Previous studies of hepatocyte spheroids have revealed many tissue-like properties. For example, spheroids have smooth surfaces and a compact structure. Hepatocytes inside spheroids often share microvilli-lined channels demarcated by tight junctions. Similar channels, called bile canaliculi exist in the liver. Bile canalicular structures are often associated with hepatocyte polarity, the segregation of proteins to specific membrane domains. To investigate polarity in spheroids, we examined the localization of bile canalicular and basolateral proteins. In vivo, they are sequestered to separate regions of the cell membrane. Using antibodies to two proteins, we labeled consecutive thin sections of plastic embedded spheroids and digitally overlaid the resulting fluorescence patterns for comparison. At the early stages of spheroid formation in spinner flasks, both proteins were present at all areas of cell-cell contact, but as the spheroids formed, the cell membranes gradually reorganized. Polarity was achieved by five days in culture, as the two proteins segregated to different regions of the membranes.

The next goal was to further characterize the structure of the channels. Use of a fluorescently-tagged dextran molecule (FITC-dextran) in conjunction with confocal microscopy revealed a highly connected network of channels inside spheroids. The channels opened through the external pores and were roughly the size of bile canaliculi. To determine if apical membrane proteins are located at the channels, a fluorogenic substrate for dipeptidyl peptidase IV (DPPIV), a bile canalicular enzyme, was employed. Results showed that DPPIV is localized heterogeneously throughout spheroids and in part to channel-like structures.