Simultaneous Isolation of Proximal and Distal Lung Progenitor Cells from Individual Mice Using a 3D Printed Guide Reduces Proximal Cell Contamination of Distal Lung Epithelial Cell Isolations

The respiratory epithelium consists of multiple, functionally distinct cell types and is maintained by regionally specific progenitor pop-ulations that repair the epithelium following injury. Several in vitro methods exist for studying lung epithelial repair using primary mu -rine lung cells, but isolation methods are hampered by a lack of surface markers distinguishing epithelial progenitors along the respiratory epithelium. Here, we developed a 3D printed lobe divider (3DLD) to aid in simultaneous isolation of proximal versus distal lung epithelial progenitors from individual mice that give rise to differentiated epithelia in multiple in vitro assays. In contrast to 3DLD-isolated distal progenitor cells, commonly used manual tracheal ligation methods followed by lobe removal resulted in co-isolation of rare proximal cells with distal cells, which altered the transcriptional landscape and size distribution of distal organoids. The 3DLD aids in reproducible isolation of distal versus proximal progenitor populations and minimizes the potential for contaminating populations to confound in vitro assays.