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.

Rheological Characterization of Three-Dimensional Neuronal Cultures Embedded in PEGylated Fibrin Hydrogels.

Three-dimensional (3D) neuronal cultures are valuable models for studying brain complexity in vitro, and the choice of the bulk material in which the neurons grow is a crucial factor in establishing successful cultures. Indeed, neuronal development and network functionality are influenced by the mechanical properties of the selected material; in turn, these properties may change due to neuron-matrix interactions that alter the microstructure of the material. To advance our understanding of the interplay between neurons and their environment, here we utilized a PEGylated fibrin hydrogel as a scaffold for mouse primary neuronal cultures and carried out a rheological characterization of the scaffold over a three-week period, both with and without cells. We observed that the hydrogels exhibited an elastic response that could be described in terms of the Young's modulus E. The hydrogels without neurons procured a stable E≃420 Pa, while the neuron-laden hydrogels showed a higher E≃590 Pa during the early stages of development that decreased to E≃340 Pa at maturer stages. Our results suggest that neurons and their processes dynamically modify the hydrogel structure during development, potentially compromising both the stability of the material and the functional traits of the developing neuronal network.

Rich dynamics and functional organization on topographically designed neuronal networks in vitro.

Neuronal cultures are a prominent experimental tool to understand complex functional organization in neuronal assemblies. However, neurons grown on flat surfaces exhibit a strongly coherent bursting behavior with limited functionality. To approach the functional richness of naturally formed neuronal circuits, here we studied neuronal networks grown on polydimethylsiloxane (PDMS) topographical patterns shaped as either parallel tracks or square valleys. We followed the evolution of spontaneous activity in these cultures along 20 days in vitro using fluorescence calcium imaging. The networks were characterized by rich spatiotemporal activity patterns that comprised from small regions of the culture to its whole extent. Effective connectivity analysis revealed the emergence of spatially compact functional modules that were associated with both the underpinned topographical features and predominant spatiotemporal activity fronts. Our results show the capacity of spatial constraints to mold activity and functional organization, bringing new opportunities to comprehend the structure-function relationship in living neuronal circuits.

Dynamic and Functional Alterations of Neuronal Networks In Vitro upon Physical Damage: A Proof of Concept.

There is a growing technological interest in combining biological neuronal networks with electronic ones, specifically for biological computation, human-machine interfacing and robotic implants. A major challenge for the development of these technologies is the resilience of the biological networks to physical damage, for instance, when used in harsh environments. To tackle this question, here, we investigated the dynamic and functional alterations of rodent cortical networks grown in vitro that were physically damaged, either by sequentially removing groups of neurons that were central for information flow or by applying an incision that cut the network in half. In both cases, we observed a remarkable capacity of the neuronal cultures to cope with damage, maintaining their activity and even reestablishing lost communication pathways. We also observed-particularly for the cultures cut in half-that a reservoir of healthy neurons surrounding the damaged region could boost resilience by providing stimulation and a communication bridge across disconnected areas. Our results show the remarkable capacity of neuronal cultures to sustain and recover from damage, and may be inspirational for the development of future hybrid biological-electronic systems.