A reproducible mechano-thermal injury method for reconstructed human epidermis to study wound re-epithelialization.

Reconstructed human epidermis (RHE) models are widely used as an ethical alternative to animal testing and are validated for cutaneous toxicity assessment. However, their application in wound healing research remains limited due to the lack of standardized methods to induce reproducible epidermal injuries. Generating localized lesions while preserving the polycarbonate membrane required for keratinocyte migration remains a technical challenge. Here we describe a method to generate controlled lesions in reconstructed human epidermis suitable for re-epithelialization studies. Several injury strategies were evaluated, including mechanical approaches using pipette tips, punches, microneedles and glass capillaries. These approaches frequently produced inconsistent lesions or disrupted the membrane. A mechano-thermal strategy proved to be the most reliable approach, generating well-defined and reproducible lesions while preserving membrane integrity. Histological analysis confirmed characteristic features of thermal injury. Longitudinal macroscopic and histological analyses, including semi-quantitative scoring, demonstrated progressive epidermal reorganization following injury. This method establishes a reproducible epidermal burn model suitable for studying epidermal regeneration and wound healing.•Comparative evaluation of mechanical and mechano-thermal strategies to induce lesions in reconstructed human epidermis.•Identification of a reproducible injury method that preserves the polycarbonate membrane required for keratinocyte migration.•Development of a standardized epidermal burn model for re-epithelialization studies.