Optimized primary organotypic culture from murine neonatal tracheal airway epithelial cells

Neonatal airway development and injury is poorly understood, in part due to challenges of studying extremes of phenotype in human pathologic samples and difficulties obtaining relevant comparator samples. Ex vivo model systems are needed to improve understanding of airway development, injury and repair in the neonatal lung. We optimized a protocol for organotypic culture of primary murine neonatal tracheal epithelial cells (MNTECs). We compared expansion and differentiation properties of MNTECs in five different media conditions, ranging from previously published “lab-made” media to commercial sources of media. We measured the success of our organotypic cultures by quantifying the relative proportions of ciliated epithelium, TP63+ basal stem cells, stromal cell contamination, as well as total cell numbers and air-liquid interface (ALI) thickness. Commercially available media performed better than standard lab-made media, with nearly 100% success and 20% success, respectively. Proliferation in commercial media improves cell proliferation of TP+63 basal cells, inhibits growth of contaminating stromal cells, and improves differentiation to a polarized, ciliated pseudostratified airway epithelium, when compared to lab-made LP media. These results provide a reliable technique for studying neonatal airway epithelial cells in wild type and genetically mutant mice.