Evaluation of gelatin-based hydrogels for colon and pancreas studies using 3D in vitro cell culture.

Biomimetic 3D models emerged some decades ago to address 2D cell culture limitations in the field of replicating biological phenomena, structures or functions found in nature. The fabrication of hydrogels for cancer disease research enables the study of cell processes including growth, proliferation and migration and their 3D design is based on the encapsulation of tumoral cells within a tunable matrix. In this work, a platform of gelatin methacrylamide (GelMA)-based photocrosslinked scaffolds with embedded colorectal (HCT-116) or pancreatic (MIA PaCa-2) cancer cells is presented. Prior to cell culture, the mechanical characterization of hydrogels was assessed in terms of stiffness and swelling behavior. Modifications of the UV curing time enabled a fine tuning of the mechanical properties, which at the same time, showed susceptibility to the chemical composition and crosslinking mechanism. All scaffolds displayed excellent cytocompatibility with both tumoral cells while eliciting various cell responses depending on the microenvironment features. Individual and collective cell migration were observed for HCT-116 and MIA PaCa-2 cell lines, highlighting the ability of the colorectal cancer cells to cluster into aggregates of different sizes governed by the surrounding matrix. Additionally, metabolic activity results pointed out to the development of a more proliferative phenotype within stiffer networks. These findings confirm the suitability of the presented platform of GelMA-based hydrogels to conduct 3D cell culture experiments and explore biological processes associated with colorectal and pancreatic cancer.

The Mechanical and Biological Performance of Photopolymerized Gelatin-Based Hydrogels as a Function of the Reaction Media.

From the first experiments with biomaterials to mimic tissue properties, the mechanical and biochemical characterization has evolved extensively. Several properties can be described, however, what should be essential is to conduct a proper and physiologically relevant characterization. Herein, the influence of the reaction media (RM) and swelling media (SM)-phosphate buffered saline (PBS) and Dulbecco's modified Eagle's medium (DMEM) with two different glucose concentrations-is described in gelatin methacrylamide (GelMA) hydrogel mechanics and in the biological behavior of two tumoral cell lines (Caco-2 and HCT-116). All scaffolds are UV-photocrosslinked under identical conditions and evaluated for mass swelling ratio and stiffness. The results indicate that stiffness is highly susceptible to the RM, but not to the SM. Additionally, PBS-prepared hydrogels exhibited a higher photopolymerization degree according to high resolution magic-angle spinning (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 are excellent for both cell lines, and Caco-2 cells displayed a characteristic apical-basal polarization based on F-actin/Nuclei fluorescence images. These characterization experiments highlight the importance of conducting mechanical testing of biomaterials in the same medium as cell culture.

Tuning of Mechanical Properties in Photopolymerizable Gelatin-Based Hydrogels for In Vitro Cell Culture Systems.

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.