195 Full Skin Equivalent Model for Burn Wound Healing Germany) were cut into square pieces of 1.44 cm2. At day one, 200,000 fibroblasts were seeded onto the matrices (for DED, on the reticular side in a metal ring), and the matrices were submerged in culture medium for 4 days at 37 °C and 5% CO2. Subsequently, 100,000 keratinocytes (from frozen stock) were seeded on the opposite side (for DED, on the papillary side in a metal ring), and the models were cultured submerged in FSE I medium containing 4 ng/mL KGF and 1 ng/mL EGF for 4 days at 37 °C and 5% CO2. Next, the FSEs were transferred to a stainless-steel grid and cultured air-exposed in FSE II medium containing 4 ng/mL KGF and 1 ng/mL EGF. From day 11, FSEs were cultured in FSE III medium containing 2 ng/mL KGF and 0.5 ng/mL EGF and from day 15 onward in FSE III medium that was refreshed twice a week. Cell numbers and culture times are based on our preliminary experiments where we optimized these settings. Ex vivo human skin model Using a dermatome (Aesculap AG & Co. KG, Tuttlingen, Germany), 0.5 mm split-thickness skin was harvested from human skin and cut into square pieces of 1.44 cm2. These models were transferred to a stainless-steel grid and cultured air-exposed at 37 °C with 5% CO2 in FSE II medium that was refreshed twice a week. Induction of burn injury A copper plate (2 mm × 10 mm) attached to a PACE intelliHeat ST50 soldering iron (PACE, Vass, NC, USA) was heated to 80 °C and applied to the epidermal side of the models for 20 s without exerting pressure. The temperature of the copper device was measured by an external digital thermometer (Farnell InOne, Utrecht, The Netherlands). FSEs were put in culture for 21 days before they were burn-injured and then cultured for 2 h (T0), 1 week, and 2 weeks. In parallel, burn injury was induced on the ex vivo human skin models, and these models were then also cultured for 2 h (T0), 1 week, 2 weeks. For both FSEs and ex vivo human skin models, the medium was refreshed twice a week. Figure 6 shows a scheme of the experiment. Figure 6. Experiment scheme showing the timing of the performed steps. 6
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