Abstract
Organs-on-chips are microfluidic cell culture devices created using microchip manufacturing techniques that contain hollow microchannels lined by living cells, which recreate specialized tissue–tissue interfaces, physical microenvironments, and vascular perfusion necessary to recapitulate organ-level physiology in vitro. Here we describe a protocol for fabrication, culture, and operation of a human lung “small airway-on-a-chip,” which contains a differentiated, mucociliary bronchiolar epithelium exposed to air and an underlying microvascular endothelium that experiences fluid flow. First, microengineering is used to fabricate a multilayered microfluidic device that contains two parallel elastomeric microchannels separated by a thin rigid porous membrane; this requires less than 1 day to complete. Next, primary human airway bronchiolar epithelial cells isolated from healthy normal donors or patients with respiratory disease are cultured on the porous membrane within one microchannel while lung microvascular endothelial cells are cultured on the opposite side of the same membrane in the second channel to create a mucociliated epithelium–endothelium interface; this process take about 4–6 weeks to complete. Finally, culture medium containing neutrophils isolated from fresh whole human blood are flowed through the microvascular channel of the device to enable real-time analysis of capture and recruitment of circulating leukocytes by endothelium under physiological shear; this step requires less than 1 day to complete. The small airway-on-a-chip represents a new microfluidic tool to model complex and dynamic inflammatory responses of healthy and diseased lungs in vitro.
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References
Rennard SI, Drummond MB (2015) Early chronic obstructive pulmonary disease: definition, assessment, and prevention. Lancet 385(9979):1778–1788. doi:10.1016/S0140-6736(15)60647-X
Pawankar R (2014) Allergic diseases and asthma: a global public health concern and a call to action. World Allergy Organ J 7(1):12. doi:10.1186/1939-4551-7-12
Benam KH, Villenave R, Lucchesi C et al (2016) Small airway-on-a-chip enables analysis of human lung inflammation and drug responses in vitro. Nat Methods 13(2):151–157. doi:10.1038/nmeth.3697
Martinez FJ, Donohue JF, Rennard SI (2011) The future of chronic obstructive pulmonary disease treatment—difficulties of and barriers to drug development. Lancet 378(9795):1027–1037. doi:10.1016/S0140-6736(11)61047-7
Bhatia SN, Ingber DE (2014) Microfluidic organs-on-chips. Nat Biotechnol 32(8):760–772. doi:10.1038/nbt.2989
Benam KH, Dauth S, Hassell B et al (2015) Engineered in vitro disease models. Annu Rev Pathol 10:195–262. doi:10.1146/annurev-pathol-012414-040418
Proudfoot AG, McAuley DF, Griffiths MJ et al (2011) Human models of acute lung injury. Dis Model Mech 4(2):145–153. doi:10.1242/dmm.006213
Hyde DM, Hamid Q, Irvin CG (2009) Anatomy, pathology, and physiology of the tracheobronchial tree: emphasis on the distal airways. J Allergy Clin Immunol 124(6 Suppl):S72–S77. doi:10.1016/j.jaci.2009.08.048
Kolaczkowska E, Kubes P (2013) Neutrophil recruitment and function in health and inflammation. Nat Rev Immunol 13(3):159–175. doi:10.1038/nri3399
Wright JL, Cosio M, Churg A (2008) Animal models of chronic obstructive pulmonary disease. Am J Physiol Lung Cell Mol Physiol 295(1):L1–15. doi:10.1152/ajplung.90200.2008
Esch EW, Bahinski A, Huh D (2015) Organs-on-chips at the frontiers of drug discovery. Nat Rev Drug Discov 14(4):248–260. doi:10.1038/nrd4539
Huh D, Kim HJ, Fraser JP et al (2013) Microfabrication of human organs-on-chips. Nat Protoc 8(11):2135–2157. doi:10.1038/nprot.2013.137
Shao J, Wu L, Wu J et al (2009) Integrated microfluidic chip for endothelial cells culture and analysis exposed to a pulsatile and oscillatory shear stress. Lab Chip 9(21):3118–3125. doi:10.1039/b909312e
Acknowledgments
We thank C. Lucchesi, J. Szabo, R. Villenave, C.D. Hinojosa, and G. Thompson for technical assistance, learning to use instruments and helpful discussions. We also thank the Wyss Microfabrication Team for their help with chip fabrication.
Potential competing interests
D.E. Ingber holds equity in Emulate, Inc. and Opsonix, Inc. and chairs their scientific advisory boards.
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Benam, K.H., Mazur, M., Choe, Y., Ferrante, T.C., Novak, R., Ingber, D.E. (2017). Human Lung Small Airway-on-a-Chip Protocol. In: Koledova, Z. (eds) 3D Cell Culture. Methods in Molecular Biology, vol 1612. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7021-6_25
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DOI: https://doi.org/10.1007/978-1-4939-7021-6_25
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Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7019-3
Online ISBN: 978-1-4939-7021-6
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