A tissue engineered approach to the modelling of alveolar epithelium and application to idiopathic pulmonary fibrosis

Abstract

Alveolar epithelial cells comprise type I (ATI) cells which facilitate gas exchange and type II (ATII) cells which play a critical role in alveolar homeostasis, innate immunity and self-renewal to replace ATI cells. Idiopathic Pulmonary Fibrosis (IPF) is a fatal fibrotic interstitial lung disease of unknown cause, however current theories implicate recurrent microinjuries to alveolar epithelium in association with activation of aberrant repair pathways. Thus, IPF research requires effective in vitro ATII cell models. Challenges of primary and stem cell culture and animal models have resulted in widespread use of the immortal A549 lung adenocarcinoma cell line, however its suitability remains unproven. It was hypothesised that modifying the culture conditions of A549 cells through three-dimensional (3D) or long term culture can enable in vitro tissue-like differentiation evidenced by expression of ATII marker genes and multilamellar body (MLB) production. Initially prolonged spheroid culture of A549 cells was tested for induction of ATII-like differentiation. RNA Microarray analysis demonstrated gene expression differences between spheroids and log phase two-dimensional (2D) cultures, suggesting the spheroids adopted a muco-secretory phenotype evidenced by markers of host defence and goblet cells. These properties were confirmed by QRT-PCR, histochemistry and transmission electron microscopy (TEM) but there was no evidence of ATII differentiation in spheroids. Next, ATII related gene expression in long term 2D cultures of A549 cells in Ham’s F12 medium was compared to freshly isolated human ATII cells using RNA microarray. Long term 2D culture cultures had patterns of gene expression similar to primary ATII cells but not of the same magnitude. MLB expression was confirmed by histology and TEM. As A549 cells may be ultimately limited by their cancerous origin, an investigation into the novel lung progenitor cell line ‘E-Cad/LGr6+’ was initiated but genotyping raised concerns about the cell line’s validity leading to the necessity to continue with A549 cells. Dexamethasone upregulated surfactant protein (SFTP) gene expression in the long term 2D A549 model, but had the undesirable side effect of downregulating genes involved in epithelial/mesenchymal communication. Finally, the ability of fibroblasts to support an ATII-like phenotype was tested using (i) conditioned medium from MRC-5 lung fibroblasts, (ii) A549 and MRC-5 co-cultures on Transwell™ and (iii) Alvetex™ 3D scaffold supports. Histochemical, gene expression and TEM analyses demonstrated that MRC-5 fibroblasts expressed factors for epithelial support driving subsequent SFTP gene expression in A549 cells. Alvetex™ scaffold co-culture initiated tissue-like organisation, ECM secretion and MLB expression. With optimisation and demonstration of reproducibility it is expected this approach will deliver a reproducible and accessible ATII model for airways research.

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ORCID for Donna Davies: orcid.org/0000-0002-5117-2991

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