GFP-KSC4 KERATINOCYTE STEM CELL LINE

Cell Line Origin

Murine keratinocyte stem cell line, genetically modified,

Cell Line Description

Keratinocytes were isolated from neonatal homozygous C57BL/6 Tg(CAG EGFP)1Osb/J mouse skin. The cell line was established by growing cells for the first 8 passages in co-culture with 3T3 J2 fibroblast feeder cells. From passage 9 onwards cells were grown without feeder cells. This line can be used for the generation of in vitro epidermal skin equivalents to be used as screening tools to look at the activity of NCEs and/or NBEs of dermatological interest, and/or to screen for such NCE and/or NBE in high throughput systems. It may also be of use to non pharmaceutical healthcare and/or cosmetic companies involved in the personal vitality and/or beauty product development segments. The spontaneously immortalized keratinocytes can be differentiated by the addition of calcium to the complete FAD medium at a final concentration of 1.2 mM (high calcium medium). Confluent cultures stratify when grown in high calcium medium.

DNA Profile

Not specified

Culture Medium

The cell line is grown in low calcium medium, DMEM/Ham′s F12 with FBS / FCS (F2442) and several supplements at 32 °C, 5% CO₂. The supplements are: 0.18 mM adenine, 0.5 μg/ml hydrocortisone, 5 μg/ml insulin,10 -10 M cholera toxin, 10 ng/ml EGF, 2 mM L-Glutamine (G7513), 1 mM pyruvate.

Subculture Routine

Wash monolayer with PBS (without Ca²⁺ and Mg²⁺), incubate with EDTA solution for 5 min at room temperature and finally incubate for 8-12 min with 0.05% trypsin/0.02% EDTA at 37 °C until they come off easily. Add complete FAD medium and resuspend the cells by pipetting up and down 3-5 times. After centrifugation for 5 min at 220 g, resuspend the pellet in complete FAD medium, and seed the cells onto collagen I-coated dishes. Seed new cultures at 1.5-3 x 10⁴ cells/cm².

Other Notes

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Disclaimer

This cell line has special release conditions: Commercial organisations are required to complete the ′Cell Line Release Authorisation for Research Use in Commercial Organisations′ release conditions form.