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Complete Growth Medium
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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.
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Subculturing
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Protocol: Remove spent medium, add fresh 0.25% trypsin, 0.53 mM EDTA solution; let the culture set incubate at 37°C for two minutes. Neutralize the trypsin with 0.1% soybean trypsin inhibitor, and gently dislodge the cells by agitating or tapping the flask. Centrifuge the cell suspension at 1000 rpm for 10 minutes, resuspend the pellet in fresh medium, aspirate and dispense into new flasks.
Subcultivation Ratio: A subcultivation ratio of 1:3 is recommended
Medium Renewal: Every 2 to 3 days
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Cryopreservation
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Freeze Medium: Complete growth medium, 85%; fetal bovine serum, 10%; DMSO, 5%
Storage Temperature: Liquid nitrogen vapor temperature
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