Immortalisation of human airway epithelial cells for genome editing applications
6 affiliations
- 1Cell Biology, Neurobiology and Biophysics, Department of Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands
- 2Cellular Disease Models, Department of Pediatrics, Regenerative Medicine Center Utrecht, University Medical Center Utrecht, Utrecht, The Netherlands
- 3These authors contributed equally
- 4Regenerative Medicine Center Utrecht, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands
- 5Centre for Living Technologies, Alliance TU/e, WUR, UU, UMC Utrecht, Utrecht, The Netherlands
- 6Utrecht Platform for Organoid Technology, UMC Utrecht, Utrecht, The Netherlands
Abstract
Aim: In vitro air–liquid interface (ALI) cultures of human airway epithelial cells are widely used to study respiratory biology and disease, as they recapitulate key features of the pseudostratified airway epithelium and reduce reliance on animal models and repeated patient biopsies. However, the limited proliferative capacity of primary airway basal cells restricts long-term studies and genome editing. This study aimed to generate stable, immortalised human airway basal cell lines that retain differentiation capacity and support genetic manipulation.
Methods: Human airway epithelial basal cells from bronchial and nasal origin were immortalised by lentiviral expression of human telomerase reverse transcriptase (hTERT) followed by polyclonal expansion. A panel of cell lines derived from three independent nasal donors was established. Differentiation capacity was assessed in ALI cultures and compared with primary airway epithelial cells. Suitability for genetic manipulation was evaluated using lentiviral overexpression and CRISPR/Cas9-mediated gene knockout and knock-in strategies.
Results: All immortalised cell lines displayed sustained proliferative capacity and efficiently differentiated into major airway epithelial cell types, including multiciliated and secretory cells. Differentiation patterns and functional characteristics closely resembled those of primary airway epithelial cultures. The cell lines supported efficient lentiviral transduction and CRISPR/Cas9-based genome editing, enabling stable gene overexpression, targeted gene disruption, and precise genomic modification.
Conclusions: hTERT-immortalised human airway basal cell lines preserve key features of primary airway epithelium while enabling long-term culture and versatile genome editing. This resource provides a robust platform for airway biology research, disease modelling, and therapeutic development.
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BibTeX
@article{grinsven2026immortalisation,
title = {{Immortalisation of human airway epithelial cells for genome editing applications}},
author = {Emma J. van Grinsven and Georgia N. Ithakisiou and Pim Cleijpool and Bram M. Bosch and Marinos Tziouvelis and Gimano D. Amatngalim and Sam F.B. van Beuningen and Wilco Nijenhuis and Bahar Yetkin-Arik and Lukas C. Kapitein and Jeffrey M. Beekman and Anna Akhmanova},
journal = {ERJ Open Research},
year = {2026},
month = sep,
doi = {10.1183/23120541.01708-2025},
url = {https://doi.org/10.1183/23120541.01708-2025},
note = {CC BY-NC 4.0},
}
RIS
TY - JOUR
TI - Immortalisation of human airway epithelial cells for genome editing applications
AU - Emma J. van Grinsven
AU - Georgia N. Ithakisiou
AU - Pim Cleijpool
AU - Bram M. Bosch
AU - Marinos Tziouvelis
AU - Gimano D. Amatngalim
AU - Sam F.B. van Beuningen
AU - Wilco Nijenhuis
AU - Bahar Yetkin-Arik
AU - Lukas C. Kapitein
AU - Jeffrey M. Beekman
AU - Anna Akhmanova
PY - 2026
DA - 2026/09/01
JO - ERJ Open Research
DO - 10.1183/23120541.01708-2025
UR - https://doi.org/10.1183/23120541.01708-2025
AB - Aim: In vitro air–liquid interface (ALI) cultures of human airway epithelial cells are widely used to study respiratory biology and disease, as they recapitulate key features of the pseudostratified airway epithelium and reduce reliance on animal models and repeated patient biopsies. However, the limited proliferative capacity of primary airway basal cells restricts long-term studies and genome editing. This study aimed to generate stable, immortalised human airway basal cell lines that retain differentiation capacity and support genetic manipulation. Methods: Human airway epithelial basal cells from bronchial and nasal origin were immortalised by lentiviral expression of human telomerase reverse transcriptase (hTERT) followed by polyclonal expansion. A panel of cell lines derived from three independent nasal donors was established. Differentiation capacity was assessed in ALI cultures and compared with primary airway epithelial cells. Suitability for genetic manipulation was evaluated using lentiviral overexpression and CRISPR/Cas9-mediated gene knockout and knock-in strategies. Results: All immortalised cell lines displayed sustained proliferative capacity and efficiently differentiated into major airway epithelial cell types, including multiciliated and secretory cells. Differentiation patterns and functional characteristics closely resembled those of primary airway epithelial cultures. The cell lines supported efficient lentiviral transduction and CRISPR/Cas9-based genome editing, enabling stable gene overexpression, targeted gene disruption, and precise genomic modification. Conclusions: hTERT-immortalised human airway basal cell lines preserve key features of primary airway epithelium while enabling long-term culture and versatile genome editing. This resource provides a robust platform for airway biology research, disease modelling, and therapeutic development.
SN - 2312-0541
ER -