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Sensory hair cell loss is a major contributor to disabling hearing and balance deficits that affect >250 million people worldwide. Sound exposures, infections, drug toxicity, genetic disorders, and aging all can cause hair cell loss and lead to permanent sensory deficits. Progress toward treatments for these deficits has been limited, in part because hair cells have only been obtainable via microdissection of the anatomically complex internal ear. Attempts to produce hair cells in vitro have resulted in reports of some success but have required transplantation into embryonic ears or coculturing with other tissues. Here, we show that avian inner ear cells can be cultured and passaged for months, frozen, and expanded to large numbers without other tissues. At any point from passage 6 up to at least passage 23, these cultures can be fully dissociated and then aggregated in suspension to induce a mesenchymal-to-epithelial transition that reliably yields new polarized sensory epithelia. Those epithelia develop numerous hair cells that are crowned by hair bundles, composed of a single kinocilium and an asymmetric array of stereocilia. These hair cells exhibit rapid permeance to FM1-43, a dye that passes through open mechanotransducing channels. Because a vial of frozen cells can now provide the capacity to produce bona fide hair cells completely in vitro, these discoveries should open new avenues of research that may ultimately contribute to better treatments for hearing loss and other inner ear disorders.balance ͉ culture ͉ hearing ͉ regeneration ͉ stem cell