INTRODUCTION

Section:

ChooseTop of pageABSTRACTINTRODUCTION <<METHODS AND MATERIALSRESULTS AND DISCUSSIONCONCLUSIONSSUPPLEMENTARY MATERIALREFERENCESCurrent drug development and testing processes are both time consuming and expensive, with high failure rates (>90%) due to safety and efficacy concerns resulting from the use of simplistic static in vitro models or complex animal models subjected to species differences.1–31. P. J. Barnes et al., “Barriers to new drug development in respiratory disease,” Eur. Respir. J. 45, 1197–1207 (2015). https://doi.org/10.1183/09031936.000079152. O. Yildirim, M. Gottwald, P. Schüler, and M. C. Michel, “Opportunities and challenges for drug development: Public-private partnerships, adaptive designs and big data,” Front. Pharmacol. 7, 1–13 (2016). https://doi.org/10.3389/fphar.2016.004613. D. E. Ingber, “Is it time for reviewer 3 to request human organ chip experiments instead of animal validation studies?,” Adv. Sci. 2002030, 1–15 (2020). https://doi.org/10.1002/advs.202002030 These impediments have led to the generation of complex and dynamic in vitro models termed “organ on a chip” (OoC) or “microphysiological systems” (MPSs), which aim to recapitulate different organ functions, interfaces, and barriers within the human body. The airway epithelium is central to the maintenance of tissue homeostasis in the lung and acts as a physical, chemical, and immunological barrier.44. E. J. Swindle, J. E. Collins, and D. E. Davies, “Breakdown in epithelial barrier function in patients with asthma: Identification of novel therapeutic approaches,” J. Allergy Clin. Immunol. 124, 23–34 (2009). https://doi.org/10.1016/j.jaci.2009.05.037 Models of the airways have been developed to replicate the bronchial barrier5–105. J. Fernandes et al., “Real-time monitoring of epithelial barrier function by impedance spectroscopy in a microfluidic platform,” Lab Chip 22, 2041–2054 (2022). https://doi.org/10.1039/D1LC01046H6. S. Elias-Kirma et al., “In situ-like aerosol inhalation exposure for cytotoxicity assessment using airway-on-chips platforms,” Front. Bioeng. Biotechnol. 8, 1–13 (2020). https://doi.org/10.3389/fbioe.2020.000917. J. Shrestha et al., “A rapidly prototyped lung-on-a-chip model using 3D-printed molds,” Organs Chip 1, 100001 (2019). https://doi.org/10.1016/j.ooc.2020.1000018. K. L. Sellgren, E. J. Butala, B. P. Gilmour, S. H. Randell, and S. Grego, “A biomimetic multicellular model of the airways using primary human cells,” Lab Chip 14, 3349–3358 (2014). https://doi.org/10.1039/C4LC00552J9. C. Blume et al., “Cellular crosstalk between airway epithelial and endothelial cells regulates barrier functions during exposure to double-stranded RNA,” Immunity Inflamm. Dis. 5, 45–56 (2017). https://doi.org/10.1002/iid3.13910. C. Blume et al., “Temporal monitoring of differentiated human airway epithelial cells using microfluidics,” PLoS ONE 10, e0139872 (2015). https://doi.org/10.1371/journal.pone.0139872 or alveolar regions11–1411. D. Huh et al., “Reconstituting organ-level lung functions on a chip,” Science 328, 1662–1668 (2010). https://doi.org/10.1126/science.118830212. O. Y. F. Henry et al., “Organs-on-chips with integrated electrodes for trans-epithelial electrical resistance (TEER) measurements of human epithelial barrier function,” Lab Chip 17, 2264–2271 (2017). https://doi.org/10.1039/C7LC00155J13. Y. Mermoud, M. Felder, J. D. Stucki, A. O. Stucki, and O. T. Guenat, “Microimpedance tomography system to monitor cell activity and membrane movements in a breathing lung-on-chip,” Sens. Actuators B: Chem. 255, 3647–3653 (2018). https://doi.org/10.1016/j.snb.2017.09.19214. X. Yang et al., “Nanofiber membrane supported lung-on-a-chip microdevice for anti-cancer drug testing,” Lab Chip 18, 486–495 (2018). https://doi.org/10.1039/C7LC01224A of the lung, primarily using two branched66. S. Elias-Kirma et al., “In situ-like aerosol inhalation exposure for cytotoxicity assessment using airway-on-chips platforms,” Front. Bioeng. Biotechnol. 