Softer lung ECM has the ability to reverse myofibroblast characteristics

Nolan Wheildon , Pierre-Simon Bellaye , Chiko Shimbori, Luke Janssen, Martin Kolb Introduction: The extracellular matrix (ECM) properties are a key component for maintenance of a pro-fibrotic microenvironment in idiopathic pulmonary fibrosis (IPF). We studied the effect that a softer ECM has on normal and fibrotic human lung myofibroblast phenotype. Methods: Decellularization was achieved using manual perfusion of 0.1% Triton X-100, 2% sodium deoxycholate (SDC), 1M NaCl, and 30 μg/mL DNase I. Decellularized lung ECMs were reseeded with normal and fibrotic primary human lung myofibroblasts within static lung slices and harvested at days 3 and 10. Static lung slices were produced by inflation of decellularized lung with a mixture of myofibroblasts and 1% agarose (w/v), which were subsequently cut into 1-2 mm thick lung slices. Normal and fibrotic myofibroblast phenotype changes were evaluated using histology, immunohistochemistry, and western blot analyses in comparison to myofibroblasts cultured on normal tissue culture plates. Results: H&E staining and Western blot analysis of α-tubulin demonstrated that full decellularization was achieved. At days 3 and 10, there was reduced expression of the apoptotic marker cleaved caspase 3. Following static lung culture for 3 days, there was no change in α-SMA expression. However, there was a significant reduction in α-SMA expression when myofibroblasts were reseeded within soft lung ECM for 10 days. Conclusion: We found that traditional cell culture plastic promoted a majority α-SMA positive myofibroblast population. Our results indicate that softer lung ECM has the ability to reverse some myofibroblast characteristics leading to the conclusion that myofibroblasts are not terminally primed.