| Primary hepatocytes, when cultured on moderately adhesive substrata or in suspension, self-assemble into three dimensional, compacted aggregates called spheroids. In order to gain a better understanding of the mechanism of spheroid formation, a cell tracking approach was developed using vital membrane dyes and confocal microscopy. Time-lapse data of labeled cells forming spheroids were processed with a deconvolution algorithm and reconstructed into 3-D images. Results verified a proposed mechanism of spheroid formation based on studies of the actin cytoskeleton.; The effects of glucocorticoids on spheroid formation were also investigated. The addition of the glucocorticoid agonist dexamethasone (DEX) to the culture medium slowed the kinetics of spheroid formation in dishes and altered the morphology of spheroids in suspension. In addition, increased albumin and urea production were observed. RT-PCR was used to further explore changes in gene expression between controls and DEX cultures.; Besides maintaining higher liver-specific functions than monolayer cultures, spheroids bear a high degree of structural similarity to native liver tissue. The surface of the spheroid is smooth, and cell-cell borders are barely distinguishable. Furthermore, adjacent hepatocytes in spheroids share bile canaliculi, microvilli-lined channels demarcated by tight junctions. Such differentiated structures are indicative of membrane surface polarization, the segregation of specific proteins to defined membrane domains. To investigate polarity, immunofluorescence labeling for an apical and basolateral protein was performed and revealed that spheroids gradually become polarized in culture. In addition, use of a fluorogenic substrate to dipeptidyl peptidase IV (DPPIV), a bile canalicular enzyme, demonstrated the presence of DPPIV in channels in spheroid. This discovery, in conjunction with prior studies of bile acid excretion, established the channels as bile canaliculi.; To extend the application of spheroids as a model of tissue self-assembly, co-culture of hepatocytes and hepatic stellate cells was explored. After liver injury, SC participate in revascularization by invading hepatocyte clusters and recruiting endothelial cells. Using the three dimensional cell tracking technique described above, the interactions of stellate cells and hepatocyte spheroids were visualized. The results indicate that this co-culture system could potentially serve as a model of liver organoid self-assembly. |