8, 1–13 (2020). https://doi.org/10.3389/fbioe.2020.00091 or non-branched5–175. J. Fernandes et al., “Real-time monitoring of epithelial barrier function by impedance spectroscopy in a microfluidic platform,” Lab Chip 22, 2041–2054 (2022). https://doi.org/10.1039/D1LC01046H7. J. Shrestha et al., “A rapidly prototyped lung-on-a-chip model using 3D-printed molds,” Organs Chip 1, 100001 (2019). https://doi.org/10.1016/j.ooc.2020.1000018. K. L. Sellgren, E. J. Butala, B. P. Gilmour, S. H. Randell, and S. Grego, “A biomimetic multicellular model of the airways using primary human cells,” Lab Chip 14, 3349–3358 (2014). https://doi.org/10.1039/C4LC00552J9. C. Blume et al., “Cellular crosstalk between airway epithelial and endothelial cells regulates barrier functions during exposure to double-stranded RNA,” Immunity Inflamm. Dis. 5, 45–56 (2017). https://doi.org/10.1002/iid3.13910. C. Blume et al., “Temporal monitoring of differentiated human airway epithelial cells using microfluidics,” PLoS ONE 10, e0139872 (2015). https://doi.org/10.1371/journal.pone.013987211. D. Huh et al., “Reconstituting organ-level lung functions on a chip,” Science 328, 1662–1668 (2010). https://doi.org/10.1126/science.118830213. Y. Mermoud, M. Felder, J. D. Stucki, A. O. Stucki, and O. T. Guenat, “Microimpedance tomography system to monitor cell activity and membrane movements in a breathing lung-on-chip,” Sens. Actuators B: Chem. 255, 3647–3653 (2018). https://doi.org/10.1016/j.snb.2017.09.19214. X. Yang et al., “Nanofiber membrane supported lung-on-a-chip microdevice for anti-cancer drug testing,” Lab Chip 18, 486–495 (2018). https://doi.org/10.1039/C7LC01224A15. K. H. Benam et al., “Small airway-on-a-chip enables analysis of human lung inflammation and drug responses in vitro,” Nat. Methods 13, 151–157 (2016). https://doi.org/10.1038/nmeth.369716. B. A. Hassell et al., “Human organ chip models recapitulate orthotopic lung cancer growth, therapeutic responses, and tumor dormancy in vitro,” Cell Rep. 21, 508–516 (2017). https://doi.org/10.1016/j.celrep.2017.09.04317. A. O. Stucki et al., “A lung-on-a-chip array with an integrated bio-inspired respiration mechanism,” Lab Chip 15, 1302–1310 (2015). https://doi.org/10.1039/C4LC01252F channels or chambers separated by a porous membrane that facilitates the culture of epithelial and/or endothelial cells on respective sides. Microfluidic systems enable the recapitulation of interstitial flow in vivo, while supplying cells with essential nutrients, removing waste and applying forces synonymous with the human microenvironment.1010. C. Blume et al., “Temporal monitoring of differentiated human airway epithelial cells using microfluidics,” PLoS ONE 10, e0139872 (2015). https://doi.org/10.1371/journal.pone.0139872 Some models of the lung incorporate the real-time analysis of barrier integrity via impedance spectroscopy5–195. J. Fernandes et al., “Real-time monitoring of epithelial barrier function by impedance spectroscopy in a microfluidic platform,” Lab Chip 22, 2041–2054 (2022). https://doi.org/10.1039/D1LC01046H12. O. Y. F. Henry et al., “Organs-on-chips with integrated electrodes for trans-epithelial electrical resistance (TEER) measurements of human epithelial barrier function,” Lab Chip 17, 2264–2271 (2017). https://doi.org/10.1039/C7LC00155J13. Y. Mermoud, M. Felder, J. D. Stucki, A. O. Stucki, and O. T. Guenat, “Microimpedance tomography system to monitor cell activity and membrane movements in a breathing lung-on-chip,” Sens. Actuators B: Chem. 255, 3647–3653 (2018). https://doi.org/10.1016/j.snb.2017.09.19218. J. Yeste et al., “A compartmentalized microfluidic chip with crisscross microgrooves and electrophysiological electrodes for modeling the blood-retinal barrier,” Lab Chip 18, 95–105 (2018). https://doi.org/10.1039/C7LC00795G19. K. Benson, S. Cramer, and H.-J. Galla, “Impedance-based cell monitoring: Barrier properties and beyond,” Fluids Barriers CNS 10, 1–11 (2013). https://doi.org/10.1186/2045-8118-10-5 and mechanical stretch to mimic cyclical pressures in the alveoli.11–1311. D. Huh et al., “Reconstituting organ-level lung functions on a chip,” Science 328, 1662–1668 (2010). https://doi.org/10.1126/science.118830213. Y. Mermoud, M. Felder, J. D. Stucki, A. O. Stucki, and O. T. Guenat, “Microimpedance tomography system to monitor cell activity and membrane movements in a breathing lung-on-chip,” Sens. Actuators B: Chem. 255, 3647–3653 (2018). https://doi.org/10.1016/j.snb.2017.09.192Airway microphysiological systems (MPSs) are a promising alternative to conventional models; however, methods need to be developed for delivery of compounds in a physiologically relevant manner, i.e., as an aerosol that better recapitulates human airway physiology with respect to respiratory diseases, as this can influence the deposition pattern and size preference of aerosols due to airway remodeling and constriction.2020. L. Y. Yeo, J. R. Friend, M. P. McIntosh, E. N. Meeusen, and D. A. Morton, “Ultrasonic nebulization platforms for pulmonary drug delivery,” Expert Opin. Drug Deliv. 