Revitalizing immortalized cancer cell line research with 3D cultures: The time is ripe for transition from 2D to 3D

Bridging the divide between simplified in vitro assays and the complex biology of living tissues remains a central objective in cancer research. Although accumulating evidence demonstrates that three-dimensional (3D) culture models provide markedly superior representations of both developmental and malignant processes, two-dimensional (2D) monolayers remain the predominant in vitro platform. This slow transition is often rooted in the assumption that only primary tissue-derived models, referred to here as primary organoids, are suitable for physiologically relevant 3D culture. However, emerging data reveal that widely available, genetically tractable, and cost-effective immortalized cell lines can recapitulate key aspects of in vivo behavior when transitioned from 2D to 3D culture environments. This review synthesizes the evidence supporting the utility of immortalized cell lines in 3D environments, articulates the conceptual and practical advantages of this approach, and highlights experimental frameworks that can facilitate broader adoption. By leveraging 3D culture systems with immortalized lines, researchers can more effectively bridge the gap between simplified in vitro assays and the physiological conditions that drive tumor development and progression.