Development of an In Vitro Airway Epithelial-Endothelial Cell Culture Model on a Flexible Porous Poly(Trimethylene Carbonate) Membrane Based on Calu-3 Airway Epithelial Cells and Lung Microvascular Endothelial Cells.

Thijs Pasman,Pieter S Hiemstra,Jos M J Paulusse,André A Poot,Roman K Truckenmüller,Robbert J Rottier,Danielle Baptista,Sander Van Riet,Naomi M Hamelmann,Dimitrios Stamatialis

doi:10.3390/membranes11030197

Abstract

Due to the continuing high impact of lung diseases on society and the emergence of new respiratory viruses, such as SARS-CoV-2, there is a great need for in vitro lung models that more accurately recapitulate the in vivo situation than current models based on lung epithelial cell cultures on stiff membranes. Therefore, we developed an in vitro airway epithelial–endothelial cell culture model based on Calu-3 human lung epithelial cells and human lung microvascular endothelial cells (LMVECs), cultured on opposite sides of flexible porous poly(trimethylene carbonate) (PTMC) membranes. Calu-3 cells, cultured for two weeks at an air–liquid interface (ALI), showed good expression of the tight junction (TJ) protein Zonula Occludens 1 (ZO-1). LMVECs cultured submerged for three weeks were CD31-positive, but the expression was diffuse and not localized at the cell membrane. Barrier functions of the Calu-3 cell cultures and the co-cultures with LMVECs were good, as determined by electrical resistance measurements and fluorescein isothiocyanate-dextran (FITC-dextran) permeability assays. Importantly, the Calu-3/LMVEC co-cultures showed better cell viability and barrier function than mono-cultures. Moreover, there was no evidence for epithelial- and endothelial-to-mesenchymal transition (EMT and EndoMT, respectively) based on staining for the mesenchymal markers vimentin and α-SMA, respectively. These results indicate the potential of this new airway epithelial–endothelial model for lung research. In addition, since the PTMC membrane is flexible, the model can be expanded by introducing cyclic stretch for enabling mechanical stimulation of the cells. Furthermore, the model can form the basis for biomimetic airway epithelial–endothelial and alveolar–endothelial models with primary lung epithelial cells.

Highlights

Lung diseases are still among the leading causes of death [1]
We aim to develop a new in vitro airway epithelial–endothelial model by co-culturing Calu-3 cells together with human lung microvascular endothelial cells (LMVECs) on flexible porous poly(trimethylene carbonate) (PTMC) membranes
Adhesion of Calu-3 cells on PTMC M0, M1, and M3 membranes was assessed by staining their nuclei (Figure 1)

Summary

Introduction

Lung diseases are still among the leading causes of death [1]. the emergence of new respiratory viruses with a huge impact on society, such as SARS-CoV-2, further emphasizes the relevance of research on lung diseases. New in vitro models for airway and alveolar epithelial–endothelial cell interactions are very useful for research of lung diseases. Primary lung epithelial cells would be used for these models. This is often not feasible due to problems concerning the availability and culturing of these cells. Advancements are made to make primary alveolar cells more accessible for research, their availability is limited and maintaining these cells in culture is difficult [2]. The majority of current lung models still relies on the use of cell lines. The widely used A549 alveolar cell line is less suited for studies on lung barriers since cultures hardly develop a barrier function.

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