The generation of induced pluripotent stem cells (iPSCs) via somatic cell reprogramming allowed to have an unlimited in vitro source of patient-specific cells.This achievement has introduced a new revolutionary way to create human in vitro models and to study human diseases starting from patient's own cells, especially important for inaccessible tissues like the brain.Recently, lab-on-a-chip technology has opened new reliable alternatives to conventional in vitro models able to replicate key aspects of human physiology, thanks to the intrinsic high surface-area-to-volume ratio, which allows fine control of the cellular microenvironment.The development of automated microfluidic platforms allowed the implementation of this technology to perform 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 iPSCs (hiPSCs) into neurons via viral-mediated overexpression of Neurogenin 2 (NGN2) 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.All operations are managed automatically, from the cell seeding, medium change, doxycycline-mediated neuronal induction, selection of the genetically engineered cells, and analysis of the output of differentiation, including immunofluorescence assay. Our results show a high-throughput, efficient and homogenous conversion of hiPSCs into neurons in 10 days, characterized by the expression of the mature neuronal 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 neurological diseases modelling in vitro and improve current preclinical models.
NGN2-based neuronal programming of hiPSCs in an automated microfluidic platform
Angiolillo, S;Laterza, C;Gagliano, O
2023
Abstract
The generation of induced pluripotent stem cells (iPSCs) via somatic cell reprogramming allowed to have an unlimited in vitro source of patient-specific cells.This achievement has introduced a new revolutionary way to create human in vitro models and to study human diseases starting from patient's own cells, especially important for inaccessible tissues like the brain.Recently, lab-on-a-chip technology has opened new reliable alternatives to conventional in vitro models able to replicate key aspects of human physiology, thanks to the intrinsic high surface-area-to-volume ratio, which allows fine control of the cellular microenvironment.The development of automated microfluidic platforms allowed the implementation of this technology to perform 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 iPSCs (hiPSCs) into neurons via viral-mediated overexpression of Neurogenin 2 (NGN2) 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.All operations are managed automatically, from the cell seeding, medium change, doxycycline-mediated neuronal induction, selection of the genetically engineered cells, and analysis of the output of differentiation, including immunofluorescence assay. Our results show a high-throughput, efficient and homogenous conversion of hiPSCs into neurons in 10 days, characterized by the expression of the mature neuronal 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 neurological diseases modelling in vitro and improve current preclinical models.
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