This study presents a novel ex vivo human skin wound model using negative pressure to induce blisters, which allows for the study of re-epithelialization and immune cell behavior. The model maintains the basement membrane intact and enables the regeneration of a multi-layered epidermis with differentiation markers. Keratinocytes migrate and proliferate to close the wound, and immune cells, including T cells, are found in higher numbers in the blister roof epidermis compared to normal epidermis. The model recapitulates key features of human epidermal regeneration and can be used to test wound healing therapies and investigate underlying mechanisms. The study also shows that dermal cells, including mast cells and immune cells, can migrate into the epidermis under negative pressure. The model is standardized, reproducible, and suitable for dermatological and immunological research. The findings highlight the importance of developing reliable human wound models to improve wound treatment techniques. The study was supported by grants from the Medical Scientific Fund of the Mayor of the City of Vienna and Schülke & Mayr GmbH. All authors contributed to the study and reviewed the manuscript. The authors declare no competing interests.This study presents a novel ex vivo human skin wound model using negative pressure to induce blisters, which allows for the study of re-epithelialization and immune cell behavior. The model maintains the basement membrane intact and enables the regeneration of a multi-layered epidermis with differentiation markers. Keratinocytes migrate and proliferate to close the wound, and immune cells, including T cells, are found in higher numbers in the blister roof epidermis compared to normal epidermis. The model recapitulates key features of human epidermal regeneration and can be used to test wound healing therapies and investigate underlying mechanisms. The study also shows that dermal cells, including mast cells and immune cells, can migrate into the epidermis under negative pressure. The model is standardized, reproducible, and suitable for dermatological and immunological research. The findings highlight the importance of developing reliable human wound models to improve wound treatment techniques. The study was supported by grants from the Medical Scientific Fund of the Mayor of the City of Vienna and Schülke & Mayr GmbH. All authors contributed to the study and reviewed the manuscript. The authors declare no competing interests.