This manuscript was supported by R01NS127829 NIH/NINDS (LTD). Figure 1 has been generated using biorender.com.

All experimental procedures demonstrated below were conducted in accordance with the ethical guidelines of the University of Michigan Medical School Institutional Review Board and the Human Pluripotent Stem Cell Research Oversight Committee. The iPSC lines used in this protocol and the representative experiments were derived from human foreskin fibroblasts obtained from a commercial source. The details of the cell lines, reagents, and equipment used in this study are listed in the Table of Materials....

Recording Network Activity in Brain Assembloids: Multi-Electrode Array Approach

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The authors have no relevant disclosures.

MEA-based methods for electrophysiological recordings of network activity in iPSC-derived assembloids have been used for in vitro modeling of epilepsy22,23. This proposed platform integrating excitatory and inhibitory synaptic connections has the potential to address mechanisms of neuronal hyperexcitability and the role of cortical interneurons in the process of epileptogenesis. Additionally, this platform allows for the stabilization of assembloids and ...

Human pluripotent stem cell (hPSC)-derived brain organoids are spatially self-organized 3D structures mirroring the tissue architecture and developmental trajectories in vivo. They are composed of multiple cell types, including progenitors (neuroepithelial cells, radial glia, neuronal progenitors, glial progenitors), neurons (cortical-like excitatory neurons and inhibitory interneurons), and glial cells (astrocytes and oligodendrocytes)1,2. Assembloids represent the next generation of brain organoids, capable of integrating multiple brain regions and/or cell lineages within a 3D culture. They provide a useful tool for modeling connections between various brain regions resembling the in vivo counterparts, capturing interactions between neurons and astrocytes to better mimic mature and complex neural networks, and to study the assembly of neural circuits. Therefore, assembloids are becoming a widely used tool to recapitulate hallmarks of epilepsy pathophysiology, in which functional measures are necessary to interrogate aberrant neural networks that may underlie the cause of the disease3,4,5,6.
To model interactions between cortical glutamatergic neurons and GABAergic interneurons, several groups developed separate organoids resembling the dorsal and ventral brain and then fused them together into a multi-region assembloid7,8,9,10. Here, a previously described assembloid culture protocol with regionally specific neural subtypes was applied9. However, a significant barrier is the lack of reproducible functional assays to monitor neural network activity during neurodevelopment. Many functional tests of the networks in organoids yield results that have high variability among batches of differentiations and cell lines. Techniques that involve slicing or dissociating organoids alter their inherent networks by severing synaptic connectivity8.
Multi-electrode arrays (MEA) provide a large-scale view of network activity over time with high temporal resolution to characterize electrophysiological properties of organoids without disrupting culture conditions or cell membrane integrity11. Compared to patch clamp electrophysiology, MEA enables high-throughput data acquisition based on large populations of neurons rather than single cells. MEA platforms vary in their electrode density, catering to different needs in brain organoid research12. The widely used systems, as shown in this protocol, record from 8 to 64 electrodes per well13,14,15. High-density MEAs with up to 26,400 electrodes per well enable increased spatial and temporal resolution, quantification of action potential propagation speed, and combination with optogenetic stimulation14,16,17. Therefore, MEA serves as a powerful tool for modeling epilepsy in vitro and a translational paradigm for anti-seizure medication screening.
One major challenge is to stabilize the large-sized assembloids on a hydrophobic metal surface for long-term recordings. This protocol outlines a detailed methodology for plating intact assembloids on MEA plates for long-term longitudinal recording together with pharmacological assays. Unique advantages of the protocol include stable attachment of assembloids to the electrode surface without losing electrical activity, use of commercially available neurophysiological basal media to accelerate functional network maturation after plating, feasibility to conduct downstream functional assays like drug treatment, and wide application to organoids generated with other region-specific protocols.
