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Abstract

Neuroscience

Production of Human Neurogenin 2-Inducible Neurons in a Three-Dimensional Suspension Bioreactor

Published: March 17th, 2023

DOI:

10.3791/65085

1Fraunhofer Project Center for Stem Cell Process Engineering, Fraunhofer Institute for Biomedical Engineering IBMT, 2Bioneer A/S, 3Fraunhofer UK Research Ltd, Technology and Innovation Centre, 4Fraunhofer Institute for Biomedical Engineering IBMT, 5Department of Molecular and Cellular Biotechnology, Saarland University, 6Facultad de Ciencias del Mar, Universidad Católica del Norte

Abstract

The derivation of neuronal lineage cells from human induced pluripotent stem cells (hiPSCs) marked a milestone in brain research. Since their first advent, protocols have been continuously optimized and are now widely used in research and drug development. However, the very long duration of these conventional differentiation and maturation protocols and the increasing demand for high-quality hiPSCs and their neural derivatives raise the need for the adoption, optimization, and standardization of these protocols to large-scale production. This work presents a fast and efficient protocol for the differentiation of genetically modified, doxycycline-inducible neurogenin 2 (iNGN2)-expressing hiPSCs into neurons using a benchtop three-dimensional (3D) suspension bioreactor.

In brief, single-cell suspensions of iNGN2-hiPSCs were allowed to form aggregates within 24 h, and neuronal lineage commitment was induced by the addition of doxycycline. Aggregates were dissociated after 2 days of induction and cells were either cryopreserved or replated for terminal maturation. The generated iNGN2 neurons expressed classical neuronal markers early on and formed complex neuritic networks within 1 week after replating, indicating an increasing maturity of neuronal cultures. In summary, a detailed step-by-step protocol for the fast generation of hiPSC-derived neurons in a 3D environment is provided that holds great potential as a starting point for disease modeling, phenotypic high-throughput drug screenings, and large-scale toxicity testing.

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Keywords 3D Suspension Bioreactor

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