Kuperwasser Lab | Basic cell culture techniques (PJ Keller)

Basic Cell Culture Techniques ( Click to Download )

Remember to follow sterile technique and use 70% ethanol liberally! Warm up media and trypsin for ~20 minutes before use.

Thawing cells

Remember: to prevent ice crystals from forming and killing your cells you want to freeze slowly and thaw quickly!

Remove cells from liquid nitrogen storage. If not immediately thawing, place on dry ice until ready to thaw.
Thaw the vial of cells quickly by immersing in a 37C water bath for 2-3 minutes; spray down tube liberally with 70% ethanol when removing from bath and wipe dry.
Transfer the contents of the tube to a 15-ml conical tube containing 10 ml of growth media for the cells you are thawing; rinse the freezer vial with an additional 1-2 ml of media and add to the 15-ml conical.
Pellet the cells at 1000-1200 rpm for 5 minutes.
Resuspend the pellet in 10 mls of growth media by pipetting up and down until no large chunks are visible, 4-7 passes usually.
Transfer the cells to a 10 cm dish labeled with the passage number (while historical passasage may not be known, keep track of your passage number for reference). Thawed cells are labeled “P0”. Check cell viablity under the microscope, healthy cells will be bright and refractile. Grow cells at 37C with the recommended CO₂ concentration (5% for our cells).

Passaging Cells

Try to trypsinize cells to passage them before they reach confluency to avoid stressing them (ideally at about 80%, though some cells do better at higher density than others). All quantities are for a 10 cm dish, scale up or down for different sized plates.

Aspirate media from the plate; rinse with 10 ml PBS to remove cellular debris and traces of serum.
Pipet 1-2 ml of trysin-EDTA (we use 0.05% Trypsin) onto the cells in the dish. Incubate at 37C for 2-5 minutes to dislodge most cells. Assist full dislodgement of cells that have rounded up on the plate by firmly tapping it on the side of your hand (but not so firmly that the trypsin gets all over the plate lid). If cells do not look rounded, return to the incubator for additional time and repeat the tapping (if really stubborn, a cell lifter may be called for but use with caution as it may damage cells).
For a 10 cm plate, pipet 8-10 ml of media onto the plate while tilting the plate. Rinse the cells off the dish moving the pipet from the top to the bottom of the tilted plate. Repeat several times. Transfer cells to a 15 ml conical tube.
Pellet cells at 1000-1200 rpm for 5 minutes.
Prepare the plate(s) you are passaging the cells onto by labeling them with the new passage number (P1, P2 etc.). Pipet 9 ml of media onto each plate.
Resuspend the pellet in the amount of growth media that corresponds to the ‘split’ you are doing. For a 1:4 split, for example, resuspend the cells in 4 mls of media. Pipet up and down until the pellet disappears; transfer 1 ml of the cells to the prepared plate(s).
Swirl and rock the plates to evenly distribute the cells and place in the incubator.

Freezing cells

Follow the passaging cells protocol through step 4.

Determine how many vials you want to freeze back, a good rule of thumb is that when thawed, it should generate a plate that is around 50% confluent (this often works out to about 1-2 million cells unless the cells are very large). Label them with the cell line, the date, the passage number, your initials and any other pertinent information.
Prepare “freezing media”, generally growth media + double the serum of the growth media + 10% DMSO (i.e. if the cells grow normally in 10% serum, make the freezing media with 20% serum). If the cells are grown in serum free media, add serum to at least 10% to protect cells during freezing.
Resuspend pelleted cells in an amount of freezing media that corresponds to the number of vials you want to freeze, usually use 1-1.5 mls of freezing media per vial. Pipet cells into vials, avoiding the ‘threads’
Screw down the caps of the vials tightly to seal the o-ring. Transfer to an isopropanol freezing container and place at –80 at least overnight and up to a few weeks (long term storage at –80 will decrease cell viability).
When cells are thoroughly frozen, transfer them to liquid nitrogen for long-term storage.

Basic cell culture techniques (PJ Keller)

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