Complete Growth Medium
The base medium for this cell line is Iscove's Modified Dulbecco's Medium (IMDM; ATCC® No. 30-2005): RPMI-1640 Medium (ATCC® No. 30-2001) at a 4 to 1 ratio. To 500 mL IMDM:RPMI 1640 (4:1) add the following components to make the complete medium:
- 56 mL FBS (ATCC® No. 30-2020), final concentration of 10%
- 1% L-glutamine (ATCC® No. 30-2214), final concentation of 1%
- 5.6 mL L-Glutamine (Gibco cat# 25030-081)
- 5.6 mL ITS-G (Gibco cat# 41400-045)
It is recommended to subculture the cells at maximum 85% confluency.
Volumes used in this protocol are for 75 cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes. Corning® T-75 flasks (catalog #430641) are recommended for subculturing this product.
- Remove and discard culture medium.
Briefly rinse the cell layer with 0.25% (w/v) Trypsin- 0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor.
Add 2.0 to 3.0 mL of 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.
Add appropriate aliquots of the cell suspension to new culture vessels.
Cultures can be established between 2 x 104 and 6 x 104 viable cells/cm2.
- Incubate cultures at 37°C.
Interval: Maintain cultures at a cell concentration between 2 X 104 and 2.0 X 105 cell/cm2.
Subcultivation Ratio: A subcultivation ratio of 1:3 to 1:8 is recommended
Medium Renewal: 2 to 3 times per week
Atmosphere: air, 95%; carbon dioxide (CO2), 5%