3D Lung Cell Aggregates Formation

3D aggregates were formed as described previously (27 (link), 28 (link)). Briefly, normal human lung fibroblasts (NHLF) and bronchial smooth muscle cells (BSMC) were isolated from anonymous donors of different ages and sexes and were purchased from Lonza (Basel, Switzerland). All cells were cultured at 37°C and 5% CO2 in primary cell culture media. NHLF, BSMC and LAM cell types were sub-cultured and mixed at 1:1 ratio then dispensed 3*105 cells/well onto a low-attachment 96-well U-bottom plates (Corning, New York, USA). The 3D aggregate co-cultures were incubated in the presence or absence of 10 nM rapamycin and/or 2 μM RA for 24 h, then collected into cryomold and sectioned for staining.