How to Choose Your 3D Assay with Confidence

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Next-generation in vitro tests boost biological insights

Traditional 2D monolayer cell cultures often fail to accuratelyreplicate the complex cellular microenvironment found in living organisms. In contrast, 3D cell-based assays allow cells to grow into three-dimensional structures that more closely mimic the architecture and interactions found in tissues and organs. This enables researchers to observe cellular behavior, such as cell-cell communication, migration, and differentiation, in a more physiologically relevant context.

Key characteristics for selecting a fit-for-purpose 3D cell-based assay

Choosing a 3D cell-based assay technology for adoption in drug discovery programs (safety and pharmacology) requires evaluating both the biological and operational aspects of an assay within adefined context of use:

1. Relevance to in vivo biology: The technology should mimic the in vivo physiological microenvironment and pathophysiological responses of test items after validation at the biochemical and cellular level.
2. Scalability and throughput: The system should allow for the required throughput and be compatible with automated liquid handling, plate readers, and compound library formats (often 384-well SBS standard) to ensure the generation of high-quality data and to minimize errors.
3. Reproducibility and reliability: The technology should demonstrate consistent performance and reproducibility, from plate to plate and batch to batch, producing standardized assays at scale matching 2D assay quality.
4. Readout compatibility: Given the wealth of biological information in a 3D model, compatibility with readouts ranging from standard biochemical assays to high-content imaging, transcriptomics, and even histopathology is key to creating information-rich, physiologically relevant, and predictive data.
5. Cost-effectiveness: The initial investment costs (such as the evaluation costs but especially the costs associated with consumables, cell consumption, and operations) must be considered for the throughput expected. Lack of automation compatibility adds to the operational cost significantly.
6. Turn around time (TAT): Often, 3D assays, especially organoids and organ-on-chip systems, come at the expense of lengthy tissue-culture protocols spanning several weeks and thus increase TAT and the risk of contamination.

For liver-safety testing, more complexity is not automatically better

The table above shows a comparison of classical 2D liver models and next-generation alternatives (spheroids, organoids, and organ-on-chip systems). While all advanced models show a superior biological relevance, there are substantial differences in scalability, reproducibility, compatibility with existing lab infrastructure and workflows, as well as cost-effectiveness. These characteristics, however, are crucial for industry adoption and are often overlooked when next-generation models are evaluated.

Operational advantages do not come at the expense of biological relevance: Spheroid-based systems provide a sensitivity up to 92% and specificity of 84%,¹ which is better or comparable with recently published data² in a much more complex organ-on-chip system.

References
1. Wolf, A. et al. J. Soc. Toxicol (2023).
2. Ewart L, Apostolou A, Briggs SA, et al. Commun. Med. 2022; 2: 154.

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