Primary hepatocytes are the main functional cells of the liver, known as parenchymal cells, and they constitute up to 80% of the liver’s volume. These specialized cells are responsible for nearly all of the organ’s diverse biochemical activities, which are fundamental for maintaining the body’s internal balance. Their unique structure and comprehensive metabolic capacity make them valuable not only for natural physiological processes but also for modern scientific investigation, particularly in the study of drug interactions and liver disease. Their use in the laboratory allows researchers to accurately model human liver function in a controlled environment, offering predictive insights into how the body processes pharmaceuticals and toxins.
The Liver’s Engine Room
Hepatocytes perform an extensive range of metabolic and synthetic functions that are necessary for life, acting as the central processing unit for nutrients and waste products entering the bloodstream. One of their principal roles involves regulating the body’s energy supply through carbohydrate metabolism, such as storing glucose as glycogen and releasing it through gluconeogenesis during periods of fasting. They are also heavily involved in lipid metabolism, oxidizing fatty acids for energy and synthesizing cholesterol, phospholipids, and bile acids.
The cells are the site of synthesis for numerous plasma proteins, including albumin, which helps maintain osmotic pressure, and various clotting factors necessary for blood coagulation. Hepatocytes execute the complex process of detoxification, where they metabolize both external substances (xenobiotics like drugs) and internal compounds (e.g., ammonia) to render them less harmful. This biotransformation process typically occurs in two phases, preparing the substances for excretion from the body in the bile or urine.
The Gold Standard for Drug Testing
Due to their comprehensive physiological functions, primary human hepatocytes are considered the most accurate in vitro model for predicting how the human body will process a new drug. These cells retain the full complement of liver-specific enzymes, most notably the Cytochrome P450 (CYP) enzyme system, which is responsible for metabolizing approximately 50% of all therapeutic drugs. Other simpler cell models often lack the complete expression of these complex enzyme pathways, leading to less reliable data.
The cells are extensively used in Absorption, Distribution, Metabolism, and Excretion (ADME) studies to determine a drug’s clearance rate and identify its metabolites. By exposing the hepatocytes to drug candidates, researchers can predict potential Drug-Drug Interactions (DDI) that might occur when multiple medications compete for the same metabolic enzymes. Hepatocytes also serve as the industry benchmark for assessing Drug-Induced Liver Injury (DILI), a major cause of drug failure during clinical trials and post-market withdrawal. The ability of these primary cells to replicate the liver’s complex reactions to toxicity makes them essential for early safety screening.
Isolation and Maintaining Viability
Obtaining functional primary hepatocytes involves a delicate process, typically starting with liver tissue obtained from surgical resections or non-transplantable donor organs. The tissue is usually perfused with a solution containing collagenase, an enzyme that digests the extracellular matrix proteins to separate the cells. This isolation process yields highly functional cells but immediately introduces challenges for long-term study.
When removed from the liver’s natural microenvironment, the cells rapidly undergo a process known as dedifferentiation, where they lose their specialized liver functions within a few days of being cultured in a standard two-dimensional monolayer. This loss of phenotype includes a significant decline in the expression and activity of the metabolic CYP enzymes, limiting the duration of experiments. Furthermore, the cells are sourced from diverse human donors, meaning there is inherent variability in metabolic capacity and genetic profile among different batches, requiring extensive characterization before use.
Moving Beyond Primary Cells
The limitations of short lifespan and donor variability have spurred the development of alternative models that can offer a more consistent and sustainable supply of liver cells. Older alternatives include immortalized cell lines, such as HepG2 cells, which are easy to culture but exhibit significantly lower levels of drug-metabolizing enzymes compared to primary cells. This functional gap has been addressed by newer technologies that better mimic the native liver architecture.
Researchers are increasingly turning to Induced Pluripotent Stem Cell (iPSC)-derived hepatocytes, sometimes called hiHeps, which are generated by reprogramming adult cells into a stem cell state and then differentiating them into liver cells. When combined with advanced three-dimensional (3D) culture systems, such as scaffold-free spheroids or organoids, these cells can maintain hepatic function for several weeks. These 3D systems allow for greater cell-to-cell contact and better mimic the liver’s tissue organization, leading to improved expression of liver-specific genes and metabolic enzymes, providing a path for long-term toxicity testing.