Characterization of acellular lung matrix. (A) 3D micro-CT of the acellular matrix airway compartment. Large airways are in green. (B) Micro-CT angiography of vascular compartment, thresholded to visualize only large vessels. In (A) and (B), voxel size is 58 μm; scale bar, 4 mm. (C) Micro-CT angiography of smaller vessels in acellular lung. Voxel size, 6.5 μm; scale bar, 500 μm. (D) Immunoblot for MHC-1, MHC-II, and β-actin in native (Nat) and decellularized (Dec) lungs, showing removal of cellular proteins. (E) Hematoxylin and eosin (H&E) stain of native rat lung. (F) H&E stain of acellular lung matrix. Scale bar, 50 μm in (E) and (F). (G) Collagen (Coll), elastin (Elas), glycosaminoglycan (GAG), and DNA contents of native lung (black bars) and acellular matrices (hatched bars). Values are mean ± SD per lung (n ≥ 4 lungs for all measures), scaled to 1 for native, with asterisk indicating P < 0.05 for difference between native and acellular matrices. (H) SEM of native rat lung. (I) SEM of acellular matrix. Scale bar, 10 μm in (H) and (I). (J) TEM of acellular lung matrix. Asterisk indicates capillaries in alveolar septa. Scale bar, 5 μm.

Implantation of engineered lungs into rats. (A) Tissue-engineered left lung was implanted into adult Fischer 344 rat recipient and photographed ~30 min later. (B) X-ray image of rat showing the implanted engineered left lung (white arrow) and the right native lung. (C) H&E stain of explanted lung. Red blood cells perfusing septa are evident, and some red blood cells are present in airspaces. Scale bar, 50 μm.

Schema for lung tissue engineering. (A) Native adult rat lung is cannulated in the pulmonary artery and trachea for infusion of decellularization solutions. (B) Acellular lung matrix is devoid of cells after 2 to 3 hours of treatment. (C) Acellular matrix is mounted inside a biomimetic bioreactor that allows seeding of vascular endothelium into the pulmonary artery and pulmonary epithelium into the trachea. (D) After 4 to 8 days of culture, the engineered lung is removed from the bioreactor and is suitable for implantation into (E) the syngeneic rat recipient.

Repopulation of the matrix with lung epithelial and endothelial cells and mechanical assessment of the engineered lungs. (A) H&E stain of mixed neonatal pulmonary epithelium seeded into acellular matrix and cultured for 8 days. (B) H&E stain of lung microvascular endothelium seeded into vascular compartment and cultured for 8 days. Scale bars, 100 μm in (A) and (B). (C) Proliferating cell nuclear antigen stain of epithelial culture after 8 days; brown nuclei are dividing. (D) Terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling stain of epithelium after 4 days of culture detects no apoptotic cells (positive cells stain brown; green indicates nuclear counterstain). Scale bars, 50 μm in (C) and (D). (E) Immunoblots for pro-SPC indicates similar expression for native (Nat) and engineered (Eng) lung. Engineered lung expresses SPB precursor proteins at 43 and 90 kD, but no mature SPB. β-actin confirms similar protein loading. (F to H) Quasi-static compliance curves for (F) typical native, (G) acellular, and (H) engineered lungs; arrows indicate inflation arm of loop. (I) Mean ultimate tensile strengths (UTS) of native (n = 4), acellular (n = 10), and engineered (n = 5) lungs. Error bars are SD; there were no significant differences between any groups.