Volumes used in this protocol are for 75 cm2 flasks; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.
- Remove and discard culture medium.
- Briefly rinse the cell layer with Ca2+/Mg2+ free Dulbecco's phosphate-buffered saline (D-PBS) or 0.025% (w/v) Trypsin-EDTA solution to remove all traces of serum which contains trypsin inhibitor.
- Add 1.0 to 2.0 mL of 0.025% Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
- Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
- Transfer cell suspension to a centrifuge tube and spin at approximately 125 x g for 5 to 10 minutes. Discard supernatant.
- Resuspend the cell pellet in fresh growth medium. Add appropriate aliquots of the cell suspension to new culture vessels. An inoculum of 1.5 X 104 to 3.0 X 104 viable cells/cm^2 is recommended.
- Incubate cultures at 37°C.
Subcultivation Ratio: A subcultivation ratio of 1:2 to 1:6 is recommended
Medium Renewal: 2 to 3 times per week
Freeze Medium: Complete growth medium supplemented with an additional 10% heat inactivated fetal bovine serum and 10% (v/v) DMSO
Storage Temperature: Liquid nitrogen vapor phase
Temperature: 35°C to 37°C
Atmosphere: air, 95%; carbon dioxide (CO2), 5%