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Design and Synthesis of an Artificial Pulmonary Pleura for High Throughput Studies in Acellular Human Lungs

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Abstract

Whole organ decellularization of complex organs, such as lungs, presents a unique opportunity for use of acellular scaffolds for ex vivo tissue engineering or for studying cell–extracellular matrix interactions ex vivo. A growing body of literature investigating decellularizing and recellularizing rodent lungs has provided important proof of concept models and rodent lungs are readily available for high throughput studies. In contrast, comparable progress in large animal and human lungs has been impeded owing to more limited availability and difficulties in handling larger tissue. While the use of smaller segments of acellular large animal or human lungs would maximize usage from a single lung, excision of small acellular segments compromises the integrity of the pleural layer, leaving the terminal ends of blood vessels and airways exposed. We have developed a novel pleural coating using non-toxic ionically crosslinked alginate or photocrosslinked methacrylated alginate which can be applied to excised acellular lung segments, permits inflation of small segments, and significantly enhances retention of cells inoculated through cannulated airways or blood vessels. Further, photocrosslinking methacrylated alginate, using eosin Y and triethanolamine at 530 nm wavelength, results in a mechanically stable pleural coating that permits effective cyclic 3-dimensional stretch, i.e., mechanical ventilation, of individual segments.

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Acknowledgments

The authors wish to thank Joseph Platz, Charles Parsons, Dino Sokocevic for decellularization, imaging, and experimental assistance; Alex Trick and Michael Bula for designing and constructing the initial LED light box; Elice Brooks for cell culture; Benjamin Cares for alginate synthesis and Marc H. Soldini for rheometry characterizations; Joseph Consiglio, PhD, and Al Correira (Harvard Apparatus, Holliston, MA) for technical assistance and assistance with the HugoSachs Minivents and DINOlite imaging; Mervin Yoder MD, Indiana University, for the CBF cells; Albert van der Vliet, PhD for the HBE cells; and FMC Biopolymer for Manugel® and Protanol® samples. These studies were supported by NIH ARRA RC4HL106625 (DJW), NHLBI R21HL094611 (DJW), NHLBI R21HL108689 (DJW), and the UVM Lung Biology Training grant T32 HL076122 from the NHLBI.

Conflict of interest

D.E. Wagner, S.L. Fenn, N.R. Bonenfant, E.R. Marks, Z.D. Borg, P.E. Saunders, R.A. Floreani, and D.J. Weiss have no conflicts of interest to declare.

Ethical Standards

All human subjects research was carried out in accordance with institutional guidelines. No animal studies were carried out by the authors for this article.

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Authors and Affiliations

  1. Department of Medicine, University of Vermont, 226 Health Sciences Research Facility Center, Burlington, VT, 05405, USA

    Darcy E. Wagner, Nicholas R. Bonenfant, Elliot R. Marks, Zachary Borg, Patrick Saunders & Daniel J. Weiss

  2. College of Engineering and Mathematical Sciences, University of Vermont, 109 Votey Hall, Burlington, VT, 05405, USA

    Spencer L. Fenn & Rachael A. Floreani

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  1. Darcy E. Wagner
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Correspondence to Daniel J. Weiss.

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Associate Editor Michael R. King oversaw the review of this article.

This article has been designated as a 2013 BMES Outstanding Contribution.

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Supplementary material 1 (DOCX 15 kb)

Supplementary material 2 Video 1 Excised segments of human lungs coated with photocrosslinked methacrylated alginate can be mechanically ventilated (WMV 7015 kb)

12195_2014_323_MOESM3_ESM.tif

Supplemental Fig. 1 Calcium alginate hydrogels can be generated by mixing 2.5% (w/v) Manugel® and 3% (w/v) CaCl2. At t0, a 2.5% (w/v) Manugel® is applied to a material or surface and allowed to equilibrate. A 3% (w/v) CaCl2 solution is added and the gel is ionically crosslinked (TIFF 46174 kb)

12195_2014_323_MOESM4_ESM.tif

Supplemental Fig. 2. Methacrylated alginate hydrogels can be photocrosslinked by exposing AA-MA solutions with eosin Y, TEOA, and 1VP to 530 nm (green) excitation. Solutions of AA-MA with eosin Y, TEOA, and 1VP are poured between two glass coverslips and exposed to 530 nm green excitation light for 10 min to complete photocrosslinking (TIFF 31678 kb)

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Wagner, D.E., Fenn, S.L., Bonenfant, N.R. et al. Design and Synthesis of an Artificial Pulmonary Pleura for High Throughput Studies in Acellular Human Lungs. Cel. Mol. Bioeng. 7, 184–195 (2014). https://doi.org/10.1007/s12195-014-0323-1

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  • DOI: https://doi.org/10.1007/s12195-014-0323-1

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