Development of a decellularized lung bioreactor system for bioengineering the lung: the matrix reloaded

Abstract

We developed a decellularized murine lung matrix bioreactor system that could be used to evaluate the potential of stem cells to regenerate lung tissue. Lungs from 2-3-month-old C57BL/6 female mice were excised en bloc with the trachea and heart, and decellularized with sequential solutions of distilled water, detergents, NaCl, and porcine pancreatic DNase. The remaining matrix was cannulated and suspended in small airway growth medium, attached to a ventilator to simulate normal, murine breathing-induced stretch. After 7 days in an incubator, lung matrices were analyzed histologically. Scanning electron microscopy and histochemical staining demonstrated that the pulmonary matrix was intact and that the geographic placement of the proximal and distal airways, alveoli and vessels, and the basement membrane of these structures all remained intact. Decellularization was confirmed by the absence of nuclear 4',6-diamidino-2-phenylindole staining and negative polymerase chain reaction for genomic DNA. Collagen content was maintained at normal levels. Elastin, laminin, and glycosaminglycans were also present, although at lower levels compared to nondecellularized lungs. The decellularized lung matrix bioreactor was capable of supporting growth of fetal alveolar type II cells. Analysis of day 7 cryosections of fetal-cell-injected lung matrices showed pro-Sp-C, cytokeratin 18, and 4',6-diamidino-2-phenylindole-positive cells lining alveolar areas that appeared to be attached to the matrix. These data illustrate the potential of using decellularized lungs as a natural three-dimensional bioengineering matrix as well as provide a model for the study of lung regeneration from pulmonary stem cells.

FIG. 1.

Maintenance of lung architecture after whole-lung decellularization. (A) Macroscopic images of lungs at different stages of decellularization process: (1) before decellularization (beginning of day 1), (2) after deoxycholate step (beginning of day 3), showing large amounts of white precipitate (likely cellular breakdown material) within the lungs, and (3) after the final rinses at the end of the process (the end of day 3). Image (4) illustrates that infusion with India ink results in complete dispersion through to the distal lung areas. (B) 4′,6-Diamidino-2-phenylindole staining of cryosections of lungs decellularized via (1) the vascular route (through the right ventricle), (2) the trachea, and (3) both the trachea and the vascular route. (C) Scanning electron micrographs are shown comparing mouse decellularized lungs with nondecellularized normal control lung. The top panels (700 × magnification) show a large bifurcating vessel surrounded by alveolar tissue that is acellular in the decellularized lung. The white arrow points to an alveolar septum lined by a capillary (on the top left panel). The top right panel shows a similar area (white arrow) still lined with matrix in the decellularized lung. Collagen fibrils are easy to distinguish surrounding the large vessel. The middle panels (700 × magnification) show the comparison of the exterior surface (the pleural equivalent of the mouse). The lower panels show low power images of the cut lungs (60 × magnification), depicting the maintenance of the characteristic spongy matrix of the lung after decellularization. Images are representative of three sets of lungs prepared for scanning electron micrographs for each condition. Color images available online at www.liebertonline.com/ten.

FIG. 2.

Setup of decellularized lung matrix bioreactor system. (A) Depiction of how the cannula is inserted through the filter cap (cap is 2.5 cm diameter), into the trachea of the decellularized lung and tied in place with silk suture. (B) The matrix is suspended in a flask filled with the small airway growth medium, and the cannula is attached to a ventilator (room air) to simulate normal, murine breathing-induced stretch (180 breaths/min; 300 μL volume) and placed in a 37°C, 5% CO₂ incubator. (C) Schematic of bioreactor setup. Supplemental Videos show the bioreactor system in action. Color images available online at www.liebertonline.com/ten.

FIG. 3.

Maintenance of lung matrix after whole-lung decellularization and ventilation in bioreactor system. (A) Lungs were decellularized and ventilated for 7 days in the lung bioreactor system. Staining by Masson's trichrome (top panels, collagen is blue, 200× magnification) and for elastin (bottom panels, elastin is black, collagen is pink) demonstrates that the decellularized lungs are acellular and the pulmonary matrix is intact. (B) Laminin staining (green, 200 × magnification) shows that laminin deposition is relatively unaffected by the decellularization and ventilation in the bioreactor. DAPI staining of nuclei (blue) is absent in the decellularized lungs. (C) Polymerase chain reaction for genomic DNA (GAPDH and β-actin), using primers spanning introns and exons, was negative in decellularized lung samples. (D) No MMP2 or MMP9 was detected in decellularized lungs by zymography. Images are representative of replicated experiments (n = 3–6 per group). DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde 3-phophate dehydrogenase.

FIG. 4.

Pulmonary function testing (PFT) can be done on decellularized whole-lung matrices. (A) Normal P/V flow loop of the lungs of an orally intubated normal B6 mouse. (B) P/V flow loop of nondecellularized lungs taken ex vivo (and suspended in air). (C) P/V loop of decellularized lungs. All PFTs were measured with a Flexivent plethysmograph set at a maximum pressure setting of 25 cm H₂O (y-axis shows volume in mL; x-axis shows pressure in cm H₂O). P/V, pressure/volume. Color images available online at www.liebertonline.com/ten.

FIG. 5.

Decellularized lung matrix bioreactor can support growth of alveolar type II cells. Cryosections of (A) normal and (B) decellularized lung infused with 3 × 10⁶ fetal lung cells and ventilated for 7 days in the bioreactor system were stained with the indicated antibodies as described in Materials and Methods. Single-color channels and merged images are shown. Magnification 600 ×. CK 18, cytokeratin 18.