Primary Hepatocytes: A Crucial Tool for Advancing Non-Clinical In Vitro Drug Research

Primary Hepatocytes: A Crucial Tool for Advancing Non-Clinical In Vitro Drug Research

Liver, as the primary organ for drug exposure, plays a crucial role in drug metabolism and toxicity processes. Primary hepatocytes possess the full spectrum of cellular characteristics and physiological levels of enzymes and cofactors, including membrane-bound enzymes such as Cytochrome P450 (a mixed-function oxidase in the smooth endoplasmic reticulum) and cytosolic esterases, encompassing all metabolic pathways found in the liver. For this reason, primary hepatocytes are widely regarded as the gold standard for constructing in vitro liver models and are favored by researchers in drug interaction, drug metabolism, and toxicity studies. This article provides an overview of 2D and 3D culture models based on primary hepatocytes and their applications in drug development.

Keywords: Primary Hepatocytes, 2D Cultivation, 3D Cultivation, Organoids, Co-Culture.

1. 1. Isolation of Primary Hepatocytes

The isolation of primary hepatocytes is a critical step in establishing in vitro liver models, with the two-step collagenase perfusion method being the most commonly used. In smaller animals, liver perfusion can be performed via the portal vein or inferior vena cava, while larger animals typically require perfusion through liver lobes or segments. Several key factors influence the successful isolation of hepatocytes: First, collagenase should be non-cytotoxic. Second, the timing of digestion is crucial—both under-digestion and over-digestion can compromise hepatocyte yield and viability. Third, the liver condition must be optimal, as hepatocytes are highly sensitive to ischemic damage. The liver used for hepatocyte preparation should be rapidly cooled to reduce metabolic rates and prevent metabolic hypoxia and subsequent ischemia.

The standards for primary hepatocytes used in drug research are as follows:

1. 2. Primary Hepatocyte 2D Culture

The suspension hepatocyte model

It contains complete drug-metabolizing enzymes and cofactors, making it suitable for studying various metabolic clearance pathways. However, the viability of suspension hepatocytes and the activity of drug-metabolizing enzymes gradually decrease as the in vitro incubation time increases, limiting the incubation time to a maximum of 4 hours. This model is typically used to estimate the clearance of drugs with moderate to high clearance rates. When the clearance rate is less than 20%, accurate clearance values cannot be determined. Traditional suspension hepatocyte-based in vitro metabolism models are insufficient for generating detectable metabolic reactions for slow-metabolizing compounds, thus limiting their ability to predict the clearance rates and metabolic products of these compounds. A suspension hepatocyte relay method (Figure 1) can be used to extend the incubation time to 20 hours or even longer.

Application: Enzyme activity and metabolic stability studies for small molecule drugs.

Figure 1. Suspension Hepatocyte Relay Method Transfer Process

Source: DRUG METAB DISPOS, 2012,40(9):1860–1865

The plateable Hepatocytes Model

Primary hepatocytes are cultured in a 2D system on collagen-coated culture plates. The hepatocytes exhibit an epithelial morphology, with protruding nuclei, often presenting in a binucleated form. The drawbacks of single hepatocyte monolayer culture include: 1. Alteration of cell polarity and function.2. Lack of other relevant cell types (i.e., non-parenchymal cells) that are required for normal function.3. Inability to provide sufficient nutrients and paracrine factors to support the hepatocytes in performing their functions (such as bile acid and serum protein biosynthesis).

Applications:

1). Evaluation of Drug-Drug Interactions: This includes enzyme induction, enzyme inhibition, and transporter studies. Firstly, when a drug acts as an inducer, it can lead to increased expression of drug-metabolizing enzymes and transporters. Strong inducers can upregulate multiple genes simultaneously, such as phenobarbital induction of CYP2B6, CYP3A4, CYP2C9, UGT, and several transport proteins like MRP2. Secondly, there are species-specific differences in how hepatocytes respond to inducers. For example, rifampicin is an effective inducer for human and rabbit hepatocytes, but has no induction effect on rat hepatocytes. Finally, suboptimal plating density of plateable hepatocytes may lead to reduced basal expression of P450s and artificially increased induction responses. As shown in Figure 3, hepatocytes at lower plating densities show lower basal activity of CYP1A2, CYP2B6, and CYP3A4, but stronger induction responses. Therefore, healthy hepatocytes at an appropriate plating density are necessary to obtain physiologically relevant data.

Figure 2. Relationship Between Plating Density of Cryopreserved Human Hepatocytes and Enzyme Induction

Source: Current Drug Discovery Technologies, 2010, 7:188-198

2). Hepatotoxicity Assessment: Observing morphological changes under a light microscope, such as cell morphology, vacuole and lipid droplet aggregation, and cell attachment/detachment. Detection of hepatocyte necrosis (as indicated by aspartate aminotransferase, lactate dehydrogenase, and alanine aminotransferase) and apoptosis (DNA fragmentation). For cytokine-mediated cytotoxicity, single monolayer cultures of hepatocytes cannot predict toxic responses due to the regulation by substances released from neighboring non-parenchymal cells, such as Kupffer cells, stellate cells, and sinusoidal endothelial cells.

