Primary human hepatocytes are cells sourced directly from human liver tissue. They are a benchmark model in biomedical research because they closely mirror the liver’s functions in a lab, allowing scientists to study how the body processes substances like nutrients and medications. This is particularly important in drug development for assessing safety and efficacy.
What Are Primary Human Hepatocytes?
Hepatocytes are the most abundant cell type in the liver, making up about 70% of its cell population. The term “primary” means they are isolated directly from liver tissue and are not immortalized or genetically modified to divide indefinitely. This distinction is important because primary cells retain the complex pathways of their origin tissue, providing a more accurate biological model.
Hepatocytes perform most of the liver’s metabolic functions, including regulating carbohydrates, lipids, and proteins to maintain the body’s energy balance. They also detoxify foreign substances like drugs through enzymatic reactions. This process is carried out by enzymes such as Cytochrome P450s (CYPs), which modify compounds to help the body excrete them.
Beyond metabolism and detoxification, hepatocytes synthesize substances like bile for fat digestion, cholesterol, and blood proteins. These proteins include albumin, which maintains osmotic pressure, and clotting factors for blood coagulation. Structurally, these cells are polygonal and have microvilli on their surface to increase the area for exchange with the bloodstream.
How Are Primary Human Hepatocytes Obtained?
Obtaining primary human hepatocytes starts with acquiring human liver tissue under strict ethical and regulatory standards. The main sources are livers from cadaveric donors that are unsuitable for transplantation, or healthy tissue from surgical resections. This repurposed tissue requires appropriate patient consent for research use.
Once procured, isolating the hepatocytes is a time-sensitive procedure. The process involves perfusing the liver tissue with an enzyme solution, like collagenase, to break down the matrix holding the cells together. This enzymatic digestion frees the individual hepatocytes from the surrounding tissue.
The resulting cell suspension contains a mixture of liver cell types. Because hepatocytes have a higher density, separation techniques like low-speed or density gradient centrifugation are used to isolate them from other cells. After isolation, the viability and quality of the cells are assessed to ensure they are suitable for experiments.
Key Uses of Primary Human Hepatocytes
Primary human hepatocytes are a valuable tool in drug discovery. They are used for drug metabolism studies (pharmacokinetics) to understand how a new drug is processed by the liver. Researchers identify metabolic pathways and resulting metabolites to predict a drug’s behavior in the body. These studies also assess potential drug-drug interactions, where one drug alters the metabolism of another.
Hepatotoxicity, or drug-induced liver injury, is a concern in pharmaceutical development. Primary hepatocytes serve as a model for screening compounds for liver toxicity. By exposing the cells to a drug, scientists can observe signs of cellular damage, creating an early warning system that helps prevent toxic compounds from reaching human clinical trials.
These cells are also used to model various liver diseases. Researchers create in-vitro models for viral infections like hepatitis B (HBV) and C (HCV) to study the viral life cycle and test new therapies. They are also used to model metabolic liver diseases like non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) to understand their mechanisms and screen for treatments.
Challenges and Advancements in Working with Primary Human Hepatocytes
Working with primary human hepatocytes presents several challenges. Their availability is limited and unpredictable, leading to high costs. Another issue is the variability between donors, as factors like age, genetics, and health status can influence cell behavior and make experimental results inconsistent.
In a standard two-dimensional (2D) culture, primary hepatocytes have a finite lifespan and rapidly dedifferentiate, meaning they lose their specialized functions within a few days. This functional decline limits their use in long-term studies. Cryopreservation (freezing) for later use is also a challenge, as thawing can impact cell viability and function.
Researchers have developed solutions to address these limitations. Advanced culture media and supplements help maintain the cells’ differentiated state and extend their functional lifespan. Three-dimensional (3D) culture systems, like spheroids or organoids, also help preserve hepatocyte function by allowing for more natural cell-to-cell interactions that better mimic the liver’s microenvironment.
Further advancements include co-culture systems, where hepatocytes are grown with other liver cell types like stellate or Kupffer cells to create a more physiologically relevant model. Additionally, “liver-on-a-chip” platforms integrate these complex culture systems into microfluidic devices to simulate liver physiology and drug responses more accurately.