Characterization an in vitro 3D alveolar model in COPD

Abstract

Introduction

New interest emerges on organoids for studying lung diseases, mainly chronic obstructive pulmonary disease (COPD). COPD is lung disease a characterize by chronic inflammation and excessive remodeling in small airway. The complexity of this disease is now widely recognized, cellular-based research remains highly challenging because of the lack of suitable experimental models that recapitulate between interaction of cells. Alveolar organoids could enable us to assess the involvement of several type cells in response to damage from cigarette smoke, one major risk factor of COPD. The aim of this study is to characterize an in vitro 3D alveolar model from mouse lungs.

Methods

We used progenitor cells isolated by filtration from pieces of mouse lung and cultivated in Matrigel. We cultivated first part of lung organoids in conditioned expansion medium for ten days. After another part of organoids were differentiated in conditioned differentiation alveolar medium during ten days. Then, we assessed several markers of alveolar cells differentiated, fibroblast markers, or stem cell markers gene expression by RT-qPCR in the two conditions.

Results

The markers used are as follow: (1) aquaporin 5 (Aqp5), homeodomain-only protein homeobox (Hopx), advanced glycation endproduct-specific receptor (Ager) and podoplanin (Pdpn) for alveolar type 1 (AT1); (2) surfactant protein b (Sfptb), surfactant protein c (Sfptc) for alveolar type 2 (AT2); (3) platelet-derived growth factor alpha (Pdgfrα) for fibroblast; (4) secretoglobin family 1A member 1 (Scgb1a1) and surfactant protein c (Sfptc) for stem cells; (5) epithelial cell adhesion molecule (Epcam) as markers of differentiated airway and alveolar epithelium. Epcam, Sfptb and Aqp5 gene expression was increased in both conditions compared to the single cell group. On the other hand, aquaporin 5 expression was greater in the differentiated medium condition compared with organoids exposed to undifferentiated medium. We also observed an increase in the expression of the Sfptc marker in the differentiated condition. Futhermore, fibroblast and stem cells appear to be poorly represented in organoids, indeed the following markers platelet-derived growth factor alpha (Pdgfrα) and secretoglobin family 1A member 1 (Scgb1a1) are weakly expressed.

Conclusion

Our results show alveolar organoids derived mouse lung present both AT1 and AT2 cells. So, these organoids can used as multicellular experimental models for studying cigarette smoke damage.