2.2. 3D Cell Culture

For generation of the 3D co-cultures, a human dermal fibroblast cell line (fHDF/TERT166, Evercyte, Vienna, Austria) and normal human epidermal skin keratinocytes (NHEK/SVTERT 3-5, Evercyte, Austria) were used. Basic cell culture was conducted according to the manufacturer’s instructions. Cells were cultured at 37 °C, 5% CO₂, and moderate humidity. Thawing of the fibroblasts and keratinocytes was conducted according to manufacturer’s instructions.

Expansion of fibroblasts was performed in T175 flasks (Greiner Bio-One, Kremsmünster, Austria) with a 1:1 medium mixture of Dulbecco’s Modified Eagle Medium (DMEM-high glucose containing phenol red, Sigma-Aldrich, St. Louis, MO, USA) and Ham’s F-12 (Lonza, Basel, Switzerland) supplemented with 10% FCS (Sigma-Aldrich, USA), 2 mM glutamine (GlutaMax, Gibco, New York, NY, USA) and 1 µg/mL G148 (InvitroGen, Waltham, MA, USA). When cells reached a confluency over 80–90%, they were split in a ratio of 1:3 or 1:4. Cells were washed twice with PBS (Lonza, Basel, Switzerland) before adding 2.5× Trypsin/EDTA (Sigma-Aldrich, USA) diluted 1:4 in PBS for 5 min at 37 °C. The reaction was stopped by adding double the amount of cell culture medium. Expansion of keratinocytes was also performed in T175 flasks with KBM-2 growth medium (Lonza, Basel, Switzerland), supplemented with KGM-2 keratinocyte medium SingleQuot kit (Lonza, Basel, Switzerland). To detach the cells, a 1:2 dilution of 2.5× Trypsin/EDTA in PBS was used after two washing steps with PBS. The reaction was stopped with equal the amount of trypsin neutralizing solution (Gibco, New York, NY, USA) and washed with 10 mL PBS. From this step onwards, the procedure was identical for both cell types. The cell suspensions were centrifuged at 100× g for 5 min in a 50 mL Falcon Tube (Greiner Bio-One, Kremsmünster, Austria). Cell pellets were resuspended in fresh medium and seeded into a T175 flask. In the case of subconfluency, cell culture medium was exchanged every third or fourth day.

To model the dermis, a collagen/fibrin hydrogel mix was prepared and subsequently loaded with fibroblasts. For this purpose, a fibroblast suspension was prepared in fetal calf serum (FCS) (Sigma-Aldrich, St. Louis, MO, USA) with an end concentration of 1 × 10⁵ cells/mL. Mixing of the gel was conducted on ice to prevent premature clotting. After preparing the cell suspension, Hank’s Balanced Salt Solution (HBSS) with phenol red (Gibco, New York, NY, USA) was added dropwise to collagen (Collagen G, type 1, solution 4 mg/mL (Sigma-Aldrich, St. Louis, MO, USA)). The pH was slowly adjusted to approximately 7.5 with 1 M sodium hydroxide solution until the mixture turned red/pink. After adjusting the pH, the fibroblast/FCS solution was added to the mixture. The proportions were one-part HBSS, one part fibroblast/FCS suspension, and eight parts collagen. Before adding the cell suspension to the collagen, thrombin (TISSEEL Lyo, Baxter, Deerfield, IL, USA) was added to the fibroblast/FCS suspension to an end concentration of 1 U/mL. To start the gelling, fibrinogen, dissolved in aprotinin, was added to the final mixture to an end concentration of 3 mg/mL. Next, 2500 μL of collagen/fibrin gel containing fibroblasts was quickly transferred without air bubbles into each insert. Plates were incubated at 37 °C, 95% humidity, with 5% CO₂ until the collagen/fibrin matrix was solid. To equilibrate the gel, 1000 μL keratinocyte growth medium was pipetted into each insert and the outer well was filled above the membrane level (appr. 4.5 mL). Equilibration for at least one hour was conducted in the incubator. Next, for the epidermal layer, keratinocytes were detached according to the standard cell culture procedure. A cell suspension of 1.5 × 10⁶ cells/mL was prepared in keratinocyte culture medium. After the removal of the equilibration medium from the inner insert, 1000 μL of keratinocyte suspension was added on top of the solid gel. The SEs were then incubated overnight at 37 °C, 5% CO₂, and moderate humidity. The next day (Day 2), the SEs were loosened by moving a 200 μL pipette tip carefully around the outer wall of the insert to avoid tension stress during the culturing. On Day 4, the medium was changed from keratinocyte growth medium to differentiation medium. For that, 500 mL KGM medium (Lonza, Basel, Switzerland) was supplemented with 25 mg ascorbic acid (Sigma-Aldrich, USA), 500 mg albumin from bovine serum (BSA) (Sigma-Aldrich, USA), 5 mg transferrin (Sigma-Aldrich, USA) and CaCl₂ (Sigma-Aldrich, USA) to an end concentration of 1.3 mM. The dry components were added to the KGM medium, and the mixture was subsequently filtrated through a 0.22 μm filter. Next, the KGM SingleQuot supplements (Lonza, Basel, Switzerland), except for the Bovine Pituitary Extract (BPE), were added. The differentiation medium was then added only to the outer wells to facilitate the ALI. For this, a volume of 4 mL per well was used. The medium was changed every other day.