Development Of High-throughput 3d Neural Cell Culture Platforms For Modelling Parkinson’s Disease

Abstract

The three-dimensional (3D) culture of neural cells in Matrigel, a thermoresponsive, self-assembling, extracellular matrix hydrogel, holds promise for modelling neurodegenerative disorders such as Parkinson’s disease (PD). 3D cell culture has been proposed as a way to bridge the cell culture vs. tissue gap by providing more realistic mass transport, cell-cell interactions, and environmental cues compared to standard two-dimensional (2D) cell culture. However, air-liquid interfacial tension and evaporation can result in inconsistent 3D cultures at low volumes. Thick-layer hydrogels can counter these factors, but large diffusion distances, high cost, and incompatibility with standard imaging tools, plate readers, and assays limit their use. To address these limitations, two low cost, high-throughput, thin-layer, Matrigel-based, 3D cell culture techniques compatible with well-established equipment and commercially available materials were developed. The first technique involved using aqueous two-phase systems (ATPSs) to confine small volumes of Matrigel containing the model human neural cell line previously used in PD research, SH-SY5Y, into thin layers. Alternatively, the Matrigel-only 3D culture method involved dispensing cell-laden Matrigel directly into culture medium. Matrigel evaporation was eliminated in both the ATPS-Matrigel 3D culture and Matrigel-only 3D culture platforms, and small volumes could form thin gels. The ATPS-Matrigel 3D culture method provided viable cell cultures with excellent control over gel shape and thickness but was discovered to generate non-adherent cell cultures that would be more applicable towards contraction assay studies. Matrigel-only 3D culture produced thin gels also capable of supporting viable cell cultures but offered less control over gel shape and size. Nonetheless, the Matrigel-only 3D culture method provided a simpler protocol and adherent cultures. Thus, this platform was ultimately selected as the foundation for a potential 3D cell culture model to study PD and assess the impacts of 3D growth environment on SH-SY5Y cell growth, differentiation, and morphology. In 3D conditions, SH-SY5Y cells extended neurite-like processes in three-dimensions when differentiated with retinoic acid (RA) and brain-derived neurotrophic factor (BDNF) and contained purer neuronal phenotypes compared to alternative cell culture platforms (e.g., standard 2D cell culture). High-throughput neurotoxin assays using compounds previously used to model PD revealed that 3D-grown RA/BDNF-differentiated SH-SY5Y cells were more robust against neurotoxins compared to 2D-grown cells indicating that growth environment significantly impacted cell differentiation outcomes and behaviour. While these results suggested that RA/BDNF-differentiated SH-SY5Y cells do not provide an appropriate foundation for modelling PD, they also revealed the influence of 3D cell culture environment on cell growth and differentiation. As both 3D culture techniques are compatible with various cell types, alternative cell lines combined with these novel technologies may provide powerful new platforms to model PD in the lab and reveal information previously masked by standard 2D cell culture methods.