Different Decellularization Methods in Bovine Lung Tissue Reveals Distinct Biochemical Composition, Stiffness, and Viscoelasticity in Reconstituted Hydrogels

Abstract

Extracellular matrix (ECM)-derived hydrogels are in demand for use in lung tissue engineering to mimic the native microenvironment of cells in vitro. Decellularization of native tissues has been pursued for preserving organotypic ECM while eliminating cellular content and reconstitution into scaffolds which allows re-cellularization for modeling homeostasis, regeneration, or diseases. Achieving mechanical stability and understanding the effects of the decellularization process on mechanical parameters of the reconstituted ECM hydrogels present a challenge in the field. Stiffness and viscoelasticity are important characteristics of tissue mechanics that regulate crucial cellular processes and their in vitro representation in engineered models is a current aspiration. The effect of decellularization on viscoelastic properties of resulting ECM hydrogels has not yet been addressed. The aim of this study was to establish bovine lung tissue decellularization for the first time via pursuing four different protocols and characterization of reconstituted decellularized lung ECM hydrogels for biochemical and mechanical properties. Our data reveal that bovine lungs provide a reproducible alternative to human lungs for disease modeling with optimal retention of ECM components upon decellularization. We demonstrate that the decellularization method significantly affects ECM content, stiffness, and viscoelastic properties of resulting hydrogels. Lastly, we examined the impact of these aspects on viability, morphology, and growth of lung cancer cells, healthy bronchial epithelial cells, and patient-derived lung organoids.

Keywords: decellularization; extracellular matrix; lung cancer; lung hydrogels; tissue engineering.

Figure 1

Schematic representation of the decellularization process and experimental characterizations.

Figure 2

Evaluation of decellularization efficiency in dECM tissues. (a) Brief summary of decellularization methods. (b) Quantification of dsDNA in native and dECM tissues, all samples were normalized to native bovine tissue. (c) Histological examination of tissues stained with haematoxylin and eosin (scale bar: 100 μm). (d) Hoechst staining of native and dECM tissues (scale bar: 100 μm). N_bovine represents native bovine lung, and N_human represents native human lung. Error bars represent sd (ns, no significance, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 3

Histological and biochemical analysis of native and decellularized lung tissues. (a) Sirius red staining for collagen. (b) Alcian blue (sGAG) staining of native and decellularized lung tissues (scale bar: 100 μm). (c) Collagen quantification, (d) sGAG quantification, and (e) elastin quantification of native and decellularized lung tissues. All samples were normalized to native bovine. N_bovine represents native bovine lung, and N_human represents native human lung. Error bars represent sd (ns, no significance, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 4

Gelation potential of dECM solutions and mechanical characterizations with oscillatory shear rheology. (a) Representative images of dECM solutions to gel transition after thermal crosslinking. (b) Rheological properties of dECM solutions, storage modulus (G). (c) Loss modulus of hydrogels (G″). (d) Temperature and time sweep showing gelation kinetics for different dECM samples. Error bars represent sd (ns, no significance, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 5

Viscoelasticity of dECM hydrogels. (a) Schematic representation of a creep curve. (b) Permanent strain preserved in dECM gels. (c) Creep recovery test. (d) Loss tangent. Error bars represent sd (ns, no significance, *p < 0.05).

Figure 6

Cell viability and cytocompatibility assessments. (a) Representative images of cells stained for calcein-AM (green) and PI (red) on days 1 and 14 (scale bars: 100 μm). Zoomed in bright field images (scale bars: 50 μm) to represent clump morphologies. (b) Metabolic activity results from days 4, 7, 11, and 14 were normalized to day 1 for each method.

Figure 7

Cell viability assessment of non-tumorigenic human lung cells. (a) Schematic representation of human lung tissue collection, isolation of pulmonary epithelium, and culturing of lung organoids in lung dECM hydrogels. (b) Representative images of lung organoids encapsulated in A-dECM and D-dECM hydrogels and stained for calcein-AM (green) and PI (red) on day 10 (scale bar: 70 μm). (c) Representative images of BEAS-2B cell line encapsulated in A-dECM and D-dECM hydrogels and stained for calcein-AM (green) and PI (red) on day 10 (scale bar: 70 μm).