The goal is to provide a functional assay with high temporal resolution to investigate network activity, examine disease-specific changes, and test drugs with therapeutic potential in epilepsy. Video instructions are provided for the most challenging steps of this protocol, showing the techniques for plating assembloids on MEA plates, as well as representative recordings from these cultures.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 cm Corning Non TC-treated culture dishes</td>
			<td>Corning</td>
			<td>08-772-32</td>
			<td>For suspension culture on the shaker</td>
		</tr>
		<tr>
			<td>100 mL Beaker&#160;</td>
			<td>Fisher Scientific</td>
			<td>FB100100</td>
			<td></td>
		</tr>
		<tr>
			<td>100% Ethanol</td>
			<td>Fisher Scientific</td>
			<td>BP28184-4L</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol (&#946;-ME)</td>
			<td>Thermo Fisher</td>
			<td>21985023</td>
			<td>Working concentration 100 &#956;M</td>
		</tr>
		<tr>
			<td>48-well cell culture plate</td>
			<td>Fisher Scientific</td>
			<td>50-202-140</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well cell culture plate</td>
			<td>Fisher Scientific</td>
			<td>07-200-83</td>
			<td></td>
		</tr>
		<tr>
			<td>Aggrewell 800</td>
			<td>Fisher Scientific</td>
			<td>501974754</td>
			<td></td>
		</tr>
		<tr>
			<td>Allegra X-14R Refrigerated Centrifuge</td>
			<td>Beckman Coulter&#160;</td>
			<td>BE-AX14R</td>
			<td></td>
		</tr>
		<tr>
			<td>Allopregnanolone</td>
			<td>Cayman</td>
			<td>16930</td>
			<td>Suspended 5mg into DMSO to get 1 mM stock solution. Aliquot and freeze at &#8722;80 &#176;C. Dilute at 1:10,000 for use. Working concentration 100 nM.</td>
		</tr>
		<tr>
			<td>Automated cell counter</td>
			<td>Thermo Fisher</td>
			<td>AMQAX2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Axion CytoView MEA 6-well plates&#160;</td>
			<td>Axion Biosystems</td>
			<td>M384-tMEA-6B</td>
			<td></td>
		</tr>
		<tr>
			<td>Axion Maestro MEA platform</td>
			<td>Axion Biosystems</td>
			<td>Maestro</td>
			<td>With temp environmental control</td>
		</tr>
		<tr>
			<td>B-27 supplement (regular, with Vitamin A)</td>
			<td>Thermo Fisher</td>
			<td>21985023</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 supplement (without Vitamin A)</td>
			<td>Thermo Fisher</td>
			<td>12587010</td>
			<td></td>
		</tr>
		<tr>
			<td>Basement membrane matrix- Geltrex</td>
			<td>Thermo Fisher</td>
			<td>A1569601</td>
			<td></td>
		</tr>
		<tr>
			<td>Bead bath</td>
			<td>Fisher Scientific</td>
			<td>10-876-001</td>
			<td>Isotemp</td>
		</tr>
		<tr>
			<td>Benchtop inverted microscope</td>
			<td>Olympus</td>
			<td>CKX53</td>
			<td>Kept in laminar flow clean bench</td>
		</tr>
		<tr>
			<td>Bicuculline</td>
			<td>Sigma-Aldrich</td>
			<td>14340</td>
			<td>Working concentration 10 &#956;M</td>
		</tr>
		<tr>
			<td>Bleach</td>
			<td>CLOROX</td>
			<td>67619-26</td>
			<td></td>
		</tr>
		<tr>
			<td>Borate buffer 20x</td>
			<td>Thermo Fisher</td>
			<td>28341</td>
			<td>Working concentration at 1x</td>
		</tr>
		<tr>
			<td>BrainPhys media</td>
			<td>StemCell Technologies</td>
			<td>5790</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell dissociation reagent (StemPro Accutase)</td>
			<td>Thermo Fisher</td>
			<td>A1110501</td>
			<td></td>
		</tr>
		<tr>
			<td>Celltron orbital shaker</td>
			<td>HT-Infors</td>
			<td>&#160;I69222&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Detergent/enzyme (Terg-A-Zyme)</td>
			<td>Sigma-Aldrich</td>
			<td>Z273287</td>
			<td>Working concentration 1% m/v</td>
		</tr>
		<tr>
			<td>DMEM/F12 + HEPES/L-Glutamine</td>
			<td>Thermo Fisher</td>
			<td>113300</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>67685</td>
			<td></td>
		</tr>
		<tr>
			<td>Dorsomorphin</td>
			<td>Sigma-Aldrich</td>
			<td>P5499</td>
			<td>Dissolve 5mg into DMSO to get 10 mM stock solution. Aliquot and freeze at &#8722;20 &#176;C. Dilute at 1:2000 for use. (working concentration 5 &#956;M)</td>
		</tr>
		<tr>
			<td>D-PBS w/o calcium or magnesium</td>
			<td>Thermo Fisher</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>Glial cell line-derived neurotrophic (GDNF)</td>
			<td>Peprotech</td>
			<td>450-10</td>
			<td>Dissolve 100 &#956;g in 1mL of PBS to 100 &#956;g/mL. Aliquot and freeze at &#8722;20 &#176;C. Dilute at 1:5000 for use. (working concentration 20 ng/mL).</td>
		</tr>
		<tr>
			<td>GlutaMAX supplement</td>
			<td>Thermo Fisher</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Election Microscopy Sciences</td>
