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Three-Dimensional Organization and Enhanced Function of HepG2 Liver Cells Cultured in a Reduced Gravity Environment.    Tammy T. Chang, M.D., Ph.D. and Millie Hughes-Fulford, Ph.D.  Department of Surgery, University of California, San Francisco and Laboratory of Cell Growth, San Francisco Veterans Affairs Medical Center, San Francisco, California.

   Liver transplantation is currently the only treatment for patients with end-stage liver disease but availability of donor organs is a major limitation.  A potential application for tissue engineering is to construct a bioartificial liver ex vivo for therapeutic purposes.  One of the first requirements for such an endeavor is to provide a 3-dimensional (3D) environment for cells to grow and form tissue structure.  In order to elucidate how 3-dimensional growth may affect cellular organization and function, we cultured HepG2 cells, a human hepatoblastoma cell line, in the 3D culture environment of the Rotating Wall Vessel (RWV) bioreactors developed from NASA technology.  We found that within 72 hours, HepG2 cells form cell aggregates up to 1mm in diameter.  In contrast to cells in 2-dimensional (2D) culture that form actin stress fibers, cell aggregates in 3D culture demonstrate cortical actin organization important for maintaining cellular function.  Albumin production and cytochrome P450 activity of HepG2 cells are significantly enhanced in the 3D cell aggregates compared to 2D culture.  This enhancement in synthetic and metabolic function is dependent upon continued culture within the RWV bioreactors.  Cell spheroids from long-term 3D cultures demonstrate decreased functional activity after transfer into a static 2D environment.  Differences in cell morphology and enhancement of cellular function in 3D cultures reflect differential gene expression between 3D and 2D cultures.  These findings are another illustration of how surrounding physical forces affect basic cellular processes.

(Supported by the American College of Surgeons Research Scholarship.)