Closing the Gap: The Benefits of Animal-Free 3D Models

In recent years, the significance of 3D cell culture in drug discovery has grown exponentially. 3D models offer a more accurate representation of human tissue compared to traditional 2D models, allowing for more physiologically-relevant insights into cellular behavior, drug responses and disease processes. Organoid models, advanced 3D cell models that can be cultured from human stem cells, further enhance this relevance by mimicking the complexity and functionality of human tissues. These models surpass traditional in vivo animal models in their ability to replicate human-specific biological processes, making them more reliable for studying disease mechanisms and testing potential treatments.

While the value of animal models is declining in place of 3D cell culture technologies, the use of animal-derived cell culture products and supplements in 3D cell culture remains prevalent. Animal-based products can be problematic, particularly in therapeutic development, bringing batch-to-batch variability, unintended experimental effects, and safety issues. These factors undermine the reliability of assays and complicate the development of therapeutics.

Animal-free 3D culture products offer a promising alternative, providing a consistent and reproducible platform for cell culture without the biological or ethical complications associated with animal-derived materials. Innovations such as animal-free hydrogels are enhancing the reliability of preclinical models and helping to bridge the gap between laboratory research and human clinical trials.

In this article, we will explore how transitioning to animal-free 3D cell culture is helping to close the gap between preclinical models and human trials, paving the way for more effective drug discovery.

Limitations of animal models in preclinical drug discovery

The pharmaceutical industry faces a perilous problem – despite on average taking 10-15 years and over $1Bn to develop a new drug, 9 out of 10 drugs that make it to human trials fail [1]. This failure rate is stark and speaks to a significant translational gap between preclinical models and human outcomes in clinical trials.

In vivo models have long been a cornerstone in drug discovery, but extracting valuable data from these models can be exceptionally challenging. Whole organism models often fail to accurately replicate human-specific physiological responses due to species-specific differences in metabolism, genetics, cellular makeup and more. As a result, drugs that appear promising in animal models frequently underperform or cause unforeseen safety issues or off-target effects in human clinical trials.

The complexity of animal models, plus the inability to probe intricate pharmacodynamics and physiological responses makes it challenging to isolate the effects of a drug compound on specific systems or cellular processes, often leading to ambiguous data. As such, many animal studies are conducted for rudimentary measures, such as determining the lethal dose (LD₅₀) of a compound, rather than providing detailed mechanistic insights. These tests, while important for safety assessments and regulations, do not contribute substantially to understanding the therapeutic potential or precise biological activity of a drug.

Consequently, over-reliance on animal models in preclinical drug development can lead to increased likelihood of failure, significant delays and ballooning costs in the development pipeline, underlining the need for more human-relevant preclinical models.

Bridging the translational gap with 3D cell culture

The shocking failure rate of new drugs entering clinical trials highlights the substantial gap between preclinical drug testing models and patient populations. As already discussed, traditional 2D cell cultures and animal models often fail to capture the biology of human systems, making them poor predictors of human responses to candidate drugs in clinical trials. To address this challenge, researchers are increasingly turning to advanced 3D human cell models like organoids and spheroids.

3D cell models unlock the potential for more accurate human-specific data by incorporating human cells, which better emulate human biological processes. Unlike 2D models, which lack the complexity of real tissues, 3D models can replicate the intricate architecture and functionality of human tissues. This makes them invaluable for studying disease mechanisms and drug responses in a more physiologically-relevant context.

Organoids, which are miniaturized versions of organs that can be created by differentiating human stem cells, offer a much higher degree of human relevance. These structures can replicate the intricate cellular heterogeneity and functionality of human tissues, providing a more accurate platform for studying disease mechanisms and drug responses: A recent study investigated the effects of thalidomide, a drug that causes birth defects in humans, upon embryo-like gastruloids, organoids with the ability to model aspects of fetal development and gastrulation. The human stem cell-derived gastruloids were found to be strong predictors of fetal toxicity caused by thalidomide, while mouse models failed to identify this toxicity [2].

The value of organoid models across drug discovery 

The role of organoids in drug discovery is becoming increasingly critical, offering unparalleled opportunities for more accurate and human-specific research in preclinical studies. And their utility spans multiple stages of the drug discovery pipeline.

