Three dimensional cell culture of human neural stem cells using polysaccharide-based hydrogels and subsequent bioanalyses

Abstract

Protocols for culturing neural stem cells (NSCs) are increasingly finding utilization for studying and growing of tissues that can appropriately model the neural regeneration processes. Two-dimensional (2D) plastic or glass surface-enabled mammalian cell cultures have been the platforms for performing in vitro cell cultures. Isolated mammalian cells, however, come from three-dimensional (3D) spaces, thus recapitulating such 3D microenvironments is among the challenges for many tissue engineering applications. Herein, we present the protocols for culturing NSCs in 3D polysaccharide-based hydrogel microenvironments that mimic, for instance, the native extracellular matrix (ECM) space (of the brain). The protocols include three key steps: (1) generation of 3D hydrogels with living cells, (2) culturing NSCs in 3D environments, and (3) characterization via immunostaining and genetic expression assay (RT-qPCR).