Isolation and Culture of Primary Keratinocytes from Human Skin Grafts

The skin, the largest organ of the human body, performs essential physiological functions including barrier protection, thermal regulation, and mechano-sensation. Despite its structural consistency, it exhibits marked regional heterogeneity, particularly under pathological conditions. Our recent investigations have revealed that in secondary lymphedema, the affected skin undergoes distinct pathomorphological changes, including hyperkeratosis, spongiosis, and altered keratinocyte polarity within the epidermis. These findings have prompted deeper studies into the molecular and epigenetic mechanisms contributing to the irreversible progression of this disease. This protocol describes a detailed, reproducible method for isolating and culturing primary keratinocytes from both full-thickness and split-thickness skin grafts obtained from lymphedematous regions. The workflow comprises tissue processing, enzymatic dissociation, selective enrichment of keratinocytes, and optimized culture conditions for downstream molecular analyses. Validation of isolated keratinocytes is performed using Western blotting, flow cytometry, and immunofluorescence histology. This approach preserves native phenotypic traits and epigenetic signatures in patient-derived keratinocytes, thereby enhancing the translational relevance of in vitro studies investigating the pathophysiology of secondary lymphedema.