The Hepatocyte Expansion Paradox: A Review of In Vitro Challenges and Advances

The adult liver exhibits remarkable regenerative capacity in vivo; however, primary hepatocytes (PHs), the principal functional cells of the liver, swiftly forfeit their proliferative potential and specialized hepatic functions when isolated from their native microenvironment and cultured in vitro. While PHs are the benchmark for studying hepatic physiology, xenobiotic metabolism, and toxicological responses, their rapid dedifferentiation resulting in the loss of hepatic functions significantly limits their further application. Recent studies suggest three converging strategies to address the challenge of long-term maintenance and expansion of PHs. First, defined chemical and growth factor-based protocols can temporarily induce hepatocytes into a proliferative, progenitor-like state, followed by a maturation phase that restores differentiated hepatic functions. In addition, the inhibition of signaling pathways linked to cellular stress responses and identity loss can postpone dedifferentiation and preserve drug-metabolizing activity for prolonged durations, thereby enhancing disease modeling and toxicology studies. Finally, three-dimensional (3D) culture platforms generally improve hepatocyte maturation and functional stability, but these are often not scalable due to matrix dependence, technical complexity, handling requirements, and cost. This review thoroughly examines innovative methodological advancements designed to facilitate the proliferation and prolonged viability of healthy PHs, focusing on their translational relevance and clinical applicability.