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We studied the effect of continuous medium flow on the viabilityand structural organization of hepatocytes high density entrapped inalginate gel beads in the first few hours after isolation.The metabolic energy status of the entrapped cells, monitored invivo by (31)P NMR spectroscopy, was stable during theexperimental time and a physiological redox ratio was reachedafter the first three hours of culture. The morphologicalanalysis revealed that the entrapped hepatocytes placed in a fixed-bed bioreactor under continuous flow showed a polyhedricalshape with numerous microvilli on cell surface and reconstitutedtight junctions as well as bile canalicular structures, closelyresembling those present in the liver.These results suggest that continuous flow allows the culture ofhepatocytes at very high cell density within a matrix withoutloss of viability and accelerates cellular tissue reconstructionat very short times after isolation. This type of culture couldrepresent a very useful model for physiological andtoxicological studies as well as a promising approach toward thedevelopment of a bioartificial hybrid support device in acuteliver failure.

Energy metabolism and re-establishment of intercellularadhesion complexes of gel entrapped hepatocytes.