Ultrasoft Alginate Hydrogels Support Long-Term Three-Dimensional Functional Neuronal Networks

Abstract

Neuron development and function are exquisitely sensitive to the mechanical properties of their surroundings. Three-dimensional (3D) cultures are therefore being explored as they better mimic the features of the native extracellular matrix. Limitations of existing 3D culture models include poorly defined composition, rapid degradation, and suboptimal biocompatibility. Here we show that ionically cross-linked ultrasoft hydrogels made from unmodified alginate can potently promote neuritogenesis. Alginate hydrogels were characterized mechanically and a remarkable range of stiffness (10–4000 Pa) could be produced by varying the macromer content (0.1–0.4% w/v) and CaCl₂ concentration. Dissociated rat embryonic cortical neurons encapsulated within the softest of the hydrogels (0.1% w/v, 10 mM CaCl₂) showed excellent viability, extensive formation of axons and dendrites, and long-term activity as determined by calcium imaging. In conclusion, alginate is an off-the-shelf, easy to handle, and inexpensive material, which can be used to make ultrasoft hydrogels for the formation of stable and functional 3D neuronal networks. This 3D culture system could have important applications in neuropharmacology, toxicology, and regenerative medicine.