longitudinal electrophysiological recording of human brain assembloids using multi-electrode arrays

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

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.

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.

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.

longitudinal-electrophysiological-recording-human-brain-assembloids

Research

JoVE Journal

Neuroscience

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.

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 ...