Complete Growth Medium
The base medium for this cell line is provided by Invitrogen (GIBCO) as part of a kit: Keratinocyte Serum Free Medium (K-SFM), Kit Catalog Number 17005-042. This kit is supplied with each of the two additives required to grow this cell line (bovine pituitary extract (BPE) and human recombinant epidermal growth factor (EGF).
To make the complete growth medium, you will need to add the following components to the base medium:
0.05 mg/ml BPE - provided with the K-SFM kit
5 ng/ml EGF - provided with the K-SFM kit.
NOTE: Do not filter complete medium.
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.
- Add 2.0 to 3.0 mL 0.25% (w/v) Trypsin-0.53 mM EDTA solution for 2-3 minutes. Add 6.0 to 8.0 ml of 0.1% Soybean Trypsin Inhibitor and gently dislodge the cells by agitating or tapping the flask.
- 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.
- Incubate cultures at 37°C
Subculture Ratio: 1:3
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, 85%; fetal bovine serum, 10%; DMSO, 5%
Storage Temperature: Liquid nitrogen vapor temperature