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mesheuropmc: embryonic structures
In these studies the ability of a three-dimensional hepatocyte-stellate cell co-culture system to preserve some key aspects of differentiated hepatocyte function in vitro is demonstrated. A poly(DL-lactic Acid) surface allows dynamic and rapid interaction of hepatocytes and stellate cells to form co-culture spheroids in a complex multistage process (shown by time lapse microscopy). After five days the spheroids have developed a substantial extracellular matrix support and hepatic ultra-structure including bile canaliculi, tight junctions, desmosomes and lipid storage. The distribution of the stellate cells in the final structure is related to their motile and aggregating role in spheroid formation, i.e. mainly central and peripheral, and provides a unique and generically applicable insight into the dynamics of multicellular spheroid formation where aggregation is induced by one cell type and imposed on another. The spheroid morphology supports enhanced cell viability relative to hepatocytes in a mono-culture mono-layer. Co-culture spheroids also have superior cytochrome P450 3A and 2B function, and increased inducibility of 2B function, relative to a range of hepatocyte monoculture techniques (HPLC detection of testosterone metabolites). Increased function in co-culture is supported by greater expression of cytochrome P450 3A23,1A2, and 2E1 mRNA relative to monoculture (RT-QPCR). Also, high hepatocyte growth factor mRNA expression in co-culture suggests a post-traumatic, or possibly regenerative, environment. A preliminary study of human hepatocytes co-cultured with rat stellate cells demonstrated prolonged function of cytochrome P450 3A4,2C19 and 2C9. The co-culture spheroids are also shown to maintain a low level of sensitivity to hepatotoxins DDC and amiodarone after seven days in culture. This study shows that stellate cells facilitate spheroid formation, influence spheroid architecture, and are an effective method of preserving some aspects of hepatocyte function in the early stage of culture.
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