Hepatocytes, which are the main functional cells of the liver, have a typical cubical shape with sides measuring between 20-30 μm, and a typical volume of 3.4 x 10−9 cm ³. Compared to a human hair, which has a diameter of 17 to 180 μm, hepatocytes are relatively small ^2. One unique feature of hepatocytes is the abundance of smooth endoplasmic reticulum they contain, which distinguishes them from most other types of cells ³.

Under a microscope, hepatocytes have an eosinophilic cytoplasm, indicating the presence of numerous mitochondria, as well as basophilic stippling caused by large amounts of smooth endoplasmic reticulum and free ribosomes ⁴. Brown lipofuscin granules may also be observed with age, along with unstained areas of cytoplasm that correspond to glycogen and lipid stores removed during histological preparation. Hepatocyte nuclei are round and contain dispersed chromatin and prominent nucleoli. Anisokaryosis, or variation in the size of the nuclei, is common and reflects the normal occurrence of polyploidy in a significant portion of adult human liver cells¹.

Hepatocytes are arranged in plates that are separated by vascular channels known as sinusoids, which are supported by a network of reticulin (collagen type III). In mammals, the hepatocyte plates are only one cell thick, while in chickens, they are two cells thick. Sinusoids have a fenestrated endothelial cell lining that is separated from the hepatocytes by the space of Disse, which drains lymph into the portal tract lymphatics. Kupffer cells, which are part of the reticuloendothelial system, are scattered between endothelial cells and phagocytose spent erythrocytes. Stellate (Ito) cells are difficult to observe by light microscopy but are also distributed among endothelial cells and store vitamin A while producing extracellular matrix and collagen.

Drug Metabolism and Pharmacokinetics (DMPK)

Drug metabolism is a crucial step in drug development, as it can affect the safety and efficacy of a drug. Understanding the mechanisms of drug metabolism is, therefore, essential for drug discovery and development. One of the primary methods used for drug metabolism studies is in vitro testing with hepatocytes. These studies fall under the umbrella of Drug Metabolism and Pharmacokinetics (DMPK), which aims to understand how drugs are absorbed, distributed, metabolized, and excreted in the body. Hepatocytes are ideal for DMPK studies because they are responsible for the majority of drug metabolism in the body(in vivo) and contain various enzymes, including cytochrome P450 enzymes (CYPs), which play a significant role in drug metabolism.

Advantages of In vitro drug testing with hepatocytes

In vitro testing with hepatocytes offers several advantages over in vivo testing, including cost-effectiveness, reduced ethical concerns, and the ability to test multiple compounds simultaneously. The use of hepatocytes in drug metabolism studies has been applied to various therapeutic areas such as oncology, infectious diseases, and cardiovascular diseases. Different types of hepatocyte-based assays are available, including incubation studies, microsomal assays, and sandwich-cultured hepatocyte assays.

The liver plays a crucial role in drug metabolism, with approximately 60% of marketed drugs being metabolized by cytochrome P450 (CYP) enzymes in the liver ⁵. Liver microsomes, which contain membrane-bound drug metabolizing enzymes, including CYP, are subcellular fractions that can be used to determine the intrinsic clearance of a compound in vitro. Species-specific microsomes can also provide insight into interspecies differences in drug metabolism ⁶. Microsomes are a convenient and cost-effective alternative to whole-cell models, as they are easy to prepare, use, and store. To minimize interindividual variability, microsomes are pooled from multiple donors. Additionally, the activity of microsomes is characterized using probe substrates to ensure consistent activity between batches.

Sandwich-cultured hepatocytes (SCH) are a valuable in vitro tool for studying various aspects of drug transport, including hepatobiliary drug transport, species differences in drug transport, transport protein regulation, drug-drug interactions, and hepatotoxicity ⁷. This review presents a comprehensive summary of the SCH model, including its history, introduction, and methodology for evaluating hepatobiliary drug disposition. It also highlights several studies that have utilized this model to examine the interplay between drug-metabolizing enzymes and transport proteins, drug-drug interactions at the transport level, and hepatotoxicity resulting from altered hepatic transport.

