Human iPSC-derived iNSCs were seeded at 6 × 104 cells in a 24-well microelectrode array (MEA) plate (Axion Biosystems, Atlanta, GA, USA) containing 16 electrodes for the assay of neuron differentiation and maturation. All procedures were conducted by following the user’s manual of Axion Biosystems. MEAs system can capture the extracellular field potentials of electrically active cells by recording their neuronal activity [21 (link)]. To examine the spontaneous electrical activity of differentiated neuron, we collected the activity data from neuronal networks for 8 min by MEA every two or three days starting from day 14 after neuronal differentiation (3–4 replicates per experiment). Spike and burst rates represented the overall activity of the neuronal network, with more spikes and bursts corresponding to a higher electric activity. Network events include synchronous network spikes and bursts in which more than 35% electrodes captured the activity simultaneously. For electrical stimulation, after differentiation for 8 weeks the neurons received current injection at 50 μA/1000 μs/1000 mV every 5 s for five consecutive stimulations.
Mea plate
Manufactured by Axion Biosystems
Sourced in United States
MEA plates are specialized cell culture plates designed for use with Axion Biosystems' multi-electrode array (MEA) systems. They provide a platform for recording electrical signals from cells, enabling researchers to study cellular electrophysiology and network activity.
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Lab products found in correlation
Laminin, by Merck Group (1 mentions)
Cho s sfm 2 medium, by Thermo Fisher Scientific (1 mentions)
Nbactiv1 media, by Transnetyx (1 mentions)
Axis software, by Axion Biosystems (1 mentions)
Laminin, by Thermo Fisher Scientific (1 mentions)
Glass bottom petri dishes, by Cellvis (1 mentions)
Polyethylenimine (pei), by Merck Group (1 mentions)
Dmem f12, by Corning (1 mentions)
Maestro mea platform, by Axion Biosystems (1 mentions)
Prism 7, by GraphPad (1 mentions)
Offline sorter, by Plexon (1 mentions)
Maestro mea system, by Axion Biosystems (1 mentions)
Neural metrics tool, by Axion Biosystems (1 mentions)
Brainphys medium, by STEMCELL (1 mentions)
Axis software spontaneous neural configuration, by Axion Biosystems (1 mentions)
Brain derived neurotrophic factor (bdnf), by Thermo Fisher Scientific (1 mentions)
L ascorbic acid, by Merck Group (1 mentions)
7 protocols using mea plate
1
Evaluating Neuron Differentiation and Maturation
2
Rat Cortical Neuron Culture Protocol
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Rat cortical cultures were prepared from E18 rat embryos (BrainBits, Springfield, IL) dissociated by trituration after papain digestion as per the manufacturer's protocol. The cells were resuspended in the NBActiv1 media (BrainBits) supplemented with 5% fetal bovine serum and 20 µg/mL laminin (Sigma-Aldrich, Natick, MA). Neurons were seeded at 80,000 cells/well on a 48-well microelectrode array (MEA) plate (Axion BioSystems, Atlanta, GA) covered with adhesion promoting molecules (PEI). For experiments on the SyncroPatch 384PE, dissociated neurons were plated in 100 mm of poly-d-lysine-coated dishes at 4 × 10E6 to 6 × 10E6 cells/dish. Cells were fed every 3-4 days by replacing approximately half of the media with serumfree media. On the day of the experiment, cells were harvested as described previously [13] [14] [15] with trypsin-EDTA solution (PCS-999-003, ATCC, Manassas, VA) for 5-10 min at 37 °C and subsequently neutralized in serum-free CHO-S-SFM II medium (Thermo Fisher, Bedford, MA). Cells were then centrifuged and resuspended in chip fill solution supplemented with 3 mM MgCl 2 and gently pipetted 10 times to break up cell clumps; cells were then prepared at 100,000 cells/mL for SyncroPatch recording.
3
Electrophysiological Profiling of Neuron-Astrocyte Co-Cultures
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WT and mutant hiPSC-derived NPCs (DIV 30) were co-cultured with hiPSC-derived WT astrocytes (DIV 60+) in 1:1 ratio and plated onto 48-well multielectrode array (MEA) plates (Axion BioSystems) coated with PLO/L. Cells were plated using drop seed method, where 15 μL of mixed cell suspension (60,000 cells in total) in a 10% dilution of matrigel in BrainPhys™ + 1X CultureOne™ media was pipetted onto the electrode array in the center of each well. The cultures were fed by partial media replacement every 3 days according to our neuronal differentiation protocol stated above. Spontaneous electrophysiological activity of the neuron-astrocyte co-cultures was recorded for 10 min at cortical neuron DIV 50 using the Axion biosystems maestro MEA at 37 °C and 5% CO2. Data analysis was performed using AxIs software (Axion BioSystems) to extract the number of spikes and bursts from the recording file. Quality criteria for the assays were defined as follows: an electrode having an average of more than 5 spikes/min and wells with less than 30% of the total electrodes active were considered inactive and excluded from the analysis.
