Strategies to control cellular spatial organization in microphysiological systems

Spatial organization is fundamental to tissue physiology, as it governs how cells migrate, grow, differentiate, and interact within their native environments. In living tissues, cells are positioned within finely tuned microarchitectures defined by chemical gradients, boundaries, and mechanical cues - features that are essential for proper tissue function and homeostasis. Microphysiological systems (MPSs) aim to replicate key aspects of human tissue in vitro, yet without appropriate spatial control, they often fail to reproduce certain aspects of tissue-level organization and function. In this review, we categorize spatial patterning strategies into two main approaches: direct methods, which involve the physical placement of cells or compartments using techniques such as 3D bioprinting, microfluidic compartmentalization, and physical trapping; and indirect methods, which rely on cellular responses to engineered environmental cues, including extracellular matrix (ECM) composition, mechanical gradients, and soluble factor distributions. While direct methods offer precision and reproducibility, indirect strategies more closely reflect natural developmental and self-organizing processes. We discuss how these approaches are applied across diverse biological structures, from cellular interfaces and barrier tissues to dynamic host-microbe systems. Enhancing spatial fidelity in MPSs is essential for recapitulating tissue complexity, and will be key to advancing disease modeling, developmental biology, and drug screening applications.