Myofibroblast-like activated hepatic stellate cells (aHSCs), which produce collagen, a major cause of liver fibrosis, are specific target cells for antifibrotic treatment. Recently, several reports have indicated that extracellular vesicles (EVs) play important roles in cell-to-cell communication through their tropism for specific cells or organs. Therefore, the present study aimed to identify aHSC-directed EVs by focusing on cell-to-cell interactions in the liver under pathological conditions. EVs were derived from the hepatocyte cell line AML12 treated with or without palmitic acid (PA) and evaluated for their physical properties and uptake by the aHSC cell line LX-2. AML12-derived EVs had a mean particle diameter of 110–130 nm, negative charge, and expressed the exosomal makers CD9 and CD63. PA-treated AML12 cells released larger EVs with higher protein levels than those without PA treatment. The intracellular uptake efficacy of EVs derived from PA-treated AML12 cells into activated LX-2 cells was significantly higher than those without PA treatment. Our study revealed that PA treatment induces hepatocytes to release EVs with aHSC-tropism. These findings may contribute to the development of an EV-based drug delivery system (DDS) for aHSC-targeted therapy and provide new insights into the role of steatotic hepatocyte-derived EVs in physiological or pathophysiological functions.

Currently, there is no effective treatment for liver fibrosis. In vitro studies on the activation of hepatic stellate cells (HSCs), which is responsible for liver fibrosis, have been used as a drug screening system for it. However, even if some drug shows an inhibitory effect on HSC activation, its anti-fibrotic effect is required to be confirmed in liver fibrosis animal models, which further requires a lot of time and cost. In the present study, we tried to establish an ex vivo model of liver fibrosis using precision-cut liver slices (PCLSs) to solve such problems. PCLSs of 250 µm thickness were prepared from male C57BL/6J mice using a vibratome and cultured in RPMI medium in a 5% CO 2 incubator. Although cellular ATP content was decreased on day 1 compared to day 0, it was then maintained until day 5, suggesting that the ex vivo model is viable for at least 5 days. Treatment with Et-OH (50, 100 mM), one of the liver injury stimuli, for 5 days increased mRNA expression of Acta2 and Col1a1, liver fibrosis markers, in PCLSs. DIF-1 (50, 100 µM), which has an anti-fibrotic effect, significantly suppressed the Et-OH-induced increases in the markers. These results suggest that the ex vivo model using PCLSs is useful as a drug screening system for the development of drugs for treatment of liver fibrosis.