Collagen-Dependent Neurite Outgrowth and Response to Dynamic Deformation in Three-Dimensional Neuronal Cultures

Abstract

Abstract-In vitro models of brain injury that use thick 3-D cultures and control extracellular matrix constituents allow evaluation of cell-matrix interactions in a more physiologically relevant configuration than traditional 2-D cultures. We have developed a 3-D cell culture system consisting of primary rat cortical neurons distributed throughout thick (>500 lm) gels consisting of type IV collagen (Col) conjugated to agarose. Neuronal viability and neurite outgrowth were examined for a range of agarose (AG) percentages (1.0-3.0%) and initial collagen concentrations ([Col] i ; 0-600 lg/ mL). In unmodified AG, 1.5% gels supported viable cultures with significant neurite outgrowth, which was not found at lower (£1.0%) concentrations. Varying [Col] 30, 150, and 300 lg/mL, which supported cultures with similar baseline viability and neurite outgrowth. Conjugation of Col to AG also increased the complex modulus of the hydrogel. Following high rate deformation, neuronal viability significantly decreased with increasing [Col] i , implicating cell-matrix adhesions in acute mechanotransduction events associated with traumatic loading. These results suggest interrelated roles for matrix mechanical properties and receptor-mediated cell-matrix interactions in neuronal viability, neurite outgrowth, and transduction of high rate deformation. This model system may be further exploited for the elucidation of mechanotransduction mechanisms and cellular pathology following mechanical insult.

Advances in diagnosis, treatments, and molecular mechanistic studies of traumatic brain injury

“…However, several studies have indicated that it may be an initiator of TBI. In an in vitro study, quick deformation of three-dimensional collagen gels led to a decrease in embedded neuronal viability when the collagen concentration increased, indicating the potential impact of cell-ECM interactions on injury (82). Another study suggested that ECM influences axonal injury by activating Rho signaling pathways; up-regulation of RhoA was accompanied by fluid percussion brain injury (83).…”

In Vitro Models for Biomechanical Studies of Neural Tissues

“…Moreover, the ECM components and cellular distribution are difficult to control in reaggregate cultures; however, these models are extremely useful for studying cell-cell interactions, growth, and function at cell densities that closely match those found in vivo. Three-dimensional cell culture models consisting of neural cells distributed throughout a matrix have also been developed [13,[15][16][17][30][31][32]. In these systems, trade-offs exist between culture thickness (i.e., surface area to volume ratio), and hence the scope of 3D spatial interactions, and cell density, necessitating that relatively thick ([500 lm) cultures use cell densities at least an order of magnitude lower than that found in brain cortices [32,33].…”

“…Three-dimensional cell culture models consisting of neural cells distributed throughout a matrix have also been developed [13,[15][16][17][30][31][32]. In these systems, trade-offs exist between culture thickness (i.e., surface area to volume ratio), and hence the scope of 3D spatial interactions, and cell density, necessitating that relatively thick ([500 lm) cultures use cell densities at least an order of magnitude lower than that found in brain cortices [32,33]. However, using these 3D scaffold-based neural cultures, key parameters governing neuronal survival and neurite outgrowth based on scaffold physical/biological properties and mass transport phenomena have been uncovered.…”

“…Also, in 3D matrices, DRG neurite growth was inhibited by both certain biochemical and mechanical transitions [38], whereas outgrowth was enhanced in engineered matrices by the presence of specific peptide sequences [39]. The survival of primary neurons from the cerebral cortex has been demonstrated within 3D matrices of collagen and/or various hydrogels (e.g., poly[N-(2-hydroxypropyl)-methacrylamide] [16], poly(acrylate) [13], agarose mixed with collagen [15], collagen covalently linked to agarose [32], and Matrigel [31]), In general, primary cortical neuronal survival and the extent of neurite outgrowth are improved by the addition of specific bioactive cell-matrix interactions [13,15,16,32]. However, neuronal survival and neurite outgrowth in 3D matrices are influenced by intrinsic (e.g., neuronal maturation, receptor expression) as well as extrinsic (e.g., matrix mechanical properties, ligand concentration) signals.…”

“…A direct relationship between neuronal plating density and neuronal survival has been found [32,49]. Primary cerebral cortical neurons were plated in thick (500-600 lm) bioactive matrices at cell densities varying over an order of magnitude (1,250-12,500 cells/mm 3 ), and culture viability was assessed at 2 and 7 days post-plating.…”