Transwell culture inserts

The method for organotypic culture was adapted from Dongari-Bagtzoglou and Kashleva (Dongari-Bagtzoglou and Kashleva, 2006 (link)). Briefly, seeding rafts were assembled by mixing Culturex 3-D Matrix (Sigma-Aldrich St. Louis, MO) with 4 × 10⁵ NIH 3 T3 fibroblasts (1:3, v/v) in 200 μL/well of DMEM/10% FBS, in 6 well plates with Transwell culture inserts (VWR) and incubated at 37oC for 2 h. Dermal gel rafts were equilibrated after 24 h using 4 mL complete DMEM/10% FBS at 37oC until gels contracted. Medium was then withdrawn and 1 × 106 keratinocytes in 200 μL of complete KSFM were seeded on top of the raft and incubated for 2 h at 37 °C. Subsequently, skin equivalent on top of the insert was covered with complete KSFM, while DMEM/10% FBS filled the bottom of the well, coming in contact with the bottom surface of the skin equivalent. Plates were cultivated for 7 days—changing the medium every 2 days. At day 7, medium was withdrawn from the top and skin equivalents lifted to air–liquid interface, with KSFM/5% FBS/ 1mM calcium chloride at the bottom of the inserts, for 16 more days at 37 °C, changing the medium every other day. At day 16, skin equivalents were processed for H&E staining.