of cell to be passed in the new vessel
V = volume in which you resuspended the trypzinized cells
S.r. = split ratio (let’s say 1/4 = 0.25, referring to the previous example)
V.r. = volume ratio (lets say 1/3 = 0.3333, referring to the previous example)
How are split ratio, population doubling (PD) and time to subculture correlated?
the number of PDs that the cell culture will do BEFORE reaching a degree of confluence equal to that of the initial culture, is equal to the square root of the split ratio.
PD=√S.r
- if the split ratio is 1: 2 then the culture will have to double before being split again, so the PD will be one (the cells will have to double once before reaching the same number of cells as the initial culture).
- if it is 1: 4, then the number of cells in culture must double twice (22) before reaching the starting point, so the PD in this case will be 2, i.e. there must be twice the population doubling before the cells reach the same number of cells as the initial culture
- if the split ratio is 1: 8 then the culture will have to triple (23) before reaching the starting point, so the PD in this case will be 2, i.e. there must be twice the population doubling before the cells reach the same number of cells as the initial culture
If, having the growth curve of your cell line, you know what the PD time of the culture is (e.g. suppose it is 24 h, for convenience), you will also know after how much time (hours or days) you will have the same number of cells of the starting trypzinized culture and I’ll have to subculture again your cells.
For example if the split ratio is 1: 2 and the PD time is 24 hours, it means that after 24 hours you will have the same number of cells of the starting trypzinized culture.
If the split ratio is 1: 4, since you will have to wait 2 PD, it means that you will have to wait at least 48 hours or so before you will have the same number of cells of the starting trypzinized culture
…..and so on.
At which split ratio is it best to pass the cells?
In general, you must divide cells in primary culture while maintaining a lower split ratio, which is 1: 2 or 1: 4, compared to immortalized cell lines, which is 1:10 / 1: 100.
Even when you are dealing with a cell line for the first time or with which you have little experience, it is good to keep a lower split ratio (1: 2 or 1: 4), then, as you gain experience, it could be useful increase the division ratio so that less trypsinize the cells.
However, remember that oversplitting can cause premature senescence and death of the cells.
