When we talk about “splitting” cells, we’re not talking about ripping cells open or anything - instead, we are taking a bunch of cells that are growing too close for comfort and splitting them up into a larger area/volume (such as multiple flasks or dishes or at least bigger ones). This allows them to continue growing and multiplying without running out of room or food.
Splitting is also referred to as sub-culturing or, more commonly, passaging. And when we add the cells to a new growth vessel, we call it “seeding.” It’s easy to do this for suspension cells (which we can simply dilute or if we prefer we can spin them down in the centrifuge, remove the old media, and add fresh media first). For adherent cells (cells growing stuck to the surface of a growth vessel such as a dish or flask) we first have to physically separate them from that surface (and from one another), typically by using trypsin and EDTA. More on this later.
We determine how much to seed based on “counting cells” (determining how concentrated the cells are in a sample and using that to calculate how many cells we have) and calculating to seed an amount that will give us a particular “seeding density” (e.g 0.1 million cells per mL or 3 million cells per 10 cm dish) or by diluting using seeding ratios (e.g 1:2 would be diluting by half by adding 1 part cell solution to 1 part new media).
The optimal seeding densities and ratios will depend on the cell type (since different cell types have different doubling times and min/max density allowances) and your plans for them. The lower you split them, the longer it will take for them to multiply back to their starting density. So if you want to use them soon, you’ll want to split them high (e.g 1:2) but if you don’t need them for a while, you’re just maintaining a stock and don’t want to have to deal with passaging them, you’ll want to split them low (e.g 1:10). Look up the recommended ranges for the cell type you’re working with!
Each time we split cells, we up the passage number for those cells. So, for example, if we start with a fresh stock of cells and we split/passage/subculture it once we have a passage number of 1, which we often write P1. In a few days (depending on the seeding density), we will need to passage it again. This will be the cells’ second passage, so we will write “P2” in our notes and on our flask/dish. Next time will be P3 etc. etc.
It’s important to keep track of the passage number because the cells continuously acquire mutations that could affect their growth and/or impact your experimental results. Therefore, even if you’re dealing with a “continuous”, “immortal” cell line, which theoretically could keep growing and dividing forever, you want to keep your passage number low and go back and start with a new stock vial if you get too high up.
This is especially true for “finite” cell lines, such as primary cell lines, which are mortal. They can’t grow forever. Instead, after a certain number of “population doublings” (PD), where, on average, each cell in the culture has divided once, they stop growing (senesce). Because this limit is related to the PD (how many times the cells are multiplying) rather than the actual passaging (how many times you are divvying up the cells) it can be more helpful to keep track of the PD for such cell lines.
Even if the cells aren’t senescing yet, they are still undergoing changes. Often, for primary cell lines, which have a distinct cellular identity (e.g a specific type of heart or brain cell) they randomly acquire mutations that make it easier to grow in cell culture but make them less like the original tissue they came from. Since these mutations provide a growth advantage, these cells get selected for and overtake the more distinctive cells. So it’s important to monitor the cells closely with a careful eye for morphological (shape) changes, etc. and to keep the passage number low. Unfortunately, for finite cell lines, early mutations are often the most drastic, as the cells adapt to growing outside their usual home. For continuous cell lines, they already have tons of mutations that enable them to grow readily so the further mutations they acquire often have less of an impact.
Regardless of cell type, a key thing to remember is that mutations occur randomly (and only then can they be selected for based on fitness advantages allowing them to “outcompete” their neighbors). Different populations of cells from the same original cell source will therefore have different combinations of mutations. The closer you are to the original cells, the less time there’s been to diverge though so by staying at a low passage number you can reduce the variability.
We’ve talked about too much passaging as being “bad,” but passaging is crucial for the cells to survive.
How do we know when to split cells? It’s best to split when the cells are in the late “log phase” of growth.
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