(512g) Investigating Inter-Cellular Signaling in Engineered Liver Mimetic Tissues

In vivo, cytokine signaling can generate both pro- and anti-inflammatory effects such as cell death, generation of reactive oxygen species, expression of cytochrome P450 enzymes, progression towards carcinomas, as well as liver regeneration.  Interleukin 6 (IL-6), a cytokine released by Kupffer cells (KCs), is commonly associated with liver injury and regeneration in hepatocytes.  In vitro studies on the effects of cytokines have been conducted primarily on hepatocyte monolayers (HMs) or collagen sandwich (CS) cultures.  However, neither culture is physiologically relevant.  Due to dedifferentiation of in vitro primary hepatic cultures, studies on cytokine signaling are often conducted within 24 h in culture, and therefore provide limited information on longer term effects. We have assembled engineered multi-cellular organotypic hepatic models that closely recapitulate the in vivo architecture and cellular composition. Since these tissues maintain hepatic phenotype for extended time periods, they are ideal to conduct detailed investigations into the effects of cytokine secretion on multiple liver cell types. 

The present study is focused upon the perturbations in liver function and signaling in the presence of exogenous and endogenous IL-6. Organotypic liver models were assembled using primary hepatic cells obtained through the surgical excision of rat livers.  Cellular models that closely mimic the architecture and in vivo composition were assembled using detachable polyelectrolyte multilayers to mimic the Space of Disse [1].  These liver models are typically assembled with primary hepatocytes, KCs, and liver sinusoidal endothelial cells (LSECs).  HM and CS cultures served as controls. All hepatic cultures were maintained for up to 12 days and hepatic functions were monitored.

The introduction of exogenous IL-6 into HM, CS and liver model cultures elicited different responses. HM and CS cultures both exhibited a reduction in albumin production upon addition of IL-6. Interestingly, CS cultures recovered and secreted basal albumin levels within 5 days. Another very interesting trend was the level of IL-6 measured in spent culture medium 24 h subsequent to the administration of this cytokine. It was observed that liver models (comprised of LSECs and hepatocytes) had approximately two-fold higher levels of IL-6 in spent culture medium in comparison to HM and CS samples.  These trends were observed at two separate time points (days 4 and 8) suggesting that LSECs in the liver models could be contributing through an endogenous release of this cytokine.  Our ongoing efforts are to investigate how exogenous administration of IL-6 activates these pathways using a combined proteomics and transcriptional approach as compared to endogenous signaling.

Cytokines are important signaling molecules that often elicit multiple effects.  We have demonstrated that the use of an organotypic hepatic model facilitates the study of cytokines at later time points.  In addition, we hypothesize that hepatic non-parenchymal cells, such as LSECs, are also playing an important role.

References: 

1. Larkin A. L. and coworkers, Tissue Engineering Part C: Methods, In Press, 2013.