3D Hepatic Cultures Simultaneously Maintain Primary Hepatocyte and Liver Sinusoidal Endothelial Cell Phenotypes

Developing in vitro engineered hepatic tissues that exhibit stable phenotype is a major challenge in the field of hepatic tissue engineering. However, the rapid dedifferentiation of hepatic parenchymal (hepatocytes) and non-parenchymal (liver sinusoidal endothelial, LSEC) cell types when removed from their natural environment in vivo remains a major obstacle. The primary goal of this study was to demonstrate that hepatic cells cultured in layered architectures could preserve or potentially enhance liver-specific behavior of both cell types. Primary rat hepatocytes and rat LSECs (rLSECs) were cultured in a layered three-dimensional (3D) configuration. The cell layers were separated by a chitosan-hyaluronic acid polyelectrolyte multilayer (PEM), which served to mimic the Space of Disse. Hepatocytes and rLSECs exhibited several key phenotypic characteristics over a twelve day culture period. Immunostaining for the sinusoidal endothelial 1 antibody (SE-1) demonstrated that rLSECs cultured in the 3D hepatic model maintained this unique feature over twelve days. In contrast, rLSECs cultured in monolayers lost their phenotype within three days. The unique stratified structure of the 3D culture resulted in enhanced heterotypic cell-cell interactions, which led to improvements in hepatocyte functions. Albumin production increased three to six fold in the rLSEC-PEM-Hepatocyte cultures. Only rLSEC-PEM-Hepatocyte cultures exhibited increasing CYP1A1/2 and CYP3A activity. Well-defined bile canaliculi were observed only in the rLSEC-PEM-Hepatocyte cultures. Together, these data suggest that rLSEC-PEM-Hepatocyte cultures are highly suitable models to monitor the transformation of toxins in the liver and their transport out of this organ. In summary, these results indicate that the layered rLSEC-PEM-hepatocyte model, which recapitulates key features of hepatic sinusoids, is a potentially powerful medium for obtaining comprehensive knowledge on liver metabolism, detoxification and signaling pathways in vitro

Blood, sphingosine-1-phosphate and lymphocyte migration dynamics in the spleen.

The spleen, the largest secondary lymphoid organ, has long been known to play important roles in immunity against blood-borne invaders. Yet how cells migrate within the spleen to ensure fast and effective responses is only now coming to light. Chemokines and oxysterols guide lymphocytes from sites of release at terminal arterioles into the lymphocyte-rich white pulp. Sphingosine-1-phosphate (S1P) and S1P-receptor-1 (S1PR1) promote lymphocyte egress from white to red pulp and back to circulation. Intravital two-photon microscopy has shown that marginal zone (MZ) B cells that are enriched between white and red pulps undergo continual oscillatory migration between the MZ and follicles, ferrying antigens. Cycles of G-protein-coupled receptor kinase-2 (GRK2) mediated S1PR1 desensitization and resensitization underlie this remarkable behavior. The findings discussed in this review have implications for understanding how splenic antibody and T-cell responses are mounted, how the immunosuppressant drug FTY720 (fingolimod) affects the spleen, and how cell shuttling behaviors contribute to immunity

Blood, Sphingosine-1-Phosphate and Lymphocyte Migration Dynamics in the Spleen

The spleen, the largest secondary lymphoid organ, has long been known to play important roles in immunity against blood-borne invaders. Yet how cells migrate within the spleen to ensure fast and effective responses is only now coming to light. Chemokines and oxysterols guide lymphocytes from sites of release at terminal arterioles into the lymphocyte-rich white pulp. Sphingosine-1-phosphate (S1P) and S1P-receptor-1 (S1PR1) promote lymphocyte egress from white to red pulp and back to circulation. Intravital two-photon microscopy has shown that marginal zone (MZ) B cells that are enriched between white and red pulps undergo continual oscillatory migration between the MZ and follicles, ferrying antigens. Cycles of G-protein-coupled receptor kinase-2 (GRK2) mediated S1PR1 desensitization and resensitization underlie this remarkable behavior. The findings discussed in this review have implications for understanding how splenic antibody and T-cell responses are mounted, how the immunosuppressant drug FTY720 (fingolimod) affects the spleen, and how cell shuttling behaviors contribute to immunity