Seeding of the decellularized matrix with mixed populations of neonatal rat lung epithelial cells (fig. S7
) into the airway compartment generally resulted in good adherence of the cells to alveolar structures, as well as to small- and medium-sized airways (). Microvascular lung endothelial cells, when injected into the pulmonary artery of acellular scaffolds, adhered throughout the scaffold vasculature (). Seeded pulmonary epithelial cells replicated rapidly and rarely displayed markers of apoptotic cell death (), despite the difficulty in culturing such cells on standard tissue culture plastic in vitro (8
). This observation suggests that substrate cues on the acellular lung matrix are important for pulmonary epithelial cell attachment and replication.
Fig. 3 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 (more ...)
In the biomimetic bioreactor, vascular perfusion greatly enhanced endothelial adhesion and survival on the lung matrix (fig. S9, A and B
). Negative pressure ventilation had multiple beneficial effects on cultured lung epithelium, including enhanced survival of epithelium in distal alveoli and clearance of epithelial secretions from the airway tree (fig. S9, C to F
). Clearance of airway secretions through ventilation indicates that the developing epithelium is in communication with the airway tree and is not growing randomly within the matrix. In addition, ventilation with air—as opposed to with culture medium—increased the numbers of type I alveolar epithelial cells, as well as the numbers of ciliated columnar epithelial cells (fig. S9, G to J
). Engineered lungs also produce pro-surfactant proteins B and C (pro-SPB, pro-SPC), although we could not detect mature SPB (). Surfactant proteins are critical for reducing alveolar surface tension and enabling lung inflation at physiologically normal pressures.
We performed compliance testing on the engineered lungs under quasi-static conditions by injecting air into the lungs and monitoring resultant pressure changes. Typical compliance curves for native lung, acellular matrix, and repopulated engineered lungs are shown in . The compliance values were, respectively, 0.35 ± 0.08 (n
= 10 measures), 0.09 ± 0.02 (n
= 4 measures), and 0.14 ± 0.06 (n
= 5 measures) mL/mmHg (mean ± SD, P
< 0.001 for difference between native and both decellularized and engineered compliances). Compliance values were measured at initial filling of the lungs (arrowheads in ), and thus lower compliances for decellularized matrix and engineered lung mean that these two tissues have higher “opening pressures,” and less functional surfactant, than does native lung. Despite this difference, the overall stress-strain relationships and the ultimate tensile stresses were similar between the three groups ( and fig. S6
). Thus, no substantial stiffening or weakening of the extracellular matrix occurs in the repopulated, engineered lungs as compared with native adult lungs.
To evaluate the distribution and phenotype of cells in the engineered lungs, we performed fluorescent immunohistochemical staining (fig. S8
). Endothelial cells seeded into the vasculature were extensively distributed and expressed CD-31, as did comparable cells in native rat lung (fig. S8A
). TEM analysis revealed the presence of tight junctions within the engineered endothelium, which is consistent with the development of some barrier function (fig. S10
). With respect to the seeded lung epithelium, Clara cell secretory protein (CCSP)–positive cells, which in native lung are found in small airways, were found primarily in very small airway structures after 4 days and in larger structures after 8 days in engineered lungs (fig. S8B
). Pro-SPC, a marker of type II alveolar epithelium, is normally present at the vertices of alveoli in native lung. In the engineered lungs, pro-SPC expression was diffuse in alveoli and in small airways after 4 days of culture, but at 8 days it showed a more native expression pattern at the vertices of alveoli (fig. S8C
). In contrast, aquaporin-5—a specific marker for type I epithelium—was found diffusely throughout native alveoli and in engineered lungs after 4 days but was largely absent after 8 days (fig. S8D
). This observation is consistent with previous work on neonatal rat development, which showed that type I cells do not fully differentiate until the post-natal period, when air breathing commences (9
). Indeed, engineered lungs that were ventilated with air, as opposed to liquid culture medium, displayed more aquaporin-5 expression in alveoli after 8 days of culture (fig. S9, G and H
) and also contained sparse ciliated epithelial cells (fig. S9, I and J
). Additional cell types noted included mesenchymal cells and the airway epithelial progenitor basal cells (fig. S8, E and F
). Hence, the engineered lungs contained many of the important cell types of native lung tissues. In addition, the spatial distribution of the various cell types was regional-specific, and with extended culture periods in the bioreactor the overall pattern of cellular distribution and differentiation became more similar to that in native lung tissue.
To determine whether the decellularization and repopulation methodologies used in our studies of rat lungs were applicable to human tissues, we obtained human lung segments from a tissue bank and treated them with decellularization solutions for up to 6 hours (7
). Histological staining showed that complete cellular removal was achieved, with preservation of alveolar architecture (fig. S11, A and B
). We seeded the acellular matrices with A549 human epithelial carcinoma cells and endothelial cells derived from human cord-blood endothelial progenitor cells (7
). The A549 cells adhered well to alveolar surfaces, and the endothelial cells adhered to the vasculature (fig. S11, C and D
), supporting the notion that these approaches may also be applicable to human lung tissues.