Haynes:SplittingCells

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Passaging Adherent Cells

Karmella Haynes, 2013

Materials

• 70% ethanol spray bottle
• 1x Phosphate-buffered saline (PBS)
• Trypsin-EDTA buffer/ media
• Complete cell culture medium (e.g., 10% FBS, 1% pen-strep)
• Sterile tissue culture-treated vent-capped T-75 flask, one per new culture
• 100% confluent (dense) cell culture (check confluency under the light microscope)

Three flask sizes: T-25 (small, for starting cultures from frozen stocks), T-75 (medium, for routine passaging and expanding cell cultures), and T-150 (large, for growing & harvesting large amounts of cells). A flask must be properly laid flat (where the opening tilts upward) so that the cells can adhere to & grow on the bottom inside surface.

Reference the chart below for suggested dilution ratios and frequency of medium renewal.

Cell Line	Dilution Ratio	Medium Renewal
HEK-293	1:6 to 1:10	every 2-3 days
K-562	1:10	every 2-3 days
SK-N-SH	1:3 to 1:8	every 3-4 days
U-2 OS	1:3 to 1:6	every 2-3 days
MCF7	1:3 to 1:6	every 2-3 days
BT-474	1:2 to 1:3	every 3-4 days
BT-549	1:2 to 1:6	every 3-4 days
MCF 10A	1:3 to 1:4	every 2-3 days

Procedure

1. Pre-warm all liquid reagents to 37°C in the bead bath.
2. After reagents are warmed, spray bottles down with 70% ethanol and prepare the biosafety hood as for routine work. Do all work in the biosafety hood.
3. Retrieve the cell culture from the incubator. Stand the flask up (see image above). Open the cap and aspirate old culture media from the bottom of the flask.
4. Add 5 mL 1x PBS to the flask. Lay the flask flat (see image above) and wash by gently tilting the flask back and forth.
5. Stand the flask up. Open the cap and aspirate off the PBS.
6. Add 2 mL trypsin-EDTA into the bottom of the culture flask. Lay the flask flat and coat the cells completely with the trypsin-EDTA. Let the flask sit in the hood at room temperature for 5 minutes.
7. After incubation, examine the cells under a microscope. Fully trypsinized cells should appear rounded up and no longer attached to the surface of the flask.
8. If the cells are not fully detached, place the flask back into the 37°C incubator. Some cells may require some mechanical agitation (including “tapping” the flask).
9. Once the cells have detached, stand the flask up and add 8 mL serum-containing medium (e.g., 10% FBS) to the cells to deactivate the trypsin. Repeatedly suck up and dispense the medium from the pipette to "wash" cells off the growth surface and into the bottom of the flask. Leave the flask standing up.
10. Get a new T-75 flask, label it with the cell line name, your initials, and the date. Include the passage number (e.g., PS-1 if you are splitting the cells for the first time, PS-2 for the second time, etc.).
    
    Note: If you need a lot of cells, and/or you are making an additional culture for a lab mate, prepare more than one new flask. For a 1:10 passage, you will be able to make up to 10 new flasks of cells.
    
    
11. Stand the new flask up. If you are splitting 1:10, add 9 mL growth medium to the new flask. In the old flask, gently resuspend the cells by pipetting up and down about 5 times. Transfer 1 mL of cells into the 9 mL of medium in the new flask. Lay the flask flat and push it back-and-forth and side-to-side to spread the cells evenly. Do not swirl it in a circular motion.
    
    Note: For less robust cells, split 1:5 by adding 2 mL cells to 8 mL fresh medium, or 1:4 by adding 2.5 mL cells to 7.5 mL medium.
    
    
12. Place the new flask into the 37°C incubator. The cells should adhere to the growth surface in a couple of hours.
13. Discard the left-over cells as liquid waste by aspirating them into the vacuum trap. Discard all plastics, etc. that have come into contact with growth medium and cells as biohazard waste.