Isolation of Alveolar Type II Epithelial Cells from Neonatal, Juvenile, and Adult Murine Lungs Adaptable to Infectious Experimental Settings

Our research aims to identify the roles of alveolar type II epithelial cells, in short AECII, for lung development, infection and inflammation. As a novelty, our protocol enables AECII isolation from neonatal and juvenile mice, including contagious samples. To begin, obtain a euthanized juvenile mouse.

After exsanguination, carefully remove the diaphragm to expose the heart and lungs. Open the rib cage using scissors. For enzymatic digestion for adult and juvenile mice, expose the trachea by removing the salivary glands and surrounding muscle.

Insert a 22-gauge indwelling cannula into the trachea. Remove the needle and advance the plastic catheter toward the lungs. Secure a piece of yarn around the trachea and catheter to hold it in place.

Instill dispase through the catheter to prepare the lung tissue. Exchange the syringe and instill low-melt agarose at 1%weight per volume in 45 degrees Celsius to maintain lung structure. Now, cover the lung with laboratory tissue paper and add ice for two minutes to allow the agarose to solidify.

Remove the ice, tissue paper, syringe, and catheter. Then tie the yarn to close the trachea and retain the enzyme in the lower airways. Remove the heart and thymus.

Cut the trachea above the yarn and remove the intact lung from the chest. Transfer the lung into a 15 milliliter tube containing pre-warmed dispase. Incubate the juvenile lung tissue in dispase at room temperature for 45 minutes.

Remove the lung from dispase and dissect the lung lobes from the bronchial stem. Place the lung lobes into a Petri dish containing seven milliliters of DMEM supplemented with HEPES and 100 microliters DNase. Using forceps, disintegrate the lung tissue and incubate for 10 minutes at room temperature to ensure efficient enzymatic digestion of the lung tissue.

To sort alveolar epithelial type II cells, install a 100 micrometer nozzle on the fluorescence-activated cell sorting device. Filter the antibody-labeled cells through a 50 micrometer cell strainer before sorting and rinse the strainer with supplemented DMEM to prevent cell loss. Resuspend the cells by vortexing immediately before sorting.

Gate for alveolar epithelial type II cells by selecting events with high side scatter, negative lineage fluorochromes, and positive CD326 signal. Exclude doublets or aggregates using forward scatter height versus area and side scatter height versus area. Collect the sorted cells into tubes containing supplemented DMEM and centrifuge for further analysis or culture.

Flow-sorting generated pure alveolar epithelial type II cell populations, with post-sort analysis validating almost 100%specificity for established cellular markers. Quantification of post-sort samples showed yields of approximately 0.15 million viable alveolar epithelial type II cells from neonatal lungs and up to 1 million cells from adult lungs per whole lung preparation. Isolated alveolar epithelial type II cells maintained typical cellular morphology after isolation.

Cellular integrity of isolated alveolar epithelial type II cells was preserved throughout the isolation process. Isolated alveolar epithelial type II cells supported ex vivo infection with influenza A virus expressing the mCherry reporter protein, demonstrating viral replication over time. High-quality RNA was successfully extracted from paraformaldehyde fixed alveolar epithelial type II cells across different processing time points.

The quality of extracted RNA showed a direct relationship with the duration of thermal treatment at 80 degrees Celsius during extraction. Quantitative reverse transcription PCR analysis confirmed successful amplification of ribosomal protein S9 and surfactant protein C transcripts from paraformaldehyde fixed and fresh alveolar epithelial type II cell samples. Our lab has previously identified crucial immunoregulatory roles and trained immunity of AECII during lung inflammation and infection.

In general, obtaining high AECII yields and high purity at the same time is an experimental challenge. This protocol paves the way for future research on the roles of AECII during respiratory infection and postnatal lung development.