(423) A novel method for isolation of small airway epithelial cells by bulk enzymatic digestion

Background: Distal small airways are an important site of early cystic fibrosis (CF) lung disease. Small airway epithelial (SAE) cell populations include secretory cells that are collectively the greatest source of CFTR expression [1]. An in vitro model of CF SAE cells would be an important advance. Prior methods for SAE cell isolation include bronchoscopy brushings or micro-dissection, both of which are challenging in CF. We introduce a method for the isolation of SAE cells using bulk enzymatic digestion of distal lung tissue followed by growth in media selective for airway epithelial cells.

Methods: Non-CF lungs were procured from de-identified organ donors without prior chronic lung disease and CF lungs were obtained following transplant. An ~5 cm x 5 cm of distal, sub-pleural lung tissue was harvested for bulk digestion. The pleura was removed, and absence of large airways was validated in the cut proximal surface. Tissue was minced in cold PBS and serially digested in: 1) Dispase II; 2) Elastase, Trypsin/EDTA, and 3) DNase. Digestion enzymes were neutralized with 10% FBS, and the sample was filtered through sterile gauze, and 100 µm and 40 µm cell strainers. ACK lysis was used to remove red blood cells. Cells were cultured on Type I/III bovine collagen coated plates in UNC BEGM media with supplemental anti-bacterial and anti-fungal antibiotics. Extra anti-microbials were removed at three days and at >70% confluence cells were sub-cultured in BEGM. Passage two cells were seeded on human placental collagen coated inserts in UNC ALI media. Well-differentiated air-liquid interface (ALI) cultures were analyzed in Ussing chambers, and cell type composition was evaluated by histology, whole mount immunohistochemistry, and transcript expression. Comparisons were made to large airway epithelial (LAE) cells from the same donors and to SAE cells obtained by micro-dissection.

Results: Bulk dissection was performed on distal lung from nine non-CF and five CF lungs. Micro-dissection of small airways from end-stage CF lungs was technically challenging due to destruction of lung architecture. Mean passage one cell yields from non-CF and CF lungs was 0.27 and 0.28 M cells/g tissue, respectively. Ussing chamber analysis was consistent with non-CF versus CF origin, and SAE cultures exhibited greater CFTR activity than LAE cells from matched non-CF donors. Histological and whole-mount immunohistochemistry revealed a thinner mucociliary epithelium in SAE versus LAE ALI cultures. SAE cells from both non-CF and CF donors preferentially expressed SPB and SCGB3A2, unique in vivo markers of SAE cells. Importantly, qRTR-PCR demonstrated consistent transcript levels of epithelial cell type markers between SAE cells from the same donor obtained by the micro-dissection and bulk digestion methods.

Conclusions: Bulk enzymatic digestion of distal lung tissue and selective media allowed isolation of SAE cells from end-stage CF lungs, where microdissection was technically challenging. Studies in progress are exploring the use of methods allowing extensive cell expansion to enhance primary SAE cell supplies. These results illustrate an important advance enabling isolation of SAE cells to study CF lung disease pathogenesis and treatment.

Acknowledgements: [Supported by CFF RDP BOUCHE19R0, NIH DK065988].

References: 1. Okuda et al. Secretory cells dominate airway CFTR expression and function in human airway superficial epithelia. Am J Respir Crit Care Med. 2021; 203(10):1275-89. doi: 10.1164/rccm.202008-3198OC