Fabrication and Characterization of 3D Bioprinted Triple-layered Human Alveolar Lung Models

Wei Long Ng, Teck Choon Ayi, Yi-Chun Liu, Swee Leong Sing, Wai Yee Yeong, Boon-Huan Tan

Article ID: 332
Vol 7, Issue 2, 2021, Article identifier:332

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The global prevalence of respiratory diseases caused by infectious pathogens has resulted in an increased demand for realistic in-vitro alveolar lung models to serve as suitable disease models. This demand has resulted in the fabrication of numerous two-dimensional (2D) and three-dimensional (3D) in-vitro alveolar lung models. The ability to fabricate these 3D in-vitro alveolar lung models in an automated manner with high repeatability and reliability is important for potential scalable
production. In this study, we reported the fabrication of human triple-layered alveolar lung models comprising of human lung epithelial cells, human endothelial cells, and human lung fibroblasts using the drop-on-demand (DOD) 3D bioprinting technique. The polyvinylpyrrolidone-based bio-inks and the use of a 300 μm nozzle diameter improved the repeatability of the bioprinting process by achieving consistent cell output over time using different human alveolar lung cells. The 3D bioprinted
human triple-layered alveolar lung models were able to maintain cell viability with relative similar proliferation profile over time as compared to non-printed cells. This DOD 3D bioprinting platform offers an attractive tool for highly repeatable and scalable fabrication of 3D in-vitro human alveolar lung models.


3D bioprinting; 3D printing; Biofabrication; Lung bioprinting; In-vitro human tissue models; Drop-on-demand

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Wu Z, McGoogan JM, 2019, Characteristics of and Important Lessons from the Coronavirus Disease 2019 (COVID-19) Outbreak in China: Summary of a Report of 72 314 Cases from the Chinese Center for Disease Control and Prevention. JAMA, 323:1239–42 https://doi.org/10.1001/jama.2020.2648

Oboho IK, Tomczyk SM, Al-Asmari AM, et al., 2015. 2014 MERS-CoV Outbreak in Jeddah a Link to Health Care Facilities. N Engl J Med, 372:846–54. https://doi.org/10.1056/nejmoa1408636

Bautista E, Chotpitayasunondh T, Gao Z, 2010, Influenza, Clinical Aspects of Pandemic 2009 Influenza A (H1N1) Virus Infection. N Engl J Med, 362:1708–19. https://doi.org/10.1056/nejmra1000449

Seto W, Tsang D, Yung RW, et al., 2003. Effectiveness of Precautions against Droplets and Contact in Prevention of Nosocomial Transmission of Severe Acute Respiratory Syndrome (SARS). Lancet, 361:1519–20. https://doi.org/10.1016/s0140-6736(03)13168-6

Steimer A, Haltner E, Lehr CM, 2005, Cell Culture Models of the Respiratory Tract Relevant to Pulmonary Drug Delivery. J Aerosol Med, 18:137–82. https://doi.org/10.1089/jam.2005.18.137

Hermanns MI, Unger RE, Kehe K, et al., 2004, Lung Epithelial Cell Lines in Coculture with Human Pulmonary Microvascular Endothelial Cells: Development of an Alveolo-Capillary Barrier In Vitro. Lab Investig, 84:736–52. https://doi.org/10.1038/labinvest.3700081

Nichols JE, Niles JA, Vega SP, et al., 2014, Modeling the Lung: Design and Development of Tissue Engineered Macro and Micro-physiologic Lung Models for Research Use. Exp Biol Med, 239:1135–69.https://doi.org/10.1177/1535370214536679

Ng WL, Chua CK, Shen YF, 2019, Print Me An Organ! Why We Are Not There Yet. Prog Polym Sci, 97:101145. https://doi.org/10.1016/j.progpolymsci.2019.101145

Sun W, Starly B, Daly AC, et al., 2020, The Bioprinting Roadmap. Biofabrication, 12:022002.

Ng WL, Chan A, Ong YS, et al, 2020, Deep Learning for Fabrication and Maturation of 3D Bioprinted Tissues and Organs. Virtual Phys Prototyp, 15:340–58.

