Ballooned hepatocytes (BH) are enlarged, abnormal hepatocytes, which are usually involved in liver diseases, in particular, nonalcoholic steatohepatitis (NASH). However, formation of BHs in vitro has been seldom reported. This study reported an in vitro strategy to produce human BHs in a cell sheet-based three-dimensional (3D) model where primary human hepatocytes were cocultured with normal human dermal fibroblasts. Enlargement of hepatocytes (2.3 times larger than normal, p < 0.01), loss of cytoplasmic keratin, appearance of Mallory-Denk bodies (MDBs), and abundant fat droplets accumulation were observed after only a few days culture. Additionally, ultrastructural characteristic findings of BHs in human NASH, including enlarged mitochondria with crystalline inclusions, dilated endoplasmic reticulum, and MDBs formation were also observed in the 3D model. Furthermore, pathophysiological features of human NASH, such as increased secretion of sonic hedgehog ligands and myofibroblast activation were found. This study reports in vitro production of human BHs by using a cell sheet-based 3D model. Similar histological, ultrastructural, and pathophysiological features to human NASH are discovered in this model. This model may facilitate study of BHs and increase our knowledge of the pathogenesis of human liver diseases. Human ballooned hepatocytes (BH), which are present in nonalcoholic steatohepatitis (NASH) are mainly studied based on human liver biopsies and animal models. In this study, human BHs can be successfully reproduced in a cell sheet-based in vitro model, which, as far as we know, is the first in vitro model that recapitulates so many histological and ultrastructural hallmarks of BHs found in human NASH. Additionally, this study also demonstrated presence of some NASH pathophysiological features. This model may facilitate the study of hepatocellular ballooning and prove beneficial in translational preclinical drug discovery in NASH.
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