3D-Cultured Primary Human Hepatocytes: Evaluating Drug-Induced Liver Damage

Drug-induced liver damage is a significant hurdle in drug development, clinical trials, and therapeutic use. Hepatotoxicity not only jeopardizes drug approvals but can also lead to severe health consequences, including liver failure. The liver is the primary organ responsible for drug metabolism, making it particularly vulnerable to toxic compounds. Thus, evaluating a drug's hepatotoxic potential before clinical trials is essential to avoid late-stage failures and ensure patient safety.

Historically, animal models have been utilized to predict liver toxicity. However, these models often fail to accurately replicate human liver responses due to species-specific biological differences. To improve the prediction of drug-induced liver damage, researchers have increasingly turned to in vitro models using primary human hepatocytes (PHHs). Unfortunately, traditional 2D cultures of PHHs face significant limitations, including short-lived viability and de-differentiation, which hinder their effectiveness in assessing long-term drug exposure.

The Need for Advanced Hepatocyte Models

Primary human hepatocytes (PHHs) are widely regarded as the gold standard for in vitro liver models due to their ability to closely mimic human liver function. However, challenges in isolating functional PHHs and their rapid loss of characteristics in traditional 2D environments limit their utility in drug testing. Typically, PHHs lose their functionality and de-differentiate within three days, making it difficult to conduct reliable hepatotoxicity assessments over time.

To overcome these limitations, researchers at Inventia Life Science have developed a novel approach to creating 3D-cultured PHHs using the RASTRUM™ Platform. This innovative technology allows for the encapsulation of PHHs in a matrix that mimics the liver’s natural extracellular environment, preserving cell viability and function for up to two weeks. This advancement provides an essential tool for studying hepatotoxicity in a more physiologically relevant context.

The RASTRUM Platform: Transforming Hepatocyte Culture

The RASTRUM platform utilizes advanced 3D cell culture technology to create a more sustainable and functional hepatocyte model. By embedding cryopreserved PHHs in a specially designed extracellular matrix (ECM), researchers can maintain the cells' integrity and functionality for extended periods. This is a significant improvement over traditional methods, which often struggle to keep PHHs viable and metabolically active.

The STAR Protocol, developed by Inventia Life Sciences, outlines the detailed process for utilizing the RASTRUM platform to create 3D-cultured PHH models. This comprehensive guide ensures that researchers can replicate the protocol and achieve consistent results in their laboratories. Key steps include preparing cell culture media, thawing, plating the hepatocytes, and using the RASTRUM platform to distribute PHHs evenly in a controlled environment.

Key Benefits of the RASTRUM Approach

1. Extended Cell Viability: The 3D-cultured PHHs created using the RASTRUM platform remain viable for up to 14 days, allowing for extensive drug testing.

2. Functional Integrity: The cells retain critical liver functions, such as drug metabolism and albumin production, making them suitable for hepatotoxicity studies.

3. Reduced Cell Usage: The RASTRUM platform requires nearly 50% fewer cells compared to traditional 2D models, enhancing the efficiency of drug testing.

Demonstrating the Functionality of RASTRUM-Printed PHHs

After successfully creating 3D-cultured PHHs, researchers conducted a series of assays to confirm the cells' viability and functionality. Key tests included live/dead cell staining assays and quantitative cell viability assays, which demonstrated that the 3D-cultured PHHs maintained their viability and characteristic hepatocyte morphology throughout the study period.

To further validate functionality, the research team performed immunofluorescent staining to quantify the expression of liver-specific markers, such as albumin and multidrug-resistant protein 2 (MRP2). The findings showed a steady increase in albumin production over 14 days, indicating that the cells retained their ability to synthesize this essential liver protein.

Moreover, the 3D-cultured PHHs exhibited CYP3A4 enzyme activity, a crucial marker for drug metabolism. This activity is vital for evaluating hepatotoxicity, as CYP3A4 is one of the primary enzymes responsible for breaking down drugs in the liver. The researchers found that treatment with Rifampicin, a non-hepatotoxic drug, upregulated CYP3A4 activity by fourfold within two days, confirming the model’s functionality.

Evaluating Hepatotoxicity: Acetaminophen and Troglitazone

Following the confirmation of PHH functionality, the research team assessed the hepatotoxicity of two well-known drugs: acetaminophen and troglitazone. Acetaminophen is a widely used analgesic that can cause liver damage at high doses, while troglitazone, an anti-diabetic medication, was withdrawn from the market due to its hepatotoxic effects.

The IC50 values for acetaminophen and troglitazone were determined to be 15.96 mM and 0.203 mM, respectively. These values are consistent with those obtained from traditional 2D models but demonstrate the advantage of using RASTRUM-printed PHHs, as they required significantly fewer cells for the same results.

1. Acetaminophen Testing: Acute drug administration was conducted five days post-printing. Cell viability was measured two days after exposure, revealing that increasing the acetaminophen dose from 1 mM to 100 mM led to a more than 50% reduction in cell viability.
2. Troglitazone Testing: Chronic exposure studies were conducted by administering troglitazone ten days post-printing. Over seven days of continuous exposure, increasing the drug dose from 0.1 mM to 0.4 mM resulted in nearly complete cell death, highlighting the drug’s potent hepatotoxicity.

These findings illustrate that RASTRUM-printed PHHs are a powerful tool for accurately assessing liver damage potential in drugs, addressing a critical barrier to market delivery.

Limitations and Future Directions

While the RASTRUM-printed PHH model offers significant advantages, researchers must be aware of its limitations. Variability in PHH quality based on the donor can impact cell behavior and responses to drugs. Therefore, calibration of printing parameters may be necessary for each batch of hepatocytes.

The current protocol also focuses on a limited set of functional markers for characterization. Future studies could expand this by incorporating additional hepatic markers, assessing glycogen synthesis, and evaluating drug clearance rates. Such enhancements would provide a more comprehensive understanding of the liver’s response to various compounds.

Lastly, increasing the number of replicates in experiments will help account for inherent variability in 3D cell structures, improving the consistency of results and further validating the utility of the RASTRUM platform in drug testing.

Conclusion

The advancement of 3D-cultured primary human hepatocytes using the RASTRUM platform marks a significant step forward in hepatotoxicity testing. By maintaining cell viability and functionality for longer periods, this model allows for more accurate assessments of drug interactions with the liver. The ability to evaluate both acute and chronic drug exposure with fewer cells enhances the efficiency of drug development pipelines, ultimately improving drug safety.

For detailed guidance on implementing this innovative protocol, refer to the full article published in STAR Protocols: "Protocol to Create Phenotypic Primary Human Hepatocyte Cultures Using the RASTRUM 3D Cell Model Platform."