An Ex Vivo Assay to Assess the Potential of Skin Keratinocytes for Wound Epithelialization (1)
In vertebrates the epithelial lining of the skin provides a crucial barrier between the environment and internal structures. The consequences of loss of barrier function are severe and potentially lethal, and include fluid loss and infection. The formation of a new stratified layer of epidermis over the lesion begins as keratinocytes located proximal to the wound undergo an activation phase.
Adult skin was used, approximately 4 cm × 4 cm of the dorsal skin was shaved using a commercial shaver. Then, the entire decapitated body was washed with 10% povidone-iodine solution, double-distilled water, and 70% ethanol in order to reduce contaminants, and the shaved skin was removed. Once this piece of skin was laid flat in a Petri dish, the procedure was continued as described above. To prepare explants without the dermis, the skin was peeled off the pup and floated on 0.25% trypsin for 5 h at 4°C. After the dermis was removed, the epidermis was washed in medium, cut with a sterile, 4 mm punch biopsy and then treated as the full-thickness explants. These were individually placed into the wells of a 24-well untreated tissue culture dish. After waiting 5 min for the skin to adhere to the plate, 200 µl medium was added, and the plates were incubated at 37°C, 5% CO2 (day 0). On day 1, the explants were submerged by adding ≈ 1.5 ml of medium to each well. Medium was replaced every 2–3 d thereafter.
Within 1 d of ex vivo culture, a number of keratinocytes could be seen protruding from the explanted skin. Cells proximal to the explant are smaller and can be seen dividing frequently (black arrowheads) as confirmed by BrdU incorporation. Cells distal to the explant (white solid outline) are much larger and more flattened, and mitoses are less frequent in that region. In an enlarged view of the distal edge of a 6 d explant, the cells appear flattened. When explants from KT1–1p and KT1–5m transgenic mice were cultured, the edge of the punch biopsy and most of the keratinocytes growing outward had β-galactosidase activity. The cells did not grow out from the punch biopsy as a single sheet and were smaller than the keratinocytes growing out of the full thickness explants. Immunofluorescence staining of these epidermis-only cells showed them to have a slightly different keratin protein profile. Skin was obtained from four adult wild-type (+/+) and four age-matched obese (ob/ob) mice and subjected to the explant assay and quantitative analysis. The ob/ob explants had an average area of 14 ± 1.2 mm2 (SEM, n = 24) compared with 24 ± 2.0 mm2 (SEM, n = 24) for the wild-type explants.
