The mechanical microenvironment plays a crucial role in the evolution of colorectal cancer, a complex disease characterized by heterogeneous tumors with varying elasticity. Toward setting up distinct scenarios, herein, we describe the preparation and characterization of gelatin methacrylamide (GelMA)-based hydrogels via two different mechanisms: free-radical photopolymerization and photo-induced thiol-ene reaction. A precise stiffness modulation of covalently crosslinked scaffolds was achieved through the application of well-defined irradiation times while keeping the intensity constant. Besides, the incorporation of thiol chemistry strongly increased stiffness with low to moderate curing times. This wide range of finely tuned mechanical properties successfully covered from healthy tissue to colorectal cancer stages. Hydrogels prepared in phosphate-buffered saline or Dulbecco’s modified Eagle’s medium resulted in different mechanical and swelling properties, although a similar trend was observed for both conditions: thiol-ene systems exhibited higher stiffness and, at the same time, higher swelling capacity than free-radical photopolymerized networks. In terms of biological behavior, three of the substrates showed good cell proliferation rates according to the formation of a confluent monolayer of Caco-2 cells after 14 days of cell culture. Likewise, a characteristic apical-basal polarization of cells was observed for these three hydrogels. These results demonstrate the versatility of the presented platform of biomimetic materials as in vitro cell culture scaffolds.

From the first experiments with biomaterials to mimic tissue properties, the mechanical and biochemical characterization have evolved extensively. A number of properties can be described, however, what should be essential is to conduct a proper and physiologically relevant characterization. Herein, we describe the influence of the reaction media and buffer media –phosphate buffer saline (PBS) and Dulbecco's modified Eagle's medium (DMEM) with two different glucose concentrations– in GelMA hydrogel mechanics and in the biological behavior of two tumoral cell lines (Caco‐2 and HCT‐116). All scaffolds were photocrosslinked using UV light under identical conditions and evaluated for mass swelling ratio and stiffness. Our results indicate that stiffness is highly susceptible to the reaction media, but not to the swelling media. In addition, PBS‐prepared hydrogels exhibited a higher photopolymerization degree as confirmed by HR‐MAS NMR. These findings correlate with the biological response of Caco‐2 and HCT‐116 cells seeded on the substrates, which demonstrated flatter morphologies on stiffer hydrogels. Overall, cell viability and proliferation were excellent for both cell lines, and Caco‐2 cells displayed a characteristic apical‐basal polarization as observed in F‐actin/Nuclei fluorescence images. These characterization experiments highlight the importance of conducting mechanical testing of biomaterials in the same medium as cell culture.This article is protected by copyright. All rights reserved