Project Summary Voice impairment (dysphonia) affects an estimated 20 million people in the United States and, despite advances in diagnosis and treatment, remains a challenging clinical problem with a negative impact on patient quality-of-life. Advances in the emerging field of VF mucosal biology have resulted in a number of powerful and widely used in vitro experimental tools, such as human VF fibroblast cell lines and vibrational bioreactors; however, there remain limited cell biology resources specific to the VF epithelium and its constituent cells, hampering advances in VF epithelial biology and the understanding of epithelial diseases. A central reason for this lack of progress is the technical challenges associated with VF epithelial cell isolation, maintenance, and expansion in vitro. A reliable and reproducible culture methodology is needed to overcome these challenges. In the proposed work, we will introduce and validate a promising in vitro technique – anchorage-independent culture – for the isolation and maintenance of VF epithelial stem cells. Our team’s prior work in other epithelial systems, and pilot data generated using VF epithelial cells, demonstrate that this technique results in the formation of three-dimensional culture spheres that are enriched for tissue- specific stem cells. In the proposed research, we will characterize the makeup, growth, maintenance, and passage of VF spheres, as well as test the capacity of sphere-contained stem cells for in vitro differentiation and as a cell source for VF tissue engineering. This work, conducted using human cells, will result in a validated and reproducible in vitro methodology that will advance progress in VF epithelial (stem) cell biology and facilitate ongoing and future research amongst the wider scientific community. This research effort represents a powerful collaboration between experts in VF mucosal biology and stem cell biology and has broad applicability to both basic scientific research and regenerative medicine of the larynx.