The liver contains specialized cells called hepatocytes, which constitute about 80% of the organ’s mass and perform its vast array of biochemical tasks. In laboratory settings, scientists work with “primary cells,” which are cells taken directly from living tissue for cultivation. This approach contrasts with immortalized cell lines, which are modified to divide indefinitely. Primary hepatocytes are therefore isolated directly from liver tissue, offering a valuable model for studying liver physiology and disease.
The Role of Hepatocytes in Liver Function
Hepatocytes are central to the body’s metabolic regulation. They manage carbohydrate balance by storing glucose as glycogen when blood sugar is high and releasing it through processes like glycogenolysis and gluogenesis when energy is needed. For lipid metabolism, these cells synthesize cholesterol, phospholipids, and the bile salts necessary for digesting fats. They are also responsible for producing most of the proteins found in blood plasma, such as albumin and clotting factors.
Another function of hepatocytes is detoxification. They contain a family of enzymes known as cytochrome P450, which chemically modify drugs and other foreign substances, preparing them for excretion from the body. This system also breaks down the body’s own metabolic waste, such as converting ammonia into urea. This filtration protects the body from both ingested and internally produced toxins.
Hepatocytes also produce and secrete bile. This substance is stored in the gallbladder and released into the small intestine to aid in the absorption of fats and fat-soluble vitamins. The liver serves as a storage depot for micronutrients. Hepatocytes hold reserves of vitamins A, D, K, and B12, as well as minerals like iron, releasing them as needed.
Sourcing and Isolation of Primary Hepatocytes
The primary hepatocytes used in research are obtained from both human and animal sources. Human hepatocytes are isolated from donor livers that are not suitable for organ transplantation or from small pieces of tissue removed during other surgical procedures. Animal-derived hepatocytes, most commonly from rodents like mice and rats, are also used, with the species chosen based on the study’s needs. These sources require strict ethical oversight and regulatory approval, particularly when human tissue is involved.
The isolation process is a multi-step procedure to separate the hepatocytes from the liver’s structural matrix. It begins with the perfusion of the liver tissue with a solution containing an enzyme called collagenase. This enzyme works to digest the connective tissue that holds the cells together. Following the enzymatic digestion, the tissue is gently agitated to free the individual cells.
Once the cells are dissociated, they must be purified to separate the hepatocytes from other liver cell types, such as endothelial and Kupffer cells. This is often accomplished through centrifugation, sometimes using a dense solution like a Percoll gradient, which separates cells based on their density. After isolation, the purified hepatocytes undergo quality control checks to confirm their viability and purity for experimental use.
Laboratory and Clinical Uses
In the laboratory, primary hepatocytes are a tool for pharmaceutical development, particularly in studies of drug metabolism and toxicology. Researchers use these cells to investigate how a potential new drug is absorbed, distributed, metabolized, and excreted (ADMET). Because primary hepatocytes retain the metabolic functions of a normal liver, they provide a reliable model for predicting a drug’s potential to cause liver injury, a major reason for the failure of compounds in clinical trials.
These cells are also used in creating models for various liver diseases. Scientists can use primary hepatocytes to study the mechanisms behind conditions like viral hepatitis, non-alcoholic fatty liver disease (NAFLD), and liver fibrosis. For instance, by infecting cultured hepatocytes with the hepatitis C virus, researchers can observe the viral life cycle and test the effectiveness of antiviral therapies. These models allow for a detailed examination of disease progression.
Primary hepatocytes are used in research to explore the liver’s basic physiological processes, including its ability to regenerate after injury. There is also experimental work involving their use in bioartificial liver devices, which are designed to provide temporary support for patients experiencing acute liver failure. In regenerative medicine, researchers are investigating the potential for transplanting healthy hepatocytes to treat certain metabolic liver disorders.
Difficulties in Working with Primary Hepatocytes
Despite their value, working with primary hepatocytes presents several practical difficulties. A primary issue is their limited lifespan and inability to proliferate in a standard laboratory culture. Unlike immortalized cell lines, these cells do not divide extensively, meaning a single isolation provides a finite supply of cells for experiments.
Another challenge is a phenomenon known as dedifferentiation. When removed from their natural environment within the liver, hepatocytes quickly begin to lose their specialized functions in culture. The activity of metabolic enzymes like the cytochrome P450 system can decrease, and the production of liver-specific proteins like albumin can decline. This limits the duration over which the cells accurately represent liver function, making long-term studies challenging.
There is also considerable variability between cell batches. Hepatocytes isolated from different donors can behave differently due to factors like the donor’s age, genetics, and health status, which can affect experimental consistency. The availability of high-quality human hepatocytes is also limited and can be costly. Maintaining their function in the lab requires advanced culture methods, such as arranging them in 3D structures or co-culturing them with other cell types to mimic the native liver environment.