Cytoview mea

Differentiation induction of cholinergic neurons from human iPSCs was carried out using the Quick-Neuron^TM Cholinergic—SeV Kit (Elixirgen Scientific, Inc., Baltimore, MD). The protocol was used because it is overall more rapid in generating differentiated MNs. This differentiation of human iPSCs into cholinergic neurons is driven by a Sendai virus (SeV)-based vector (ID Pharma Co., Ltd., Tokyo, Japan) which allows forced overexpression of neural inducing factors (Goparaju et al., 2017 (link)) in a temperature-dependent manner (Ban et al., 2011 (link)). Briefly, human iPSCs were infected with the SeV vector and incubated at 33°C, 5% CO₂ for 2 days in differentiation medium for 2 days. Then, the cultures were transferred to 37°C, 5% CO₂ to inactivate the SeV vector and further incubated for 16–18 h. Finally, immature neurons were passaged onto a coated microelectrode array plate, CytoView MEA (Axion Biosystems), after being mixed with human primary astrocytes (Thermo Fisher Scientific). This protocol has been shown to yield cholinergic MNs as identified by islet1 and Hb9 staining in combination with choline acetyltransferase (ChAT) staining (Goparaju et al., 2017 (link)).

Neurons were dissociated after 45 days of directed differentiation and plated at 200,000 cells/well into a minimum of 4 wells of a 48-well (16 electrode) CytoView MEA (Axion Biosystems, Atlanta, GA, USA; M768-tMEA-48W). The neurons were cultured on the plate for an additional 10 days and recorded at day 55. Briefly, the MEA plate was recorded using the Axion Integrated Studio (AxIS) software, version 2.1 (Axion Biosystems, Atlanta, GA, USA) on the Axion Maestro (Axion Biosystems, Atlanta, GA, USA). The plate was recorded for 5 min at 37 °C and 5% CO₂. The 5% CO₂ was obtained from a compressed gas bottle (Linde, Munich, Germany) and was regulated to the MEA at 0.2 bar. Re-recording of the MEA was performed using AxIS, software version 2.5, with a Butterworth (200 Hz–3 kHz) filter. The Spike Detector program was used to measure activity, the Adaptive Threshold Crossing method was used to detect crossings at 6 × Standard Deviation. The neural metrics were analysed using the Neural Metric Tool, software version 2.6.1 (Axion Biosystems, Atlanta, GA, USA). The threshold for an active electrode was set to five spikes per minute.

SymNs for the extracellular spike measurements were plated on 96-well BioCircuit or CytoView MEA plate (Axion BioSystems), coated with PO/LM/FN, at 1 × 10⁵ cells/cm² density. If BioCircuit plates were used, it is recommended that additional wells on a regular cell culture plate with visible bottoms (Corning, CLS35) are used at the same condition to monitor the growth of symNs. Neural activity was measured at the desired timepoints using the Maestro Pro multiwell plate reader under neural detection mode accordingto manufacturer’s instructions.