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; RUclips: ruclips.net/video/0BgGRKdVOVM/видео.html 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 RUclips: ruclips.net/video/NQxOMicpsi8/видео.html 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
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 ; RUclips: ruclips.net/video/tLtKMvZ2z5U/видео.html
more about culture media: blog: bit.ly/cell_culture_media ; RUclips: ruclips.net/video/8GKN60J9mC4/видео.html 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
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?
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!
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?
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 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
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
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; RUclips: ruclips.net/video/0BgGRKdVOVM/видео.html
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 RUclips: ruclips.net/video/NQxOMicpsi8/видео.html
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 ; RUclips: ruclips.net/video/tLtKMvZ2z5U/видео.html
more about culture media: blog: bit.ly/cell_culture_media ; RUclips: ruclips.net/video/8GKN60J9mC4/видео.html
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
I work in the upstream MFG department of a gene therapy site and this information is very accurate. Glad you’re teaching others!
Great to hear. Thanks!
I am working with HEK293 cells rn and your cell culture videos are really helpful for budding Researcher. Good work!
So glad to hear. Best of luck!
Amazing! Discover your channel today and I am loving it! Just started to work with cells and your content is super informative! Thank you :)
Thanks so much! Good luck with your work!
Thank you so much for these videos, they are extremely helpful!
Glad to hear it!
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?
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!
So helpful. Could you please help with how to know when to freeze your cells, do you freeze at 80%?
bit.ly/cryopreserving_cells RUclips: ruclips.net/video/NQxOMicpsi8/видео.html
can you suggest any book for passage number, seeding density, maintenance of neuronal cell lines?
I don't know sorry!
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?
Glad to hear! Yes - you will at least start with the same number but they might then grow differently
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
Good 'ole C1V1 = C2V2! bit.ly/c1v1equalsc2v2 Happy I could help!
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?
It would be passage 16 when you thaw it
@@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
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
@@thebumblingbiochemist perfect, thank you
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