This study presents a novel ex vivo human skin model for wound healing research, specifically focusing on re-epithelialization and immune cell behavior. The model uses negative pressure to create suction blisters, which preserve the basement membrane and dermal-epidermal junctions. Key findings include: 1. **Re-epithelialization**: Keratinocytes migrated and proliferated to close the wound within 6 days, forming a multi-cell thick layer. By day 8, a multi-layered epidermis expressing differentiation markers (K10, filaggrin, DSG-1, CDSN) was observed. 2. **Immune Cell Behavior**: The blister roof epidermis had more T cells compared to normal epidermis. Several cell populations, including CD11c+ cells and CD83+ cells, were identified in the blister roof epidermis and suction blister fluid, indicating dermal efflux upon negative pressure. 3. **MMP-9 Expression**: Dermal cells expressed MMP-9, which is crucial for keratinocyte migration and wound closure. MMP-9+ cells had a "vessel-like" shape but did not co-express CD31, suggesting they are not endothelial cells. 4. **Clinical Relevance**: The model recapitulates the main features of epithelial wound regeneration and can be used to test wound healing therapies and investigate underlying mechanisms. The study highlights the potential of this ex vivo model for advancing wound healing research and improving therapeutic approaches.This study presents a novel ex vivo human skin model for wound healing research, specifically focusing on re-epithelialization and immune cell behavior. The model uses negative pressure to create suction blisters, which preserve the basement membrane and dermal-epidermal junctions. Key findings include: 1. **Re-epithelialization**: Keratinocytes migrated and proliferated to close the wound within 6 days, forming a multi-cell thick layer. By day 8, a multi-layered epidermis expressing differentiation markers (K10, filaggrin, DSG-1, CDSN) was observed. 2. **Immune Cell Behavior**: The blister roof epidermis had more T cells compared to normal epidermis. Several cell populations, including CD11c+ cells and CD83+ cells, were identified in the blister roof epidermis and suction blister fluid, indicating dermal efflux upon negative pressure. 3. **MMP-9 Expression**: Dermal cells expressed MMP-9, which is crucial for keratinocyte migration and wound closure. MMP-9+ cells had a "vessel-like" shape but did not co-express CD31, suggesting they are not endothelial cells. 4. **Clinical Relevance**: The model recapitulates the main features of epithelial wound regeneration and can be used to test wound healing therapies and investigate underlying mechanisms. The study highlights the potential of this ex vivo model for advancing wound healing research and improving therapeutic approaches.