The human skin is a remarkable organ capable of extensive regeneration, especially after severe injuries such as burns and related wounds. The de-epidermized dermis (DED) model has become a valuable in vitro tool for skin regeneration studies, particularly for testing the mechanism of action and the efficacy of clinical cutaneous cell therapies. To further improve the quality and robustness of these applications, our study focused on optimizing and standardizing DED tissue preparation and storage, enhancing its effectiveness for clinical testing. Therefore, we optimized the air-liquid interfacial culture medium composition by simplifying the historical formulation without compromising keratinocyte (therapeutic cell model) viability or proliferation. Furthermore, we investigated the impacts of adding burn wound exudates in the model by focusing on cell behavior for enhanced translational significance. The results revealed notable differences in keratinocyte adhesion and proliferation between burn wound exudates collected at the early stages and late stages of acute patient treatment, providing new information on a possible therapeutic window to apply cell therapies on burn patients. Generally, this study reported a robust method for the preclinical in vitro assessment of keratinocyte-based cutaneous cell therapies using DED models. Overall, the study underscored the importance of using in vitro models with enhanced translational relevance to better predict the clinical effects of cutaneous cell therapies in burn patient populations.