Immortalized Mouse Epidermal Cells (COCA)

Serum Affects Keratinization and Tight Junctions in 3D Cultures of the Mouse Keratinocyte Cell Line COCA through Retinoic Acid Receptor-Mediated Signaling

Vitamin A in serum affects keratinocyte proliferation, differentiation, and keratinization. While mouse oral, esophageal, and forestomach epithelia are keratinized, human equivalents are not. Ozaki et al. evaluated serum effects on morphology, differentiation markers, tight junction proteins, and paracellular permeability in 3D cultures of mouse keratinocytes.

A new 3D medium (CnT-PR 3D Barrier) replaced the previous formulation, which did not induce keratinized stratified epithelium-like structures in COCA cultures. Epidermal analogs were generated by adding calcium, KGF, and APM to CnT-PR. COCA formed epidermis-like structures within 1 week, maintained for at least 3 weeks, though the cornified layer became loosely packed (Fig. 1). Serum effects were examined after 2 weeks (Fig. 2), given its essential role in skin equivalent models. Control cultures formed keratinized stratified squamous epithelium (SSE) (Fig. 2a). With 0.01% charcoal-stripped FBS (ch-FBS), nuclei persisted in the cornified layer (Fig. 2b, arrowheads). Keratinization was inhibited by 0.1%, 1%, and 10% ch-FBS (Fig. 2c-e). Non-keratinized SSE with 0.1% ch-FBS showed 2-3 superficial squamous cell layers with detached nucleated cells (Fig. 2c). Hyperplastic rather than flat superficial cells appeared with 1% or 10% ch-FBS, with some detaching (Fig. 2d, e, arrows). Flattened cell layers were observed beneath hyperplastic surfaces (Fig. 2d, large arrow), and large intercellular gaps occurred at 10% ch-FBS (Fig. 2e).

Immunofluorescence revealed LOR (late keratinization marker) in cells beneath the cornified layer without ch-FBS, but K4 (non-keratinization marker) was absent from nucleated cells (Fig. 3a). With ≥0.1% ch-FBS, K4 replaced LOR in all suprabasal cells of non-keratinized SSE (Fig. 3b-d). Antibody specificity was confirmed in porcine oral mucosa.