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Comparative Study
. 2013 Sep;96(3):1046-55; discussion 1055-6.
doi: 10.1016/j.athoracsur.2013.04.022. Epub 2013 Jul 18.

Decellularization of Human and Porcine Lung Tissues for Pulmonary Tissue Engineering

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Free PMC article
Comparative Study

Decellularization of Human and Porcine Lung Tissues for Pulmonary Tissue Engineering

John D O'Neill et al. Ann Thorac Surg. .
Free PMC article

Abstract

Background: The only definitive treatment for end-stage organ failure is orthotopic transplantation. Lung extracellular matrix (LECM) holds great potential as a scaffold for lung tissue engineering because it retains the complex architecture, biomechanics, and topologic specificity of the lung. Decellularization of human lungs rejected from transplantation could provide "ideal" biologic scaffolds for lung tissue engineering, but the availability of such lungs remains limited. The present study was designed to determine whether porcine lung could serve as a suitable substitute for human lung to study tissue engineering therapies.

Methods: Human and porcine lungs were procured, sliced into sheets, and decellularized by three different methods. Compositional, ultrastructural, and biomechanical changes to the LECM were characterized. The suitability of LECM for cellular repopulation was evaluated by assessing the viability, growth, and metabolic activity of human lung fibroblasts, human small airway epithelial cells, and human adipose-derived mesenchymal stem cells over a period of 7 days.

Results: Decellularization with 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) showed the best maintenance of both human and porcine LECM, with similar retention of LECM proteins except for elastin. Human and porcine LECM supported the cultivation of pulmonary cells in a similar way, except that the human LECM was stiffer and resulted in higher metabolic activity of the cells than porcine LECM.

Conclusions: Porcine lungs can be decellularized with CHAPS to produce LECM scaffolds with properties resembling those of human lungs, for pulmonary tissue engineering. We propose that porcine LECM can be an excellent screening platform for the envisioned human tissue engineering applications of decellularized lungs.

Keywords: 11.

Figures

Figure 1
Figure 1. Overall Approach
Decellularized slices from human and porcine lungs were compared with respect to their histomorphology, biochemical composition, mechanical properties and ability to support the growth and metabolism of cultured cells. Three different methods of decellularization and three different types of human cells (lung fibroblasts, small airway epithelial cells, mesenchymal cells) were evaluated.
Figure 2
Figure 2. Characterization of Lung Decellularization using the SDS, CHAPS, and 3 Step Methods
(A) Macroscopic images of human and porcine lung tissue slices before and after decellularization. Scale bar: 5 mm. (B) DNA quantification before and after decellularization. Data represent mean ± SE (n=9 for each group). * indicates p < 0.05. (C) H&E stain of three decellularization methods at 40X. Scale bar: 50 μm.
Figure 3
Figure 3. Characterization of collagen, sulfated glycosaminoglycans, and elastin using SDS, CHAPS, and 3 Step Method
(A) Collagen content; (B) Sulfated glycosaminoglycan content; (C) Elastin content. Data represent mean ± SE (n=9 for each group). * indicates p < 0.05. (D) Biochemical compositions of the human and porcine lung tissues, in their native state and following decellularization by three different methods.
Figure 4
Figure 4. Histological evaluation of human and porcine lung tissues following decellularization by one of the three methods
Masson's Trichrome (collagen, blue), Alcian Blue (sGAG, blue), and Van Gieson's (elastic fibers, black) staining of decellularized human and porcine lung tissue at 20X objective. Scale bar represents 100 μm.
Figure 5
Figure 5. Distributions of Extracellular Matrix Proteins
Representative immunohistochemical stains are shown for: (A) Collagen IV, (B) Laminin, (C) Fibronectin, and (D) Elastin. All images were acquired with a 10X objective. Scale bar: 100μm.
Figure 6
Figure 6. Ultrastructure of Decellularized Lung Tissues
Representative scanning electron micrographs are show for all experimental groups. Scale bar: 50μm.
Figure 7
Figure 7. Preparation of Lung Samples for Mechanical Characterization
Slices were obtained from the lower left lobes of human and porcine lungs and decellularized by three different methods. Samples were randomly obtained from the transverse sections of the lung and tested in uniaxial tensile strain.
Figure 8
Figure 8. Mechanical Properties of Lung Tissue
Representative uniaxial stress-strain curves for (A) human and (B) porcine lung ECM, in their native state and following decellularization. Linear correlation was detected between the elastin content and the maximum stress (C) and tangential modulus (D) at 20% strain for decellularized human and porcine lung ECM. Data represent Mean ± SE (n ≥ 9).
Figure 9
Figure 9. Growth Curves of Three Human Cell Types on CHAPS-Decellularized Lung Scaffolds
Growth curves for 7 days culture of three different types of cells on CHAPS-decellularized ■ human and porcine lung ECM (A) Δ human lung fibroblasts (hMRC-5s), (B) human small airway epithelial cells (hSAECs), and (C) human adipose-derived mesenchymal stem cells (hMSCs). Data represent Mean ± SE (n ≥ 9). * p < 0.05.
Figure 10
Figure 10. Viability and Metabolic Activity of Three Human Cell Types on CHAPS-Decellularized Lung Scaffolds
(A-B) Cell viability (live cells stained green for calcein-AM, dead cells stained red for ethidium homodimer-1) for human lung fibroblasts (hMRC-5s), human small airway epithelial cells (hSAECs), and human adipose-derived mesenchymal stem cells (hMSCs) after 1 and 7 days of culture on decellularized lung matrix. Scale bar: 40 μm. (C-D) Metabolic cell activity measured after 1 and 7 days of culture. Data represent Mean ± SE (n = 9).

Comment in

  • Invited commentary.
    Ott HC. Ott HC. Ann Thorac Surg. 2013 Sep;96(3):1056. doi: 10.1016/j.athoracsur.2013.04.067. Ann Thorac Surg. 2013. PMID: 23992696 No abstract available.

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