The ability to tailor mechanical properties and architecture is crucial in creating macroporous hydrogel scaffolds for tissue engineering. In the present work, an approach for tailoring the pore size and stiffness of acrylamide-based cryogels via the regulation of an electron beam irradiation dose is demonstrated. The samples were characterized by equilibrium swelling measurements, light and scanning electron microscopy, mercury porosimetry, Brunauer-Emmett-Teller surface area analysis and stiffness meas-urements, and their properties were compared to those of cryogels prepared by a standard redox-initiated radical polymerization. A model reaction with 125I radiolabeled azidopentanoyl-GGGRGDSGGGY-NH2 peptide was performed to determine the capacity for biomimetic modification. The performance of the prepared substrates was tested in cell culture of adipose-derived stem cells.. Furthermore, the feasibility of preparing layered hydrogels was demonstrated, which may be key to the future preparation of complex hydrogel-based scaffolds that mimic the extracellular matrix for a wide range of tasks.