Effective NGN2-based neuronal programming of hiPSCs in an automated microfluidic platform

Abstract: Neurodegenerative diseases represent an increasing health burden, with a worrying lack of models recapitulating the hallmarks of the pathology. Recently, lab-on-a-chip technology has opened new reliable alternatives to conventional in vitro models able to replicate key aspects of human physiology. For instance, microfluidics allows to mimic the extracellular accumulation of misfolded proteins in the finely controlled microenvironment, thanks to the intrinsic high surface-area-to-volume ratio. Automated microfluidic platforms offer advantages in implementing high-throughput, standardized and parallelized assays, suitable for drug screenings and developing new therapeutic approaches in a cost-effective way. However, the major challenges in the broad application of automated lab-on-a-chip in biological research are the lack of production robustness and ease of use of the devices. Here, we present an automated microfluidic platform able to host the rapid conversion of human induced pluripotent stem cells (hiPSCs) into neurons via NGN2 viral programming in a user-friendly manner. The design of the platform, built with multilayer soft-lithography techniques, shows easiness in the fabrication and assembly thanks to the simple geometry and experimental reproducibility at the same time. The all operations are automatically managed from the cell seeding, medium change, doxycycline-mediated neuronal induction, and selection of the genetically engineered cells, to the analysis, including immunofluorescence assay. Our results show a high-throughput, efficient and homogenous conversion of hiPSCs in neurons in 10 days showing the expression of mature marker MAP2, and calcium signaling. The neurons-on-chip model here described represents a fully automated loop system able to address the challenges in the field of neurodegenerative diseases and improve current preclinical models.