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Hydrogels are crosslinked polymeric gels of great interest in the field of tissue engineering, particularly as biocompatible cell or drug carriers. Reagent-free electron irradiated gelatin is simple to manufacture, inexpensive and biocompatible. Here, the potential to micropattern gelatin hydrogel surfaces during electron irradiation crosslinking was demonstrated as a promising microfabrication technique to produce thermally stable structures on highly relevant length scales for bioapplications. In the present work, grooves of 3.75 to 170 µm width and several hundred nanometers depth were transferred onto gelatin hydrogels during electron irradiation and characterized by 3D confocal microscopy after exposure to ambient and physiological conditions. The survival and influence of these microstructures on cellular growth was further characterized using NIH 3T3 fibroblasts. Topographical modifications produced surface structures on which the cultured fibroblasts attached and responded by adapting their morphologies. This developed technique allows for simple and effective structuring of gelatin and opens up new possibilities for irradiation crosslinked hydrogels in biomedical applications in which cell attachment and contact guidance are favored. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 320-330, 2018. [ABSTRACT FROM AUTHOR]