Polyacrylamide hydrogels are interesting materials for studying cells and cell–material interactions, thanks to the possibility of precisely adjusting their stiffness, shear modulus and porosity during synthesis, and to the feasibility of processing and manufacturing them towards structures and devices with controlled morphology and topography. In this study a novel approach, related to the processing of polyacrylamide hydrogels using soft-lithography and employing microstructured templates, is presented. The main novelty relies on the design and manufacturing processes used for achieving the microstructured templates, which are transferred by soft-lithography, with remarkable level of detail, to the polyacrylamide hydrogels. The conceived process is demonstrated by patterning polyacrylamide substrates with a set of vascular-like and parenchymal-like textures, for controlling cell populations. Final culture of amoeboid cells, whose dynamics is affected by the polyacrylamide patterns, provides a preliminary validation of the described strategy and helps to discuss its potentials.