7, 663–679 (2010). https://doi.org/10.1517/17425247.2010.485608 Conventional methods of drug delivery such as inertial or electrostatic impactors and impingers cannot be easily miniaturized, and consequently, compounds are generally deposited in a liquid suspension.7–217. J. Shrestha et al., “A rapidly prototyped lung-on-a-chip model using 3D-printed molds,” Organs Chip 1, 100001 (2019). https://doi.org/10.1016/j.ooc.2020.1000019. C. Blume et al., “Cellular crosstalk between airway epithelial and endothelial cells regulates barrier functions during exposure to double-stranded RNA,” Immunity Inflamm. Dis. 5, 45–56 (2017). https://doi.org/10.1002/iid3.13910. C. Blume et al., “Temporal monitoring of differentiated human airway epithelial cells using microfluidics,” PLoS ONE 10, e0139872 (2015). https://doi.org/10.1371/journal.pone.013987211. D. Huh et al., “Reconstituting organ-level lung functions on a chip,” Science 328, 1662–1668 (2010). https://doi.org/10.1126/science.118830213. Y. Mermoud, M. Felder, J. D. Stucki, A. O. Stucki, and O. T. Guenat, “Microimpedance tomography system to monitor cell activity and membrane movements in a breathing lung-on-chip,” Sens. Actuators B: Chem. 255, 3647–3653 (2018). https://doi.org/10.1016/j.snb.2017.09.19214. X. Yang et al., “Nanofiber membrane supported lung-on-a-chip microdevice for anti-cancer drug testing,” Lab Chip 18, 486–495 (2018). https://doi.org/10.1039/C7LC01224A21. K. H. Benam et al., “Matched-comparative modeling of normal and diseased human airway responses using a microengineered breathing lung chip,” Cell Syst. 3, 456–466.e4 (2016). https://doi.org/10.1016/j.cels.2016.10.003 MPSs have been integrated with a collision-type atomizer66. S. Elias-Kirma et al., “In situ-like aerosol inhalation exposure for cytotoxicity assessment using airway-on-chips platforms,” Front. Bioeng. Biotechnol. 8, 1–13 (2020). https://doi.org/10.3389/fbioe.2020.00091 or a mesh nebulizer22–2422. K. Domansky, M. Karpelson, R. J. Wood, and D. E. Ingber, “On-chip aerosol generation for organs-on-chips,” in 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences (CBMS, 2012), pp. 302–304.23. A. Artzy-Schnirman et al., “Advanced in vitro lung-on-chip platforms for inhalation assays: From prospect to pipeline,” Eur. J. Pharm. Biopharm. 144, 11–17 (2019). https://doi.org/10.1016/j.ejpb.2019.09.00624. D. C. Leslie et al., “Aerosol drug delivery for lung on a chip,” in the 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011, Seattle WA, 2-6 October 2011 (CBMS, 2011). with polydimethylsiloxane (PDMS) channels mimicking airway bifurcations66. S. Elias-Kirma et al., “In situ-like aerosol inhalation exposure for cytotoxicity assessment using airway-on-chips platforms,” Front. Bioeng. Biotechnol. 8, 1–13 (2020). https://doi.org/10.3389/fbioe.2020.00091 or a large stagnant chamber (ALICE-Cloud) where drugs deposit via sedimentation onto an open-plate device.2323. A. Artzy-Schnirman et al., “Advanced in vitro lung-on-chip platforms for inhalation assays: From prospect to pipeline,” Eur. J. Pharm. Biopharm. 144, 11–17 (2019). https://doi.org/10.1016/j.ejpb.2019.09.006 However, issues with these devices include the use of PDMS,66. S. Elias-Kirma et al., “In situ-like aerosol inhalation exposure for cytotoxicity assessment using airway-on-chips platforms,” Front. Bioeng. Biotechnol. 8, 1–13 (2020). https://doi.org/10.3389/fbioe.2020.00091 which can leach or absorb compounds, excessive sample use due to non-specific deposition2323. A. Artzy-Schnirman et al., “Advanced in vitro lung-on-chip platforms for inhalation assays: From prospect to pipeline,” Eur. J. Pharm. Biopharm. 144, 11–17 (2019). https://doi.org/10.1016/j.ejpb.2019.09.006 or to facilitate adequate dosing, sample solubility, viscosity, and degradation via shearing or unfolding in mesh nebulizers22–2422. K. Domansky, M. Karpelson, R. J. Wood, and D. E. Ingber, “On-chip aerosol generation for organs-on-chips,” in 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences (CBMS, 2012), pp. 302–304.23. A. Artzy-Schnirman et al., “Advanced in vitro lung-on-chip platforms for inhalation assays: From prospect to pipeline,” Eur. J. Pharm. Biopharm. 