Optimizing the in vitro neuronal microenvironment to mitigate phototoxicity in live-cell imaging

Abstract

Long-term imaging formats are ideal for capturing dynamic neuronal network formation in vitro, yet fluorescent techniques are often constrained by the impact of phototoxicity on cell survival. Here we present a live-imaging protocol that was optimised via quantitative analysis of 3 target culturing conditions on neuromorphological health: extracellular matrix (human- versus murine-derived laminin), culture media (Neurobasal™ versus Brainphys™ Imaging media), and seeding density (1 × 105 versus 2 × 105 cells/cm2). A cortical neuron reporter line was differentiated from human embryonic stem cells by transduction of Neurogenin-2 and green fluorescent protein, then fluorescently imaged in 8 diff..