Three-dimensional (3D) in vitro models have emerged as powerful tools that bridge the gap between conventional two-dimensional (2D) cultures and in vivo systems. Traditional 2D cultures fail to replicate the natural cellular microenvironment due to limited cell–cell and cell–matrix interactions [1], whereas 3D models overcome these limitations, making them essential for tumor research, tissue engineering, and fundamental biological studies [2,3]. Moreover, 3D systems provide an accurate and reproducible platform for drug testing, enabling optimized compound selection and improved tumor distribution analyses, while simultaneously reducing costs and minimizing animal use, thereby promoting efficient and ethical research practices. In our group, we have optimized and established a series of 3D models that progressively increase in structural and biological complexity, offering a robust platform suitable for highthroughput (HTS) and high-content screening (HCS) applications. Starting from simple homospheroids lacking extracellular matrix (ECM) components, we have systematically optimized each stage of our 3D modeling approach to enhance reproducibility, scalability, and physiological relevance. Using multiple formation platforms (ULA plates from Revvity, ThermoFisher, or Akura plates from InSphero), we refined culture conditions to ensure consistent HT compatibility. The subsequent incorporation of collagen matrices further improved the structural integrity and biological fidelity of the models [6]. By integrating two cell types within collagen, we established heterospheroid systems optimized for studying intercellular communication and tumor microenvironment dynamics, particularly in cancer research.
Building upon these optimized 3D systems, we developed advanced organoid models that recapitulate higher-order tissue organization and functionality [7]. These organoids extend the advantages of 3D culture by faithfully mimicking the architecture and physiological responses of native tissues, providing a robust and predictive platform for HTS and HCS. Finally, our prototype skin model represents the culmination of this optimization pipeline, integrating multiple cell types in a spatially organized 3D architecture that closely replicates native skin structure and function [8]. This level of complexity and refinement enables precise assessment of transdermal drug delivery, toxicity, and regenerative processes, underscoring the potential of our optimized 3D systems as a comprehensive screening platform. Collectively, these optimized 3D models highlight our capacity to recreate progressively complex and physiologically relevant biological systems. By integrating structural and cellular complexity at each stage, they form a robust platform for HCS and HTS, enabling efficient drug evaluation and generating results that more accurately reflect human tissue responses.