144, 11–17 (2019). https://doi.org/10.1016/j.ejpb.2019.09.00624. D. C. Leslie et al., “Aerosol drug delivery for lung on a chip,” in the 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011, Seattle WA, 2-6 October 2011 (CBMS, 2011). as highlighted recently.2525. N. Karra, E. J. Swindle, and H. Morgan, “Drug delivery for traditional and emerging airway models,” Organs Chip 1, 100002 (2019). https://doi.org/10.1016/j.ooc.2020.100002 Thus, there is a necessity to develop a new compact airway barrier MPS compatible drug delivery device without the need for size sorting, with direct deposition of a correctly sized aerosol droplet containing compounds.Surface acoustic wave (SAW) devices generate aerosols of respirable sizes (1–10 μm) without meshes, blockages, frequent cleaning, or damage to sample integrity found with mesh nebulizers.26–2926. L. Alhasan, A. Qi, A. R. Rezk, L. Y. Yeo, and P. P. Y. Chan, “Assessment of the potential of a high frequency acoustomicrofluidic nebulisation platform for inhaled stem cell therapy,” Integr. Biol. 8, 12–20 (2016). https://doi.org/10.1039/C5IB00206K27. C. Cortez-Jugo, A. Qi, A. Rajapaksa, J. R. Friend, and L. Y. Yeo, “Pulmonary monoclonal antibody delivery via a portable microfluidic nebulization platform,” Biomicrofluidics 9, 052603 (2015). https://doi.org/10.1063/1.491718128. A. E. Rajapaksa et al., “Effective pulmonary delivery of an aerosolized plasmid DNA vaccine via surface acoustic wave nebulization,” Respir. Res. 15, 60 (2014). https://doi.org/10.1186/1465-9921-15-6029. P. C. L. Kwok et al., “In vivo deposition study of a new generation nebuliser utilising hybrid resonant acoustic (HYDRA) technology,” Int. J. Pharm. 580, 119196 (2020). https://doi.org/10.1016/j.ijpharm.2020.119196 SAW devices can nebulize monoclonal antibodies, plasmid DNA, yeast and mesenchymal stem cells without causing cavitation, degradation, or impacting cellular viability while suppressing large shear forces.26–2826. L. Alhasan, A. Qi, A. R. Rezk, L. Y. Yeo, and P. P. Y. Chan, “Assessment of the potential of a high frequency acoustomicrofluidic nebulisation platform for inhaled stem cell therapy,” Integr. Biol. 8, 12–20 (2016). https://doi.org/10.1039/C5IB00206K27. C. Cortez-Jugo, A. Qi, A. Rajapaksa, J. R. Friend, and L. Y. Yeo, “Pulmonary monoclonal antibody delivery via a portable microfluidic nebulization platform,” Biomicrofluidics 9, 052603 (2015). https://doi.org/10.1063/1.491718128. A. E. Rajapaksa et al., “Effective pulmonary delivery of an aerosolized plasmid DNA vaccine via surface acoustic wave nebulization,” Respir. Res. 15, 60 (2014). https://doi.org/10.1186/1465-9921-15-60 It is considered a gentler process of nebulization,2626. L. Alhasan, A. Qi, A. R. Rezk, L. Y. Yeo, and P. P. Y. Chan, “Assessment of the potential of a high frequency acoustomicrofluidic nebulisation platform for inhaled stem cell therapy,” Integr. Biol. 8, 12–20 (2016). https://doi.org/10.1039/C5IB00206K,2727. C. Cortez-Jugo, A. Qi, A. Rajapaksa, J. R. Friend, and L. Y. Yeo, “Pulmonary monoclonal antibody delivery via a portable microfluidic nebulization platform,” Biomicrofluidics 9, 052603 (2015). https://doi.org/10.1063/1.4917181 with dose controlled through nebulization time2828. A. E. Rajapaksa et al., “Effective pulmonary delivery of an aerosolized plasmid DNA vaccine via surface acoustic wave nebulization,” Respir. Res. 15, 60 (2014). https://doi.org/10.1186/1465-9921-15-60,2929. P. C. L. Kwok et al., “In vivo deposition study of a new generation nebuliser utilising hybrid resonant acoustic (HYDRA) technology,” Int. J. Pharm. 580, 119196 (2020). https://doi.org/10.1016/j.ijpharm.2020.119196 offering a potential drug delivery method that can be integrated with MPS technology.SAWs are generated by piezoelectric materials such a lithium niobate. The surface is patterned with an array of interdigitated electrodes and the application of a high-frequency AC voltage creates mechanical perturbation generating a surface travelling wave, with the electrode width and spacing determining the frequency. Upon interaction with a liquid interface (droplets or thin films), the wave destabilizes the liquid boundary through a refracted longitudinal pressure wave at the Rayleigh angle. This generates unstable capillary waves at the liquid boundary, leading to the formation of jets that break off to form aerosolized droplets of pre-determined size,30–3330. Y. J. Guo et al., “Nebulization of water/glycerol droplets generated by ZnO/Si surface acoustic wave devices,” Microfluid. Nanofluidics 19, 273–282 (2015). https://doi.org/10.1007/s10404-014-1501-031. X. Ding et al., “Surface acoustic wave microfluidics,” Lab Chip 13, 3626 (2013). https://doi.org/10.1039/c3lc50361e32. L. Y. Yeo and J. R. Friend, “Surface acoustic wave microfluidics,” Annu. Rev. Fluid Mech. 46, 379–406 (2014). https://doi.org/10.1146/annurev-fluid-010313-14141833. E. Nazarzadeh et al., “Confinement of surface waves at the air-water interface to control aerosol size and dispersity,” Phys. Fluids 29, 112105 (2017). https://doi.org/10.1063/1.4993793 as illustrated in Fig. 1.

