Spheroid-based three-dimensional liver-on-a-chip to investigate hepatocyte–hepatic stellate cell interactions and flow effects

TLDR

The authors built a 3D liver‑on‑a‑chip that co‑cultures hepatocyte spheroids and hepatic stellate cells without direct contact and incorporates continuous medium flow via osmotic pumping, eliminating external power and simplifying handling. The chip enhances spheroid formation, maintenance, and liver function—flow improves spheroid stability, HSCs promote tight contacts and hepatocyte activity, leading to higher albumin, urea, and CYP450 outputs—showing promise as a platform for studying hepatic interactions and extending hepatocyte viability.

Abstract

We have developed a three-dimensional (3D) liver-on-a-chip to investigate the interaction of hepatocytes and hepatic stellate cells (HSCs) in which primary 3D hepatocyte spheroids and HSCs are co-cultured without direct cell–cell contact. Here, we show that the 3D liver chip offers substantial advantages for the formation and harvesting of spheroids. The most important feature of this liver chip is that it enables continuous flow of medium to the cells through osmotic pumping, and thus requires only minimal handling and no external power source. We also demonstrate that flow assists the formation and long-term maintenance of spheroids. Additionally, we quantitatively and qualitatively investigated the paracrine effects of HSCs, demonstrating that HSCs assist in the maintenance of hepatocyte spheroids and play an important role in the formation of tight cell–cell contacts, thereby improving liver-specific function. Spheroids derived from co-cultures exhibited improved albumin and urea secretion rates compared to mono-cultured spheroids after 9 days. Immunostaining for cytochrome P450 revealed that the enzymatic activity of spheroids co-cultured for 8 days was greater than that of mono-cultured spheroids. These results indicate that this system has the potential for further development as a unique model for studying cellular interactions or as a tool that can be incorporated into other models aimed at creating hepatic structure and prolonging hepatocyte function in culture.