Preparation and Mechano-Functional Characterization of PEGylated Fibrin Hydrogels: Impact of Thrombin Concentration.

Three-dimensional (3D) neuronal cultures grown in hydrogels are promising platforms to design brain-like neuronal networks in vitro. However, the optimal properties of such cultures must be tuned to ensure a hydrogel matrix sufficiently porous to promote healthy development but also sufficiently rigid for structural support. Such an optimization is difficult since it implies the exploration of different hydrogel compositions and, at the same time, a functional analysis to validate neuronal culture viability. To advance in this quest, here we present a combination of a rheological protocol and a network-based functional analysis to investigate PEGylated fibrin hydrogel networks with gradually higher stiffness, achieved by increasing the concentration of thrombin. We observed that moderate thrombin concentrations of 10% and 25% in volume shaped healthy networks, although the functional traits depended on the hydrogel stiffness, which was much higher for the latter concentration. Thrombin concentrations of 65% or higher led to networks that did not survive. Our results illustrate the difficulties and limitations in preparing 3D neuronal networks, and stress the importance of combining a mechano-structural characterization of a biomaterial with a functional one.