This paper describes a simple and compact SAW device for direct aerosolized compound-delivery to polarized or differentiated human bronchial epithelial cell cultures grown in a microfluidic MPS at a liquid–liquid (LLI) or air–liquid (ALI) interface. Airway barrier formation and disruption were monitored in real-time using transepithelial electrical resistance (TER) measurements and confirmed by immunofluorescent staining for tight junctional proteins. Cells were challenged with aerosolized poly I:C, a dsRNA analog that mimics viral infection, and with basolateral tumour necrosis factor alpha (TNF-α) as a model of inflammation in the presence or absence of aerosolized steroids.

METHODS AND MATERIALS

Section:

ChooseTop of pageABSTRACTINTRODUCTIONMETHODS AND MATERIALS <<RESULTS AND DISCUSSIONCONCLUSIONSSUPPLEMENTARY MATERIALREFERENCES

Airway barrier MPS

The airway barrier MPS55. J. Fernandes et al., “Real-time monitoring of epithelial barrier function by impedance spectroscopy in a microfluidic platform,” Lab Chip 22, 2041–2054 (2022). https://doi.org/10.1039/D1LC01046H (see supplementary material 10) consists of eight individual microfluidic chips and manifolds, housed in a custom polymethylmethacylate (PMMA) stand. Media are perfused through the microfluidic chip with a syringe pump and commercial bubble traps (Darwin Microfluidics). The platform has easily exchangeable chips (held with magnets), connected to an impedance analyzer with plug-and-play functionality. The entire platform is controlled using a web-enabled interface. For further details, see Fernandes et al. (2022).55. J. Fernandes et al., “Real-time monitoring of epithelial barrier function by impedance spectroscopy in a microfluidic platform,” Lab Chip 22, 2041–2054 (2022). https://doi.org/10.1039/D1LC01046H

Each microfluidic chip comprises a glass substrate onto which pairs of electrodes are patterned to record the TER of the cells. A 10 mm high PMMA structure forms the apical chamber, and the microfluidic channel is made from a 275 μm thick laser cut tape and PMMA. The two halves are separated by a high porosity (12 μm thickness, 0.4 μm pore diameter, 1 × 108 pores/cm2, PVP coating) polyester membrane (it4ip). The microfluidic manifold was milled out of polyetheretherketone (PEEK).

Prior to incorporation of cells, the system was cleaned with 1:50 bleach (<5% sodium hypochlorite), followed by copious rinsing with sterile de-ionized (DI) water.

