A Human Liver‐on‐a‐Chip Platform for Modeling Nonalcoholic Fatty Liver Disease

Abstract

The liver possesses a unique microenvironment with a complex internal vascular system and cell–cell interactions. Nonalcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease, and although much effort has been dedicated to building models to target NAFLD, most in vitro systems rely on simple models failing to recapitulate complex liver functions. Here, an in vitro system is presented to study NAFLD (steatosis) by coculturing human hepatocellular carcinoma (HepG2) cells and umbilical vein endothelial cells (HUVECs) into spheroids. Analysis of colocalization of HepG2–HUVECs along with the level of steatosis reveals that the NAFLD pathogenesis could be better modeled when 20% of HUVECs are presented in HepG2 spheroids. Spheroids with fat supplements progressed to the steatosis stage on day 2, which could be maintained for more than a week without being harmful for cells. Transferring spheroids onto a chip system with an array of interconnected hexagonal microwells proves helpful for monitoring functionality through increased albumin secretions with HepG2–HUVEC interactions and elevated production of reactive oxygen species for steatotic spheroids. The reversibility of steatosis is demonstrated by simply stopping fat‐based diet or by antisteatotic drug administration, the latter showing a faster return of intracellular lipid levels to the basal level.

In Vitro Human Liver Model of Nonalcoholic Steatohepatitis by Coculturing Hepatocytes, Endothelial Cells, and Kupffer Cells

“…After overnight incubation, the steatosis inducing diet was given for 8 days, and the media was refreshed and collected every 2 days. In line with our previous report and other studies, spheroids supplemented with FFAs showed higher levels of lipid accumulation over 8 days compared to those cultured with healthy diet (Figure D,E), indicating the proposed system could provide an ideal model for the study on the progress of the disease from NAFL to NASH.…”

“…Here, different cell types, human hepatocytes (HepG2 or HepaRG), HUVECs, and HKCs, were mixed in appropriate ratios to generate the multicellular spheroids in the chip system. The spheroid size (200 cells per spheroid), that was previously optimized by controlling the seeding density, was chosen due to the absence of a necrotic core as well as good circularity and low aspect ratio which are necessary to sustain proper oxygen tension in the spheroid. The chip system is based on an array of interconnected honeycomb structures which have a similar size to the liver lobules, which are the functional units of the liver, and allow for molecular communications between the spheroids, mimicking the in vivo environment of the liver physiology.…”

“…In this work, we hypothesized engineering an in vitro human liver model by coculturing human hepatocytes, endothelial cells, and KCs as spheroids, could be used for understanding the progression from steatosis to NASH with better communications between those different cell types. To verify this hypothesis, HepG2, HUVECs, and HKCs were mixed at a specific ratio (70% HepG2, 15% HUVECs, and 15% HKCs) based on our previous report showing around 80% of HepG2 and 20% of HUVECs could be a better system for the NAFLD pathogenesis as well as this ratio is close to the in vivo percentages of the different cell types . First, to confirm the presence of all three cell types in spheroids, each cell type was incubated with a specific cell tracking dye (red for HepG2, green for HUVECs, and blue for HKCs).…”

“…To explore our hypothesis about the engineered strategy to build a liver disease‐on‐a‐chip model from steatosis to NASH, we showed that human hepatocytes, endothelial cells, and KCs could be cocultured as viable spheroids. Next, to examine the functionality and the disease progression of spheroids with the presence of HKCs on the chip system, the spheroids cultured for 4 d with and without HKCs in the inverse pyramid‐shaped microwells were harvested and transferred into the chip with an array of interconnected‐hexagonal microwells as previously reported . Here, the suggestion is that the inclusion of HKCs to the system could lead to inflammation, when the steatosis inducing diet was given, thus NASH can be studied.…”

“…We have previously designed a liver‐on‐a‐chip platform to model steatosis where multicellular spheroids composed of HepG2 and human umbilical vein endothelial cells (HUVECs) were cocultured in honeycomb microwells mimicking the hepatic lobules architecture . Multicellular spheroids represent interesting 3D cell culture models especially for the liver where the oxygen tension and nutrients gradients are naturally reproduced mimicking an important key feature of the native liver, the hepatic lobule zonation.…”