Odorant Binding Causes Cytoskeletal Rearrangement, Leading to Detectable Changes in Endothelial and Epithelial Barrier Function and Micromotion.

Non-olfactory cells have excellent biosensor potential because they express functional olfactory receptors (ORs) and are non-neuronal cells that are easy to culture. ORs are G-protein coupled receptors (GPCRs), and there is a well-established link between different classes of G-proteins and cytoskeletal structure changes affecting cellular morphology that has been unexplored for odorant sensing. Thus, the present study was conducted to determine if odorant binding in non-olfactory cells causes cytoskeletal changes that will lead to cell changes detectable by electric cell-substrate impedance sensing (ECIS). To this end, we used the human umbilical vein endothelial cells (HUVECs), which express OR10J5, and the human keratinocyte (HaCaT) cells, which express OR2AT4. Using these two different cell barriers, we showed that odorant addition, lyral and Sandalore, respectively, caused an increase in cAMP, changes in the organization of the cytoskeleton, and a decrease in the integrity of the junctions between the cells, causing a decrease in cellular electrical resistance. In addition, the random cellular movement of the monolayers (micromotion) was significantly decreased after odorant exposure. Collectively, these data demonstrate a new physiological role of olfactory receptor signaling in endothelial and epithelial cell barriers and represent a new label-free method to detect odorant binding.

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.