Zhang B, Gao L, Ma L, et al., 2019, 3D Bioprinting: A Novel Avenue for Manufacturing Tissues and Organs. Engineering, 5:777–94. https://doi.org/10.1016/j.eng.2019.03.009

Ren KF, Hu M, Zhang H, et al., 2019, Layer-by-Layer Assembly as a Robust Method to Construct Extracellular Matrix Mimic Surfaces to Modulate Cell Behavior. Prog Polym Sci, 92:1–34. https://doi.org/10.1016/j.progpolymsci.2019.02.004

Liu F, Liu C, Chen Q, et al., 2018, Progress in Organ 3D Bioprinting. Int J Bioprint, 4:128–42.

Ng WL, Lee JM, Zhou M, et al., 2020, Hydrogels for 3-D bioprinting-based tissue engineering. In: R. Narayan, editor. Rapid Prototyping of Biomaterials, Elsevier, Chapel Hill, NC, pp. 183–204. https://doi.org/10.1016/b978-0-08-102663-2.00008-3

Lee JM, Suen SK, Ng WL, et al., 2020, Bioprinting of Collagen: Considerations, Potentials, and Applications. Macromol Biosci, 21:2000280. https://doi.org/10.1002/mabi.202000280

Osidak EO, Kozhukhov VI, Osidak MS, et al., 2020, Collagen as Bioink for Bioprinting: A Comprehensive Review. Int J Bioprint, 6:270. https://doi.org/10.18063/ijb.v6i3.270

Lee JM, Ng WL, Yeong WY, 2019, Resolution and Shape in Bioprinting: Strategizing Towards Complex Tissue and Organ Printing. Appl Phys Rev, 6:011307. https://doi.org/10.1063/1.5053909

Mir TA, Iwanaga S, Kurooka T, et al., 2019, Biofabrication Offers Future Hope for Tackling Various Obstacles and Challenges in Tissue Engineering and Regenerative Medicine: A Perspective. Int J Bioprint, 5:153–63. https://doi.org/10.18063/ijb.v5i1.153

Kang HW, Lee SJ, Ko IK, et al., 2016, A 3D Bioprinting System to Produce Human-Scale Tissue Constructs with Structural Integrity. Nat Biotechnol, 34:312–9. https://doi.org/10.1038/nbt.3413

Kim W, Kim G, 2019, Collagen/Bioceramic-based Composite Bioink to Fabricate a Porous 3D hASCs-laden Structure for Bone Tissue Regeneration. Biofabrication, 12:015007. https://doi.org/10.1088/1758-5090/ab436d

Ahlfeld T, et al., 2018, Bioprinting of Mineralized Constructs Utilizing Multichannel Plotting of a Self-setting Calcium Phosphate Cement and a Cell-laden Bioink. Biofabrication, 10:045002. https://doi.org/10.1088/1758-5090/aad36d

Noor N, Shapira A, Edri R, et al., 2019, 3D Printing of Personalized Thick and Perfusable Cardiac Patches and Hearts. Adv Sci, 6:1900344. https://doi.org/10.1002/advs.201900344

Jang J, Park HJ, Kim SW, et al., 2017, 3D Printed Complex Tissue Construct Using Stem Cell-laden Decellularized Extracellular Matrix Bioinks for Cardiac Repair. Biomaterials, 112:264–74. https://doi.org/10.1016/j.biomaterials.2016.10.026

Izadifar M, Chapman D, Babyn P, et al., 2018, UV-assisted 3D Bioprinting of Nanoreinforced Hybrid Cardiac Patch for Myocardial Tissue Engineering. Tissue Eng Part C Methods, 24:74–88. https://doi.org/10.1089/ten.tec.2017.0346

You F, Chen X, Cooper D, et al., 2018, Homogeneous Hydroxyapatite/Alginate Composite Hydrogel Promotes Calcified Cartilage Matrix Deposition with Potential for Three-dimensional Bioprinting. Biofabrication, 11:015015. https://doi.org/10.1088/1758-5090/aaf44a

Nguyen D, Hägg D, Forsman A, et al., 2017, Cartilage Tissue Engineering by the 3D Bioprinting of iPS Cells in a Nanocellulose/Alginate Bioink. Sci Rep, 7:658. https://doi.org/10.1038/s41598-017-00690-y

