Comparative analysis of surface coatings for human neural co-cultures identifies fibronectin as a robust MEA substrate.

Human induced pluripotent stem cell (hiPSC)-derived neurons offer a promising, physiologically relevant alternative to animal-based neurotoxicity models. However, the influence of the surface coating, a basic but important variable, has often been neglected. Here, the impact of commonly used surface coatings including polymers (polyethylenimine, poly-L-ornithine, poly-D-lysine, poly-L-lysine) as well as extracellular matrix proteins (laminin, fibronectin, Matrigel) on NGN2 neurons, primary human astrocytes, and neuron/astrocyte co-cultures was systematically investigated. Neurons cultured on polymer-only coatings exhibited a less mature neuronal network. Interestingly, neurons on polymers with laminin, laminin only, fibronectin and Matrigel exhibited no differences in protein and gene expression but displayed different electrophysiological profiles in co-culture with human primary astrocytes. The peak in number of network bursts was reached at different time points, with fibronectin and Matrigel as early as week 2. Co-cultures on fibronectin exhibited stable cell-electrode coupling and robust activity patterns up to week 6, despite being rarely used in MEA. In a proof-of-principle experiment, laminin, fibronectin and Matrigel were further evaluated in a donor-matched hiPSC-derived co-culture system, supporting the observed effects. This study highlights the importance of the coating selection for establishing neural cultures, ultimately improving the maturation and robustness of hiPSC-derived neuronal models for drug development.