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

• Published in: Stem cell reports Vol. 17; no. 12; pp. 2718 - 2731
• Main Authors: Alsafadi, Hani N., Stegmayr, John, Ptasinski, Victoria, Silva, Iran, Mittendorfer, Margareta, Murray, Lynne A., Wagner, Darcy E.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 13.12.2022; Elsevier
• Subjects: 3D printing; air-liquid interface; Animals; Basic Medicine; Cell and Molecular Biology; Cell Differentiation; cell isolation; Cell Separation; Cell- och molekylärbiologi; Epithelial Cells - metabolism; Lung; lung epithelium; lung progenitors; Medical and Health Sciences; Medicin och hälsovetenskap; Medicinska och farmaceutiska grundvetenskaper; Mice; murine alveolar type II cells; murine tracheal epithelial cells; organoids; Printing, Three-Dimensional; Resource; RNA-seq deconvolution; Stem Cells; surgical guide; surgical guide; murine tracheal epithelial cells; RNA-seq deconvolution; air-liquid interface; cell isolation; organoids; lung epithelium; murine alveolar type II cells; lung progenitors; 3D printing
• ISSN: 2213-6711; 2213-6711
• DOI: 10.1016/j.stemcr.2022.11.002

The respiratory epithelium consists of multiple, functionally distinct cell types and is maintained by regionally specific progenitor populations that repair the epithelium following injury. Several in vitro methods exist for studying lung epithelial repair using primary murine 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. [Display omitted] •3DLD reproducibly separates lung lobes and extrapulmonary airways (bronchi/trachea)•3DLD cell isolation yields consistent isolation of distal epithelial cells (DECs)•Contamination of proximal cells in classic DEC isolations may alter in vitro results•3DLD allows simultaneous isolation of proximal and DECs from single animals Alsafadi et al. describe a method for simultaneous isolation of viable and functional lung epithelial proximal and distal progenitors using a 3D printed device (3DLD). The 3DLD guide minimized contamination of proximal cells in distal cell isolations whose presence altered the transcriptional and morphological landscape of epithelial organoid cultures composed predominantly of distally derived Epcam+, CD45−, CD31− progenitor cells.