Bringing a new drug to market is an expensive and time-intensive process, with high costs and low clinical success rates (10-20%)1,2. Hepatotoxicity is a major cause of drug failures due to the liver’s central role in metabolism, yet accurate prediction of liver toxicity in preclinical studies remains a significant challenge³.  

Animal models, while widely used, often fail to accurately reflect human toxicity due to differences in physiology, metabolic pathways, and disease adaptations, limiting their clinical predictiveness3. As a result, in vitro human hepatic models are preferred as a cost-effective high-throughput alternative for preclinical drug metabolism and pharmacokinetics (DMPK) screening, including studies that support regulatory submissions. 

Primary human hepatocytes and subcellular fractions (i.e., liver microsomes) are commonly used in vitro systems for liver metabolism modeling. Each system has unique advantages and limitations, varying in physiological relevance, metabolic activity, and suitability for specific applications. A novel alternative model/approach leverages permeabilized hepatocytes to combine the key advantages of both models, offering robust metabolic function with greater ease of use and consistency. 

Primary Human Hepatocytes

Primary human hepatocytes are the gold standard in vitro model for studying the liver’s in vivo metabolic processes. The primary benefits of adherent hepatocytes are elevated liver-specific activity, expression of cytochrome P450 (CYP) and phase II enzymes, short-term inducibility of CYP enzymes, and efficient transferability of data to human hepatocyte data4. Alternatively, primary hepatocyte suspensions may offer better estimates of in vivo clearance and toxicity than monolayer cultures4.  

Derived from whole livers or resected liver tissue, primary hepatocytes can be difficult to obtain due to limitations in the availability of high-quality human livers. Other limitations include short-term preservation of functionality, limited lifespan in culture, and donor-to-donor variability, complicating their use for long-term studies3. 

The introduction of cryopreservation has helped address this challenge, cementing the routine use of cryopreserved primary hepatocytes for preclinical studies5. Additionally, commercial vendors can produce pooled donor human hepatocytes, which are primary hepatocytes from different donors combined into a single lot. For example, Gentest® Human Pooled Hepatocytes help reduce genetic variability, which improves their predictability of drug metabolism, clearance, and inhibition studies. These pooled lots are available in plateable and suspension formats, expanding their applicability across different drug metabolism studies.

Human Liver Microsomes

Hepatic subcellular fractions, such as liver microsomes, are derived from the endoplasmic reticulum and contain a rich array of drug-metabolizing enzymes. They are widely used in pharmaceutical research to screen new chemical entities for metabolic stability, estimate in vivo hepatic clearance, and evaluate CYP–related drug properties, and UDP-glucuronosyltransferase (UDPGT)-mediated drug metabolism5. 

Liver microsomes offer advantages like being readily accessible, easily prepared, are highly enriched in metabolic enzymes, and are generally less expensive to use than other in vitro liver models. However, they lack the full complement of drug-metabolizing enzyme (DME) pathways present in intact hepatocytes. Further, microsomes do not contain the full range of hepatic transporters, which limits their ability to assess drug efflux and uptake mechanisms 5. As a result, microsomal data may not fully capture the in vivo hepatic metabolic fate of a drug compound. 

Permeabilized Hepatocytes – A Novel Approach

Discovery Life Sciences’ Gentest® MetMax® pooled human hepatocytes are a novel in vitro liver model that combines the desirable properties of both primary hepatocytes and subcellular fractions5,6. These permeabilized, cofactor-supplemented cells provide direct access to intracellular enzymes, enabling even poorly permeable drug compounds to access the interior of the hepatocyte without the need for transporter-assisted entry—a significant benefit of this system. 

Unlike subcellular fractions, Gentest® MetMax® hepatocytes retain all major metabolic functions and subcellular structures of primary hepatocytes. Functionally, comparative studies have demonstrated their equivalence to primary hepatocytes in key drug-metabolizing enzyme (DME) pathways, including phase I oxidation and phase II conjugation5.  

Gentest® MetMax® hepatocytes offer several ease-of-use advantages over traditional hepatocyte models. They are robust, easy to handle, and do not require cell culture maintenance. No cell thawing and recovery is required, and they remain functional and stable in suspension for extended periods, making them a practical choice for high-throughput screening.

Comparing Metabolism Models

Because each model has its strengths and trade-offs, selecting the right in vitro liver model for drug metabolism studies will largely depend on your study objectives, throughput needs, and desired endpoints. Table 1 provides a summary of each system and recommendations for use:

Table 1. Comparing human hepatocyte, liver microsome and Gentest® MetMax® hepatocyte models
Primary Hepatocytes Liver Microsomes Gentest® MetMax® Hepatocytes
Advantages
• High physiological relevance

• Expresses full complement of metabolic enzymes

• Available in suspension or plateable formats
• Enriched in phase I and phase II metabolic enzymes

• Easy to prepare and store

• Cost-effective
• Permeabilized to provide direct access to intracellular enzymes

• Stored at -80°C and readily available

• No culture maintenance required

• Consistent and stable
Disadvantages
• Limited tissue availability

• Donor variability

• Limited life span in culture

• Requires cell maintenance
• Non-viable

• Lacks phase III transporters

• Not representative of whole-cell hepatic metabolism
• Non-viable

• Lack functional phase III transporters
Recommended For:
• Cellular toxicity

• Comprehensive DMPK studies (phase I, II & III)

• Transporter assays

• Evaluation of drug-drug interactions (DDI)

• Long-term hepatotoxicity
• High-throughput screening

• Intrinsic Clearance

• CYP inhibition/induction

• Phase I/Phase II (UGT) metabolism

• Metabolite profiling
• High-throughput Phase I/II metabolism and clearance studies

• Evaluation of DDI

• CYP enzyme profiling and induction studie
While primary human hepatocytes offer the most physiologically relevant system, they are constrained by donor variability and short-term functionality. Liver microsomes, however, provide a cost-effective, high-throughput option enriched in metabolic enzymes but lack cellular complexity and full DME pathways. Gentest® MetMax® hepatocytes bridge the gap by retaining key metabolic capabilities of primary hepatocytes and microsomes and, because they are permeabilized, they allow direct access to metabolic enzymes greatly simplifying preclinical ADMET screening and liver metabolism assessments. 
Conclusion
Recent advances in 3D cell culture methods, such as liver organoids, and more sophisticated flow-based liver systems, like liver-on-a-chip technology, are pushing the boundaries of in vitro drug safety testing7,8. These models are contributing valuable data for drug safety assessments. However, they remain more complex to use and do not yet match the throughput capabilities of traditional 2D models.   Despite these innovations, 2D hepatocyte models remain a workhorse for early-stage DMPK evaluations, supported by historical preclinical data. However as mentioned here, 2D primary hepatocyte and liver microsome models have limitations. The Gentest® MetMax® hepatocyte system overcomes common limitations by combining the high throughput capabilities of traditional models with enhanced metabolic functionality, while also offering streamlined functionality and greater consistency.   The need for reliable preclinical ADMET testing remains essential in the drug development process. Continued investments, such as the development of novel models like MetMax® hepatocytes, will help drive drug metabolism research forward, enabling drug developers to reduce costs, improve clinical success rates, and accelerate the path to market. 
References
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