Izadifar Z, Chang T, Kulyk W, et al., 2016, Analyzing Biological Performance of 3D-Printed, Cell-impregnated Hybrid Constructs for Cartilage Tissue Engineering. Tissue Eng Part C Methods, 22;173–88. https://doi.org/10.1089/ten.tec.2015.0307

Norona LM, Nguyen DG, Gerber DA, et al., 2019, Bioprinted Liver Provides Early Insight into the Role of Kupffer Cells in TGF-β1 and Methotrexate-induced Fibrogenesis. PloS One, 14:e0208958. https://doi.org/10.1371/journal.pone.0208958

Nguyen DG, Funk J, Robbins JB, et al., 2016, Bioprinted 3D Primary Liver Tissues Allow Assessment of Organ-level Response to Clinical Drug Induced Toxicity In Vitro. PloS One, 11:e0158674. https://doi.org/10.1371/journal.pone.0158674

Mazzocchi A, Devarasetty M, Huntwork R, et al., 2018, Optimization of Collagen Type I-hyaluronan Hybrid Bioink for 3D Bioprinted Liver Microenvironments. Biofabrication, 11:015003. https://doi.org/10.1088/1758-5090/aae543

Horváth L, Umehara Y, Jud C, et al., 2015, Engineering an In Vitro Air-blood Barrier by 3D Bioprinting. Sci Rep, 5:7974 https://doi.org/10.1038/srep07974

Park JY, Ryu H, Lee B, et al., Development of a Functional Airway-on-a-chip by 3D Cell Printing. Biofabrication, 11:015002.

Ng WL, Wang S, Yeong WY, et al., 2016, Skin Bioprinting: Impending Reality or Fantasy? Trends Biotechnol, 34:689–99. https://doi.org/10.1016/j.tibtech.2016.08.009

Cubo N, Garcia M, del Cañizo JE, et al., 2016, 3D Bioprinting of Functional Human Skin: Production and In Vivo Analysis. Biofabrication, 9:015006. https://doi.org/10.1088/1758-5090/9/1/015006

Kim BS, Lee JS, Gao G, et al., 2017, Direct 3D Cell printing of Human Skin with Functional Transwell System. Biofabrication, 9:025034. https://doi.org/10.1088/1758-5090/aa71c8

Ng WL, Tan ZQ, Yeong WY, et al., 2018, Proof-of-concept: 3D Bioprinting of Pigmented Human Skin Constructs. Biofabrication, 10:025005. https://doi.org/10.1088/1758-5090/aa9e1e

Ng WL, Yeong WY, 2019, The Future of Skin Toxicology Testing 3D Bioprinting Meets Microfluidics. Int J Bioprint, 5:237. https://doi.org/10.18063/ijb.v5i2.1.237

Kathawala MH, Ng WL, Liu D, et al., 2019, Healing of Chronic Wounds an Update of Recent Developments and Future Possibilities. Tissue Eng Part B Rev, 25:429–44. https://doi.org/10.1089/ten.teb.2019.0019

Zhuang P, Ng WL, An J, et al., 2019, Layer-by-layer Ultraviolet Assisted Extrusion-Based (UAE) Bioprinting of Hydrogel Constructs with High Aspect Ratio for Soft Tissue Engineering Applications. PLoS One, 14:e0216776. https://doi.org/10.1371/journal.pone.0216776

Ozbolat IT, Hospodiuk M, 2016, Current Advances and Future Perspectives in Extrusion-based Bioprinting. Biomaterials, 76:321–43. https://doi.org/10.1016/j.biomaterials.2015.10.076

Ng WL, Yeong WY, Naing MW, 2016, Development of Polyelectrolyte Chitosan-gelatin Hydrogels for Skin Bioprinting. Procedia CIRP, 49:105–12. https://doi.org/10.1016/j.procir.2015.09.002

Ng WL, Yeong WY, Naing MW, 2016, Polyelectrolyte Gelatin-chitosan Hydrogel Optimized for 3D Bioprinting in Skin Tissue Engineering. Int J Bioprint, 2:53–62. https://doi.org/10.18063/ijb.2016.01.009

Ng WL, Yeong WY, Naing MW, 2014, Potential of Bioprinted Films for Skin Tissue Engineering. Proceedings of the 1st International Conference on Progress in Additive Manufacturing, pp. 441–6. https://doi.org/10.3850/978-981-09-0446-3_065

