The excisional 3D in vitro wound model using precast EFT cultures and DEs. (A) Hematoxylin and eosin–stained paraffin sections of the EFT cultures. The epidermis (E) displayed basal, spinous, granular, and cornified keratinocyte layers (C), and the dermis (D) distributed fibroblasts. Broken lines denote dermal–epidermal junction. (B) Precast EFT skin cultures were wounded twice with a 2-mm biopsy punch. (C) After wounding, cultures were placed into a 12-well ThinCert insert. (D) Cultures were halved with one punch on every tissue sample and cut until the wound was microscopically visible. Sections were marked as layer 1 (0 µm). Based on that, further defined sections in layer 2 (150 µm) and in layer 3 (300 µm) were produced. (E) Histology of the wound cultures. Hematoxylin and eosin–stained sections showed punched wounds with clearly defined wound margins (arrowheads). The underlying DE was seamlessly attached, providing the wound matrix (dotted line shows basal membrane). (F) Hematoxylin and eosin reepithelialization kinetics of the wound cultures over a period of 10 d. Broken lines denote wound margins. (G) The relative wound closure was quantified as the ratio of the migration distance (length of the EET; l_EET) to the size of the wound in the respective section (l_Wound). The data shown are from two independent experiments; n = 8 (day 0 [d0]), 10 (day 1), 8 (day 2), 10 (day 3), 9 (day 4), 4 (day 5), and 3 (day 6); 100 slides were analyzed in total; data are means ± SEM; **, P ≤ 0.01; ***, P ≤ 0.001, Student’s t test. Bars, 500 µm.