DEVELOPMENT OF COCHLEAR CELL CULTURES

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Our long-term research objective is elucidation of basic cellular and molecular mechanisms whereby cells in the lateral wall of the inner ear regulate ion flux and generate the EP. The major factor limiting inner ear cell biology is the difficulty of studying lateral wall cell types in vivo. The development of well characterized cultures of several different highly specialized lateral wall cell types would help to rectify this problem by providing the ability to study these cell types in isolation under defined conditions. Research proposed here aims to investigate the environmental conditions (support matrix, growth medium and supplements) needed to establish homogeneous long-term cell cultures from a variety of lateral wall cell types. Ultrastructural and immunocytochemical analysis will be used to ascertain that specialized in vivo characteristics are retained in vitro. The study has two specific aims. The first is to establish primary cell cultures from tissue explants of the cochlear lateral wall. A series of basal growth media and support matrices will be assessed to determine which combination best promotes sufficient differential outgrowth of epithelial cells and/or fibrocytes to allow their subculture. The second goal is to define the optimal conditions for the proliferation of secondary cultures. A variety of growth media supplements will be analyzed for their ability to promote cells that: 1 )retain in vivo characteristics; 2) can be subcultured for a sufficient number of generations to allow cryopreservation of cells for future use; and 3) produce adequate number of cells to allow assessment and experimental manipulation using molecular, biochemical and electrophysiological techniques. Future work will address the functional properties of the secondary cultures and the viability of cryopreserved cell stocks.