Equine lung decellularization: a potential approach for in vitro modeling the role of the extracellular matrix in asthma

Abstract

Contrary to conventional research animals, horses naturally develop asthma, a disease in which the extracellular matrix of the lung plays a significant role. Hence, the horse lung extracellular matrix appears to be an ideal candidate model for in vitro studying the mechanisms and potential treatments for asthma. However, so far, such model to study cell-extracellular matrix interactions in asthma has not been developed. The aim of this study was to establish a protocol for equine lung decellularization that maintains the architecture of the extracellular matrix and could be used in the future as an in vitro model for therapeutic treatment in asthma. For this the equine lungs were decellularized by sodium dodecyl sulfate detergent perfusion at constant gravitational pressure of 30 cmH₂O. Lung scaffolds were assessed by immunohistochemistry (collagen I, III, IV, laminin, and fibronectin), scanning electron microscopy, and DNA quantification. Their mechanical property was assessed by measuring lung compliance using the super-syringe technique. The optimized protocol of lung equine decellularization was effective to remove cells (19.8 ng/mg) and to preserve collagen I, III, IV, laminin, and fibronectin. Moreover, scanning electron microscopy analysis demonstrated maintained microscopic lung structures. The decellularized lungs presented lower compliance compared to native lung. In conclusion we described a reproducible decellularization protocol that can produce an acellular equine lung feasible for the future development of novel treatment strategies in asthma.

Keywords: Equine lung; asthma; decellularized; extracellular matrix; respiratory diseases.

Figure 1.

Schematic illustration of the system for equine lung decellularization.

Figure 2.

Representative example of (a) an intact and (b) a decellularized equine lung.

Figure 3.

(a) DNA quantification before and after equine lung decellularization. (b) Lung compliance before and after decellularization. (c) Pressure/volume (P/V) values assessed using the super-syringe technique with inflation of the lungs in steps of 100 mL up to 1 L. Native lung is represented as [—] and decellularized lung as [—]. Data are represented as mean ± SE.

Figure 4.

Representative native and decellularized equine lung tissue, as visualized by hematoxylin and eosin (H&E), colloidal iron stain, and SEM images. Sections indicate maintenance of tissue architecture, ECM, removal of debris and blood, and lack of visible nuclear material.

Figure 5.

Representative histological analysis for collagen quantification of (a) native and (b) decellularized equine lung tissue. The lung tissue samples were stained with picrosirius red (red = collagen). Bar graph shows collagen area (%). Asterisk indicates significance of difference between the groups (*p < 0.005; **p < 0.05).

Figure 6.

Immunohistochemistry images of native and decellularized equine lung slices stained for different components of the extracellular matrix (collagen I, III, IV, fibronectin, and elastin). Scale bar = 100 µm.

Figure 7.

Immunocytochemistry of equine lung scaffolds with YSVEGF. (a) DAPI nuclei YSVEGF cells in scaffold of equine lung; (b) marked presence of YSVEGF cells expressing eGFP; (c) scaffold of lung expressing fibronectin in red; (d) presence of these cells in the scaffold in red that expresses fibronectin is very clear, proving the efficient recellularization.

Figure 8.

Immunocytochemistry of equine lung scaffolds with fibroblast cells. (a) DAPI nuclei of fibroblast cells; (b) marked presence of fibroblast cells expressing CD90 in green; (c) marked presence in lung sacffold of expressing fibronectin in red; (d) presence of these cells in the scaffold that expresses proving the efficient recellularization.

Figure 9.

Immunofluorescence of equine lung scaffolds for expression of N-cadherin, a biomarker for adhesion cell. (a) DAPI nuclei of fibroblast cells; (b) expression of N-cadherin; (c) presence in equine lung scaffold of DAPI and N-cadherin; (d) DAPI nuclei of YSVEGF; and (e) expression of N-cadherin; (f) presence in equine lung scaffold of DAPI and N-cadherin.

Figure 10.

Immunofluorescence of equine lung scaffolds for expression of CD31, a biomarker for adhesion cell. (a) DAPI nuclei of fibroblast cells and (b) expression of CD31; (c) presence in equine lung scaffold of DAPI and CD31.