A multi-dimensional culture system model for hippocampal NSCs on PLLA microwell pattern

Poly-L-lactic acid (PLLA) microwell patterns were fabricated using a lithography-based replica molding method to develop neural stem cell-based assays. Hippocampal neural stem cells (NSCs) were cultured on microwell patterns to construct a multi-dimensional culture system model in which cells within the microwells were mainly cultured with three-dimensional cellular aggregates (MW-3D cells), and cells on the top surfaces were mainly cultured with two-dimensional single-layer adherent cultures (TS-2D cells). It was found that self-renewal and MW-3D cell-directed migration and transformation regulate the construction of this model. Patterns without channel connections occurred earlier in the construction of this model than those with channel connections, due to the tendency of NSCs to extend to both sides along the direction of the channels. Self-renewal and stemness maintenance of NSCs within the microwells were promoted by the patterns with the channel connections as a suitable microenvironment for a prolonged period of time, and can be used to build quasi-one-dimensional neural networks within the microwells. This will become a practical model for studying the functional behavior of NSCs, with different culture systems dynamically assembled on the same platform for stem cell research and the development of stem cell-based assays.

Multi-dimensional culture system model of the hippocampal NSCs on microwell patterns. (a-b) Hippocampal NSCs were stained with Nestin and DAPI after 7 days on the (a) 100–0 μm and (b) 120–40 μm patterns. Images were obtained by multi-slice scanning with CLSM, volume rendering of the image sequence, and overlaying of the confocal fluorescence images with phase-contrast micrographs. (c-d) Cross-sectional views of the 3D reconstructed confocal microscopic images of DAPI and Nestin were used to analyze the distribution of cells inside the microwells.