PerfuPul-A Versatile Perfusable Platform to Assess Permeability and Barrier Function of Air Exposed Pulmonary Epithelia.

Patrick Carius,Aurélie Dubois,Claus-Michael Lehr,Josué Sznitman,Arbel Artzy-Schnirman,Morvarid Ajdarirad,Nicole Schneider-Daum

doi:10.3389/fbioe.2021.743236

Abstract

Complex in vitro models, especially those based on human cells and tissues, may successfully reduce or even replace animal models within pre-clinical development of orally inhaled drug products. Microfluidic lung-on-chips are regarded as especially promising models since they allow the culture of lung specific cell types under physiological stimuli including perfusion and air-liquid interface (ALI) conditions within a precisely controlled in vitro environment. Currently, though, such models are not available to a broad user community given their need for sophisticated microfabrication techniques. They further require systematic comparison to well-based filter supports, in analogy to traditional Transwells®. We here present a versatile perfusable platform that combines the advantages of well-based filter supports with the benefits of perfusion, to assess barrier permeability of and aerosol deposition on ALI cultured pulmonary epithelial cells. The platform as well as the required technical accessories can be reproduced via a detailed step-by-step protocol and implemented in typical bio-/pharmaceutical laboratories without specific expertise in microfabrication methods nor the need to buy costly specialized equipment. Calu-3 cells cultured under liquid covered conditions (LCC) inside the platform showed similar development of transepithelial electrical resistance (TEER) over a period of 14 days as cells cultured on a traditional Transwell®. By using a customized deposition chamber, fluorescein sodium was nebulized via a clinically relevant Aerogen® Solo nebulizer onto Calu-3 cells cultured under ALI conditions within the platform. This not only allowed to analyze the transport of fluorescein sodium after ALI deposition under perfusion, but also to compare it to transport under traditional static conditions.

Highlights

Animal models have undoubtedly been essential for the development of oral inhalation drug products, especially for demonstrating safety as well as, at least for some diseases, efficacy in preclinical research
The same dose of fluorescein sodium was used for the transport studies under air-liquid interface (ALI) conditions and liquid covered conditions (LCC), we reduced the duration of the transport studies under ALI conditions to 5 h due to an increased concentration gradient
It encompasses an apical compartment, which is open to the top, and a basolateral compartment, which can be perfused via two lateral channels (Figure 1A)

Summary

Introduction

Animal models have undoubtedly been essential for the development of oral inhalation drug products, especially for demonstrating safety as well as, at least for some diseases, efficacy in preclinical research. While forced inhalation or tracheobronchial instillation may still allow to draw some conclusion about pulmonary toxicity, the problem becomes more challenging for efficacy studies This is especially true for inhalable anti-infective drugs, where the available animal models fail to adequately replicate how such diseases affect the human respiratory tract (Lorenz et al, 2016). This can be attributed to evident speciesspecies variations between humans and model organisms (e.g., in lung anatomy, airway histology, cellular composition of epithelial and sub epithelial compartments) that amongst other reasons eventually slow down the development of orally inhaled drug products (Barnes et al, 2015; Artzy-Schnirman et al, 2019a; Jimenez-Valdes et al, 2020). Various simple pulmonary in vitro models, usually consisting of bronchial or alveolar epithelial cell monocultures, and complex models comprising pulmonary epi- and/or endothelial cells in co-culture with other cell types like immune cells (e.g., dendritic cells, macrophages or neutrophils) or fibroblasts have been extensively reviewed (Gordon et al, 2015; Hittinger et al, 2015; Hittinger et al, 2017; Ehrmann et al, 2020)

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Conclusion