While 2D culture models have been used extensively to elucidate the cell-to-cell communication, they do not recapitulate fully the 3D characteristics of microenvironment in vivo, e.g., polarized cell attachment and generally confer a considerably stiffer substrate than the endogenous extracellular matrix. Development of fibrous scaffolds that can better mimic the native microenvironment and improve the spatial arrangement of seeded cells should foster experimental strategies to monitor and determine the 3D cell-to-cell communication. In this study, poly(ε- caprolactone) (PCL) fibers were fabricated in different sizes using a microfluidic platform and spatially arranged to create a suitable 3D microenvironment in order to investigate the cell viability and calcium signaling in mouse astrocytes. A powerful algorithm, referred to as wavelet transform coherence (WTC), was applied to establish the correlation between astrocytes that were seeded on the PCL fiber. As expected, two astrocytes that appeared to be in physical contact showed high correlation, whereas two astrocytes seeded within a few cell lengths but not in physical contact showed negligible correlation. The WTC correlation analysis of a cluster of six astrocytes seeded on a single PCL fiber led to surprising results that the cells can communicate over many cell lengths without being in physical contact. More systematic studies using spatially controlled 3D microenvironment will likely help unravel the intricate cell communication mechanisms.