Ali Doryab 1,2, Mehmet Berat Taskin 3, Philipp Stahlhut 3, Andreas Schröppel 1,2, Sezer Orak 1,2, Carola Voss 1,2, Arti Ahluwalia 4,5, Markus Rehberg 1,2, Anne Hilgendorff 1,2,6, Tobias Stöger 1,2, Jürgen Groll 3 and Otmar Schmid 1,2
1 Comprehensive Pneumology Center Munich, Member of the German Center for Lung Research, Munich, Germany,
2 Helmholtz Zentrum München—German Research Center for Environmental Health, Institute of Lung Biology and Disease, Munich, Germany,
3 Department of Functional Materials in Medicine and Dentistry, Bavarian Polymer Institute, University of Würzburg, Würzburg, Germany,
4 Research Center “E. Piaggio”, University of Pisa, Pisa, Italy,
5 Department of Information Engineering, University of Pisa, Pisa, Italy,
6 Center for Comprehensive Developmental Care (CDeCLMU), Dr. von Haunersches Children’s Hospital University, Hospital of the Ludwig-Maximilians University, Munich, Germany
We have recently introduced a novel porous and elastic membrane for in vitro cell-stretch models of the lung cultured under ALI conditions (Doryab et al., 2020). This innovative hybrid biphasic membrane, henceforth referred to as Biphasic Elastic Thin for Air-liquid culture conditions (BETA) membrane, was developed to optimize membrane characteristics for the two phases of cell-stretch experiments under ALI conditions, namely the initial cell seeding, attachment and growth phase under submerged cell culture conditions (phase I) followed by an ALI acclimatization and cell-stretch phase at the ALI (phase II). This patented aerosol-cell exposure unit has recently been made commercially available as VITROCELL® Cloud MAX.
Evolution has endowed the lung with exceptional design providing a large surface area for gas exchange area (ca. 100 m2) in a relatively small tissue volume (ca. 6 L). This is possible due to a complex tissue architecture that has resulted in one of the most challenging organs to be recreated in the lab. The need for realistic and robust in vitro lung models becomes even more evident as causal therapies, especially for chronic respiratory diseases, are lacking. Here, we describe the Cyclic In VItro Cell-stretch (CIVIC) “breathing” lung bioreactor for pulmonary epithelial cells at the air-liquid interface (ALI) experiencing cyclic stretch while monitoring stretch-related parameters (amplitude, frequency, and membrane elastic modulus) under real-time conditions. The previously described biomimetic copolymeric BETA membrane (5 µm thick, bioactive, porous, and elastic) was attempted to be improved for even more biomimetic permeability, elasticity (elastic modulus and stretchability), and bioactivity by changing its chemical composition. This biphasic membrane supports both the initial formation of a tight monolayer of pulmonary epithelial cells (A549 and 16HBE14o−) under submerged conditions and the subsequent cell-stretch experiments at the ALI without preconditioning of the membrane. The newly manufactured versions of the BETA membrane did not improve the characteristics of the previously determined optimum BETA membrane (9.35% PCL and 6.34% gelatin [w/v solvent]). Hence, the optimum BETA membrane was used to investigate quantitatively the role of physiologic cyclic mechanical stretch (10% linear stretch; 0.33 Hz: light exercise conditions) on size-dependent cellular uptake and transepithelial transport of nanoparticles (100 nm) and microparticles (1,000 nm) for alveolar epithelial cells (A549) under ALI conditions. Our results show that physiologic stretch enhances cellular uptake of 100 nm nanoparticles across the epithelial cell barrier, but the barrier becomes permeable for both nano- and micron-sized particles (100 and 1,000 nm). This suggests that currently used static in vitro assays may underestimate cellular uptake and transbarrier transport of nanoparticles in the lung.