From Primary Cells to Organoids: Expanding the Biological Relevance of In Vitro Cellular Models

Abstract

Highlights What are the main findings? In vitro models form a continuum from primary cells and established cell lines to iPSC-derived organoids and advanced bioengineered systems, with distinct strengths and limitations in modeling human biology. Biological relevance must be balanced with reproducibility, scalability, experimental control, and fit-for-purpose characterization and validation. What are the implications of the main findings? Model selection should be driven by the biological question and intended context of use rather than by model complexity alone. Standardization, bioengineering, automation, and AI will be important for translating advanced cellular models into robust NAMs for biomedical research and drug development. Abstract Advanced in vitro cellular models have become indispensable tools for investigating development, disease mechanisms, drug discovery, regenerative medicine, and precision therapeutics. Recent advances in primary cell isolation, cell immortalization, induced pluripotent stem cell (iPSC) technology, and three-dimensional (3D) organoid culture have substantially improved the physiological relevance and versatility of these experimental systems. However, selecting, developing, and validating appropriate in vitro models remain challenging due to differences in biological complexity, reproducibility, and functionality. In this review, we summarize the major strategies for establishing advanced in vitro cellular models, highlighting the principles underlying primary cell derivation, cell immortalization, iPSC reprogramming, and organoid generation. We further discuss critical factors that influence model development, including cell source selection, signaling pathways, microenvironmental regulation, and tissue self-organization, together with current approaches for characterization and functional validation. Finally, we examine emerging bioengineering technologies, key limitations, and future directions for developing increasingly robust, reproducible, and physiologically relevant in vitro models. This review provides a practical framework for researchers seeking to develop and apply advanced cellular model systems for basic research and translational biomedical applications.