Long term culture of the a549 cancer cell line promotes multilamellar body formation and differentiation towards an alveolar type II Pneumocyte phenotype

James Ross Cooper*, Muhammad Bilal Abdullatif, Edward C. Burnett, Karen Kempsell, Franco Conforti, Howard Tolley, Jane E. Collins, Donna E. Davies

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

68 Citations (Scopus)


Pulmonary research requires models that represent the physiology of alveolar epithelium but concerns with reproducibility, consistency and the technical and ethical challenges of using primary or stem cells has resulted in widespread use of continuous cancer or other immortalized cell lines. The A549 'alveolar' cell line has been available for over four decades but there is an inconsistent view as to its suitability as an appropriate model for primary alveolar type II (ATII) cells. Since most work with A549 cells involves short term culture of proliferating cells, we postulated that culture conditions that reduced proliferation of the cancer cells would promote a more differentiated ATII cell phenotype. We examined A549 cell growth in different media over long term culture and then used microarray analysis to investigate temporal regulation of pathways involved in cell cycle and ATII differentiation; we also made comparisons with gene expression in freshly isolated human ATII cells. Analyses indicated that long term culture in Ham's F12 resulted in substantial modulation of cell cycle genes to result in a quiescent population of cells with significant up-regulation of autophagic, differentiation and lipidogenic pathways. There were also increased numbers of up- and down-regulated genes shared with primary cells suggesting adoption of ATII characteristics and multilamellar body (MLB) development. Subsequent Oil Red-O staining and Transmission Electron Microscopy confirmed MLB expression in the differentiated A549 cells. This work defines a set of conditions for promoting ATII differentiation characteristics in A549 cells that may be advantageous for studies with this cell line.

Original languageEnglish
Article numbere0164438
JournalPLoS ONE
Issue number10
Publication statusPublished - Oct 2016

Bibliographical note

Funding Information:
This study was funded by the Pipeline Funding Board of Public Health England (PHE) (at that time the organization was known as the Health Protection Agency (HPA)) as part of its 2012/13 call to fund studies which had the potential to aid life science research. The original bid title was: "Use of microarray analysis in the characterisation of HPACC cell lines for the presence of cell biomarkers". PHE like its predecessor HPA is an executive agency sponsored by the Department of Health (England). The funding was awarded to Dr Edward Burnett of the Culture Collections of PHE; a not for profit Bio-Resource within the PHE National Infections Service (NIS). Five of the authors (James R Cooper, Muhammad Abdullatif, Edward C Burnett, Karen E Kempsell and Howard Tolley) were employed by HPA/PHE during the study but they were not and never have been members of the Pipeline Funding Board. The HPA/PHE Pipeline Funding Board had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Dr Franco Confort's contribution was funded by the British Lung Foundation reference number IPFPG12-2.

Publisher Copyright:
© 2016 Cooper et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


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