Primary hepatocytes self-assemble into spheroids that possess tight junctions and microvilli-lined channels. We hypothesized that polarity develops gradually and that the channels structurally and functionally resemble bile canaliculi. Immunofluorescence labeling of apical and basolateral proteins demonstrated reorganization of the membrane proteins into a polarized distribution during spheroid culture. By means of fluorescent dextran diffusion and confocal microscopy, an extensive network of channels was revealed in the interior of the spheroids. These channels connected over several planes and opened to pores on the surface. To examine the content of apical proteins in the channel membranes, the bile canalicular enzyme dipeptidyl peptidase IV (DPPIV) was localized using a fluorogenic substrate, Ala-Pro-cresyl violet. The results show that DPPIV activity is heterogeneously distributed in spheroids and localized in part to channels. Bile acid excretion was then investigated to demonstrate functional polarity. A fluorescent bile acid analogue, fluorescein isothiocyanate-labeled glycocholate, was taken up into the spheroids and excreted into bile canalicular channels. Due to the structural polarity of spheroids and their ability to excrete bile into channels, they are a unique three-dimensional model of in vitro liver tissue self-assembly. (Video animations of some results are available at http://hugroup.cems.umn.edu/research_movies).
Bibliographical noteFunding Information:
The antibodies were a gift from Dr. Ann Hubbard. We are also grateful to Dr. C. J. Van Noorden for providing the fluorogenic substrate and thoughtful comments on the manuscript. The consultation of Dr. Stan Erlandsen was instrumental in the success of the immunofluorescence studies. The authors also thank Dr. Vinayak Hosagrahara and Dr. Rory Remmel for their assistance with the FITC-GC synthesis and Muriel Gavin and the University of Minnesota Medical School EM Facility for assistance with the glycol methacrylate embedding and sectioning. The technical assistance of Kristine Groehler, Diane Tobolt, and Josh Wilhelm is also greatly appreciated. This material is based upon work supported under a National Science Foundation graduate fellowship (S.F.A.) S.F.A. and J.R.F. were also supported by a biotechnology training grant from NIGMS (GM08347). The project was funded by NASA (NAG8-134) and NIH (DK453).
- 3D culture
- Bile acid excretion
- Dipeptidyl peptidase IV
- Multicellular aggregates
- Tissue engineering