Model-Dependent EV Biomarker Signatures: From Culture Systems to Translational Perspectives

Extracellular Vesicles (EVs) have emerged as key mediators of intercellular communication and, more recently, as promising biomarkers for disease diagnosis, prognosis, and therapeutic monitoring. In vitro cellular models are essential tools for investigating EV biogenesis, cargo composition, and biological functions. This review provides an overview of EV biology, including their classification, molecular content, biological functions and clinical relevance. It summarizes the main two-dimensional (2D) and three-dimensional (3D) culture systems currently used in EV research. The literature suggests that 3D models more closely recapitulate in vivo architecture, whereas conventional 2D cultures may introduce biological biases that affect experimental outcomes. While these aspects are presented from a simplified perspective to provide necessary background, the review primarily evaluates whether and how culture model selection influences EV production and features. In the final sections, we summarize and discuss evidence comparing EV yield, molecular cargo, functional properties, and biomarker profiles from 2D and 3D systems, and how they reflect in vivo settings. By examining these differences, we discuss their implications for interpreting EV-based studies and the importance of model choice in identifying and validating reliable EV biomarkers.