Optimized primary organotypic culture from murine neonatal tracheal airway epithelial cells

Neonatal airway development and injury are poorly understood, in part due to challenges of studying extremes of phenotype in human pathological 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, and 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 expansion of TP63+ basal cells, inhibits growth of contaminating stromal cells, and improves differentiation to a polarized, ciliated pseudostratified airway epithelium, when compared with lab-made LP media. These results provide a reliable technique for studying neonatal airway epithelial cells in wild-type and genetically mutant mice.NEW & NOTEWORTHY In this study, we optimized murine neonatal tracheal epithelial cell (MNTEC) growth and differentiation for mechanistic studies of disrupted airway epithelial cell development, injury, and repair. Our protocol removes significant complexity and historical variability of murine air-liquid interface (ALI) cultures and is specifically designed for neonatal airway epithelial cell cultures with limited cell numbers. We also compared the transcriptomes of well-differentiated and poorly differentiated organotypic airway epithelial cultures to identify key genes for epithelial growth and polarization.