			<td>63510-20</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Thermo Fisher</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>Heraguard ECO Clean Bench</td>
			<td>Thermo Fisher</td>
			<td>51029692</td>
			<td></td>
		</tr>
		<tr>
			<td>Humidity controlled cell culture incubator</td>
			<td>Thermo Fisher</td>
			<td>370</td>
			<td>set to 37 &#176;C, 5% CO2</td>
		</tr>
		<tr>
			<td>IWP-2</td>
			<td>Selleckchem</td>
			<td>S7085</td>
			<td>Aliquot and freeze at &#8722;80 &#176;C. It will precipitate if thawed at room temp. Frozen aliquots should be placed directly into 37 &#176;C before use.</td>
		</tr>
		<tr>
			<td>Knockout serum replacement (KOSR)</td>
			<td>Thermo Fisher</td>
			<td>10828010</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSRplus (medium + supplements)</td>
			<td>StemCell Technologies</td>
			<td>100-0276</td>
			<td>cGMP, stabilized feeder-free medium for human iPSC cells</td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Thermo Fisher</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal A</td>
			<td>Thermo Fisher</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-essential amino acids (NEAA)</td>
			<td>Thermo Fisher</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>NT3</td>
			<td>Peprotech</td>
			<td>450-03</td>
			<td>Dissolve 100 &#956;g in 1mL of PBS to 100 &#956;g/mL. Aliquot and freeze at &#8722;20 &#176;C. Dilute at 1:5000 for use. (working concentration 20 ng/mL).</td>
		</tr>
		<tr>
			<td>NuFF Human neonatal foreskin fibroblasts</td>
			<td>MTI-GlobalStem</td>
			<td>GSC-3002</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>PARAFILM</td>
			<td>P7793</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilin/Streptomycin</td>
			<td>Thermo Fisher</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette (P10, P200, P1000)</td>
			<td>Eppendorf</td>
			<td>EP4926000034</td>
			<td>Autoclaved cut P1000 tips for organoid collection</td>
		</tr>
		<tr>
			<td>Poly (Ethyleneimine) (PEI)</td>
			<td>Sigma-Aldrich</td>
			<td>P3143</td>
			<td>Dilute stock in sterile borate buffer. Working concentration 0.07%. See details in manuscript.</td>
		</tr>
		<tr>
			<td>Recombinant human epidermal growth factor (EGF)</td>
			<td>R&#38;D Systems</td>
			<td>236-EG-200</td>
			<td>Suspended in PBS. Aliquot and freeze at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Recombinant Human fibroblast growth factor (FGF)-basic&#160;</td>
			<td>Peprotech</td>
			<td>100-18B</td>
			<td>Suspended in PBS. Aliquot and freeze at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Recombinant human-brain-derived neurotrophic factor (BDNF)</td>
			<td>Peprotech</td>
			<td>450-02</td>
			<td>Centrifuge briefly before reconstitution. Dissolve 100 &#956;g in 1 mL of PBS to 100 &#956;g/mL. Aliquot and freeze at &#8722;20 &#176;C. Dilute at 1:5000 for use. (working concentration 20 ng/mL).</td>
		</tr>
		<tr>
			<td>Retinoic acid (RA)</td>
			<td>Sigma-Aldrich</td>
			<td>R2625</td>
			<td>Dissolve 100 mg into 3.3 mL of DMSO to get 100 mM stock solution. Aliquot the stock 100 &#956;L/tube and freeze at &#8722;80 &#176;C. Take 200 &#956;L of 100 mM stock and dilute 10x (add 1.8 mL of DMSO) to make 10 mM stock. Aliquot 50 &#956;L/tube and store at &#8722;80 &#176;C. Dilute at 1:100,000 for use. (working concentration 100 nM).</td>
		</tr>
		<tr>
			<td>ROCK inhibitor Y-27632</td>
			<td>Tocris</td>
			<td>1254</td>
			<td>1:200 from 10 mM stock</td>
		</tr>
		<tr>
			<td>SAG (smoothened agonist)</td>
			<td>Selleckchem</td>
			<td>S7779</td>
			<td>Aliquot and freeze at &#8722;80 &#176;C. Stock concentration 1mM. Use at 1:10,000 dilution (working concentration 100 nM).</td>
		</tr>
		<tr>
			<td>SB-431542</td>
			<td>Tocris</td>
			<td>1614</td>
			<td>Dissolve 5mg into 1.3 mL of DMSO to get 10 mM stock solution. Aliquot and freeze at &#8722;80 &#176;C. Dilute at 1:1000 for use. (working concentration 10 &#956;M)</td>
		</tr>
		<tr>
			<td>Serological pipette filler</td>
			<td>Fisher Scientific</td>
			<td>14-387-166</td>
			<td></td>
		</tr>
		<tr>
			<td>Steriflip vacuum tube top filter</td>
			<td>Sigma-Aldrich</td>
			<td>SE1M179M6</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile cell culture hoods</td>
			<td>Baker Company</td>
			<td>SG-600</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue solution (0.4%)</td>
			<td>Thermo Fisher</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%)</td>
			<td>Thermo Fisher</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>Zoom stereomicroscope</td>
			<td>Olympus</td>
			<td>SZ61/SZ51</td>
			<td>Kept in laminar flow clean bench</td>
		</tr>
	</tbody>
</table>

