Sterilization of lung matrices by supercritical carbon dioxide

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.