Cellular and Molecular Bioengineering

, Volume 7, Issue 2, pp 184–195 | Cite as

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

  • Darcy E. Wagner
  • Spencer L. Fenn
  • Nicholas R. Bonenfant
  • Elliot R. Marks
  • Zachary Borg
  • Patrick Saunders
  • Rachael A. Oldinski
  • Daniel J. WeissEmail author


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.


Lung Alginate Decellularization Ventilation Artificial pleura 



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. Oldinski, 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.

Supplementary material

12195_2014_323_MOESM1_ESM.docx (16 kb)
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 (45.1 mb)
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 (30.9 mb)
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)


  1. 1.
    Bonenfant, N. R., D. Sokocevic, D. E. Wagner, et al. The effects of storage and sterilization on de-cellularized and re-cellularized whole lung. Biomaterials 34:3231–3245, 2013.CrossRefGoogle Scholar
  2. 2.
    Bonvillain, R. W., S. Danchuk, D. E. Sullivan, et al. A nonhuman primate model of lung regeneration: detergent-mediated decellularization and initial in vitro recellularization with mesenchymal stem cells. Tissue Eng. Part A 18:2437–2452, 2012.CrossRefGoogle Scholar
  3. 3.
    Booth, A. J., R. Hadley, A. M. Cornett, et al. Acellular normal and fibrotic human lung matrices as a culture system for in vitro investigation. Am. J. Respir. Crit. Care Med. 186:866–876, 2012.CrossRefGoogle Scholar
  4. 4.
    Burdick, J. A., C. Chung, X. Jia, M. A. Randolph, and R. Langer. Controlled degradation and mechanical behavior of photopolymerized hyaluronic acid networks. Biomacromolecules 6:386–391, 2004.CrossRefGoogle Scholar
  5. 5.
    Cortiella, J., J. Niles, A. Cantu, et al. Influence of acellular natural lung matrix on murine embryonic stem cell differentiation and tissue formation. Tissue Eng. Part A 16:2565–2580, 2010.CrossRefGoogle Scholar
  6. 6.
    Daly, A. B., J. M. Wallis, Z. D. Borg, et al. Initial binding and recellularization of decellularized mouse lung scaffolds with bone marrow-derived mesenchymal stromal cells. Tissue Eng. Part A 18:1–16, 2012.CrossRefGoogle Scholar
  7. 7.
    Gilpin, S. E., J. P. Guyette, G. Gonzalez, et al. Perfusion decellularization of human and porcine lungs: bringing the matrix to clinical scale. J. Heart Lung Transpl. 2013. doi: 10.1016/j.healun.2013.10.030
  8. 8.
    Gombotz, W. R., and S. F. Wee. Protein release from alginate matrices. Adv. Drug Deliv. Rev. 31:267–285, 1998.CrossRefGoogle Scholar
  9. 9.
    Jay, S. M., and W. M. Saltzman. Controlled delivery of VEGF via modulation of alginate microparticle ionic crosslinking. J. Controlled Release 134:26–34, 2009.CrossRefGoogle Scholar
  10. 10.
    Jensen, T., B. Roszell, F. Zang, et al. A rapid lung de-cellularization protocol supports embryonic stem cell differentiation in vitro and following implantation. Tissue Eng. Part C 18:632–646, 2012.CrossRefGoogle Scholar
  11. 11.
    Jeon, O., K. H. Bouhadir, J. M. Mansour, and E. Alsberg. Photocrosslinked alginate hydrogels with tunable biodegradation rates and mechanical properties. Biomaterials 30:2724–2734, 2009.CrossRefGoogle Scholar
  12. 12.
    Kim, J., Y. Park, G. Tae, et al. Characterization of low-molecular-weight hyaluronic acid-based hydrogel and differential stem cell responses in the hydrogel microenvironments. J. Biomed. Mater. Res. A 88A:967–975, 2009.CrossRefGoogle Scholar
  13. 13.
    Kuo, C. K., and P. X. Ma. Ionically crosslinked alginate hydrogels as scaffolds for tissue engineering: part 1. Structure, gelation rate and mechanical properties. Biomaterials 22:511–521, 2001.CrossRefGoogle Scholar
  14. 14.
    Lee, K. Y., and D. J. Mooney. Alginate: properties and biomedical applications. Prog. Polym. Sci. 37:106–126, 2012.CrossRefGoogle Scholar
  15. 15.
    Lemoine, D., F. Wauters, S. Bouchend’homme, and V. Préat. Preparation and characterization of alginate microspheres containing a model antigen. Int. J. Pharm. 176:9–19, 1998.CrossRefGoogle Scholar
  16. 16.
    Liu, W. F., M. Ma, K. M. Bratlie, T. T. Dang, R. Langer, and D. G. Anderson. Real-time in vivo detection of biomaterial-induced reactive oxygen species. Biomaterials 32:1796–1801, 2011.CrossRefGoogle Scholar
  17. 17.
    Longmire, T. A., L. Ikonomou, F. Hawkins, et al. Efficient derivation of purified lung and thyroid progenitors from embryonic stem cells. Cell Stem Cell 10:398–411, 2012.CrossRefGoogle Scholar
  18. 18.
    Macchiarini, P., J. Wain, S. Almy, and P. Dartevelle. Experimental and clinical evaluation of a new synthetic, absorbable sealant to reduce air leaks in thoracic operations. J. Thorac. Cardiovasc. Surg. 117:751–758, 1999.CrossRefGoogle Scholar
  19. 19.
