Splitting (aka passaging, subculturing) cells - what, why, & how

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.
blog form: bit.ly/passagi...
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.
finished in comments

[@IsaacColbert]

I work in the upstream MFG department of a gene therapy site and this information is very accurate. Glad you’re teaching others!

[@thebumblingbiochemist]

Great to hear. Thanks!

[@thebumblingbiochemist]

Basically, although we can talk of “doubling time” (which will differ for different cell lines, often is about 24 h for some of the continuous ones) the growth rate isn’t constant. Instead, it will start slow as the cells settle down, establish connections to the dish, work out some hormonal signaling and only then can actually “feel comfortable enough” to divide. Once they do, however, they’ll start growing rapidly, at a “logarithmic” rate where the doubling time is constant. But then they’ll slow down again because they will start running out of resources and they’ll be swimming in cell waste and stuff, so the growth will plateau. You want to split them before that plateau. How do you know? For adherent cells, this is when the cells are about 80% confluent, meaning that cells cover about 80% of the growth area. For suspension cells, you just have to sample and count, then compare to the published guidelines for the cell type. Now for some practical details Suspension cells are nice in that you can monitor their growth without actually having to passage them. A downside is you have to actually open up the flask and take a sample rather than just looking at a closed plate, so you’ll need to sterilize your hood and everything and you risk contaminating your culture every time you open the flask. But you can check the cell count without committing yourself to passaging them. With adherent cells, on the other hand, since you’re actually removing all the cells from their home, rather than just taking a small sample, you have to split them (or waste them). Speaking of “waste” - since the cells grow so readily we often end up with more than we need. So we often just seed a portion of what we collect after trypsinization (and maybe share with labmates). Speaking of trypsinization, I talk about it in depth in another post, linked to below, but here’s an overview. Trypsin is a protease (protein cutter) that cuts the connections adherent cells have to one another and the surface. Many of these connections are mediated by calcium-dependent proteins called cadherins. EDTA is a chelator (metal binder) that hides calcium ions (charged particles) from these proteins, weakening the connections and making trypsin’s job easier. Then, once trypsin’s done its job, we inhibit it by adding growth media (which naturally contains trypsin inhibitors). much more here: bit.ly/trypsin_edta; KZbin: kzbin.info/www/bejne/ZnPKeIWBmbuCjK8   but typically, the protocol is something like this:  - check cells under the microscope to see if they look like they need a passaging  - aspirate (suck off) old media  - wash with DPBS  - aspirate DPBS  - add enough trypsin/EDTA to coat surface (often a 0.05% solution)  - stick in incubator  - check in a couple min to see if detached (hold up to light and see if they’re still stuck or flowing nicely)  - check every 30s or so until they are  - as soon as they’re all detached, add media (about twice as much as you added trypsin/EDTA)  - pipet out the cell/trypsin/edta/media mix into a falcon tube & centrifuge (I typically do 1000g 5 min)  - aspirate trypsin/edta/media mix  - resuspend in smaller volume fresh media  Once we’ve collected our cells (or simply by sucking up a small amount of suspension cells) we can then determine how concentrated our cells are using a hemocytometer (a microscope slide with a grid to help you count) or an automated cell counter such as a “Countess” machine. If we want to know how many cells we have in total, we just multiply that concentration by the volume. 
Then we just have to calculate, dilute, and plate where required. Regardless of when we think they’ll be ready, the cells will be ready when they think so! So it’s good to check your cells visually every day. This also helps you make sure they’re not contaminated or anything. more on cryopreserving cell stocks: bit.ly/cryopreserving_cells KZbin: kzbin.info/www/bejne/hILbgICfmNWmn5o    Some helpful resources:  Useful numbers for cell culture, ThermoFisher Scientific/Gibco: www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/cell-culture-protocols/cell-culture-useful-numbers.html   Cell line passage numbers explained, UK Health Security Agency: www.culturecollections.org.uk/technical/cell-line-passage-numbers-explained.aspx Mammalian cell tissue culture techniques protocol, abeam: www.abcam.com/protocols/mammalian-cell-tissue-culture-techniques-protocol   Subculturing Adherent Cells, ThermoFisher Scientific/Gibco: www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/cell-culture-protocols/subculturing-adherent-cells.html    Cell Dissociation Protocol using Trypsin, Millipore Sigma: www.sigmaaldrich.com/US/en/technical-documents/protocol/cell-culture-and-cell-culture-analysis/mammalian-cell-culture/cell-dissociation-with-trypsin     more on HEK293 cells: blog form: bit.ly/hekcells ; KZbin: kzbin.info/www/bejne/qn3XfICsj5eta7c      more about culture media: blog: bit.ly/cell_culture_media ; KZbin: kzbin.info/www/bejne/bniuf2lmf56geZY   more cell culture posts: bit.ly/cell_culture      more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 bit.ly/2OllAB0 or search blog: thebumblingbiochemist.com

