What question did this study set out to answer?

This research aims to establish stable and immortalized human airway epithelial cell lines necessary for studying chronic respiratory diseases.

June 12, 2026Open Access

Immortalization of Human Airway Epithelial Cells Using BMI1 and hTERT: Development and Optimization of Small and Large Airway Cell Culture Models

Key Points

This research aims to establish stable and immortalized human airway epithelial cell lines necessary for studying chronic respiratory diseases.
Developed cell culture protocols for immortalizing small and large airway epithelial cells using BMI1 and hTERT.
Conducted three experimental phases with primary cell sources to optimize growth conditions and measure cell viability.
Analyzed population doubling rates and protein expression through western blot techniques.
D19V LAE cells at P4 exhibited a population doubling rate of 1.74-fold higher than controls (3.43 vs 1.98).
Phase 2 showed successful passaging of LAE cells to Passage 7, confirming the effectiveness of the optimized culture conditions.
Findings establish proof-of-concept for airway cell immortalization with implications for disease modeling.

Abstract

Human small airway epithelial (SAE) cells play a central role in numerous chronic respiratory diseases, including cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), asthma, and idiopathic pulmonary fibrosis (IPF), yet stable, well-characterized immortalized SAE cell lines are extremely limited. This gap represents a significant limitation for respiratory medicine research. This study describes the development and optimization of cell culture protocols for generating stable, immortalized cell lines from primary SAE and large airway epithelial (LAE) cells via lentiviral transduction of B lymphoma Mo-MLV insertion region 1 (BMi1) and human Telomerase Reverse Transcriptase (hTERT). Three experimental phases were conducted using primary cell sources DD22U, D19V, and D01W. Phase 1 (DD22U) established that feeder cell support is essential for sustained proliferation. Phase 2 (D19V) employed a refined four-condition design and demonstrated successful passaging to Passage 7 (P7) for LAE cells, with western blot confirming BMI1 and hTERT protein expression. Phase 3 utilized two donor samples (D01W and D02W): D01W showed immediate proliferative failure attributed to poor initial viability, while D02W exhibited transient initial proliferation followed by rapid collapse by passages P3–P5, representing a partially viable but unstable outcome. Population doubling analysis revealed that LAE cells exhibit consistently higher proliferative capacity than SAE cells across passages, and that BMI1/hTERT-infected cells maintain substantially superior growth kinetics at late passages (P4–P5), where control cells undergo replicative senescence. The most striking example was D19V LAE cells at P4, where infected cells showed a 1.74-fold higher population doubling than controls (3.43 vs. 1.98). While some donor samples showed limited or transient proliferation, these findings establish proof-of-concept for airway epithelial cell immortalization and define key parameters influencing long-term growth. This work provides a foundation for developing stable SAE and LAE models for studying respiratory disease mechanisms and therapeutic development.

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