Beyond monolayers: a comparative analysis of 2D cell cultures and 3D in vitro models as new approach methodologies.

Two-dimensional (2D) cell cultures have been used as in vitro models in biomedical research as fundamental tools for investigating biological processes. However, their limited capacity to recapitulate the complexity of human tissues has driven the search for more physiologically relevant in vitro systems. In parallel, limitations in the predictive capacity, costs, and ethical concerns associated with animal models have reinforced the importance of the 3Rs principles (Replacement, Reduction, and Refinement), introduced in 1959, and more recently contributed to the emergence of New Approach Methodologies (NAMs). In this context, three-dimensional (3D) cell culture models, such as spheroids, organoids, and bioprinted models have been developed. This literature review focuses on studies that directly compare spheroids, organoids, and 3D-printed models with their 2D culture counterparts, highlighting their potential and limitations as human-relevant NAMs.