[@paulineaugust]

Amazing! Discover your channel today and I am loving it! Just started to work with cells and your content is super informative! Thank you :)

[@thebumblingbiochemist]

Thanks so much! Good luck with your work!

[@Jasbindarkaur]

Thank you so much for these videos, they are extremely helpful!

[@thebumblingbiochemist]

Glad to hear it!

[@lexiedrueco9268]

Thank you so much for the helpful information! I'm currently waiting form my scholarship outcome, I just took a leap of faith without any background in cell culture/passaging, and angiogenesis study. I'm just a medical laboratory scientist, so this field is quite new to me. Again, thank you! Hoping to see more videos :D Question: If it says that I need to passage 3 times, does it mean i have to do everything again for 3 times?

[@thebumblingbiochemist]

Best of luck with the scholarship! Yes, if it says to passage 3 times, you need to do the whole process 3 times (split, grow, split, grow, split, grow). Good luck!

[@BongiCekuse]

So helpful. Could you please help with how to know when to freeze your cells, do you freeze at 80%?

[@thebumblingbiochemist]

bit.ly/cryopreserving_cells KZbin: kzbin.info/www/bejne/hILbgICfmNWmn5o

[@iot3136]

❤❤❤

[@BongiCekuse]

Im new to TC and this is very helpful. I have a question, say I want to subculture 500000 cells in a T75 flask and from my cell suspension I use 100ul, will the amount of cells stay the same no matter how much media I add in the flask? For instance 100ul cs + 15ml fresh media (in a flask) and 100ul cs + 10ml fresh media (in a dish) will they both have 500000 cells?

[@thebumblingbiochemist]

Glad to hear! Yes - you will at least start with the same number but they might then grow differently

[@Nada-KatarinaKasic]

Thank you for the calculation! Can you please clarify how did you get .35 viable/ml for 70mL media? You are saving me in the lab

[@thebumblingbiochemist]

Good 'ole C1V1 = C2V2! bit.ly/c1v1equalsc2v2 Happy I could help!

[@preeti97rox]

can you suggest any book for passage number, seeding density, maintenance of neuronal cell lines?

[@thebumblingbiochemist]

I don't know sorry!

[@marlaelianaladeragomez2235]

Thank you for the video! I have a question about what is the passage number that you should record. For example, I have a frozen vial in passage 16, now if I thaw it, is the first passage passage 1 or passage 17?

[@thebumblingbiochemist]

It would be passage 16 when you thaw it

[@marlaladera5038]

@@thebumblingbiochemist ok, so when they ask you in an assay to be in low passages, are they referring to the first passages from when the cell line was established or the first passages after thawing? How do we keep the cells in low passages? Thank youu

[@thebumblingbiochemist]

you typically freeze stock vials when you are at an early passage number and can then thaw a new vial to return to a lower passage number which I assume is what you are referring to for that method. hope that helps

[@marlaladera5038]

@@thebumblingbiochemist perfect, thank you