SAW device

The SAW drug delivery device comprises a patterned black lithium niobate chip, a Peltier cooler, a fluid delivery mechanism, and a custom 3D holder (Fig. 2). The chip is patterned with curved interdigitated electrodes following the single-phase unidirectional transducer (SPUDT) design of Shilton et al. and Qi et al.3535. R. Shilton, M. K. Tan, L. Y. Yeo, and J. R. Friend, “Particle concentration and mixing in microdrops driven by focused surface acoustic waves,” J. Appl. Phys. 104, 014910 (2008). https://doi.org/10.1063/1.2951467,3636. A. Qi, L. Yeo, J. Friend, and J. Ho, “The extraction of liquid, protein molecules and yeast cells from paper through surface acoustic wave atomization,” Lab Chip 10, 470–476 (2010). https://doi.org/10.1039/B915833B to enable greater focusing of acoustic energy.3535. R. Shilton, M. K. Tan, L. Y. Yeo, and J. R. Friend, “Particle concentration and mixing in microdrops driven by focused surface acoustic waves,” J. Appl. Phys. 104, 014910 (2008). https://doi.org/10.1063/1.2951467 The device has 25 pairs of electrodes with a width of 17 or 49 μm and a spacing of 17 μm, generating an oscillation with a wavelength of 132 μm and an operating frequency of 30.62 MHz [see Figs. 2(a)–2(c)]. Electrode arrays were fabricated on a Y-cut, X propagating 128° black lithium niobate wafer (Roditi) from a layer of 10 nm chromium and 60 nm gold patterned by standard photolithography. The wafer was diced into individual chips, which were then soldered to a ribbon cable and bonded to a Peltier cooler with heat sink using a pressure-sensitive tape and mounted in a custom 3D-printed holder with the glass fiber filter.A Peltier cooler and fan [Fig. 2(d)] maintained the chip surface at ∼38 °C (see supplementary material 1). The device was driven with voltages ranging from 28 to 40 Vp-p at 30.62 MHz, with modulation at 1 kHz to reduce the total power and increase nebulization efficiency.3737. A. Rajapaksa, A. Qi, L. Y. Yeo, R. Coppel, and J. R. Friend, “Enabling practical surface acoustic wave nebulizer drug delivery via amplitude modulation,” Lab Chip 14, 1858–1865 (2014). https://doi.org/10.1039/C4LC00232F The temperature remained constant during 5 min of continuous actuation. A continuous supply of liquid was provided to the chip from a glass fiber filter, and the nebulization rate was ∼20 μl/min via Schlichting streaming3232. L. Y. Yeo and J. R. Friend, “Surface acoustic wave microfluidics,” Annu. Rev. Fluid Mech. 46, 379–406 (2014). https://doi.org/10.1146/annurev-fluid-010313-141418 and Eckart streaming.32–3832. L. Y. Yeo and J. R. Friend, “Surface acoustic wave microfluidics,” Annu. Rev. Fluid Mech. 46, 379–406 (2014). https://doi.org/10.1146/annurev-fluid-010313-14141838. A. Qi, J. Friend, and L. Yeo, “A miniaturized surface acoustic wave atomizer with a disposable pump-free liquid supply system for continuous atomization,” in NEMS 2011—6th IEEE International Conference on Nano/Micro Engineered and Molecular Systems (IEEE, 2011), pp. 289–292. https://doi.org/10.1109/NEMS.2011.6017350The SAW chip was mounted on commercial nanoporous membrane inserts (Transwell™ supports) and also on the airway barrier MPS previously described in Fernandes et al.55. J. Fernandes et al., “Real-time monitoring of epithelial barrier function by impedance spectroscopy in a microfluidic platform,” Lab Chip 22, 2041–2054 (2022). https://doi.org/10.1039/D1LC01046H using custom 3D printed holders so that aerosols were directly introduced into the apical chamber (Fig. 3). The complete SAW device (Fig. 2) was connected a high-power amplifier (Mini Circuits, ZHL-5W-1+) and a signal generator (Tektronix TDS2014C). Prior to use, the SAW device was decontaminated with 70% ethanol and assembled in a MSCII.

