Primary cell cultures versus immortalized cell lines: critical comparative insights into biological fidelity and translational applications

In vitro cell culture models are fundamental to biomedical research, yet the choice between primary cell cultures and immortalized cell lines profoundly influences experimental outcomes. Immortalized lines offer convenience and unlimited proliferative capacity but frequently acquire genetic, epigenetic, and phenotypic alterations that compromise tissue-specific functions and overall biological fidelity. In contrast, primary cell cultures preserve native morphology, signaling pathways, and functional characteristics of their tissue of origin, providing a more physiologically relevant model. While previous reviews have outlined the general advantages of primary cells, few critically synthesize comparative data across cell types or evaluate strategies to mitigate inherent limitations such as finite lifespan and donor variability. This review critically examines the comparison between primary and immortalized cells across different cell types in terms of preserved tissue-specific characteristics, genomic and epigenetic fidelity, predictive value in drug testing and disease modeling, and emerging roles in personalized medicine. We further discuss innovative approaches, including conditional reprogramming, optimized donor selection, pooled-donor strategies, and standardized culture protocols, that enhance reproducibility and functional stability. By integrating comparative evidence with methodological advances, this review provides a critical framework for selecting appropriate in vitro models, highlighting the translational significance of primary cells in drug discovery, precision medicine, and biomedical research.