Yet the ethical question is becoming harder to ignore: if researchers can obtain meaningful biological information without immediately moving into live-animal experiments, should they?

The principle is consistent with the long-established 3Rs framework—replacement, reduction, and refinement—which encourages researchers to minimize animal use and distress while maintaining scientific quality. This does not mean that every in vivo experiment can or should disappear. Rather, it creates a strong incentive to develop experimental systems capable of answering more questions before animal studies begin.

This is where animal 3D cell culture models are gaining importance. Positioned between simple two-dimensional cultures and whole-organism models, 3D systems can reproduce selected aspects of tissue architecture, cellular organization, and microenvironmental conditions that are difficult to study in conventional monolayers. Veterinary research is increasingly exploring organoids and other 3D systems as tools for disease modeling, drug evaluation, infectious-disease research, and precision medicine.

The Technical Challenge Is Not Simply "Going 3D"

A 3D culture is not automatically a better model. Its scientific value depends on how closely its design matches the biological question.

One of the first decisions is the model format. Scaffold-based systems use natural or synthetic matrices to provide structural support, whereas scaffold-free approaches allow cells to aggregate or self-organize. Spheroids offer relatively accessible multicellular structures for screening, while organoids can reproduce selected architectural and functional characteristics of their tissue of origin. Tumoroids provide another specialized format for studying tumor biology and treatment responses.

Cell source is equally important. Immortalized cell lines can improve scalability and consistency, but primary cells may retain more biologically relevant characteristics. Stem-cell-derived systems can enable differentiation and self-organization, although their protocols may require tighter control of growth factors, extracellular matrices, and culture conditions.

Researchers also need to consider seeding density, nutrient availability, oxygen gradients, matrix stiffness, medium composition, culture duration, and the accessibility of analytical endpoints. Excessive spheroid size, for example, can produce strong diffusion limitations and central necrosis that may or may not reflect the intended biology. Matrix composition can also influence cell signaling and introduce batch-to-batch variability.

Consequently, a successful 3D model should be defined by measurable performance rather than appearance alone. Morphology, viability, phenotype, molecular markers, functional assays, imaging, and treatment response can all contribute to model qualification.

Where 3D Models Can Add the Most Value

Drug discovery is one of the most compelling areas for 3D cell culture because cellular responses observed in flat monolayers do not always translate directly to organized tissues. Three-dimensional architecture changes cell-cell interactions, extracellular-matrix signaling, diffusion, polarity, and gene-expression patterns. This can affect how a candidate penetrates a cellular structure and how different cell populations respond to treatment.

The implications extend well beyond conventional small-molecule screening. In veterinary R&D, 3D models may help investigate infectious disease, gastrointestinal biology, liver function, reproductive biology, mammary-gland physiology, oncology, regenerative medicine, and species-specific therapeutic responses. Recent reviews describe veterinary organoids derived from livestock and companion-animal tissues and highlight their expanding role in disease and drug research.

They may also support emerging therapeutic modalities. During animal-derived therapeutic peptide development, for example, an appropriately designed 3D assay may provide an intermediate platform for investigating biological activity, tissue response, penetration, or cytotoxicity before a smaller number of prioritized candidates advance to more complex studies.

Importantly, such models do not reproduce systemic absorption, metabolism, immune responses, organ-to-organ interactions, or whole-body pharmacokinetics. Their greatest value therefore lies not in claiming to replace every existing model, but in generating better evidence for deciding what should happen next.

Breaking the Translational Barrier with a Model Hierarchy

One of the persistent barriers in preclinical development is the gap between experimental simplicity and biological reality. A 2D assay may be highly controllable but overly reductionist. A complex animal study can provide systemic information but is costly, resource-intensive, and ethically consequential. Moving directly from one to the other may leave important mechanistic questions unresolved.

A more rational strategy is to construct a hierarchy of models.

Early feasibility can begin in conventional cell culture, followed by a purpose-built 3D system for tissue-level or multicellular questions. Only candidates that meet predefined criteria then progress toward customized animal model development.

At the in vivo stage, the research question can expand from local cellular effects to systemic exposure, pharmacokinetics, pharmacodynamics, dose-response relationships, pathology, biomarkers, imaging, and other endpoints available through integrated preclinical in vivo research solutions.

For some programs, rodents or other small-animal models may not adequately reproduce the anatomy, physiology, disease characteristics, or experimental procedures required for the next decision. Carefully selected preclinical large-animal research solutions can then serve as a later translational layer when species and model characteristics are scientifically justified.

The goal is not greater complexity at every stage. It is appropriate complexity at the appropriate stage.

Standardization May Be the Next Major Hurdle

Despite their promise, 3D systems still face important barriers. Model-to-model variability, differences in extracellular matrices, incomplete vascularization, limited immune-cell representation, difficulties in long-term culture, imaging constraints, and inconsistent protocols can complicate comparisons between laboratories. Recent veterinary reviews identify standardization and reproduction of vascular and immune microenvironments among the major technical challenges still facing organoid research.

This makes quality control essential. Researchers should document cell origin, passage number, matrix composition, culture conditions, model dimensions, differentiation state, acceptance criteria, and analytical methods. Wherever possible, results should be benchmarked against established biological data or corresponding tissues.

Emerging combinations of organoids, microfluidics, organ-on-chip systems, bioprinting, gene editing, advanced imaging, and computational analysis may address some of these limitations. However, adding technological sophistication only creates value when it improves the ability to answer a defined research question.

From Animal Replacement to Better Experimental Design

The most important contribution of 3D cell culture may ultimately be more nuanced than "replacing animal experiments."

Its greater potential lies in changing how researchers decide when an animal experiment is necessary.

A well-designed 3D system can provide a biologically richer screening environment, expose unsuitable candidates earlier, clarify mechanisms, and help refine the hypotheses carried into subsequent studies. When integrated with carefully selected in vivo models rather than treated as an isolated technology, it can support a more evidence-driven progression from molecular hypothesis to tissue-level response and, where necessary, whole-organism validation.

For veterinary and biomedical scientists, that represents both an ethical opportunity and a technical one: not simply doing fewer experiments, but extracting more relevant information from each experimental stage.

Beyond 2D: How 3D Cell Culture Models Are Reshaping Preclinical Research

For decades, animal studies have occupied a central position in biomedical and veterinary research.