Harvesting cell culture - basics and practical tips

It’s always harvest season in the lab! Because “harvest” is a term we use to describe separating cells we’re growing in cell culture from the media (liquid food) we’re growing them in and keeping the cells to do fun things with. Here’s the basics of how we can harvest cells - both suspension cells (cells growing suspended in liquid, often shaking in an Erlenmeyer flask) - and adherent cells (cells growing while stuck to a plate or dish).
blog: bit.ly/harvesting_cells
For suspension cells, we typically centrifuge them to pellet out the cells, pour off the media, optionally wash them by resuspending them in clean liquids often PBS), then freeze the pellet or resuspend it in the buffer we plan to later lyse them in, containing protease inhibitors to protect the stuff that will come out of the cells when they lyse (break open).
note: You need to be much gentler with animal cells than bacterial ones. Bacterial cells have more protection, thanks to their cell walls, but animal cells are delicate, so take care to avoid premature lysis. It’s a good thing bacteria can take more stress because we have to use higher centrifugation speeds to pellet them out since they’re smaller than animal cells. Typically we use ~4000-5000 x g to spin down bacterial cells & ~100-1000 x g to spin down mammalian cells (use the low end (100-500) if you want the cells to stay alive, higher end when you’re just trying to harvest them to lyse or something and would rather have the firmer pellet and better chance to make sure you collect all the cells).
more on centrifugation: blog: bit.ly/lab_centrifuges ; CZcams: • Working with laborator...  
more on pelleting: bit.ly/pelleting_practical ; CZcams:    • Pelleting tips and tricks
For adherent cells, we have to get them off the dish - but first we take advantage of the fact that they’re stuck… We can simply aspirate (suck off) the media, rinse them with PBS, suck that off, then, now that they’re clean, scrape them off or use an enzymatic method like trypsinization to sever their connections to the dish and to other cells.
more on trypsinization: blog: bit.ly/trypsin_edta ; CZcams: • Cell detachment with t...  
more on cryopreserving cell stocks: blog form: bit.ly/cryopreserving_cells CZcams: • Cryopreserving cell st...  
more posts on cell culture: bit.ly/cell_culture
more on osmosis and tonicity: blog form: bit.ly/osmolarityandmore ; CZcams: • Osmolarity, tonicity, ...
more on cell culture media: blog: bit.ly/cell_culture_media ; CZcams: • Mammalian cell culture...
more (hopefully) helpful random practical lab tips & tricks: bit.ly/lab_tricks_page    
  
more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 bit.ly/2OllAB0 or search blog: thebumblingbiochemist.com

[@kuldeepbiotech4014]

Hi.. its a great pleasure to get so much information from your videos.

[@thebumblingbiochemist]

Glad they're helpful!

[@funny11744]

Question : Regarding the stem cell culture in T 75 flask - using media without CO2 : Can we use a closed cap for T75 flask ?

[@thebumblingbiochemist]

I haven't tried to do cell culture without CO2 control, sorry

[@kosheeka]

It's not advisable to seal a T 75 flask when growing stem cells, in CO2 media. Here's why;
• CO2 regulation; In vitro cells thrive in an environment with levels of CO2 for growth. Media lacking CO2 may not be able to maintain the pH balance needed for cell well being. Using a vented cap permits gas exchange allowing the cells produced CO2 to escape preventing the acidification of the media.
• Oxygen supply; For cultures that don't need CO2 sealing the flask can impede oxygen flow. Vented caps ensure oxygen enters the flask for respiration.