3). "Relay Method" for Study of Slow Metabolizing Compound Clearance and Its Metabolites: The activity of drug-metabolizing enzymes in plateable hepatocytes begins to decrease after 24 hours of plating. After incubating plateable hepatocytes with serum-free medium containing the compound of interest for 24 hours, the medium is collected and mixed, then transferred to new plateable hepatocytes for further study (Figure 3).

Figure 3. Plateable Hepatocyte Relay Method Transfer Process

Source: Drug Metabolism Letters, 2016, 10: 3-15

3). Evaluate the cellular uptake, endocytosis, endosomal escape, and silencing effects on the target gene of hepatocyte-targeted small nucleic acid drugs.

Sandwich Cultivation Model

In vitro, hepatocytes can be cultured between two layers of collagen or Matrigel to reconstruct in vivo structures, known as sandwich cultivation. Hepatocytes cultured between two layers of gel-collagen (sandwich structure) can improve the morphology and viability of the cells, and maintain their functionality for a longer period. Furthermore, hepatocytes in sandwich culture can restore polarity, allowing for proper localization of basolateral and canalicular transporters, as well as the formation of functional bile duct networks (Figure 4).

Figure 4. Polarized Expression of Transporters in the Human Hepatocyte Sandwich Model

Source: Current Drug Discovery Technologies, 2010, 7, 188-198

Applications:

1. 1). Estimating biliary excretion of compounds.
2. 2). Evaluating the hepatic and biliary distribution of endogenous and exogenous compounds and metabolites.
3. 3). Estimating clearance mediated by metabolism and transporters, and constructing physiology-based pharmacokinetic models.
4. 4). Studying hepatotoxicity and providing mechanisms for clinical drug-induced liver injury. Data is integrated into systems pharmacology models to predict potential drug-induced liver injury in humans.
5.  
   1. 3. Primary Hepatocyte 3D Culture

6. In 3D culture systems, hepatocytes are cultured in a three-dimensional matrix, which better mimics the in vivo liver architecture compared to 2D monolayer cultures. These systems promote cell-cell and cell-matrix interactions, which can restore more of the liver’s physiological functions, including drug metabolism, protein secretion, and bile formation. Primary hepatocyte 3D cultures can be used to study liver-specific functions in a more in vivo-like environment, offering advantages for drug testing, toxicity assessment, and disease modeling.

   Spheroid Model

Through techniques such as ultra-low attachment culture, hanging drop culture, and magnetic cell culture (Figure 5), primary hepatocytes can aggregate into spheroidal aggregates with a diameter of up to 150-175 µm without relying on external matrices. One advantage of spheroid culture is that each spheroid requires only 1,330-2,000 cells, significantly reducing the number of cells compared to other 3D culture techniques. Recent studies have shown that primary hepatocyte spheroid cultures can sustain for up to 5 weeks, with CYP enzyme activity remaining almost unchanged between day 8 and day 35. Proteomics analysis revealed that, compared to sandwich culture, enzymes responsible for drug absorption, distribution, metabolism, and excretion are better preserved for 14 days in spheroid culture. However, the liver is much more complex than a cell aggregate. The basolateral side of hepatocytes interacts with blood, while bile flows out from the apical side, which is a key feature of the complex liver lobule structure, and this cannot yet be replicated in the spheroid model.

Applications:

1. 1). Study of slow metabolizing compound clearance and its metabolites.
2. 2). Hepatotoxicity research.
3. 3). Evaluation of cellular uptake, endocytosis, endosomal escape, and silencing effects on target genes of hepatocyte-targeted small nucleic acid drugs.

Figure 5. Spheroid Culture Method

Liver Organoids Model

The consensus definition of organoids is: 3D structures derived from stem cells, progenitor cells, or differentiated cells, capable of reproducing certain functions and structures of the native tissue in vitro, effectively mimicking the in vivo microenvironment and cell-to-cell interactions. Liver organoids have been identified as the most advanced model for human liver biology research.

Cell Sources for Liver Organoid Construction:

① Pluripotent Stem Cells (PSCs):

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) possess high pluripotency, plasticity, and unlimited proliferative capacity. Under the influence of specific signaling factors, they differentiate into hepatocyte-like cells with activity and function. However, liver organoids derived from PSCs may undergo epigenetic and genetic alterations, exhibiting chromosomal aneuploidy changes during amplification.

