Most methods to culture cells in three-dimensions depend on a cell-seedable biomaterial to define the global structure of the culture and the microenvironment of the cells. Efforts to tailor these scaffolds have focused on the chemical and mechanical properties of the biomaterial itself. Besides these efforts, techniques for sensing local oxygen level within the extracellular microenvironment have been developed to elucidate roles of major metabolites in the biological and physiological context. This study presents a strategy to control the distribution of soluble chemicals and to monitor oxygen concentrations within the bulk of a three-dimensional tissue scaffold. The presentation of this strategy includes: 1) a lithographic technique to build functional microfluidic structures directly within a calcium alginate hydrogel seeded with cells, 2) synthesis of oxygen sensing microbeads, 3) characterization of the lithographic process with respect to microstructural fidelity and cell viability, 4) characterization of convective and diffusive mass transfer of small and large solutes, and oxygen within this microfluidic scaffold, and 5) demonstration of temporal and spatial control of the distribution of non-reactive solutes and reactive solutes within the bulk of the scaffold. In vitro culture of primary chondrocytes and HepG2/C3A cell lines will be presented to illustrate the use of this new culture format to control and monitor cellular function.