In the early phases, organoids can be employed for target identification and validation. They allow researchers to observe how potential drug targets behave in a human-like environment, leading to more relevant and accurate identification of novel drug targets.

Organoid models can also be adapted for automation and high-throughput screening (HTS), presenting a more rapid and reliable platform for drug compound screening versus 2D models or in vivo systems, since they provide a more accurate reflection of how a drug will perform in the human body. This enables a more rapid selection of lead compounds and better outcomes further down the pipeline. A group from University of Southern California (USC) recently developed a scalable organoid platform able to generate thousands of kidney organoids in microwell plate format. In a recent study, they successfully screened hundreds of enzyme inhibitors for effectiveness against a mutation found in people with autosomal dominant polycystic kidney disease [3].

Lead optimization is another fruitful arena for organoids in drug discovery, whereby more human-relevant modelling and a greater data yield can allow for more reliable assessment of drug efficacy and safety, reducing the likelihood of late-stage failures and associated costs.

Live cell imaging and isolation of organoids

Conference speaker Andris Abramenkovs, from Sartorius AG, highlighted the advantages of using organoids in drug discovery, and particularly in screening applications. By incorporating organoid models into technologies that can handle automation and large-scale assays, researchers can conduct extensive screenings with human-relevant models, significantly improving the predictability of drug efficacy and target activation.

Figure 1: Live cell imaging system demonstrated by Andris Abramenkovs. The system is employed to track the growth of organoid cultures prior to screening, enabling the standardization and QC of organoid cultures for HTS in drug discovery.

Andris highlighted the importance of standardization on organoid models for screening, and the critical practice of confirming normal organoid morphology and behavior in test cultures prior to screening. To confirm this, automated imaging systems can continuously monitor organoid growth and behavior, providing real-time data on changes in size, shape, and function. This not only enhances the precision and reproducibility of drug testing but also allows for early detection of abnormalities, improving the reliability of study data.

Patient-derived organoids and personalized medicine

An exciting avenue unlocked by organoid models is the ability to create ex vivo organoid models from patient-derived cells. This has significant implications in drug discovery and personalized medicine. By generating organoids from patient populations, researchers can tailor drug testing to specific patient populations, or test on a wide variety of patient-derived organoids to capture population heterogeneity more closely during HTS and other efficacy and safety tests during preclinical drug development.

By adopting this tailored approach to 3D cell culture, drug development programs can more closely align with the patient populations that they are seeking to treat – providing enhanced predictability and better chances of successful translation to human trials. This is particularly valuable for broadly heterogenous diseases, cancer being a prime example. In oncological drug development organoids can be leveraged to interrogate tumor mutations and responses to treatment, leading to more targeted and effective cancer therapies.

Animal-derived versus animal-free 3D culture products in drug discovery

Another major topic of discussion at this year’s annual UPM conference was the benefits of using animal-free 3D cell culture products – especially in the arena of drug discovery and therapeutic development.

Despite their widespread use in 3D cell culture, animal-based 3D culture products come with a range of inherent drawbacks that can impede the scientific progress. One major issue is the significant batch-to-batch variability of animal-derived materials, which can lead to inconsistent experimental outcomes. This is particularly critical in screening applications where reproducibility is paramount. Animal products can also introduce unintended biological effects that may alter cell behavior, compromising the reliability of any data obtained.

Safety is another area of concern, since animal-derived components can introduce pathogens or contaminants (adventitious agents) into the culture. In cell therapy development, this could carry over into the final product, posing a risk to patients. As such, animal-derived products are not permitted for use in cell and gene therapy production.

Practicality can also be hindered by animal-based culture products – automation, imaging and detection, drug diffusion and downstream processing can all be made more challenging by the presence of animal contaminants in the test culture. In contrast, animal-free culture solutions offer an unparalleled level of product consistency and practicality, enhancing the reliability and reproducibility of study data.

Compatibility with automation technologies

When it comes to 3D culture applications in drug discovery, automation technology is key to generating scalable cultures capable of HTS or high content screening (HCS) applications. In addition to organoid models, spheroid models are highly popular platform for screening applications. Spheroids are more basic than organoids, manifesting as compact, rounded 3D cellular structures, normally comprising only one cell type. Spheroid models are often favored in screening applications due to their simplicity and reproducibility.