a multi-electrode array platform for modeling epilepsy using human pluripotent stem cell-derived brain assembloids

Human brain organoids are three-dimensional (3D) structures derived from human pluripotent stem cells (hPSCs) that recapitulate&#160;aspects of fetal brain development. The fusion of dorsal with ventral regionally specified brain organoids in vitro generates assembloids, which have functionally integrated microcircuits with excitatory and inhibitory neurons. Due to their structural complexity and diverse population of neurons, assembloids have become a useful in vitro tool for studying aberrant network activity. Multi-electrode array (MEA) recordings serve as a method for capturing electrical field potentials, spikes, and longitudinal network dynamics from a population of neurons without compromising cell membrane integrity. However, adhering assembloids onto the electrodes for long-term recordings can be challenging due to their large size and limited contact surface area with the electrodes. Here, we demonstrate a method to plate assembloids onto MEA plates for recording electrophysiological activity over a 2-month span. Although the current protocol utilizes human cortical organoids, it can be broadly adapted to organoids differentiated to model other brain regions. This protocol establishes a robust, longitudinal, electrophysiological assay for studying the development of a neuronal network, and this platform has the potential to be used in drug screening for therapeutic development in epilepsy.

Author Spotlight: Advancing Genetic Epilepsy Studies with Multi-Electrode Array-Based Long-Term Electrophysiological Monitoring of Human Brain Assembloids

A Multi-Electrode Array Platform for Modeling Epilepsy Using Human Pluripotent Stem Cell-Derived Brain Assembloids

In the past few decades, an explosion of gene discovery has identified hundreds of genes that cause or are associated with epilepsy. However, the pathogenic mechanisms are not as well explored. Our laboratory studies how genetic changes affect early brain development and result in brain malformations and epilepsy, a debilitating disease characterized by chronic seizures.Genetic epilepsies have been typically studied using animal models, including mice, zebrafish, drosophila, and rabbits. Human stem cells have more recently been used to model genetic epilepsies as techniques to differentiate stem cells into neuro tissue have advanced. With the advent of brain organoids, you can recapitulate structure aspects of brain development.Measuring electrophysiological activity and determining biomarkers of epilepsy related activity from brain organoids and assembloids is challenging. Partly, this technique is limited because brain organoids cannot have seizures like an intact animal can. Nonetheless, finding electrophysiological differences in this in vitro model and responses to drug treatments may help determine pathological mechanisms and therapeutic responses in genetic epilepsies.Electrophysiological activity can be assessed using traditional patch climb recordings, local field recordings with electrodes, and optical techniques like calcium and memory voltage imaging. Using multielectrode array recordings has the advantage of being able to do launch two recordings over time and recording from multiple locations of an assembloid simultaneously.

