Development of a Decellularized Lung Bioreactor System for Bioengineering the Lung: The Matrix Reloaded

Abstract

We developed a decellularized murine lung matrix bioreactor system that could be used to evaluate the potential of stem cells to regenerate lung tissue. Lungs from 2-3-month-old C57BL/6 female mice were excised en bloc with the trachea and heart, and decellularized with sequential solutions of distilled water, detergents, NaCl, and porcine pancreatic DNase. The remaining matrix was cannulated and suspended in small airway growth medium, attached to a ventilator to simulate normal, murine breathing-induced stretch. After 7 days in an incubator, lung matrices were analyzed histologically. Scanning electron microscopy and histochemical staining demonstrated that the pulmonary matrix was intact and that the geographic placement of the proximal and distal airways, alveoli and vessels, and the basement membrane of these structures all remained intact. Decellularization was confirmed by the absence of nuclear 4',6-diamidino-2-phenylindole staining and negative polymerase chain reaction for genomic DNA. Collagen content was maintained at normal levels. Elastin, laminin, and glycosaminglycans were also present, although at lower levels compared to nondecellularized lungs. The decellularized lung matrix bioreactor was capable of supporting growth of fetal alveolar type II cells. Analysis of day 7 cryosections of fetal-cell-injected lung matrices showed pro-Sp-C, cytokeratin 18, and 4',6-diamidino-2-phenylindole-positive cells lining alveolar areas that appeared to be attached to the matrix. These data illustrate the potential of using decellularized lungs as a natural three-dimensional bioengineering matrix as well as provide a model for the study of lung regeneration from pulmonary stem cells.

Quantification of Extracellular Matrix Proteins from a Rat Lung Scaffold to Provide a Molecular Readout for Tissue Engineering

“…Similarly, cellular proteins such as cytoskeletal actins, tubulins, and mitochondrial ATP synthase subunit alpha-1 appear to be inefficiently removed by the decellularization process according to the peptide spectral match comparisons in Table I. These and the above examples are universally inconsistent with previous reports that indicate near complete depletion of intracellular components, while retaining key components of the ECM (4,44,46,47). Due to these significant qualitative and quantitative inconsistencies utilizing untargeted LC-MS/MS, we deemed this method insufficient to accurately measure differences in protein levels between the two lung types.…”

Control of lung vascular permeability and endotoxin-induced pulmonary oedema by changes in extracellular matrix mechanics

“…The detergentextracted lungs extracted all of the cellular contents from the lung as detected by removal of 4 0 ,6-diamidino-2-phenylindole (DAPI) staining ( Supplementary Fig. S2b), but retained normal microarchitecture 25,26 , including ECMs delineating large vessels surrounded by alveoli, alveolar septae, the characteristic spongy matrix of the lung (Fig. 2a, top) and fine ECM fibrils containing collagen I, III, IV, VI, major collagens in the lung, when analysed by immunofluorescence microscopy ( Fig.…”

“…We treated the mice with LPS for the last 2 days. For decellularization of lung, we perfused the lung with decellularization buffer (0.5% SDS/PBS) for 1 h and cryosectioned it and analysed by Haematoxylin and eosin (H & E) staining and immunohistochemistry 25,26 . We confirmed that the collagen immunofluorescence staining we observed is specific and not due to autofluorescence using a secondary only control (rabbit IgG secondary used for all anti-collagen antibodies) ( Supplementary Fig.…”

Stem Cells and Cell Therapies in Lung Biology and Diseases: Conference Report

“…Angela Panoskaltsis-Mortari, Ph.D., from the University of Minnesota, gave a presentation entitled "Using Decellularized Matrices for Bioengineering the Lung," in which she presented her work on acellular ECM scaffolds and their use in bioreactors to evaluate the potential of multipotent stem cells to regenerate lung tissue (42). When seeded into these scaffolds under the appropriate growth conditions, iPS can express several lung markers, which illustrates the potential for regenerating lung tissue using autologous iPS cells on decellularized matrices to avoid immune-mediated rejection on subsequent transplantation.…”