The coordinated behavior of living cells is responsible for tissue development, growth, and regeneration. Understanding the mechanisms by which external and intrinsic cues regulate cell fate and function is cmcial to the success of engineered tissues and cellbased therapies in the treatment of human disease. These cellular microenvironment signals have recently been investigated using 2-D micropatterning tools. However, there is growing evidence that microenvironment cues expressed in a 3-D context are critical to reflect in vivo cell responses, such that tools to control cellular organization and local chemistry in 3-D are greatly needed. This dissertation aims to develop a novel tool for cell patterning within hydrogels that is:​ (1) high resolution to define microscale cell organization and cell-cell interactions, (2) versatile and compatible with many biomaterials and cell types, and (3) compatible with standard assays of cell fate and function.

In this work, mammalian cells were rapidly organized within a photopolymerizable poly(ethylene glycol) (PEG) hydrogel using dielectrophoretic (DEP) forces. First, patterning kinetics were mathematically modeled and validated to optimize experimental conditions. Next, various electrode configurations were explored to create well-defined cell organizations that modulate cell-cell interactions, particularly linear patterns and cluster arrays, and design rules were developed for the reproducible fabrication of cell electropattemed hydrogel (CEPH) constructs. This platform was then applied to the study of 3-D cell organization within articular cartilage, the low-friction, load-bearing, wear-resistant material that allows for normal joint motion. Cartilage is often damaged or diseased but has little intrinsic healing capacity;​ thus, engineering strategies attempt to recapitulate native tissue properties by coaxing the regeneration of the extracellular matrix (ECM). Biosynthesis of glycosaminoglycan, a principal component of the ECM, was downregulated nearly two-fold by clustered chondrocytes relative to randomly dispersed cells, independent of volumetric cell density, viability, mass transfer, and hydrogel chemistry. However, chondrocytes surrounded by a thin matrix were insensitive to cell organization. These findings suggest that chondrocyte organization may be an important regulator of biosynthetic function, carrying implications for engineered tissues as well as in vivo cartilage growth, homeostasis and degeneration, and the methods developed herein may have similar impact in other organ systems.