Cell culture

Two human bronchial epithelial cell lines were used: the 16HBE14o- cell line and the basal cell immortalized non-smoker cell line BSi-NS1.1, chosen for their ability to form a polarized3939. A. L. Cozens et al., “CFTR expression and chloride secretion in polarized immortal human bronchial epithelial cells,” Am. J. Respir. Cell Mol. Biol. 10, 38–47 (1994). https://doi.org/10.1165/ajrcmb.10.1.7507342 or polarized and differentiated epithelial barrier4040. M. S. Walters et al., “Generation of a human airway epithelium derived basal cell line with multipotent differentiation capacity,” Respir. Res. 14, 26–30 (2013). https://doi.org/10.1186/1465-9921-14-135 on nanoporous membranes, respectively. The 16HBE14o- cell line was maintained in a minimal essential medium (MEM) with Glutamax (Gibco) supplemented with 1% penicillin–streptomycin and 10% foetal bovine serum (FBS) (Life Technologies). The BCi-NS1.1 cell line was maintained in Pneumacult™ Ex Plus Complete media [PneumaCult™-EX Plus Medium supplemented with PneumaCult™-Ex Plus supplement (1×), 1% penicillin–streptomycin, 20 U/ml nystatin and 0.096 μg/ml hydrocortisone] solution.

Cells were seeded at densities of 7.6 × 105 cells/cm2 (16HBE14o- cells) or 4.5 × 105 cells/cm2 (BCi-NS1.1 cells) onto collagen coated [30 μg/ml collagen (Advanced Biomatrix)] 0.4 μm PET membranes (12 μm, 0.4 μm pore diameter, 1 × 108 pores/cm2, PVP coated) in the microfluidic chip, or 0.4 μm PET membranes (10 μm, 0.4 μm pore diameter, 4 × 106 pores/cm2, cell culture treated) in Transwell™ inserts (Corning) for 1 h to facilitate cell adhesion (without flow). After cell adhesion, fluid was pumped through the chips (30 μl/h) and the electrical impedance was measured every 17 min. For Transwells™, TER measurements were performed using chopstick electrodes and an ERS-2™ Millicell Voltohmmeter (MERS00002, Merck). Data were recorded daily for 16HBE14o- cells and weekly for BCi-NS1.1 cells [after a 15-min incubation at 37 °C with Hanks buffered saline solution (100 μl)]. Apical and basolateral media were replaced on days 2 and 4 for 16HBE14o- cells while basolateral media were replaced 3× weekly for BCi.NS1.1 cells.

Air–liquid interface cell culture

For optimal growth and polarization, the 16HBE14o- cells prefer a liquid–liquid interface.4141. C. Ehrhardt et al., “Influence of apical fluid volume on the development of functional intercellular junctions in the human epithelial cell line 16HBE14o- : Implications for the use of this cell line as an in vitro model for bronchial drug absorption studies,” Cell Tissue Res. 308, 391–400 (2002). https://doi.org/10.1007/s00441-002-0548-5 In order to challenge cells at ALI, after barrier formation (>300 Ω cm2) on day 5, the apical volume was removed, and cells were incubated at ALI for between 0 and 4 h to determine the optimal time. The TER was measured by submerging the cells in equilibrated media and taking readings hourly thereafter up to 5 h and then again at 24 h. BCi-NS1.1 cells were taken to ALI after 2–3 days of submersion in Pneumacult™ Ex Plus Complete media by removing the apical media and replacing the basolateral media with Pneumacult™ ALI Maintenance media [PneumaCult™-ALI base medium supplemented with PneumaCult™ ALI supplement (1×), PneumaCult™ ALI maintenance supplement (1×), 1% penicillin–streptomycin, 20 U/ml nystatin, 4 μg/ml heparin solution, and 0.48 μg/ml hydrocortisone solution] and cultured for four weeks with basolateral media replaced 3× weekly.

dsRNA analog or tumour necrosis factor-α challenge with steroid treatment

Cells were challenged on day 5 (16HBE14o-) or day 28 (BCi-NS1.1) after formation of an epithelial barrier (>300 Ω cm2). All aerosolized challenges were performed in an MSCII and the deposition amount controlled by the nebulization rate (20 μl/min). The cells are temperature sensitive,4242. P. J. Callaghan, B. Ferrick, E. Rybakovsky, S. Thomas, and J. M. Mullin, “Epithelial barrier function properties of the 16HBE14o- human bronchial epithelial cell culture model,” Biosci. Rep. 40, 1–16 (2020). https://doi.org/10.1042/BSR20201532 so only one or two chips/culture were removed at a time.

Liquid-liquid interface

The 16HBE14o- cells were challenged by removing 20 μl of the medium from the apical compartment and replacing it with 20 μl of poly I:C (25 μg/ml, a dsRNA analog as a mimic of viral replication), fluticasone propionate (100 nM) or poly I:C, and fluticasone propionate together or media alone as a control. In each case, samples were either pipetted or nebulized (onto the bulk liquid).

