Full-Thickness Skin Model Development

Full thickness models (FTMs) were generated in two steps and cultured on inert membranes using a transwell system (Corning Transwell cell culture inserts, membrane diameter 24 mm, pore size 3 μm; Corning Life Sciences, The Netherlands) as described before^{9 (link),42 (link)}. In the first step, the dermal equivalents were generated using primary fibroblasts (1.2–1.5 × 10⁵) in passage number 2–5, hydrated rat-tail tendon collagen (4 mg/mL), 1 M NaOH, Hank’s Balanced Salt Solution, and 5% fetal bovine serum (FBS; GE Healthcare, Chicago, IL, USA). After polymerization at 37 °C, the dermal equivalents were cultured 7 days as described elsewhere^{9 (link),42 (link)}. In the second step, the epidermal equivalents were generated by seeding primary keratinocytes (2.5 × 10⁵/model) in passage number 1 directly onto each dermal equivalent as reported before^{9 (link),57 (link)}. The FTMs were kept submerged for total of four days. Hereafter, the FTMs were lifted to the air-liquid interface and cultured for 14–15 days under normoxia (20%) in the Memmert INC153med CO₂ incubator (Memmert, Schwabach, Germany) or under hypoxia (3%) in the Heracell™ 240 CO₂ incubator (ThermoFisher, Waltham, MA, USA). FTM batches were generated with 5 different primary cell donors.