Complete Growth Medium
These cells are grown in Keratinocyte-Serum Free Medium (Gibco 17005-042) with added Keratinocytes Supplements (Gibco 37000-015) including Bovine Pituitary Extract (BPE; Gibco 13028-014) and human recombinant epidermal growth factor (EGF; Gibco 10450-013) further supplemented with:
- Additional 30 ng/ml human recombinant epidermal growth factor (EGF; BD cat# 354052)
Do not filter the complete medium.
This medium is formulated for use with a 5% CO2 in air atmosphere.
Volumes used in this protocol are for 75 cm2 flask; 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 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.
- To remove trypsin-EDTA solution, transfer cell suspension to centrifuge tube and spin at approximately 125 x g for 5 to 10 minutes.
- Discard supernatant and resuspend cells in fresh growth medium. Add appropriate aliquots of cell suspension to new culture vessels.
- Place culture vessels in incubators at 37°C.
Subculture Ratio: 1:3 to 1:5
Medium Renewal: Twice a week.
Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 10 in Culture of Animal Cells, a manual of Basic Technique by R. Ian Freshney, 3rd edition, published by Alan R. Liss, N.Y., 1994.
Freeze Medium: Ham's F12 medium 85%; DMSO, 5%; FBS 10%
Storage Temperature: Liquid nitrogen vapor phase
Add fresh medium twice per week