(K-435) Fabrication of a Vascularized Liver Model to Support Hepatocytes in 3D Culture

Almost 1 million people were diagnosed with liver cancer globally in 2020, and this number is projected to rise more than 55% by 2040. The primary treatment for liver cancer is a partial hepatectomy, in which up to two-thirds of the liver is removed and then subsequently allowed to regenerate. The liver is the only organ that has this regenerative capacity, and while intracellular pathways of liver regeneration have been thoroughly studied, the triggers of these pathways are not clear. A further understanding of this process could be beneficial to the improvement of liver disease treatment. One primary hypothesis suggests the increased shear stress on liver sinusoidal endothelial cells (LSECs) that results from a partial hepatectomy, as portal vein flow is channeled through the reduced liver area, could trigger this regeneration. However, current in vitro models that have been created to replicate liver regeneration do not have the ability to study the effects of flow on the liver, due to unrepresentative vasculature, lack of incorporation of a controllable and constant  flow element, and a lack of organ specific endothelial cells. The development of a representative in vitro model to display this process is key, and advances in this area could further our understanding of liver regeneration and thus improve disease therapies.

We have developed a fabrication process to create perfusable, vascularized, collagen “microvessels.” Soft lithography is used to imprint a thirteen by thirteen grid pattern onto a collagen layer on a top housing device, representing a vascular network in vitro. Inlet and outlet holes are punched, and this patterned layer is  placed on top of a bottom housing device, coated in a flat layer of collagen, creating a fully perfusable network.  The collagen has primary rat hepatocyte spheroids suspended within, and after construct formation we injected HUVECs into the inlet to endothelialize the vasculature. We compared these perfusable tissues to a control vessel, which contains no fabricated perfusable vasculature and only hepatocytes within the collagen. Both sets of vessels were cultured for seven days with periodic media collection. We evaluated the survival of the hepatocytes within the vessels with immunohistochemistry to visualize the cells and the vasculature. As primary liver endothelial cells (ECs) are a more relevant cell type for this model, we then cultured hepatocytes in 2D with various donor liver ECs in our lab to determine which ones best support hepatocyte function. We compared the albumin production within these cultures to the albumin production of a control culture with only hepatocytes using an ELISA. We also visualized the cultures through immunohistochemistry to assess phenotypic differences.

Using confocal microscopy to compare our microvessels and non-vascularized controls, we found that the controls showed less staining of hepatocyte spheroids within the vessels, implying lower levels of hepatocyte survival in our control condition. We also found that there was a larger number of hepatocytes in the grid as compared to away from the  grid in our microvessels, suggesting that vasculature supports hepatocyte survival. Our 2D co-culture of primary hepatocytes and liver endothelial cells  showed that rat hepatocytes co-cultured with liver endothelial cells exhibit higher albumin production than rat hepatocytes alone. By using confocal microscopy we identified that some donor cell populations showed a more pure endothelial cell phenotype than others, and found that the purity of the donors correlated with increased albumin production.  

Through these experiments we have identified that there is higher survival of hepatocytes within our microvessels as compared to non-vascularized controls. We have also identified several donor liver endothelial cells that help support hepatocyte function in culture. Our future work aims to combine these findings, utilizing primary liver endothelial cells within our microvessel model. Our work gives a strong foundation to new work in in vitro modeling of liver regeneration, which has wide applications, including liver diseases related to flow. In addition, future modifications of this vessel model could incorporate heterogeneous tissue, and thus be used to create a more physiologically relevant model of the liver.

We acknowledge the Lynn and Mike Garvey Imaging Laboratory in the Institute of Stem Cell and Regenerative Medicine at the University of Washington, the Mary Gates Research Scholarship, and Dr. Kelly Stevens, Olivia Prado, and Fredrick Johansson for cells and assistance in animal work.