Improved AECs viability translates to a better performance in <i>in vitro</i> culture.

Cold digestion culture yields a greater number of basal cell colonies, which occupy a larger area. (a) Workflow diagram representing the steps involved in digesting the murine lungs and MACS sorting AECs for in vitro culture. (b) Representative immunofluorescent micrographs (cold and hot digestions) of P63 (red), KRT5 (green), Hoechst (blue) and E-Cadherin (magenta) used for quantification of the number of P63 and KRT5 double positive colonies. Scale bar 0.1mm. 25% of each 24-WP well imaged, which equals to area of 52.6mm2. Each well was imaged using 20x objective starting from the centre. (c) 20x magnification phase-contrast (grey) image overlayed with immunofluorescent KRT5 (green) and Hoechst (blue) images to present morphology of KRT5+ cells following cold digestion. Scale bar (white bar), 100μm (d) Quantification of the number (left) and surface area (right) of basal AECs colonies. Colony was defined as ten E-cadh/P63/KRT5+ cells in immediate vicinity. Colony numbers were quantified based on E-cadh/P63/KRT5+ cells, while colony surface was quantified using only KRT5 pixels. Unpaired t-test (n = 8), median ± min/max. Each data point is a single well cultured from MACS sorted CD45-CD31- EpCAM+ isolated from a single murine lung.</p

Flow cytometry antibodies.

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.</div

Flow cytometric analysis of murine lung digests with lung inflation, comparing the number of cells after hot (1h, 37°C) or cold (20h, 4°C) digests.

(a) Representative gating strategy following debris exclusion (FSC/SSC), singlets (FSC-H/FSC-A) and dead cells exclusion (LIVE/DEAD fixable near-IR). CD45-CD31- cells are gated, followed by EpCAM+ gating. (b) Number of CD45-CD31-EpCAM+ AECs, CD31+ endothelial cells or CD45+ leukocytes. (c) Comparison of relative MFI (median) values for EpCAM, CD45 and CD31 between cold and hot digests within respective CD45-CD31-EpCAM+, CD45+ and CD31+ gates. Each data point represents a lung from a single mouse. Unpaired t-test, median ± min/max.</p

Murine AECs can be passaged (P0-2) following cold digestion and CD45^-CD31^-EpCAM⁺ MACS sorting.

(a) Following cold digestion, murine AECs (CD34-CD31-EpCAM+) were MACS sorted and 2x105 cells were seeded into a 24-well plate and cultured in a Stemcell PneumaCult Ex-Plus medium with 10μM Y-27632, 3μM CHIR99021, 1μM A 83–01 for 14 days. Cells were passaged twice. Each time cells were detached using TrypLE and split 1:10 into a larger well plate starting from 24-well plate (P0), followed by 12-well plate (P1) and lastly a 6-well plate (P2). Once cells reached confluency at the end of passage 2, cells were lifted up using TrypLE and 1.4x106 live cells (trypan blue exclusion) were counted using haemocytometer. Six 10x objective phase-contrast single fields of view (3x2) were stitched together. Scale bar 400μm. N>2. (b) All cells express KRT5 (green) after three passages of CD45-CD31-EpCAM+ MACS sorted AECs. Scale bar, 300μm. (TIF)</p

Step-by-step protocol including materials and recipes.

Step-by-step protocol including materials and recipes.</p

Digestion type does not affect cellular oxidative stress based on LDHa (lactate dehydrogenase) levels.

Following either hot or cold digestion CD45-CD31-EpCAM+ cells were MACS sorted and RNA isolated using Trizol and chloroform-based RNA isolation. SYBR green qPCR was performed, with Rpl37a as endogenous control. N = 3–4. (TIF)</p

Cold dispase II digest improves the viability and yield of highly pure MACS sorted CD45^-CD31^-EpCAM⁺ cells.

(a) Representative gating strategy for hot and cold digestion following debris exclusion (FSC/SSC) and singlets (FSC-H/FSC-A) for analysis of viability of CD45-CD31-EpCAM+ AECs. (b) Comparison of frequency of viable FACS-sorted CD45-CD31-EpCAM+ through live/dead near-IR fixable dye. Unpaired t-test (n = 21–33), median ± min/max. (c) Comparison of cell yield between the digest methods after CD45, CD31 MACS depletion and EpCAM+ MACS selection. Quantification using haemocytometer and trypan blue live/dead exclusion. Unpaired t-test (n = 8), median ± min/max. (d) Evaluation of cell suspension purity after MACS sorting CD45-CD31-EpCAM+ cells using flow cytometry. Each data point corresponds to a single murine lung. (e) Validating the CD45-CD31-EpCAM+ cells identity. CD45-CD31-EpCAM+ cells also express CD49f and CD24. Presented gating followed debris exclusion (FSC/SSC), singlets gating (FSC-H/FSC-A), live cells gating (LIVE/DEAD fixable near-IR/FSC-H), CD45 and CD31 exclusion (CD45/CD31) and EpCAM+ gating (EpCAM/FSC-H).</p

Cold digestion with dispase II does not remove MHC-I, MHC-II or CD24 from the surface of CD45^-CD31^-EpCAM⁺ cells.

Following debris exclusion (FSC/SSC), singlets gating (FSC-H/FSC-A), live cells gating (LIVE/DEAD fixable near-IR/FSC-H), CD45 and CD31 exclusion (CD45/CD31) and EpCAM+ gating (EpCAM/FSC-H) expression of MHC-I, MHC-II and CD24 was evaluated. Gating was based on fluorescence minus one (FMO) controls. (TIF)</p

Immunofluorescent microscopy antibodies.

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.</div

Step-by-step protocol, also available on protocols.io.

https://dx.doi.org/10.17504/protocols.io.rm7vzxo68gx1/v1. (PDF)</p