A novel in vitro model to study alveologenesis

Abstract

Many pediatric pulmonary diseases are associated with significant morbidity and mortality due to impairment of alveolar development. The lack of an appropriate in vitro model system limits the identification of therapies aimed at improving alveolarization. Herein, we characterize an ex vivo lung culture model that facilitates investigation of signaling pathways that influence alveolar septation. Postnatal Day 4 (P4) mouse pup lungs were inflated with 0.4% agarose, sliced, and cultured within a collagen matrix in medium that was optimized to support cell proliferation and promote septation. Lung slices were grown with and without 1D11, an active transforming growth factor-β-neutralizing antibody. After 4 days, the lung sections (designated P4 + 4) and noncultured lung sections were examined using quantitative morphometry to assess alveolar septation and immunohistochemistry to evaluate cell proliferation and differentiation. We observed that the P4 + 4 lung sections exhibited ex vivo alveolarization, as evidenced by an increase in septal density, thinning of septal walls, and a decrease in mean linear intercept comparable to P8, age-matched, uncultured lungs. Moreover, immunostaining showed ongoing cell proliferation and differentiation in cultured lungs that were similar to P8 controls. Cultured lungs exposed to 1D11 had a distinct phenotype of decreased septal density when compared with untreated P4 + 4 lungs, indicating the utility of investigating signaling in these lung slices. These results indicate that this novel lung culture system is optimized to permit the investigation of pathways involved in septation, and potentially the identification of therapeutic targets that enhance alveolarization.

Figure 1.

Schematic representation of organ culture model and tissue handling. Mouse pups of the indicated postnatal age were killed and the heart and lungs were perfused with sodium citrate. The lungs were inflated with 0.4% low-melt agarose in modified M199 media at 20 mm water for 10 minutes, and then placed in ice-cold PBS. The left lung was isolated and sectioned by hand into 1-mm-thick slices. One slice of the Postnatal Day 4 (P4) lung was fixed immediately as P4 control; the remainders were placed into culture in 1-mg/ml type 1 collagen matrices. Similarly processed lung slices from P8 and P12 pups were fixed immediately as controls. After cultured P4 lung slices had grown for 4 days, the slices were fixed and processed for structural work. For morphometric analysis, Hart’s stained sections were imaged in a nonbiased fashion by taking three sequential, nonoverlaping images starting at the left upper portion of the section along the pleura and moving counter-clockwise.

Figure 2.

Hart’s stained images of lungs grown in vivo and in vitro. P4, P8, and P12 lungs show the expected increase in complexity of the parenchyma that accompanies alveolarization that has occurred in lungs growing in vivo. After 4 days, P4 (P4 + 4) in vitro–grown lungs show an increased complexity when compared with P4 lungs, and a similar level compared with P8. Higher-power images reveal increased parenchymal complexity and more septa (arrows) and accumulation of elastin in the septae (arrowheads) as alveolarization progresses from P4 through P12. P4 + 4 lungs also show increased complexity, septal formation, and elastin organization when compared with P4 lungs, and appear similar to P8 lungs. Original magnifications of image capture are indicated.

Figure 3.

Septal density and mean linear intercept (Lm) of lungs grown in vivo and in vitro. (A) Septal density increases with advancing age (P4–P12) in lungs growing in vivo. Lungs grown in culture (P4 + 4) show a significant increase in septal density compared with P4 lungs (*P = 0.0035) and a similar density to lungs grown in vivo (P8). (B) Lm decreases as lung complexity and septation advance with increased age. Cultured (P4 + 4) lungs show a significant decrease in Lm when compared with P4 lungs (*P = 0.0253), but no difference when compared with P8 lungs. (C) Alveolar wall thickness decreases as postnatal age increases. Cultured lungs have significantly thinner walls than P4 uncultured controls (*P < 0.001), but are of similar thickness as P8 in vivo control lungs. WT, wild type.

Figure 4.

Protein expression in lungs compared with in vivo grown lungs. (A) Immunohistochemical staining for proliferating cells (Ki67), myofibroblast differentiating marker (calponin), differentiatied myofibroblasts (α-smooth muscle actin [α-SMA]), type 2 pneumocytes (pro–surfactant C [Pro-Surf C]), type 1 pneumocytes (popoplanin, podo), and endothelial cells (aquaporin-5) in P4, P4 + 4 cultured lungs, and P8 lungs. P4 + 4 lungs show ongoing proliferation comparable to P8 lungs and markers of cell differentiation similar to that seen in P8 lungs. Calponin staining appears similar in the P4 + 4 and P8 lungs when compared with P4. SMA localizes to septal tips in cultured and uncultured lungs (arrows). Type 2 pneumocytes are seen scattered throughout the parenchyma in P4, P4 + 4, and P8 lungs. Aquaporin-5 in the cell membrane of type 1 pneumocytes is seen in cells of the alveolar walls in P4 lungs with increased reactivity in P4 + 4 and P8 lungs (asterisks). IB₄ lectin identifying capillary endothelial cells is seen in cells of alveolar walls in P4 lungs (arrowheads). In P4 + 4 lungs, the lectin identifies the double capillary network in secondary septa, but appears in a more lacy and diffuse pattern in the parenchyma than in the P8 lungs. Original magnification, ×20, except IB₄ lectin at ×100. Within each antibody group, images were color matched to match the P4 image. (B) Western blot analysis reveals ongoing expression of cell type–specific proteins in cultured and uncultured lungs. Myofibroblast proteins, calponin and α-SMA, are expressed similarly in the cultured and uncultured controls. Pro-Surf C, a protein expressed in type two pneumocytes, is also found in P4 + 4 cultured lungs, as well as control in vivo lungs. The type 1 pneumocyte–specific protein, aguaporin-5, shows increased expression in P4 + 4 cultured lungs and the P8 in vivo control when compared with P4 uncultured lungs. Expression of CD31 in endothelial cells is diminished when compared with P4 and P8 controls.

Figure 5.

Histology and morphometrics of neutralizing transforming growth factor (TGF)-β antibody (TGF-βAb)–treated lungs. (A) Hart’s stained sections from P4 in vivo lungs and P4 + 4 cultured lungs compared with P4 + 4 lungs cultured with neutralizing TGF-β antibody. The antibody-treated lungs have fewer septa and thickened alveolar walls (indicated by “H”) when compared with the P4 and P4 + 4 controls. (B) Septal density measurements show decreased septation in lungs treated with the TGF-β–neutralizing antibody, 1D11, when compared with the P4 + 4 control cultured lung (*P = 0.0002). (C) Lm in the lungs treated with neutralizing TGF-β antibody is not significantly different than the Lm of P4 lungs or P4 + 4 lungs. (D) Wall thickness is increased in lungs grown in the presence of neutralizing TGF-β antibody when compared with P4 + 4 cultured lungs (*P < 0.001). No differences in morphometric parameters were identified between P4 + 4 lungs and controls (IgG isotype control antibody [G3A1] or antibody diluent [PBS]).