Keratinocyte Primary Isolation, Cultivation, and Air-Liquid Interface Culture

This study establishes a stable, reproducible, and biologically relevant system for the isolation, identification, and cultivation of primary keratinocytes from mouse tails, aiming to provide reliable in vitro models for investigating psoriasis pathogenesis and drug screening. Primary cells were isolated using a sequential digestion protocol involving Dispase II and trypsin, followed by purity validation via Krt14 immunofluorescence and flow cytometry. Using primary cells and human keratinocyte cell line (HaCaT), two-dimensional (2D) and three-dimensional (3D) psoriasis-like cell models were successfully established. The 2D model was induced with the M5 cocktail, a cytokine mixture consisting of TNF-α, IL-1α, IL-6, IL-17A and IL-22 to mimic the psoriatic inflammatory microenvironment, and utilized for assessing cell proliferation and inflammatory factor expression. The 3D model was generated using the air-liquid interface (ALI) culture technique, reconstructing a physiologically stratified epidermal structure, and its features were characterized by hematoxylin-eosin (H&E) staining after M5 stimulation to evaluate epidermal hyperplasia and differentiation. This dual-model system integrates the high-throughput capacity of the 2D platform with the physiological relevance of the 3D platform, offering an important experimental tool for deciphering the pathological mechanisms of psoriasis and screening candidate therapeutic agents.