223 Immune Cell Model for Burn Immune Reactions Figure 6 Development of human full skin equivalent (burn wound) model with monocytes or T cells. (A) Development of FSE. (B) Incorporating monocytes into FSE. (C) Incorporating T cells into FSE. Induction of burn injury A copper plate (2 × 10 mm) attached to a PACE intelliHeat ST50 soldering iron (Vass, USA) was heated to 80-90 °C and applied to the epidermal side of the models for 20 sec without exerting pressure (Figure 6A). The temperature of the copper device was measured by an external digital thermometer (Farnell InOne, Utrecht, the Netherlands). PBMC isolation from human buffy coat PBMCs were isolated from buffy coats obtained from healthy donors (Sanquin, Amsterdam, the Netherlands) by density gradient centrifugation using Lymphoprep (Stemcell Technologies, Vancouver, Canada). The buffy coat was diluted in PBS and layered over the density gradient medium. After centrifugation at 1000 × g for 15 min (without brakes), the PBMCs were collected in FSE I medium. Cells were resuspended in 50% fetal bovine serum (Gibco) + 40% FSE I medium + 10% dimethyl sulfoxide. After 24 h storage in Mr. Frosty (ThermoFisher scientific) with isopropanol at -80 °C, cells were stored in liquid nitrogen until use. Monocyte FSE PBMCs were incubated with anti-CD14 beads (Invitrogen, Waltham, MA, USA) at a bead/ cell ratio of 2.5:1 at 2-8 °C for 20 min on a tube roller. Monocytes were isolated from the PBMCs using a magnet (Invitrogen Dynal AS, Oslo, Norway). Monocytes were resuspended in FSE I medium and 2.5 × 105 cells were added to the dermal side of FSEs. Inverted FSEs with monocytes were incubated at 37 °C for 2 h and subsequently placed back onto 7
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