Wound healing is an essential physiological process for tissue homeostasis, involving multiple types of cells, extracellular matrices, and growth factor/chemokine interactions. Many in vitro studies have investigated the interactions between cues mentioned above; however, most of them only focused on a single factor. In the present study, we design a wound healing device to recapitulate in vivo complex microenvironments and heterogeneous cell situations to investigate how three types of physiologically related cells interact with their microenvironments around and with each other during a wound healing process. Briefly, a microfluidic device with a micropillar substrate, where diameter and interspacing can be tuned to mimic the topographical features of the 3D extracellular matrix, was designed to perform positional cell loading on the micropillar substrate, co-culture of three types of physiologically related cells, keratinocytes, dermal fibroblasts, and human umbilical vein endothelial cells, as well as an investigation of their interactions during wound healing. The result showed that cell attachment, morphology, cytoskeleton distribution, and nucleus shape were strongly affected by the micropillars, and these cells showed collaborative response to heal the wound. Taken together, these findings highlight the dynamic relationship between cells and their microenvironments. Also, this reproducible device may facilitate the in vitro investigation of numerous physiological and pathological processes such as cancer metastasis, angiogenesis, and tissue engineering.