(451) Robust and Efficient Workflow to Establish, Culture, and Functionally Assess Primary-Isolated Airway Epithelial Cells

Thursday, November 3, 2022

12:45 PM – 1:25 PM ET

Presenting Author(s)

Tyler Brown (he/him/his)

Sr. Research Associate
STEMCELL Technologies Inc., British Columbia, Canada

Co-Author(s)

RS

Rick Swayze

Technical Scientist
STEMCELL Technologies Inc., British Columbia, Canada
NR

Natalie Ronaghan

Scientist
STEMCELL Technologies Inc., British Columbia, Canada
AE

Allen C. Eaves

President and CEO
STEMCELL Technologies Inc., British Columbia, Canada
SL

Sharon A. Louis

Senior Vice President, Research & Development
STEMCELL Technologies Inc., British Columbia, Canada
WC

Wing Chang

Director, Tissue Stem Cell Biology
STEMCELL Technologies Inc., British Columbia, Canada
EJ

Eric Jervis

Senior Principal Scientist
STEMCELL Technologies Inc., British Columbia, Canada
PK

Philipp Kramer

Senior Scientist
STEMCELL Technologies Inc., British Columbia, Canada

Background: Air-liquid interface (ALI) and organoid culture are key techniques for differentiating human airway epithelial cells (HAECs), and the efficiency and robustness of these assays often depends on the quality of primary-isolated cells. However, primary cell isolation workflows, where the user controls the choice of isolation method, cell culture medium, and culture format, may lead to reduced reproducibility. Therefore, an optimized and standardized workflow can further enhance and support the isolation of epithelial cells from diseased donors with potentially rare cystic fibrosis (CF) mutations, or particularly sensitive cell populations. Thus, we have developed a standardized workflow for the isolation and culture of freshly-derived airway epithelial cells.

Methods: Briefly, HAECs isolated from primary tissue were expanded in PneumaCult™-Ex Plus Medium for 1 week, and then seeded into Corning® Transwell® inserts and expanded until confluency. The cells were then differentiated in PneumaCult™-ALI Medium for at least 4 weeks. To assess differentiation efficiency in ALI culture, the cells were immunostained to detect Muc5AC, acetylated tubulin, and ZO-1 to identify goblet cells, ciliated cells, and apical tight junctions, respectively, as well as SARS-CoV-2 cell entry targets ACE2 and TMPRSS2. Ion transport and barrier function of the ALI cultures, as well as response to cystic fibrosis transmembrane conductance regulator (CFTR) correctors were also measured. In addition, freshly-derived HAECs were seeded into Corning® Matrigel® domes in the presence of PneumaCult™ Airway Organoid Seeding Medium. One week later, the medium was changed to PneumaCult™ Airway Organoid Differentiation Medium and maintained for an additional 3 weeks to promote cell differentiation. These airway organoids were then treated with CFTR corrector VX-809 for 24 hours, followed by a 6-hour treatment with amiloride, forskolin, and genistein to induce organoid swelling.

Results: Our results demonstrate that ALI cultures derived from CF donors displayed partial rescue of CFTR across multiple passages after treatment with VX-809 compared to a vehicle control. Airway organoids were found to express functional CFTR, as evidenced by forskolin treatment, which induced a 64 ± 14% (mean ± SD, n = 1 donor) increase in organoid area compared to a vehicle control-treated airway organoids. Airway organoids derived from CF donors displayed a loss of forskolin-induced swelling, which could be partially re-established with VX-809 treatment (29 ± 9%, n = 3).

Conclusions: In summary, the PneumaCult™ workflow supports robust and efficient culture of primary airway epithelial cells that can be used as physiologically relevant models suitable for cystic fibrosis research, CFTR corrector screening, and studying airway biology.

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