Multi-cellular transitional organotypic models to investigate liver fibrosis

Hepatic fibrosis is the result of wound healing and inflammation resulting in organ dysfunction. Hepatocytes, liver sinusoidal endothelial cells (LSECs), Kupffer cells (KCs), and hepatic stellate cells (HSCs) play critical roles in fibrogenesis. As the liver undergoes fibrosis, there are populations of cells that are healthy, fibrotic as well as those undergoing fibrosis. We investigated how a varying mechanical environment could induce changes in hepatic cells. In this study, a gradient in the mechanical properties of the microenvironment resulted in transitioning phenotypes in hepatic cells. We have designed detachable polyelectrolyte multilayers (PEMs) whose elastic moduli ranged from 21 to 43 kPa to serve as Space of Disse mimics. We assembled novel 3D organotypic liver models comprised of hepatocytes, LSECs, HSCs, KCs, and the Space of Disse mimic. We demonstrate how cells in contact with a mechanical gradient exhibit different properties compared to cells cultured using non-gradient PEMs. Significant differences were observed in HSC and KC proliferation between 3D cultures assembled with gradient and non-gradient PEMs. While HSCs on the stiffer regions of the gradient PEMs expressed both GFAP and α-SMA, cells in cultures assembled with homogeneous 43 kPa multilayers primarily expressed α-SMA. Over an 8-day culture, the elastic modulus in the 21 and 43 kPa regions of the gradient PEMs increased by 1.6 and 3.7-fold, respectively. This was accompanied by a 4-fold increase in hydroxyproline. Such in vitro tissues can be used to investigate the effects of liver fibrosis. STATEMENT OF SIGNIFICANCE: We have assembled a liver model assembled with four major primary hepatic cell types to investigate how a varying mechanical environment induces changes in hepatic cells. In this study, a gradient in the mechanical properties of the microenvironment results in transitioning phenotypes in hepatic cells. Our goal was to investigate the interplay between mechanical properties and a multi-cellular engineered liver tissue. In these models, Kupffer cell proliferation and hepatic stellate cell activation occurred due to mechanical cues and inter-cellular signaling across a distance of 2000 μm. These models are unique, in that, fibrosis was initiated purely through changes to the microenvironment. These models were not exposed to fibrogenic factors nor were the models assembled with cells from fibrotic rats. To the best of our knowledge, these are the first liver models that capture how a gradient microenvironment can result in transitioning cellular phenotypes.