    Muller, R., E. Gerard, P. Dugand, P. Rempp, and Y. Gnanou. Rheological characterization of the gel point: a new interpretation. Macromolecules 24:1321–1326, 1991.CrossRefGoogle Scholar
  20. 20.
    Nichols, J. E., J. Niles, M. Riddle, et al. Production and assessment of decellularized pig and human lung scaffolds. Tissue Eng. Part A 19:2045–2062, 2013.CrossRefGoogle Scholar
  21. 21.
    Ohta, S., M. Hirose, and H. Ishibashi. Pleural covering method of polyglycolic acid felt with sodium alginate water solution for prevention of postoperative pulmonary fistula. Kyobu Geka 61:561–563, 2008.Google Scholar
  22. 22.
    O’Neill, J. D., R. Anfang, A. Anandappa, et al. Decellularization of human and porcine lung tissues for pulmonary tissue engineering. Ann. Thorac. Surg. 96:1046–1056, 2013.CrossRefGoogle Scholar
  23. 23.
    Ott, H. C., B. Clippinger, C. Conrad, et al. Regeneration and orthotopic transplantation of a bioartificial lung. Nat. Med. 16:927–933, 2010.CrossRefGoogle Scholar
  24. 24.
    Park, Y. D., N. Tirelli, and J. A. Hubbell. Photopolymerized hyaluronic acid-based hydrogels and interpenetrating networks. Biomaterials 24:893–900, 2003.CrossRefGoogle Scholar
  25. 25.
    Pawar, S. N., and K. J. Edgar. Alginate derivatization: a review of chemistry, properties and applications. Biomaterials 33:3279–3305, 2012.CrossRefGoogle Scholar
  26. 26.
    Petersen, T. H., E. A. Calle, L. Zhao, et al. Tissue-engineered lungs for in vivo implantation. Science 329:538–541, 2010.CrossRefGoogle Scholar
  27. 27.
    Price, A. P., K. A. England, A. M. Matson, B. R. Blazar, and A. Panoskaltsis-Mortari. Development of a decellularized lung bioreactor system for bioengineering the lung: the matrix reloaded. Tissue Eng. Part A 16:2581–2591, 2010.CrossRefGoogle Scholar
  28. 28.
    Smeds, K. A., A. Pfister-Serres, D. Miki, et al. Photocrosslinkable polysaccharides for in situ hydrogel formation. J. Biomed. Mater. Res. 55:254–255, 2001.CrossRefGoogle Scholar
  29. 29.
    Smidsrød, O. Solution properties of alginate. Carbohydr. Res. 13:359–372, 1970.CrossRefGoogle Scholar
  30. 30.
    Sokocevic, D., N. R. Bonenfant, D. E. Wagner, et al. The effect of age and emphysematous and fibrotic injury on the re-cellularization of de-cellularized lungs. Biomaterials 34:3256–3269, 2013.CrossRefGoogle Scholar
  31. 31.
    Song, J. J., S. S. Kim, Z. Liu, et al. Enhanced in vivo function of bioartificial lungs in rats. Ann. Thorac. Surg. 92:998–1006, 2011.CrossRefGoogle Scholar
  32. 32.
    Tonnesen, H. H., and J. Karlsen. Alginate in drug delivery systems. Drug Dev. Ind. Pharm. 28:621–630, 2002.CrossRefGoogle Scholar
  33. 33.
    Wagner, D. E., N. R. Bonenfant, C. S. Parsons, et al. Comparative decellularization and recellularization of normal versus emphysematous human lungs. Biomaterials 35:3281–3297, 2014.CrossRefGoogle Scholar
  34. 34.
    Wagner, D. E., N. R. Bonenfant, D. Sokocevic, et al. Three-dimensional scaffolds of acellular human and porcine lungs for high throughput studies of lung disease and regeneration. Biomaterials 35(9):2664–2679, 2014.CrossRefGoogle Scholar
  35. 35.
    Wagner, D. E., R. W. Bonvillain, T. Jensen, et al. Can stem cells be used to generate new lungs? Ex vivo lung bioengineering with decellularized whole lung scaffolds. Respirology 18:895–911, 2013.CrossRefGoogle Scholar
  36. 36.
    Wallis, J. M., Z. D. Borg, A. B. Daly, et al. Comparative assessment of detergent-based protocols for mouse lung de-cellularization and re-cellularization. Tissue Eng. Part C 18:420–432, 2012.CrossRefGoogle Scholar
  37. 37.
    Winter, H. H. Can the gel point of a cross-linking polymer be detected by the G′–G″ crossover? Polym. Eng. Sci. 27:1698–1702, 1987.CrossRefGoogle Scholar
  38. 38.
    Yang, D., and K. S. Jones. Effect of alginate on innate immune activation of macrophages. J. Biomed. Mater. Res. A 90A:411–418, 2009.CrossRefGoogle Scholar
  39. 39.
    Yankaskas, J. R., J. E. Haizlip, M. Conrad, et al. Papilloma virus immortalized tracheal epithelial cells retain a well-differentiated phenotype. Am. J. Physiol. 264:C1219–C1230, 1993.Google Scholar
  40. 40.
    Zar, J. Biostatistical Analysis. Upper Saddle River, NJ: Prentice-Hall, 2009.Google Scholar

Copyright information

© Biomedical Engineering Society 2014

Authors and Affiliations

  • Darcy E. Wagner
    • 1
  • Spencer L. Fenn
    • 2
  • Nicholas R. Bonenfant
    • 1
  • Elliot R. Marks
    • 1
  • Zachary Borg
    • 1
  • Patrick Saunders
    • 1
  • Rachael A. Oldinski
    • 2
  • Daniel J. Weiss
    • 1
    Email author
  1. 1.Department of MedicineUniversity of VermontBurlingtonUSA
  2. 2.College of Engineering and Mathematical SciencesUniversity of VermontBurlingtonUSA

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