Dual-Crosslinked Alginate-Based Hydrogels with Tunable Mechanical Properties for Cultured Meat

Abstract

Cultured meat refers to the production of animal tissue by utilizing the same techniques as tissue engineering through cell culture. Various biomaterials have been designed to serve as in vitro supports for cell viability, growth, and migration. In this study, visible light and dual-crosslinked alginate hydrogels were designed to enable control of the physical and mechanical properties needed for the fabrication of cultured meat scaffolds. We hypothesized that a difference in hydrogel stiffness would influence cell behavior, indicating the efficacy of our processing methods to benefit the cultured meat field. Herein, we synthesized and created: (1) methacrylated alginate (AlgMA) to enable covalent crosslinking via visible light exposure, (2) Methacrylated alginate and arginyl-glycyl-aspartic acid RGD conjugates (AlgMA-RGD), using carbodiimide chemistries to provide cell-binding sites on the material, and (3) designer hydrogels incorporating different crosslinking techniques. The material and mechanical properties were evaluated to determine the structural integrity of the hydrogels, and in vitro cell assays were conducted to verify cytocompatibility and cell adhesion. Gelation, swell ratio, and weight loss calculations revealed longer gelation times for the AlgMA scaffolds and similar physical properties for all hydrogel groups. We showed that by adjusting the polymer concentration and the crosslinking methodology, the scaffold’s mechanical properties can be controlled and optimized within physiological ranges. Incorporating dual crosslinking significantly increased the compressive moduli of the AlgMA hydrogels, compared to visible-light crosslinking alone. Moreover, the muscle satellite cells responded favorably to the AlgMA scaffolds, with clear differences in cell density when cultured on materials with significantly different mechanical properties. Our results indicate the usefulness of the dual-crosslinking alginate hydrogel system to support in vitro meat growth.