222 Chapter 7 point. Split-thickness samples of 0.3 mm were harvested using a dermatome (Aesculap AG & Co. KG, Tuttlingen, Germany). Isolation of human keratinocytes and fibroblasts See Supplementary Table 1 for the contents of culture media. Harvested skin was incubated in 0.25% dispase (Gibco, ThermoFisher Scientific, Paisley, UK) at 37 °C for 45 min. The epidermis was separated from the dermis using forceps. For fibroblast isolation, the dermal part of the split skin was cut into small pieces and submerged into a 0.25% collagenase A (Roche, Basel, Switzerland) solution at 37 °C for 2 h. After addition of 1 mM EDTA (Life Technologies, Paisley, UK) + PBS (Gibco) to inhibit enzyme activity, the cell suspension was poured through a 500 µm cell strainer (PluriSelect, Leipzich, Germany) and centrifuged for 10 min at 360 × g. The cell pellet was resuspended in culture medium and poured through a 70 µm cell strainer (PluriSelect) and cultured at 37 °C with 5% CO2. For keratinocyte isolation, the epidermis was transferred into 0.05% trypsin (Gibco) and incubated for 20 min at 37 °C. The cell suspension was poured through a 70 µm cell strainer and centrifuged for 10 min at 110 × g. Next, the cell pellet was washed in culture medium and centrifuged for 10 min at 160 × g. The cell pellet was then resuspended in CnT-07 medium (CELLnTEC Advanced Cell Systems AG, Bern, Switzerland) and keratinocytes were transferred onto a 1 µg/cm2 collagen type IV (Sigma-Aldrich, Saint Louis, MO, USA)-coated culturing flasks (Starstedt AG & Co. KG, Nümbrecht, Germany) at 37 °C with 5% CO2. Human Full skin equivalents MatriDerm® (MedSkin Solutions Dr. Suwelack AG, Billerbeck, Germany) with a thickness 3 mm was cut into circular pieces of 1.13 cm2. At day one, 2 × 105 fibroblasts were seeded onto the matrix and the matrix were submerged in culture medium containing 65 µg/mL ascorbic acid for 4 days at 37 °C with 5% CO2 (Figure 6A). Subsequently, 1 × 105 keratinocytes were seeded on the opposite side and the models were cultured submerged in FSE I medium containing 2 ng/ml KGF (ImmunoTools GmbH, Friesoythe, Germany) and 0.5 ng/ml EGF (R&D Systems, Inc., Minneapolis, MN, USA) for 4 days at 37 °C with 5% CO2. Next, the FSEs were transferred to transwells (Starstedt) and cultured airexposed in deep well plates (Greiner Bio-One BV, Alphen aan den Rijn, the Netherlands) with FSE II medium containing 4 ng/ml KGF and 1 ng/ml EGF. From day 11 onward FSEs were cultured in FSE III medium containing 4 ng/ml KGF and 1 ng/ml EGF and from day 15 onward in FSE III medium that was refreshed twice a week. Cell numbers and culture conditions are based on preceding experiments [30]. At day 22, immune cells were added to the FSEs.
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