Human dorsal-ventral assembloids were plated on a 6-well MEA plate (n = 6 per differentiation, 3 differentiations), with each well containing 64 electrodes (Figure 2A). Nine out of ten assembloids planned for longitudinal recording were firmly attached to the electrodes for over 50 days in vitro (Figure 2D). 6 out of 8 assembloids designated for pharmacological assays were successfully settled through the wash-out phase ...

This protocol aims to stably plate dorsal-ventral fused assembloids on multi-electrode arrays for modeling epilepsy in vitro.

Human brain organoids are three-dimensional (3D) structures derived from human pluripotent stem cells (hPSCs) that recapitulate&#160;aspects of fetal ...

a-multi-electrode-array-platform-for-modeling-epilepsy-using-human

Research

JoVE Journal

Neuroscience

Longitudinal Electrophysiological Recording of Human Brain Assembloids Using Multi-Electrode Arrays

To begin, generate virus labeled assembloids using fate-specific brain organoids. To prepare reagents for MEA surface pre-treatment, dissolve 0.5 grams of detergent enzyme powder in 50 milliliters of deionized water. Vortex the mixture thoroughly until the powder is fully dissolved and sterilize the solution using a sterile tube top vacuum filter inside the biosafety cabinet.Dilute 500 microliters of the 7%polyethylenimine, or PEI stock solution with 49.5 milliliters of 1X borate buffer to prepare a 0.07%PEI solution. To sterilize the solution, filter it through a 0.22 micrometer filter unit inside the biosafety cabinet. Next, dispense one milliliter of the 1%detergent enzyme solution to each well of a sterile MEA plate and incubate it at 37 degrees Celsius for two hours.Then remove the detergent enzyme solution from each well and wash the wells five times with 1.5 milliliters of sterile deionized water per well. Fill each well with one milliliter of culture media for preconditioning. Place the MEA plates back into a 37 degrees Celsius, 5%carbon dioxide incubator for two days.After incubation, remove the culture media from the wells and add 50 microliters of 0.07%PEI solution to cover the MEA electrodes for primary coating. Incubate the plate at 37 degrees Celsius for one hour. Then aspirate the PEI solution completely from each well.Wash each well three times with one milliliter of sterile deionized water. After aspirating the water, dry the plates at room temperature for 15 minutes in the biosafety cabinet with the lid open. Now cover the electrode area in each well with 50 microliters of one to 20 volume to volume basement membrane matrix diluted in culture media for secondary coating.Place the MEA plates back into the 37 degrees Celsius incubator overnight. The next day, aspirate the diluted matrix solution, leaving a thin layer on the surface. If a thick layer forms, spray the electrode contact area forcefully with culture media using a P1000 pipette tip.Then using a P1000 tip with a wide cut, collect an assembloid from the dish and carefully place it on top of the electrodes in a well while transferring as little media as possible. Place the plate under a dissection microscope in a laminar flow hood and using a sterile P 20 tip, carefully push the assembloid to the desired location. Use a P 20 tip to remove excess liquid around the assembloid.After incubation, remove the plate from the incubator. Then observing under a dissection microscope, apply two to three small drops of one to 50 basement membrane matrix diluted in culture media on top of the assembloids, and return the plate to the incubator for another 30 minutes. Afterward, carefully add three drops of culture media close to the assembloids using a dissection microscope.The next day, check if the assembloids have settled and stabilized under a dissection microscope. Add 750 microliters of culture media to bring the total volume to one milliliter per well and leave the plate undisturbed for at least two days in the incubator. Every week, carefully remove 500 microliters of culture media from each well, and introduce fresh 700 microliters of neurophysiological basal media to each well.Increased network bursting duration was observed on day 92 compared to day 78, likely due to neuron maturation. The cellular and network activity slowly faded by day 125.

457 Views.  Michigan Medicine, Ann Arbor.  This protocol aims to stably plate dorsal-ventral fused assembloids on multi-electrode arrays for modeling epilepsy in vitro.

Recording Network Activity in Brain Assembloids: Multi-Electrode Array Approach

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