Miruna-Maria Furtuna 1 *, Camelia Dascalu 1, Bogdan-Ionel Tamba 1
1 Prof. Ostin C. Mungiu Advanced Research and Development Center for Experimental Medicine – CEMEX, Grigore T. Popa University of Medicine and Pharmacy, Iasi, Romania
* Correspondence to: Miruna-Maria Furtuna, Prof. Ostin C. Mungiu Advanced Research and Development Center for Experimental Medicine – CEMEX, Grigore T. Popa University of Medicine and Pharmacy, Mihail Kogălniceanu Street 9-13, Iasi 700259, Romania. E-mail: miruna.furtuna@umfiasi.ro
Abstract
Two-dimensional monolayer cultures remain the most commonly used preclinical systems in anticancer drug discovery, yet their oversimplified structure fails to recapitulate the biochemical, mechanical, and architectural complexity of solid tumors. In 2D platforms, cells grow on rigid plastic surfaces under homogeneous conditions, with unrestricted access to nutrients, oxygen, and therapeutic agents. These artificial conditions distort key biological features such as proliferation dynamics, gene expression patterns, metabolic adaptation, extracellular matrix (ECM) signaling, and drug response heterogeneity. In contrast, three-dimensional multicellular tumor spheroids provide a physiologically relevant model that mimics essential characteristics of the tumor microenvironment, including oxygen and nutrient diffusion gradients, ECM remodeling, spatially stratified proliferation, and barriers to drug penetration. Rapid progress between 2021 and 2025 has expanded the technological landscape of spheroid generation—ranging from scaffold-free aggregation methods to ECM-rich scaffold-based systems, microfluidic tumor-on-a-chip platforms emulating vascular perfusion, and bioprinted architectures allowing precise spatial organization. This narrative review summarizes recent advances in spheroid generation, characterization, and application across therapeutics, pharmacology, and toxicology. We examine scaffold-free and scaffold-based approaches, microfluidic technologies, bioprinting strategies, and analytical readouts including high-content confocal microscopy, live imaging, and mass spectrometry imaging. We highlight the unique capacity of spheroids to provide realistic insights into drug penetration, chemotherapeutic efficacy, nanocarrier distribution, resistance mechanisms, and immunotherapy performance. Spheroids support organ-specific toxicity testing, long-term safety assessment, and alignment with the 3R principles by reducing animal use. Key limitations—variability, lack of vascularization, and analytical complexity—are critically assessed. Finally, we introduce the Pharmacological Relevance Index, a multidimensional descriptive framework that captures the conceptual and translational significance of spheroid models by integrating cellular complexity, ECM context, geometric control, analytical throughput, translational linkage, and toxicological breadth. Ultimately, tumor spheroids should be understood not merely as improved in vitro tools, but as customizable 3D micro-ecosystems capable of bridging early drug discovery and clinical translation.