Cytoview mea 24

iPSC-CMs were plated (as above) at 100,000 cells per well directly on the electrode array of 24-well MEA plates (CytoView MEA 24, Axion Biosystems) pre-coated with matrigel. RPMI-B27 was exchanged every 2 days. MEA analysis occurred at day 35 post differentiation (2 weeks post-plating on MEA plates). MEA data was acquired at 37 °C and 5% CO₂ and recordings were acquired for 5 min using the Maestro MEA system (Axion Biosystems) using standard recording settings for spontaneous cardiac field potentials. Automated data analysis was focused on the 30 most stable beats within the recording period. The beat detection threshold was dependent on the individual experiment, and the FPD was manually annotated to detect the T-wave. The FPD was corrected for the beat period according to Fridericia’s formula: FPDc = FPD/(beat period)^{1/371 (link)–73 (link)}. Results for individual wells were calculated by averaging all of the electrodes. For each MEA experiment, 4 technical replicates per condition were averaged and reported results represent the average of three distinct biological replicates. For statistical significance testing, one-way ANOVA with a Dunnett’s correction for multiple comparisons was performed.

All experiments with hiPSC-derived sensory neurons were performed with Reprocell.Inc's hiPSC-derived sensory neurons (cat #RCDN004N, Reprocell.Inc). HiPSC-sensory neurons were differentiated from the StemRNA Human iPSCs line 771-3G (RCRP003N, Reprocell.Inc) and we followed the differentiation method referenced as Young et al.^{8 (link)}. The expression pattern of sensory neuron-related genes showed features similar to those in the referenced paper. One vial of frozen sensory neurons was placed in a 37 °C-water bath and warmed for about 4 min, until completely thawed. Using a P1000 pipette, the cell suspension was gently added to a 50 mL sterile conical tube. 9 mL wash medium was added dropwise into this tube. The conical tube was centrifuged at 300 × g for 5 min at room temperature. When using a 24 well plate coated with iMatrix-511 silk (Nippi), the sensory neurons were seeded at 1.5 × 10⁵ cells per well at a density of 3 × 10⁵ cells/mL. When using a CytoView MEA 24 (AXION BIOSYSTEMS) plate coated with iMatrix-511 silk, sensory neurons were centrifuged and seeded at 1.5 × 10⁵ cells/5 µL per spot at a density of 30 × 10⁶ cells/mL. They were maintained in Sensory Neuron Culture Medium (cat#RCDN103, Reprocell.Inc) at 37 °C, 5% CO₂. Half medium change was performed every 3–4 days.

iCell Neurons were plated at 100,000 cells per well on 24-well MEA plates (Cytoview MEA24, Axion Biosystems, Atlanta, GA, USA) previously coated with Polyethyleneimine (Sigma). Spontaneous electrical activity was acquired using the Axion Maestro Edge MEA system and Axion’s Integrated Studio (AXIS) software, v. 2.0.4.21 (Axion Biosystems) over 28 days. All recordings were performed at a constant temperature of 37 °C and 5% CO₂. Each well of the 24-well MEA plate contains 16 individual microelectrodes, giving a total of 384 integrated recording electrodes per plate. Prior to a 10 min baseline recording period, the MEA plates were placed in the Maestro MEA platform and equilibrated for 5 min. AXIS software was used to control the heating system and to monitor the recordings, which involves simultaneous sampling of the channels at 12.5 kHz per channel with a gain of 1200× and band pass filters of 200–3 KHz. After recording, the RAW files were re-recorded with AXIS to convert the data into Microsoft Excel files (version 16), which includes spike timing and profile information. Spikes were detected using a threshold set to 6 times the estimated standard deviation of the rms-noise on each channel. Electrodes with activity higher than 0.05 spikes/s at least once over the recording time were only included for data analysis.