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

Abstract

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.

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

“…This result may be attributed to the absence of living cells within its structure. This hypothesis is supported by our previous work [17], where we examined the evolution of T2-like hydrogels with and without cells. For matrices without cells we measured a Young's modulus of ≃420 Pa that did not change during development.…”

“…All hydrogel samples contain an ensemble of cortical neurons and glia cells that self-organize into a living neuronal network (Figure 1a), and will be referred to as T1, T2, and T3 PEGylated fibrin, respectively, (Figure 1b). We note that the mechanical characterization of T2 hydrogels was previously examined in a publication from our group [17]. In order to establish a relationship between the mechanical properties of the hydrogel scaffolds and the functional organization of the resultant neuronal network (Figure 1c), we present first the rheological characterization of the hydrogels with neurons, and then describe their functional organization as a living neuronal network.…”

“…We followed the protocol described in López-León et al [17] with minor modifications, as detailed in Methods. Hydrogels were first studied at day in vitro (DIV) 1, i.e., 24 h after culturing the cells, and their mechanical properties were monitored every 4-5 days up to 3 weeks.…”

“…A total of three rheological tests were performed to determine the Young's modulus E of the T1-T3 PEGylated fibrin hydrogels [17]. These tests were carried out every 3-4 DIVs, over a three-week period, and on each of the T1 to T3 hydrogels placed on the 8 mm PDMS cavities.…”

“…In the experiments presented here, even though rheological quantities slightly changed with γ 0 in this range of strain, this value of γ 0 = 5% was used for the strain amplitude to characterize the dependence in frequency. [17]. The instrument has a lower measurable limit of 2 nN•m of the oscillation torque, as illustrated in Figure 1.…”