Surface pattern-embedded microfluidic chip for long-term maintenance of hepatocytes

Abstract

Functional hepatocytes play a crucial role in drug screening and cytotoxicity studies. However, primary hepatocytes quickly lose their differentiated state and specialized functions within hours to days after seeding, limiting the utility of in vitro models for long-term drug response and toxicity assessments. To overcome this limitation, we developed a microfluidic system designed to sustain the long-term culture of hepatocytes by optimizing flow conditions and incorporating microtopography to support cell-specific functions. A microfluidic chip with integrated topography was fabricated using soft lithography and characterized through numerical studies. Flow parameters were refined using HepaRG cells, which served as a model to optimize conditions. Hepatocyte-specific functions of HepaRG cells and primary macaque hepatocytes were evaluated under static and flow conditions over time using albumin and urea assays. The system demonstrated its ability to support HepaRG cells for up to 25 days and primary macaque hepatocytes for up to 5 days. Notably, flow conditions enhanced metabolic activity, with HepaRG cells showing a 70-fold increase in albumin secretion and a 40% rise in urea production by day 14, compared to static cultures. Similarly, primary macaque hepatocytes exhibited a 120-fold increase in albumin secretion under flow by day 8 relative to static conditions. These results highlight the potential of this optimized microfluidic platform for long-term hepatocyte culture, making it a valuable tool for future applications in drug screening and toxicity testing.

Keywords

• Surface patterns
• shear stress
• organ-on-chip
• hepatocytes
• long-term cell culture

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