Development of an in vitro model of reactive gliosis using stabilized collagen gels with defined and monitored stiffness environments

INTRODUCTION: Following injury or insult, failure of the injured CNS to repair is in part attributed to the inhibitory environment of the lesion site, most notably the formation of the glial scar. This consists predominantly of astrocytes, which exhibit a reactive hypertrophic phenotype exemplified by upregulation of various markers, including GFAP. In vitro models have been employed to explore the cellular mechanisms occurring during reactive gliosis but there is little understanding of the relationship between astrocytes and their mechanical environment. Astrocytes within soft 3D hydrogel cultures have been shown to adopt a less reactive phenotype than in stiff 2D cultures and the progression of reactivity can be monitored, however there are difficulties in maintaining and monitoring soft gels that may discourage wider adoption. The aim of this study therefore was to investigate whether stiffer stabilised collagen gels containing astrocytes could be used as a high throughput tool for the study of reactive gliosis, and also to develop methods for measuring gel stiffness to enable the mechanical environment to be controlled and monitored.