Air–liquid interface

For 16HBE14o- cells, the medium was replaced the day before challenge. Challenge required removal of the entire apical volume, followed by addition of 20 μl poly I:C (either nebulized or pipetted directly onto cells) followed by the addition of 180 μl (static Transwell™ cultures) or 80 μl (airway barrier MPS) equilibrated media without compounds to provide a final poly I:C concentration of 25 μg/ml. Media alone were used as a control.

For BCi-NS1.1 cells, the basolateral medium was replaced with Pneumacult™ ALI Maintenance media without hydrocortisone the day before challenge. The following day, 20 μl of dexamethasone (100 nM) was either nebulized or pipetted into the apical compartment, with or without TNF-α (1 ng/ml) pipetted in the basolateral compartment. Controls consisted of apically pipetted starvation media (Pneumacult™ ALI Maintenance media without hydrocortisone) or nebulized starvation media with TNF-α.

Laser diffraction

The size of aerosol droplets produced by the SAW device was measured using the Malvern Spraytech system STP5315. The Spraytech system measured for 1 min of continuous nebulization following initial background measurements, in which laser obscuration over 5% was recorded. The SAW device was placed 10 mm below the center of the laser and the device was actuated at 30.62 MHz with a modulation frequency of 1 kHz and 20% depth at voltages of 28/40 Vp-p (min/max). Triplicate measurements were recorded per each chip (three chips), where the values 30 s after signal actuation were averaged. Generation of a sustained plume was achieved through the use of a glass fiber filter (5 × 30 mm strip) positioned at the end of the chip, enabling a meniscus to be drawn out and nebulized when prewetted with 200 μl DI water, from which the excess was removed and replaced with 20 μl DI water, prior to actuation.

Immunofluorescence staining and imaging

At the end of a challenge, cells were fixed in 4% PFA solution (Merck), washed, and then stored in 1× phosphate-buffered saline (PBS) at 4 °C. Cells were permeabilized (0.1% Triton X-100 in PBS, 30 min at room temperature) followed by blocking (PBS with 2% bovine serum albumin (BSA) and 0.1% Tween 20, 60 min at room temperature) before overnight incubation with Acti-stain555-phalloidin (Cytoskeleton, PHDH1-A) and AlexaFluor®488-conjugated mouse anti-human occludin antibody (Life Technologies, Clone OC-3F10) at 4 °C in a humidified chamber. Samples were then washed 3× (PBS with 0.1% Tween-20), counterstained with 4′,6-diamidino-2-phenylindole (DAPI) nuclear stain (Merck, 10236276001) for 15 min, and washed 3× (PBS with 0.1% Tween-20 solution) and 1× (dH2O) before mounting onto coverslips with Mowiol (Merck, 81381). Images were captured at 63× using confocal microscopy in the xyz mode (a Leica TCS-SP8 laser scanning microscope) with excitation wavelengths of 405 nm (DAPI), 561 nm (Actin), and 488 nm (Occludin). A Z-projection stack created with the Leica application suite using sequential scans to limit spectral bleeding.

Data analysis

Results are presented as mean ± standard deviation (SD). Normality was assessed using a Shapiro–Wilk test. For 16HBE14o- cells at LLI, paired t-tests were used to compare day 0 and day 5 TER values and delivery methods (pipetted vs nebulized) while a one-way analysis of variance (ANOVA) was used to compare challenge conditions within delivery methods (Media, poly I:C, fluticasone, and combination). For the 16HBE14o- cell pipetted and nebulized at ALI, a Mann Whitney test was used to compare between conditions. For the challenge within the airway barrier-on-a-chip platform at ALI, an unpaired t-test was used. For the BCi-NS1.1 challenge, comparison between pipetted conditions used a paired t-test, while comparisons between pipetted and nebulized or within nebulized conditions used an unpaired t-test. Results were considered statistically significant when p ≤ 0.05.

ACKNOWLEDGMENTS

The work was funded by the EPSRC (DTP Studentship: No. 1953215). We would like to thank Katie Chamberlain for her assistance with fabrication of microfluidic glass chips; Charlie Turner for dicing of lithium niobate chips; Mark Long, Anthony Gardener, and Jamie Stone for their technical assistance with micro-milling and 3D printing; and David Johnstone for his support with confocal microscopy. Jonathan James and Cornelia Blume provided 16HBE14o- and BCi-NS1.1 cell stocks, respectively. We acknowledge Wendy Rowan, Theresa Pell, Pelin Candarlioglu, Melanie Hamilton, Edward Taylor, and Martin Hingle of GlaxoSmithKline for use of equipment for the aerosol analysis studies, technical support, and continued guidance.