Microfluidic devices have been used in the last two decades to study in vitro cell chemotaxis, but few existing devices generate gradients in flow-free conditions. Flow can bias cell directionality of adherent cells and precludes the study of swimming cells like naïve T lymphocytes, which only migrate in a non-adherent fashion. We developed two devices that create stable, flow-free, diffusion-based gradients and are adapted for adherent and swimming cells. The flow-free environment is achieved by using agarose gel barriers between a central channel with cells and side channels with chemoattractants. These barriers insulate cells from injection/rinsing cycles of chemoattractants, they dampen residual drift across the device, and they allow co-culture of cells without physical interaction, to study contactless paracrine communication. Our devices were used here to investigate neutrophil and naïve T lymphocyte chemotaxis.