Gudupati H, Dey M, Ozbolat I, 2016, A Comprehensive Review on Droplet-based Bioprinting: Past, Present and Future. Biomaterials, 102:20–42. https://doi.org/10.1016/j.biomaterials.2016.06.012

Ng WL, Lee JM, Yeong WY, et al., 2017, Microvalve-based Bioprinting Process, Bio-inks and Applications. Biomater Sci, 5:632–47. https://doi.org/10.1039/c6bm00861e

Ng WL, Yeong WY, Naing MW, 2016, Microvalve Bioprinting of Cellular Droplets with High Resolution and Consistency. Proceedings of the International Conference on Progress in Additive Manufacturing, pp. 397–402.

Koch L, Deiwick A, Franke A, et al., 2018, Laser Bioprinting of Human Induced Pluripotent Stem Cells the Effect of Printing and Biomaterials on Cell Survival, Pluripotency, and Differentiation. Biofabrication, 10:035005. https://doi.org/10.1088/1758-5090/aab981

Ng WL, Lee JM, Zhou M, et al., 2020, Vat Polymerization based Bioprinting Process, Materials, Applications and Regulatory Challenges. Biofabrication, 12:022001. https://doi.org/10.1088/1758-5090/ab6034

Kim SH, Yeon YK, Lee JM, et al., 2018, Precisely Printable and Biocompatible Silk Fibroin Bioink for Digital Light Processing 3D Printing. Nat Commun, 9:1620. https://doi.org/10.1038/s41467-018-04517-w

Mandt D, Gruber P, Markovic M, et al., 2018, Fabrication of Biomimetic Placental Barrier Structures within a Microfluidic Device Utilizing Two-photon Polymerization. Int J Bioprint, 4:144–55. https://doi.org/10.18063/ijb.v4i2.144

Lim KS, Levato R, Costa PF, et al., 2018, Bio-resin for High Resolution Lithography-based Biofabrication of Complex Cell-laden Constructs. Biofabrication, 10:034101. https://doi.org/10.1088/1758-5090/aac00c

Ito Y, Correll K, Schiel JA, et al., Lung Fibroblasts Accelerate Wound Closure in Human Alveolar Epithelial Cells through Hepatocyte Growth Factor/c-Met Signaling. Am J Physiol Lung Cell Mol Physiol, 307:L94–105. https://doi.org/10.1152/ajplung.00233.2013

Panganiban RA, Day RM, 2011, Hepatocyte Growth Factor in Lung Repair and Pulmonary Fibrosis. Acta Pharmacol Sin, 32:12–20. https://doi.org/10.1038/aps.2010.90

Kanaji N, et al., 2017, Hepatocyte Growth Factor Produced in Lung Fibroblasts Enhances Non-small Cell Lung Cancer Cell Survival and Tumor Progression. Respir Res, 18:118. https://doi.org/10.1186/s12931-017-0604-z

Carterson A, et al., 2005, A549 Lung Epithelial Cells Grown as Three-dimensional Aggregates: Alternative Tissue Culture Model for Pseudomonas aeruginosa Pathogenesis. Infect Immun, 73:1129–40. https://doi.org/10.1128/iai.73.2.1129-1140.2005

Paczosa MK, Fisher ML, Maldonado-Arocho FJ, Mecsas J, 2014, Yersinia Pseudotuberculosis Uses A il and YadA to Circumvent Neutrophils by Directing Y op Translocation during Lung Infection. Cell Microbiol, 16:247–68. https://doi.org/10.1111/cmi.12219

Davidson DJ, Dorin JR, McLachlan G, et al., 1995, Lung Disease in the Cystic Fibrosis Mouse Exposed to Bacterial Pathogens. Nat Genet, 9:351–7. https://doi.org/10.1038/ng0495-351

Essaidi-Laziosi M, Brito F, Benaoudia S, et al., 2018, Propagation of Respiratory Viruses in Human Airway Epithelia Reveals Persistent Virus-specific Signatures. J Allerg Clin Immunol, 141:2074–84. https://doi.org/10.1016/j.jaci.2017.07.018

Weibel ER, 2015, On the Tricks Alveolar Epithelial Cells Play to Make a Good Lung. Am J Respir Crit Care Med, 191:504–13. https://doi.org/10.1164/rccm.201409-1663oe

