Preprint Article Version 1 Preserved in Portico This version is not peer-reviewed

Cold Dispase Digestion of Murine Lungs Improves Recovery and Culture of Airway Epithelial Cells

Piotr P. Janas
ORCID logo ,
Caroline Chauché
ORCID logo ,
Patrick Shearer
,
Georgia Perona-Wright
,
Henry J. McSorley
,
Jürgen Schwarze
*
Version 1 : Received: 10 July 2023 / Approved: 11 July 2023 / Online: 12 July 2023 (08:57:55 CEST)

A peer-reviewed article of this Preprint also exists.

Chauché, C., Shearer, P., Perona-Wright, G., McSorley, H. J., & Schwarze, J. (2023). Cold dispase digestion of murine lungs improves recovery and culture of airway epithelial cells. Chauché, C., Shearer, P., Perona-Wright, G., McSorley, H. J., & Schwarze, J. (2023). Cold dispase digestion of murine lungs improves recovery and culture of airway epithelial cells.

Abstract

Airway epithelial cells (AECs) play a key role in maintaining lung homeostasis, epithelium regeneration and the initiation of pulmonary immune responses. To isolate and study murine AECs investigators have classically used short and hot (1h 37°C) digestion protocols. Here, we present a workflow for efficient AECs isolation and culture, utilizing long and cold (20h 4°C) dispase II digestion of murine lungs. This protocol yields a greater number of viable AECs compared to an established 1h 37°C dispase II digestion. Using a combination of flow cytometry and immunofluorescent microscopy, we demonstrate that compared to the established method, the cold digestion allows for recovery of a 3-fold higher number of CD45-CD31-EpCAM+ cells from murine lungs. Their viability is increased compared to established protocols, they can be isolated in larger numbers by magnetic-activated cell sorting (MACS), and they result in greater numbers of KRT5+p63+ colonies in vitro. Our findings demonstrate that temperature and duration of murine lung enzymatic digestion have a considerable impact on AEC yield, viability, and ability to form colonies in vitro. We believe this workflow will be helpful for studying lung AECs and their role in the biology of lung.

Keywords

airway epithelial cells; cell culture; MACS; dispase; cold lung digestion

Subject

Biology and Life Sciences, Cell and Developmental Biology

Copyright: This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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