Sterilization of Lung Matrices by Supercritical Carbon Dioxide

Abstract

Lung engineering is a potential alternative to transplantation for patients with end-stage pulmonary failure. Two challenges critical to the successful development of an engineered lung developed from a decellularized scaffold include (i) the suppression of resident infectious bioburden in the lung matrix, and (ii) the ability to sterilize decellularized tissues while preserving the essential biological and mechanical features intact. To date, the majority of lungs are sterilized using high concentrations of peracetic acid (PAA) resulting in extracellular matrix (ECM) depletion. These mechanically altered tissues have little to no storage potential. In this study, we report a sterilizing technique using supercritical carbon dioxide (ScCO2) that can achieve a sterility assurance level 10(-6) in decellularized lung matrix. The effects of ScCO2 treatment on the histological, mechanical, and biochemical properties of the sterile decellularized lung were evaluated and compared with those of freshly decellularized lung matrix and with PAA-treated acellular lung. Exposure of the decellularized tissue to ScCO2 did not significantly alter tissue architecture, ECM content or organization (glycosaminoglycans, elastin, collagen, and laminin), observations of cell engraftment, or mechanical integrity of the tissue. Furthermore, these attributes of lung matrix did not change after 6 months in sterile buffer following sterilization with ScCO2, indicating that ScCO2 produces a matrix that is stable during storage. The current study's results indicate that ScCO2 can be used to sterilize acellular lung tissue while simultaneously preserving key biological components required for the function of the scaffold for regenerative medicine purposes.

FIG. 1.

Sterility assessment of decellularized lung matrix sterilized with peracetic acid (PAA) and supercritical carbon dioxide (ScCO₂). Native, decellularized, ScCO₂- and PAA-treated rat lung tissue (n ≥ 4) was crushed and streaked on agar plates without antibiotic. ScCO₂-treated and PAA-treated tissues were tested 24 h poststerilization. The plates were incubated at 37°C for 7 days, after which the number of bacterial colony-forming units (CFUs) on each plate was recorded. Error bars represent ± standard deviation.

FIG. 2.

Tissue characterization of decellularized lung matrix sterilized with PAA and ScCO₂. Native (A), decellularized (B), PAA- (C) and ScCO₂ (D)-treated rat lung tissue stained with Hematoxylin and Eosin (H&E) shows maintenance of tissue architecture in both ScCO₂- and PAA-treated lung tissue immediately after sterilization. Insets (B–D) are at 400×. H&E images of ScCO₂ (E, F) and PAA (G, H)-treated tissue 6 months after treatment shows compromised tissue in PAA-treated tissue (indicated by arrow) and not ScCO₂-treated lung tissue after 6 months of storage. Scale bar = 50 μm applies to all panels. Color images available online at www.liebertpub.com/tec

FIG. 3.

Mechanical properties of sterilized, decellularized lung tissue. Representative stress–strain curves of native, freshly decellularized and acellular rat lung tissue sterilized through (A, B) ScCO₂ and (C, D) PAA, tested immediately after treatment and after 6 months of storage. Stress–strain curves of native and decellularized lung tissue (n ≥ 5 for all groups). (E) Biochemical and mechanical composition of tissues in native and decellularized conditions. Error bars show mean ± standard deviation, and “*” indicates significance from decellularized matrix at p ≤ 0.05. Color images available online at www.liebertpub.com/tec

FIG. 4.

Quantifying impact of sterilization on extracellular matrix retention. Quantification of (A) collagen, (B) elastin, and (C) sulfated GAGs (sGAGs) in native (n ≥ 5), decellularized tissue (n = 15), PAA-treated and ScCO₂-treated tissue immediately after processing. Data demonstrate retention of collagen, elastin, and sulfated GAGs in ScCO₂-treated tissue and elastin loss in PAA-treated tissue. “*” Indicates significance from decellularized matrix at p ≤ 0.05. Error bars show mean ± standard deviation.

FIG. 5.

Characterization of sterilized acellular lung matrix. (A–C) Representative micrographs of native, decellularized, and ScCO₂-treated lung tissue stained with Verhoeff's Van Gieson (EVG) to examine elastin, (D–F) Masson's Trichrome to examine tissue architecture and collagen preservation, (G–I) and Alcian Blue to observe GAG presence. The EVG, Trichrome, and Alcian Blue stain show maintenance of general tissue architecture, collagen type I and elastin fibers, and total GAGs content throughout the tissue after treatment with ScCO₂. Representative fluorescence micrographs of (J) native, (K) decellularized, and (L) ScCO₂-treated lung tissue show maintenance of laminin content throughout the tissue after treatment with ScCO₂. Scale bar = 100 μm applies to all panels. Color images available online at www.liebertpub.com/tec

FIG. 6.

Recellularization of ScCO2-treated rat lung tissue slices. Representative H&E stained tissue slices seeded with (A) human epithelial (A549) and (B) rat endothelial (rat microvascular lung endothelial cells [RLMVECs]) cells. Cells were seeded at a concentration of 500,000 cells/slice and cultured for 3 days. Tissue was ScCO₂ treated and stored in phosphate-buffered saline for 6 months before seeding. All cells homogenously engrafted in the tissue demonstrating the capacity for recellularization of sterilized acellular matrix. Scale bar = 50 μm. Color images available online at www.liebertpub.com/tec