Cnt pr 3d

Primary human KC isolation and generation of 3D HEEs were performed as described previously (Rikken et al., 2020 (link)) and according to the principles of the Declaration of Helsinki. Briefly, primary human KCs were seeded with a density of 150,000 cells into a 24-well transwell system (Nunc; Thermo Fischer Scientific, Waltham, MA) and cultured submerged for 2 days in a proliferation medium (CnT-PR; CELLnTEC, Bern, Switzerland). After 1 more day of submerged culture in differentiation promoting medium (CnT-PR-3D; CELLnTEC), the cultures were lifted to the air‒liquid interface and cultured thereafter for a total of 8 days, with refreshing of the medium every other day (Niehues et al., 2017 (link)). Cytokines and other tested factors supplemented to the culture medium during days 5‒8 of the culture, with refreshing at day 7, in different concentrations are depicted in Table 2. Concentrations of all tested factors were chosen on the basis of experience as well as literature. For signaling studies, inhibitors were added 24 hours before cytokine stimulation and left for costimulation until day 8. To label KC proliferation in time, 10 μM EdU was added into the medium for 24 hours before harvesting at the indicated time points.