Applications:

1. 1). Genetic Liver Disease Models
2. 2). Infectious Liver Disease Models
3. 3). Drug Cytotoxicity Testing

② Liver Tissue-Derived Cells: These include cholangiocytes and hepatocytes. Mature hepatocytes retain stem cell potential and proliferative ability in specific environments. Compared to PSC-derived organoids, organoids derived from primary tissue are more mature, with more stable genomes, and maintain phenotypic and genetic stability during long-term in vitro culture. However, the long-term proliferative capacity of mature human hepatocyte organoids is limited compared to fetal human hepatocytes or adult mouse primary hepatocytes. Culturing adult hepatocyte organoids remains challenging.

Culture Method (Figure 6): Liver tissue is digested into single cells, and a mixture of Matrigel and cells is seeded in a 24-well plate to form dome-shaped structures. Incubate in a cell culture incubator (37°C) for 15 minutes. After solidification, add specific culture medium. Passage after approximately 14 days. Replace the original medium with differentiation medium after 7-10 days.

Applications:

1. 1). Hepatotoxicity Models
2. 2). In Vitro Metabolism Disorder Studies
3. 3). Non-Alcoholic Fatty Liver Disease
4. 4). Drug Development for Benign and Malignant Liver Diseases

Figure 6. Culture and Passage Process of Tissue-Derived Liver Organoids

Source: Cell & Bioscience (2023) 13:197

Comparison of Spheroid Culture and Organoid Culture

4. Primary Hepatocyte Co-culture Model

1. 1. 2D Primary Hepatocyte Co-culture Model
      

      In the 2D primary hepatocyte co-culture model, two or more different cell types are mixed and cultured in a two-dimensional environment. The key feature of this model is the direct interaction between different cell types, the interaction between cells and the extracellular matrix, or the indirect signal transmission via cytokines and chemical communications. Primary hepatocyte functions, such as albumin production and drug metabolism ability, can be maintained for up to three weeks.

      Applications:

      1. 1). Primary Hepatocytes Co-cultured with Fibroblasts: This model is used for studying the clearance rate of slow-metabolizing compounds and their metabolites.
      2. 2). Primary Hepatocytes Co-cultured with Non-parenchymal Liver Cells (e.g., Stellate Cells, Sinusoidal Endothelial Cells): This model is useful for researching drug-induced liver injury (DILI), as it helps to investigate the role of cytokines, chemokines, and growth factors in modulating liver adaptive responses after drug exposure.
      3. 3). Primary Hepatocytes Co-cultured with T Cells: This model is used to detect liver drug metabolism-specific T cell responses.
      4. 4). IPHASE's HepatoMax™ Co-culture System: IPHASE has developed a co-culture system with primary hepatocytes from different species, known as HepatoMax™. By co-culturing human primary hepatocytes with stromal cells, it is possible to maintain good drug-metabolizing enzyme activity in human hepatocytes for up to 3 weeks. This system is suitable for studying slow-metabolizing compound clearance rates and their metabolites.

      This co-culture model provides a more physiologically relevant platform for assessing drug metabolism, toxicity, and liver-related disease processes, offering insights into how different cell types contribute to liver function and disease.
      
      

      3D Primary Hepatocyte Co-culture Model

      Direct 3D Co-culture: This model involves mixing two or more different types of liver cells (e.g., primary hepatocytes, sinusoidal endothelial cells, hepatic stellate cells, Kupffer cells) to form self-assembled spheroids or co-culturing them in a 3D environment constructed with materials such as collagen, fibrin, alginate, or hydrogels. Direct 3D co-culture allows close interaction between different liver cells through mechanisms like cell-to-cell adhesion, paracrine signaling via soluble cytokines, and extracellular matrix adhesion, enabling communication between hepatocytes.

      Applications:

      1. 1). Liver Fibrosis Model: Used to study the mechanisms and progression of liver fibrosis.
      2. 2). Drug-Induced Liver Injury (DILI) Model: Helps simulate and assess liver damage caused by drugs.
      3. 3). Drug Interactions, Drug Metabolism, and Enzyme Induction: Evaluates how different drugs interact, how they are metabolized, and how they induce liver enzymes.

      Indirect 3D Co-culture: This method uses a physical separation system (e.g., Transwell or other materials) to culture two or more types of cells (such as primary hepatocytes with NIH/3T3 cells or sinusoidal endothelial cells) in a 3D environment, where direct cell-to-cell contact is prevented. The communication between the cells occurs via soluble cytokines.

      Applications:
      Used for studying non-contact communication between liver cells in the body.

      
      In summary, IPHASE, as a leader in in vitro biological research, provides comprehensive solutions for non-clinical drug testing. From the isolation and culture of primary hepatocytes from various species to the development of supporting products such as culture media for specific applications or multi-specification collagen-coated plates, IPHASE is dedicated to offering the best in vitro research tools for drug discovery and development. We are a trusted partner for clients in the pharmaceutical industry, committed to providing cutting-edge solutions for non-clinical research.