This article describes a microfluidic device for cell culture and chemotaxis studies under various temporal and spatial concentration gradients of the medium or chemoattractant. Vertical membranes formed using in situ fabrication are employed to avoid fluid flow inside the cell observation chamber. Thus, the medium and chemoattractants are primarily provided by diffusion, maintaining cell-cell communication via secreted factors. Neutrophils were used to demonstrate the capability of the device for chemotaxis research. Experiments exhibited successful migration up a concentration gradient of interleukin 8.