Complete Growth Medium
Keratinocyte-Serum Free medium (GIBCO-BRL 17005-042) with 5 ng/ml human recombinant EGF, 0.05 mg/ml bovine pituitary extract, 0.005 mg/ml insulin and 500 ng/ml hydrocortisone.
Volumes used in this protocol are for 75 cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.
Note: The flasks used should be precoated with a mixture of 0.01 mg/mL fibronectin, 0.03 mg/mL bovine collagen type I and 0.01 mg/mL bovine serum albumin. Subculture when 80% confluent.
- Remove and discard culture medium.
- Briefly rinse the cell layer with 0.05% Trypsin – EDTA (GIBCO cat# 25300-054).
- 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 with 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 fresh 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 to10 minutes.
- Discard supernatant and resuspend cells in fresh growth medium. Add appropriate aliquots of cell suspension to new coated culture vessels.
- Place culture vessels in incubators at 37°C.
Subcultivation Ratio: A subcultivation ratio of 1:2 to 1:4 is recommended when 80% confluent
Medium Renewal: Every 2 to 3 days.
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: complete growth medium supplemented with 7.5% (v/v) DMSO
Storage temperature: liquid nitrogen vapor phase
Atmosphere: air, 95%; carbon dioxide (CO2), 5%