Costa A, de Souza Carvalho-Wodarz C, Seabra V, et al., 2019, Triple co-culture of Human Alveolar Epithelium, Endothelium and Macrophages for Studying the Interaction of Nanocarriers with the Air-blood Barrier. Acta Biomater, 91:235–47. https://doi.org/10.1016/j.actbio.2019.04.037

Klein SG, Serchi T, Hoffmann L, Blömeke B, et al., 2013, An Improved 3D Tetraculture System Mimicking the Cellular Organisation at the Alveolar Barrier to Study the Potential Toxic Effects of Particles on the Lung. Part Fibre Toxicol, 10:31. https://doi.org/10.1186/1743-8977-10-31

Bengalli R, Mantecca P, Camatini M, et al., 2012, Effect of Nanoparticles and Environmental Particles on a Cocultures Model of the Air-blood Barrier. BioMed Res Int, 2013:801214. https://doi.org/10.1155/2013/801214

Short KR, et al., 2016, Influenza Virus Damages the Alveolar Barrier by Disrupting Epithelial Cell Tight Junctions. Eur Respir J, 47:954–66.

Dekali S, Gamez C, Kortulewski T, et al., 2014, Assessment of an In Vitro Model of Pulmonary Barrier to Study the Translocation of Nanoparticles. Toxicol Rep, 1:157–71 https://doi.org/10.1016/j.toxrep.2014.03.003

Wang G, Zhang X, Liu X, et al., 2019, Ambient Fine Particulate Matter Induce Toxicity in Lung Epithelialendothelial Co-culture Models. Toxicol Lett, 301:133–45 https://doi.org/10.1016/j.toxlet.2018.11.010

Alfaro-Moreno E, Nawrot TS, Vanaudenaerde BM, et al., 2008, Co-cultures of Multiple Cell Types Mimic Pulmonary Cell Communication in Response to Urban PM10. Eur Respir J, 32:1184–94. https://doi.org/10.1183/09031936.00044008

Herzog F, Clift MJ, Piccapietra F, et al., 2013, Exposure of Silver-nanoparticles and Silver-ions to Lung Cells In Vitro at the Air-liquid Interface. Part Fibre Toxicol, 10:11. https://doi.org/10.1186/1743-8977-10-11

Walter MN, Kohli N, Khan N, et al., Human Mesenchymal Stem Cells Stimulate EaHy926 Endothelial Cell Migration: Combined Proteomic and In Vitro Analysis of the Influence of Donor-donor Variability. J Stem Cells Regen Med, 11:18. https://doi.org/10.46582/jsrm.1101004

Yang Z, Yang X, Xu S, et al., 2017, Reprogramming of Stromal Fibroblasts by SNAI2 Contributes to Tumor Desmoplasia and Ovarian Cancer Progression. Mol Cancer, 16:1–15. https://doi.org/10.1186/s12943-017-0732-6

Ng WL, Yeong WY, Naing MW, 2017, Polyvinylpyrrolidone-Based Bio-Ink Improves Cell Viability and Homogeneity during Drop-On-Demand Printing. Materials, 10:190. https://doi.org/10.3390/ma10020190

Ng WL, Goh MH, Yeong WY, et al., Applying Macromolecular Crowding to 3D Bioprinting: Fabrication of 3D Hierarchical Porous Collagen-based Hydrogel Constructs. Biomater Sci, 6:562–74. https://doi.org/10.1039/c7bm01015j

Wu J, Wang Y, Liu G, et al., 2018, Characterization of Air liquid Interface Culture of A549 Alveolar Epithelial Cells. Braz J Med Biol Res, 51:e6950.

Meenach SA, Tsoras AN, McGarry RC, et al., 2016, Development of Three-dimensional Lung Multicellular Spheroids in Air-and Liquid-interface Culture for the Evaluation of Anticancer Therapeutics. Int J Oncol, 48:1701–9. https://doi.org/10.3892/ijo.2016.3376

Abdelwahab EM, et al., 2019, Wnt Signaling Regulates Transdifferentiation of Stem Cell Like Type 2 Alveolar Epithelial Cells to Type 1 Epithelial Cells. Respir Res, 20:1–9. https://doi.org/10.1186/s12931-019-1176-x

DOI: http://dx.doi.org/10.18063/ijb.v7i2.332


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