4
Culturing Hb9 ESC-derived Neurons and Glia
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Mouse Hb9 ESC-derived neuron and glial cells (ArunA Biomedical, GA; Cat 7025) were cultured in AB2 basal neural medium (ArunA Biomedical, GA) and Dulbecco’s Modified Eagles Medium/F12 (DMEM/F12; Corning, NY) following a previously published protocol55 . Briefly, each well of 12 well/64 electrode per well containing MEA plates (Axion Biosystems, GA), or glass bottom petri dishes (Cellvis, CA) were coated with polyethylenimine (PEI) (Sigma-Aldrich, MO) and incubated for 1 h at 37 °C and 5% CO2, then washed with deionized water and allowed to air dry overnight. The surfaces were subsequently coated with 20 µg/ml laminin (ThermoFisher Scientific, MA) for 2 h at 37 °C and 5% CO2 immediately prior to seeding. The cells were subsequently seeded in triplicate on either glass-bottom petri dishes, or in 12 well MEA plates. MEA plates (12-well, 1.43 × 1.43 mm surface) were seeded at a density of 40,000 cells per well, and glass-bottom petri dishes were seeded at a density of ~125,000 per well to maintain comparable cell seeding density to MEAs.
5
Astrocyte-Neuron Co-culture Electrophysiology
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Astrocytes at Stage 3 were thawed into flasks and grown for a week before passage. HD (CS09iHD109n1) and control (CTR; CS83iCTR33n2) astrocytes were then seeded onto 48-well transparent microelectrode array (MEA) plates (Axion Biosystems) at between 25 and 200 thousand cells per well. Astrocytes were allowed up to 2 days in Stage 3 media after seeding before switching to Stage 4 media for maturation. After 1 week in maturation, control and HD neural progenitors (∼day 16 of differentiation) were seeded on top of the astrocytes at between 50 and 400 thousand cells per well and the co-culture experiment was considered at Day 0. Daily recordings of activity were measured using the Maestro MEA platform and recording software (Axion Biosystems) for 5 min durations. Wave form events were identified using adaptive spike threshold crossing with a standard deviation of noise set at 5.5, and then further validated and sorted to individual neurons using Offline Sorter (Plexon). A minimum of 1 spike per min was used to include neuronal waveforms for analysis, and PCA analysis of each unit cluster was used to exclude noise artifacts. Data was compiled in excel and statistics run using GraphPad Prism 7.04 and SigmaPlot software.
6
Culturing Human iPSC-Derived Cardiomyocytes on MEA
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Human iPSC-CMs (Pluricyte Cardiomyocytes, Ncardia, Leiden, The Netherlands) were stored in liquid nitrogen until thawed and cultured onto 96 well MEA plates (Axion Biosystems, Inc., Atlanta, USA) according to manufacturer instructions. Briefly, the MEA plates were coated with fibronectin (50μg/ml in PBS [+ Ca2+ & Mg2+], Sigma-Aldrich, St. Louis, MO, USA; Cat. No. F-1141) for 3 hours at 37°C and 5% CO2. After 3 hours of incubation time, the excess of fibronectin coating solution was removed and cells were plated in a 5 μl droplet at a density of 25000 cells/well. After 1 hour of incubation (37°C and 5% CO2), 100μl pre-warmed (37°C) medium (Pluricyte Cardiomyocyte Medium, Ncardia, Leiden, The Netherlands) was carefully added to each well. Cells were maintained in Pluricyte Cardiomyocyte Medium for 8 days and refreshments took place at day 1 post-thaw and subsequently every other day. MEA recordings were performed at day 8 post-thaw. The choice of these different parameters of the experimental setup was presented and commented in [16 ].
7
Multielectrode Array Recording of Cortical Organoids
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To obtain MEA recordings,35 (link),43 (link),64 (link),66 (link) six-week-old cortical organoids were plated in 6-, 12-, or 48-well (each well with an 8×8 grid of electrodes) MEA plates (Axion Biosystems, Atlanta, GA, USA) pre-coated with 100 μg/mL poly-L-ornithine and 10 μg/mL laminin. Cellular cultures were fed twice per week with Medium 2 (see above) and, 7–14 days after plating, were incrementally switched to BrainPhys medium (StemCell Technologies67 (link)) supplemented with 10 ng/mL of BDNF (PeproTech) and 200 μM L-ascorbic acid (Sigma-Aldrich). Recordings were conducted in a Maestro MEA system with AxIS Software Spontaneous Neural Configuration (Axion Biosystems). Using Axion Biosystems’ Neural Metrics Tool, active electrodes required at least five spikes/min. Bursts/electrode used an inter-spike interval (ISI) threshold requiring minimally five spikes with a maximum ISI of 100 ms. Network bursts required at least 10 spikes under the same ISI with >25% active electrodes in the well. The synchrony index utilized a cross-correlogram window of 20 ms.
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