Division of large, immature alveolar structures into smaller, more numerous alveoli increases the surface area available for gas exchange. Alveolar division requires precise epithelial-mesenchymal interactions. However, few experimental models exist for studying how these cell-cell interactions produce changes in 3-dimensional structure. Here we report an epithelial-mesenchymal cell co-culture model where 3-dimensional peaks form with similar cellular orientation as alveolar structures in vivo. Co-culturing fetal mouse lung mesenchyme with A549 epithelial cells produced tall peaks of cells covered by epithelia with cores of mesenchymal cells. These structures did not form when using adult lung fibroblasts. Peak formation did not require localized areas of cell proliferation or apoptosis. Mesenchymal cells co-cultured with epithelia adopted an elongated cell morphology closely resembling myofibroblasts within alveolar septa in vivo. Because inflammation inhibits alveolar formation, we tested the effects of E. coli lipopolysaccharide on 3-dimensional peak formation. Confocal and time-lapse imaging demonstrated that lipopolysaccharide reduced mesenchymal cell migration, resulting in fewer, shorter peaks with mesenchymal cells present predominantly at the base. This epithelial-mesenchymal co-culture model may therefore prove useful in future studies of mechanisms regulating alveolar morphogenesis.
Keywords: 3-D, 3-dimensional; ATCC, American Type Culture Collection; BALB/cJ, Bagg Albino; BMP4, bone morphogenetic protein 4; CO2, carbon dioxide; DAPI, 4′, 6-Diamidino-2-Phenylindole, Dihydrochloride; DEVD, acetyl-Asp-Glu-Val-Asp p-nitroanilide; DMEM, Dulbecco's modified eagle medium; DiI, 1, 1′-dioctadecyl-3, 3, 3′3′-tetramethylindocarbocyanine perchlorate; E-cad, e-cadherin; E. coli, Escherichia coli; E15, embryonic day 15; FBS, fetal bovine serum; FGF, fibroblast growth factor; LPS, lipopolysaccharide; PDGF, platelet derived growth factor; SHH, sonic hedgehog; TGF-β, transforming growth factor beta; TO-PRO-3, 4-[3-(3-methyl-2(3H)-benzothiazolylidene)-1-propenyl]-1-[3-(trimethylammonio)propyl]-, diiodide; VEGF, vascular endothelial growth factor; Z-VAD-FMK, Z-Val-Ala-Asp-CH2F; alveolarization; bronchopulmonary dysplasia; lung development; myofibroblast; α-SMA, alpha-smooth muscle actin.