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

Alsafadi, H. N.; Stegmayr, J.; Ptasinski, V.; Silva, I.; Mittendorfer, M.; Murray, L.; Wagner, D. E.

doi:10.1101/2022.05.10.491312

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bioRxiv · 10.1101/2022.05.10.491312

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

Alsafadi, H. N.·Stegmayr, J.·Ptasinski, V.·Silva, I.·Mittendorfer, M.·Murray, L.·Wagner, D. E.

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Abstract

The respiratory epithelium consists of multiple, functionally distinct cell-types and is maintained by regionally-specific progenitor populations which repair the epithelium following injury. Several in vitro methods exist for studying lung epithelial repair using primary murine lung epithelial 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 which give rise to differentiated epithelia in multiple in vitro assays. In contrast to 3DLD-isolated distal progenitor cells, classic manual tracheal ligation methods followed by lobe removal resulted in co-isolation of rare proximal progenitors with distal cells which altered the transcriptional landscape of distal organoid cultures. Thus, cell isolation with the 3DLD generates reproducible distal versus proximal progenitor populations and minimizes the potential for contaminating populations to confound in vitro assays. HighlightsO_LI3DLD reproducibly separates lung lobes and extrapulmonary airways (bronchi/trachea) C_LIO_LI3DLD cell isolation yields consistent isolation of distal epithelial cells (DECs) C_LIO_LIContamination of proximal cells in classic DEC isolations may alter in vitro results C_LIO_LI3DLD allows for simultaneous isolation of proximal and DECs from single animals C_LI eTOC blurbAlsafadi et al. describes a new method for simultaneous isolation of lung epithelial proximal and distal progenitors using the aid of a 3D printed device (3DLD). Both isolated cell types differentiate in multiple in vitro assays. The 3DLD guide minimized contamination of proximal cells in distal cell isolations whose presence can alter the transcriptional landscape of distal epithelial organoids.

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Alsafadi, H. N., Stegmayr, J., Ptasinski, V., Silva, I., Mittendorfer, M., Murray, L., Wagner, D. E.. 2022-05-10. 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. https://doi.org/10.1101/2022.05.10.491312

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