Cold dispase digestion of murine lungs improves recovery and culture of airway epithelial cells.
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...
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Public Library of Science (PLoS)
2024-01-01
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| Series: | PLoS ONE |
| Online Access: | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0297585&type=printable |
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| author | Piotr Pawel Janas Caroline Chauché Patrick Shearer Georgia Perona-Wright Henry J McSorley Jürgen Schwarze |
| author_facet | Piotr Pawel Janas Caroline Chauché Patrick Shearer Georgia Perona-Wright Henry J McSorley Jürgen Schwarze |
| author_sort | Piotr Pawel Janas |
| collection | DOAJ |
| description | 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 distal airway stem cell (DASC) 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. |
| format | Article |
| id | doaj-art-7eef7a8a675940f1a051061377e10cbb |
| institution | Kabale University |
| issn | 1932-6203 |
| language | English |
| publishDate | 2024-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj-art-7eef7a8a675940f1a051061377e10cbb2025-08-20T03:25:46ZengPublic Library of Science (PLoS)PLoS ONE1932-62032024-01-01191e029758510.1371/journal.pone.0297585Cold dispase digestion of murine lungs improves recovery and culture of airway epithelial cells.Piotr Pawel JanasCaroline ChauchéPatrick ShearerGeorgia Perona-WrightHenry J McSorleyJürgen SchwarzeAirway 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 distal airway stem cell (DASC) 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.https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0297585&type=printable |
| spellingShingle | Piotr Pawel Janas Caroline Chauché Patrick Shearer Georgia Perona-Wright Henry J McSorley Jürgen Schwarze Cold dispase digestion of murine lungs improves recovery and culture of airway epithelial cells. PLoS ONE |
| title | Cold dispase digestion of murine lungs improves recovery and culture of airway epithelial cells. |
| title_full | Cold dispase digestion of murine lungs improves recovery and culture of airway epithelial cells. |
| title_fullStr | Cold dispase digestion of murine lungs improves recovery and culture of airway epithelial cells. |
| title_full_unstemmed | Cold dispase digestion of murine lungs improves recovery and culture of airway epithelial cells. |
| title_short | Cold dispase digestion of murine lungs improves recovery and culture of airway epithelial cells. |
| title_sort | cold dispase digestion of murine lungs improves recovery and culture of airway epithelial cells |
| url | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0297585&type=printable |
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