Spheroid cultures are typically generated using scaffold-free U-ULA plates or scaffold-based matrices, in which the spheroids can grow and develop in 3D without clumping. Hydrogels are the most popular scaffold for spheroids, and these can come from animal sources, or natural animal-free sources like nanofibrillar cellulose (NFC). Animal-derived hydrogels are temperature-sensitive, complicating their use in automated workflows, particularly in automatic dispensing – a critical component of large-scale screens. In contrast, animal-free hydrogels like GrowDex^® are not temperature-sensitive, allowing for streamlined workflows and accurate dispensing at room temperature.

Liver organoids and microphysiological systems

Conference speaker Jonathan Sheard of UPM Biomedicals, gave an interesting talk on the application and benefits of adopting animal-free hydrogel GrowDex^® in the development of liver organoids towards their use in an advanced multicellular 3D human liver model known as a microphysiological system (MPS). 3D culture is especially critical for hepatocytes, since in traditional 2D culture, human hepatocytes lose their phenotype after approximately 2 weeks. 3D culture has unlocked the ability to retain hepatocyte phenotypes and functionality, and the ability to perform longer-term studies valuable in research and development.

The teams aiming to develop the MPS liver model spans multiple stakeholders from the pharmaceutical industry—including Merck, Janssen, Bristol-Myers Squibb, Roche, Sanofi Genentech—all seeking to develop more robust preclinical drug screening models.

Figure 2: Albumin expression from primary human hepatocytes cultured in GrowDex^® animal-free hydrogel, demonstrating stability over a 28-day culture period.

After trialing a number of cell seeding methods and growth matrices, embedding the hepatocytes into a GrowDex^® animal free hydrogel matrix was found to produce consistent growth, and a long-term culture that could remain stable for 28 days. Along with the ease of use and streamlined culture and retrieval workflows, this gives researchers an animal-free culture system, upon which to further develop their liver MPS [4].

This animal-free culture process can therefore provide hepatocytes with a microenvironment with minimal non-specific binding, minimal effect on hepatic cell function with long-term stability and viability.

Turning the tide against clinical failures

The 9th annual UPM Biomedicals conference highlighted the transformative potential of 3D cell culture in drug discovery and development. As the pharmaceutical industry grapples with the high failure rates and costs associated with drug development, the shift towards more human-relevant models like organoids and spheroids offers a promising path forward. These advanced 3D models, particularly when developed using animal-free materials, provide a more accurate representation of human biology, enhancing the predictability and reliability of preclinical studies.

By leveraging technologies such as organoids and spheroids, researchers can gain deeper insights into disease mechanisms, drug efficacy and safety, ultimately bridging the gap between preclinical models and human clinical trials. The adoption of animal-free cell culture material, such as GrowDex^® hydrogels, can further enhance this process by providing a consistent, reproducible, and sustainable alternative to traditional animal-derived materials.

As evidenced by the discussions and case studies presented at the conference, the integration of 3D cell culture into the drug discovery pipeline is not just a trend, but a significant leap towards more effective and human-centric therapeutics. The continuing advancements in this field are helping to accelerate drug discovery, reduce costs and improve success rates, paving the way for new groundbreaking therapies.

All the presentations from UPM Biomedicals 9^th annual conference are available to view online. Stay tuned as we continue to cover the highlights from the conference in our article series!

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References

1. Wouters, O. J., McKee, M., Luyten, J. (2020). Estimated Research and Development Investment Needed to Bring a New Medicine to Market, 2009-2018. JAMA, 323(9), 844–853. https://doi.org/10.1001/jama.2020.1166
2. Mantziou, V., Baillie-Benson, P., Jaklin, M., et al. (2021). In vitro teratogenicity testing using a 3D, embryo-like gastruloid system. Reproductive toxicology, 105, 72–90. https://doi.org/10.1016/j.reprotox.2021.08.003
3. Tran, T., Song, C. J., Nguyen, T., et al. (2022). A scalable organoid model of human autosomal dominant polycystic kidney disease for disease mechanism and drug discovery. Cell stem cell, 29(7), 1083–1101.e7. https://doi.org/10.1016/j.stem.2022.06.005
4. Baudy, A.R., Otieno, M.A., Hewitt, P., et al. Liver microphysiological systems development guidelines for safety risk assessment in the pharmaceutical industry (2020). Lab on a Chip, 20(2):215-225. https://doi.org/10.1039/c9lc00768g