An in vitro outgrowth culture system for normal human keratinocytes

Normal human epidermal keratinocytes usually proliferate in low-calcium and differentiate in high-calcium without a feeder layer, but they stop proliferating and differentiate at confluency even in low-calcium, serum-free medium.

We speculated that this contact inhibition would be mediated in part by mechanical tension. To prove this, we created a new assay system.

A 10 mm diameter cloning ring was put on the center of a 60 mm dish coated with type I collagen. Keratinocytes were plated in the ring and incubated for 4h, then we had a circular epidermal monolayer sheet. We changed the mechanical tension by removing the ring and measured the diameter of the sheet under various conditions.

When we used keratinocyte-serum free medium (SFM) whose calcium concentration is below 0.1 mM as a medium, the keratinocytes in the perimeter migrated individually, and the keratinocytes in the center portion started differentiation. However, when we added calcium chloride to SFM (final concentration more than 0.5 mM), keratinocytes at the periphery showed marked lamellipodia without losing contact with the surrounding cells. These keratinocytes showed coordinate sheet-like outgrowth as a whole even in high concentrations of calcium.

These results suggest that other than calcium concentration, change of the mechanical tension would be one of the factors that mediate proliferation or differentiation of keratinocytes and that this new assay can be useful in analyzing proliferation, differentiation, and migration of keratinocytes.