The SCH model provides a unique opportunity to study drug transport across the hepatocyte, which is an essential function for the liver in maintaining systemic drug concentrations. The sandwich configuration of the model allows for better preservation of hepatocyte morphology, function, and polarity than traditional monolayer cultures. SCH has been used to investigate species-specific differences in drug transport, which is crucial for predicting drug efficacy and safety in preclinical studies. Moreover, SCH has been used to study drug-drug interactions and assess potential hepatotoxicity caused by alterations in hepatic transport.

Xenobiotic metabolism by hepatocytes

Xenobiotics are foreign substances that enter the body, including environmental toxins and pollutants. Hepatocytes are responsible for metabolizing and detoxifying xenobiotics, making them essential for understanding the effects of these substances on human health. Xenobiotic metabolism involves the conversion of lipophilic compounds to hydrophilic metabolites that can be excreted from the body. Phase I metabolism involves the addition of functional groups, while phase II metabolism involves the conjugation of the xenobiotic or its metabolites with endogenous molecules. This process is catalyzed by enzymes such as cytochrome P450 (CYP) enzymes and flavin-containing monooxygenases (FMOs). Hepatocytes can be used to study xenobiotic metabolism in vitro, providing valuable information on the metabolic fate of a compound and its potential toxicity.

Enzymatic testing by hepatocytes

Enzymatic testing is another important application of hepatocytes. The activity of specific enzymes can impact drug metabolism and drug-drug interactions. Hepatocytes can be used to test the activity of various enzymes, including CYPs, to determine their role in drug metabolism and potential drug interactions. In addition to CYP enzymes, hepatocytes can also be used to test the activity of other enzymes, such as FMOs, UDP-glucuronosyltransferases (UGTs), and sulfotransferases (SULTs). These enzymes are involved in phase I and II metabolism and can impact the metabolic fate of xenobiotics.

Cholesterol, Lipid, and Steroid Synthesis by hepatocytes

Hepatocytes are also involved in the synthesis of cholesterol, lipids, and steroids, which play essential roles in maintaining cellular structure, regulating cellular processes, and hormone production. Hepatocytes produce cholesterol, which is essential for the formation of cell membranes and the synthesis of steroid hormones. They also synthesize lipids, including triglycerides and phospholipids, which are necessary for energy storage and membrane synthesis.

References

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2. Qin, L. & Crawford, J. M. Anatomy and cellular functions of the liver. Zakim and Boyer's Hepatology (2018). doi:10.1016/b978-0-323-37591-7.00001-x

3. Pavelka, Margit; Roth, J. (Cell and molecular pathologist) (2010). Functional ultrastructure : Atlas of tissue biology and pathology. Wien: SpringerWeinNewYork. ISBN 978-3-211-99390-3. OCLC 663096046.

4. Kose, E. et al. Beneficial effects of montelukast against methotrexate-induced liver toxicity: A biochemical and histological study. The Scientific World Journal 2012, 1–6 (2012).

5. Bibi, Z. Role of cytochrome P450 in drug interactions. Nutr Metab (Lond) 5, 27 (2008). https://doi.org/10.1186/1743-7075-5-27

6. Keefer, C. et al. Mechanistic insights on clearance and inhibition discordance between liver microsomes and hepatocytes when clearance in liver microsomes is higher than in hepatocytes. European Journal of Pharmaceutical Sciences 155, 105541 (2020).

7. Swift B, Pfeifer ND, Brouwer KL. Sandwich-cultured hepatocytes: an in vitro model to evaluate hepatobiliary transporter-based drug interactions and hepatotoxicity. Drug Metab Rev. 2010 Aug;42(3):446-71. doi: 10.3109/03602530903491881. PMID: 20109035; PMCID: PMC3097390.