Abstract

Background: Human pluripotent stem cell (PSC) derived hepatocyte-like cells and primary human hepatocytes applications for clinical or research uses is dependent on the ability to generate phenotypically and functionally relevant hepatocytes or hepatocyte-containing constructs. We have developed a scalable multi-well platform that enables the rapid generation of three-dimensional (3D) multicellular spheroids composed of primary and stem cell derived human liver parenchymal cell types including hepatocytes, liver sinusoidal endothelial cells (LSEC), stellate cells (HSC), and Kupffer cells. This 3D system is able to maintain hepatocellular function long-term and can be used to model the complex interactions and host responses between hepatocytes and nonparenchymal cells (LSEC, HSC, and Kupffer cells) during HBV Infection.

Methods: Multicellular spheroids containing hepatocytes and nonparenchymal cells alone were generated from primary human liver tissue or from human PSCs. This robust human multicellular platform maintains hepatocellular and nonparenchymal cell differentiated function for greater that 6 weeks and allows for extended experiments. Given the longevity and stability of the platform we examined whether such a platform could be used to model the complex interactions and host responses between hepatocytes, LSEC, HSC, and Kupffer cells in vitro during HBV infection.

Results: We generated multicellular spheroids and examined the acute and chronic changes due to HBV infection in multicellular spheroids. We found that multicellular spheroids maintain long-term infection in contrast to traditional hepatocyte-culture systems. Moreover, we found that metabolic perturbations led to impaired higher viral loads, hepatocyte injury, and macrophage and stellate cell activation. We evaluated the cross-talk between these different cell types early and late after HBV infection and found that in unexpectedly the presence of macrophages led to higher viral loads than when they were not present in the multicellular spheroids. We identified that Kupffer cell cytokine production is responsible for increased viral loads and neutralizing antibodies against IL-6 were able to mitigate this effect.

Conclusion: Our work demonstrates that multicellular spheroids composed of primary and stem cell derived human liver parenchymal cell types including hepatocytes, LSEC, stellate cells, and Kupffer cells have stable function and can model complex disease phenotypes in vitro. Leveraging these technologies focused on HBV enabled studies of viral infection and can be leveraged in mechanistic studies of infection.