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Outgrown the Roost: Passaging Adherent Cells, the Basic Process


Splitting, passaging, subculturing… whatever you call it, while the specifics of passaging adherent cells will depend upon the individual cell type, the basic process involves four simple steps. These basic steps are outlined below.

Rinse Cells With a Balanced Salt Solution (BSS)

Prior to detaching cells from the dish, it is important to aspirate off the old, spent media and rinse cells with a balanced salt solution (BSS). Rinsing the cells will help eliminate proteins and ions found in the media that might inhibit the action of cell releasing solutions. BSS are used because they maintain a physiological pH and salt concentration. Typical salt solutions include Phosphate Buffered Salines (PBS), Hanks’ Buffered Salt Solutions (HBSS) and Earle’s Balanced Salt Solutions (EBSS). Calcium- and magnesium-free salt solutions should be used when subculturing cells, as both calcium and magnesium promote cell clumping.

Detach Cells From the Bottom of Dish/Flask

Cells are released from the dish by breaking the cell protein interactions with the surface of the dish. Different cell types have different properties when it comes to adhering to the bottom of the dish. Some cells remain like little balls barely flirting with the dish while others flatten out and slather down multiple layers of proteins that bind them to the dish. Your goal is to detach the cells from the dish using the least damaging procedure for each cell type. How you choose to release the cells from the dish will depend upon the adherence property of the cells.

Mechanical detachment.  Some lightly adherent cells will begin lifting from the dish upon addition of BSS (calcium/magnesium free). In this case, simply spraying the cells directly with the BSS and tapping the plate will be sufficient to remove the cells.

EDTA.  EDTA is a calcium chelator that will remove the Ca2+ ions that integrins require to maintain cell adhesion. EDTA (1-10mM, depending upon cell type) is one of the gentler ways to detach cells from the dish, but EDTA alone is not potent enough for most cell types. EDTA is most effective when prewarmed to 37°C, however for very sensitive cells use EDTA that is at room temperature or 4°C.

Enzymatic release.  Proteolytic enzymes are used to digest the proteins that adhere cells to the dish. Proteolytic digestion can also damage the integrity of the cell by cleaving cell surface proteins; therefore, treatment should be limited to the amount of time required to just achieve detachment of cells. Treatment will need to be determined empirically for each cell type and will also depend on the length of time cells have been in culture and the confluency of the culture. Trypsin is the most frequently used enzyme for passaging cells. Trypsin cleaves after lysine or arginine residues that are not followed by prolines. Working trypsin concentrations range from 0.025-0.5% and trypsin solutions are commonly made with EDTA to enhance cell detachment.

Dissociate and Inactivate

To prevent clumping and uneven disbursement of cells, cells should be in a single cell suspension. If they are not, add a small volume of liquid to the cells and pipet the liquid into and out of a 5ml pipette. Some labs add warmed growth media while others add BSS containing 10% fetal bovine serum (FBS). The addition of warmed growth media or BSS+FBS will inactivate the agent used to detach cells from the dish and provide more volume for pipetting. For complete inactivation, collect the cells in a larger volume of growth media or BSS+FBS and centrifuge the cells. If cells are going to be diluted into a large volume of growth media for replating and residual trypsin/EDTA does not affect cell attachment, then the centrifugation step can be omitted.

Set Up New Dishes/Flasks

While the cells are detaching from the dish or are in the centrifuge, I usually set up the plates I am going to move my cells into. Label the required number of plates and place the proper amount of warmed growth media in the dishes. We’ll discuss seeding concentrations and volumes in another article. Resuspend the centrifuged cells in a small volume of growth media and count the cells using a hemocytometer. Dilute the cells to the appropriate density and aliquot cells into the new dishes. I like to add enough media to pipet 1-2 mls into each recipient dish. Gently swirl and shake the dishes to disperse the cells evenly throughout the dish.


These basic steps will work for most cell types. I also always try to talk to someone who has worked with a particular cell type to determine the appropriate cell growth media, cell releasing solution and length of time required to detach cells from the dish. Alternatively, the ATCC is an excellent resource for cell line specific protocols.

Image Credit: Living Off Grid

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