TITLE: Cold dispase digestion of murine lungs improves recovery and culture of  airway epithelial cells
AUTHORS: piotr.janas, Caroline Chauché, Patrick Shearer, Georgia Perona-Wright, Henry J McSorley, Jürgen Schwarze

[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.

[STEPS]
SECTION: Cold dispase digestion of murine lungs improves recovery and culture of  airway epithelial cells
1. Euthanise a mouse according to local protocols and regulations (excluding carbon dioxide).
SECTION: Cold dispase digestion of murine lungs improves recovery and culture of  airway epithelial cells
2. Using scissors, cut the skin and open the abdomen. Follow through by cutting the skin along the sternum and neck.
SECTION: Cold dispase digestion of murine lungs improves recovery and culture of  airway epithelial cells
3. Cut the abdominal aorta.
SECTION: Cold dispase digestion of murine lungs improves recovery and culture of  airway epithelial cells
4. Expose the trachea by removing salivary glands. Support the trachea by releasing curved tweezers underneath the trachea.
SECTION: Cold dispase digestion of murine lungs improves recovery and culture of  airway epithelial cells
5. Make a small incision below the larynx using spring bow scissors.
SECTION: Cold dispase digestion of murine lungs improves recovery and culture of  airway epithelial cells
6. Cannulate the trachea using a blunt needle and secure it using elastic thread.
SECTION: Cold dispase digestion of murine lungs improves recovery and culture of  airway epithelial cells
7. Perform bronchoalveolar lavage (BAL) three times with pre-warmed (37°C) DMEM/F12 (0.8ml/BAL/mouse).
SECTION: Cold dispase digestion of murine lungs improves recovery and culture of  airway epithelial cells
8. Remove the ribcage and flush the cardiovascular system by injecting 10ml of ice-cold DMEM/F12 into the right ventricle.
SECTION: Cold dispase digestion of murine lungs improves recovery and culture of  airway epithelial cells
9. Using the tracheal cannula inject 1.5-2ml of ice-cold digestion solution into the lungs.
SECTION: Cold dispase digestion of murine lungs improves recovery and culture of  airway epithelial cells
10. Remove the lungs by dissecting them from thoracic cavity. Cut at the primary bronchial bifurcation and quickly place lungs in remaining ice-cold 3ml of digestion mixture before the lungs deflate.
SECTION: Cold dispase digestion of murine lungs improves recovery and culture of  airway epithelial cells
11. Incubate the lungs for 20h at 4°C.
SECTION: Cold dispase digestion of murine lungs improves recovery and culture of  airway epithelial cells
12. Pour the digestion solution together with the lungs onto a 70µm strainer and mash the lungs using 5ml syringe plunger by gently agitating the lungs on the mesh (lungs should dissolve without a need for excessive force).
SECTION: Cold dispase digestion of murine lungs improves recovery and culture of  airway epithelial cells
13. Wash the strainer with 10ml of dispase wash and then centrifuge each sample for 15min, 4°C, 130 relative centrifugal force (RCF).
SECTION: Cold dispase digestion of murine lungs improves recovery and culture of  airway epithelial cells
14. Remove the supernatant and add 2ml of ice-cold ACK lysing buffer (red blood cell lysis) to each tube. Swirl the samples for 90s and then add 5ml of MACS buffer.
SECTION: Cold dispase digestion of murine lungs improves recovery and culture of  airway epithelial cells
15. Pass the samples through 40µm strainer, wash the strainer with 5ml of MACS buffer and centrifuged for 5min, 4°C at 300 RCF. 
SECTION: Cold dispase digestion of murine lungs improves recovery and culture of  airway epithelial cells
17. Count the number of cells using trypan blue and a haemocytometer.
SECTION: Cold dispase digestion of murine lungs improves recovery and culture of  airway epithelial cells
18. After incubation, centrifuge cells for 5min, 4°C, 300 RCF and resuspend in 85µl of MACS buffer + 5µl of anti-CD31 microbeads and 10µl of anti-CD45 microbeads per 107 total cells.
SECTION: Cold dispase digestion of murine lungs improves recovery and culture of  airway epithelial cells
19. Incubate the samples for 30 minutes on ice.
SECTION: Cold dispase digestion of murine lungs improves recovery and culture of  airway epithelial cells
20. Wash each sample with 1ml/107 cells of MACS buffer and centrifuge for 5min, 4°C at 300 RCF.
SECTION: Cold dispase digestion of murine lungs improves recovery and culture of  airway epithelial cells
21. Pass the samples through activated LS MACS columns on a QuadroMACS separator and collect the flow-through.
SECTION: Cold dispase digestion of murine lungs improves recovery and culture of  airway epithelial cells
22. Centrifuge collected cells for 5min, 4°C, 300 RCF and remove supernatant.
SECTION: Cold dispase digestion of murine lungs improves recovery and culture of  airway epithelial cells
23. Resuspended cells in left-over liquid
SECTION: Cold dispase digestion of murine lungs improves recovery and culture of  airway epithelial cells
24. Add 15µl of anti-EpCAM microbeads and incubate samples on ice for 30 minutes.
SECTION: Cold dispase digestion of murine lungs improves recovery and culture of  airway epithelial cells
25. Wash samples with 1ml MACS buffer, and centrifuge for 5min, 4°C, 300 RCF.
SECTION: Cold dispase digestion of murine lungs improves recovery and culture of  airway epithelial cells
26. Discard supernatant and resuspend cells in 500µl of MACS buffer.
SECTION: Cold dispase digestion of murine lungs improves recovery and culture of  airway epithelial cells
27. Pass the cell suspension through activated MS MACS columns using an OctoMACS separator.
SECTION: Cold dispase digestion of murine lungs improves recovery and culture of  airway epithelial cells
28. Flush out EpCAM+ cells into a fresh test tube from the column by applying 2ml of MACS buffer and inserting the plunger into the column.
SECTION: Cold dispase digestion of murine lungs improves recovery and culture of  airway epithelial cells
29. Centrifuge cells for 5min at 4°C, 300 RCF, remove supernatant, and resuspend cell pellet in 5ml of supplemented airway epithelial growth media.
SECTION: Cold dispase digestion of murine lungs improves recovery and culture of  airway epithelial cells
30. Count the number of live cells using trypan blue and a haemocytometer.
SECTION: Cold dispase digestion of murine lungs improves recovery and culture of  airway epithelial cells
31. Coat the well plate with well-plate coating solution and incubate for at least 4-8h at 37°C before use.
SECTION: Cold dispase digestion of murine lungs improves recovery and culture of  airway epithelial cells
32. Wash the coated 24-well plate with DPBS before adding 0.4ml of pre-warmed airway epithelial growth medium.
SECTION: Cold dispase digestion of murine lungs improves recovery and culture of  airway epithelial cells
33. Add at least 2x105 live MACS sorted cells per well and change media after 24h, followed by media changes every two days thereafter. Within 7 days p63+ KRT5+ colonies will appear.
SECTION: Cold dispase digestion of murine lungs improves recovery and culture of  airway epithelial cells
16. Resuspend each sample in 1ml of MACS buffer and block with 5µl of 0.5mg/ml anti-mouse CD16/32 antibody for at least 20min.