in-vitro culture of human-induced pluripotent stem cells (ipsc) on high-density microelectrode array (hd-mea)

Multimodal Large-Scale Neural Recordings and Analysis on HD-MEA 

Neuronal ensembles, often termed cell assemblies, are pivotal in neural coding, facilitating intricate computations for processing multiscale neural information1,2,3. These ensembles underpin the formation of expansive neuronal networks and their nuanced microcircuits4. Such networks and their oscillatory patterns drive advanced brain functions, including perception and cognition. While extensive research has explored specific neuronal types and synaptic pathways, a deeper understanding of how neurons collaboratively form cell assemblies and influence spatiotemporal information processing across circuits and networks remains elusive5.
Acute, ex-vivo brain slices are pivotal electrophysiological tools for studying intact neural circuits, offering a controlled setting to probe oscillatory activity patterns of neural function, synaptic transmission, and connectivity, with implications in pharmacological testing and disease modeling6,7,8. This study protocol highlights two key brain circuits - the hippocampal-cortical (HC) involved in learning and memory processes9,10, and the olfactory bulb (OB) responsible for odor discrimination11,12,13. In these two regions, new functional neurons are continuously generated by adult neurogenesis throughout life in mammalian brains14. Both circuits demonstrate multidimensional dynamic neural activity patterns and inherent plasticity that participate in rewiring the existing neural network and facilitate alternative information processing strategies when required15,16.
Acute, ex-vivo brain slice models are indispensable for delving into brain functionality and understanding disease mechanisms at the microcircuit level. However, in-vitro cell cultures derived from human induced pluripotent stem cells (iPSCs) neuronal networks offer a promising avenue of translational research, seamlessly connecting findings from animal experiments to potential human clinical treatment17,18. These human-centric in-vitro assays serve as a reliable platform for assessing pharmacological toxicity, enabling precise drug screening, and furthering research into innovative cell-based therapeutic strategies19,20. Recognizing the pivotal role of the iPSC neuronal model, we have dedicated the third module of this protocol study to thoroughly investigate the functional characteristics of its derived networks and to fine-tune the associated cell culture protocols.
These electrogenic neural modules have been commonly studied using techniques like calcium (Ca2+ imaging), patch-clamp recordings, and low-density microelectrode arrays (LD-MEA). While Ca2+ imaging offers single-cell activity mapping, it is a cell-labeling-based method hindered by its low temporal resolution and challenges in long-term recordings. LD-MEAs lack spatial precision, while patch-clamp, being an invasive single-site technique and laborious, often yields a low success rate21,22,23. To address these challenges and effectively probe network-wide activity, large-scale simultaneous neural recordings have emerged as a pivotal approach for understanding the computational principles of neural dynamics underlying brain complexity and their implications in health and disease24,25.
In this JoVE protocol, we demonstrate a large-scale neural recording method based on the high-density MEA (HD-MEA) for capturing spatiotemporal neuronal activity across various brain modalities, including hippocampal and olfactory bulb circuits from ex-vivo mouse brain acute slices (Figures 1A-C) and in-vitro human iPSC-derived neuronal networks (Figures 1D-E), previously reported by our group and other colleagues26,27,28,29,30,31,32,33,34,35. The HD-MEA, built on complementary-metal-oxide-semiconductor (CMOS) technology, boasts on-chip circuitry and amplification, allowing sub-millisecond recordings across a 7mm2 array size36. This non-invasive approach captures multi-site, label-free extracellular firing patterns from thousands of neuronal ensembles simultaneously using 4096 microelectrodes at a high spatiotemporal resolution, revealing the intricate dynamics of local field potentials (LFPs) and multiunit spiking activity (MUA)26,29.
Given the vastness of the data generated by this methodology, a sophisticated analytical framework is essential, yet poses challenges37. We have developed computational tools that encompass automatic event detection, classification, graph theory, machine learning, and other advanced techniques (Figure 1F)26,29,38,39. Integrating the HD-MEA with these analytical tools, a holistic approach is devised to probe the intricate dynamics from individual cell assemblies to broader neural networks across diverse neural modalities. This combined approach deepens our grasp of the computational dynamics in normal brain functions and offers insights into anomalies present in pathological conditions28. Moreover, insights from this approach can propel advancements in brain-inspired modeling, neuromorphic computing, and neural learning algorithms. Ultimately, this method holds promise in uncovering the core mechanisms behind neural network disruptions, potentially identifying biomarkers, and guiding the creation of precise diagnostic tools and targeted treatments for neurological conditions.

Author Spotlight: Advancing Large-Scale Neural Dynamics Through HD-MEA Technology

Recording and Analyzing Multimodal Large-Scale Neuronal Ensemble Dynamics on CMOS-Integrated High-Density Microelectrode Array

Our research explores the frontiers of neural technology by integrating high-density CMOS-based microelectrode array for decoding neural communication and large networks. We aim to answer how neural information across scales is encoded in unique detail, enhancing our understanding of brain function and dysfunction in health and disease. Navigating the complex area of neuronal ensemble research, we confront challenges like achieving precise signal resolution amidst brain activity, and ensuring the biocompatibility of our CMOS-based microelectrode arrays.These hurdles are pivotal in accurately capturing and interpreting the rich tapestry of neural interaction using multimodal recordings. Our research addresses a critical gap in neuroscience, the lack of a comprehensive method to recode and analyze the dynamics of larger scale neuronal ensemble with high spatial and temporal resolution. This gap hinders our understanding of complex brain networks and function in health and disease.Our protocol enables multimodal, label-free, high-resolution recordings across the hippocampus, olfactory bulb, and human IPSC-derived neurons, providing a versatile tool for diverse experiments. This unique approach facilitates unparalleled insight into neuronal dynamics, bridging the research gap between various brain regions and model systems, significantly advancing our understanding of neural function and disorder. Future endeavors in our lab will deeply investigate neural computations and dynamics from genes to networks, aiming to bridge molecular and functional signatures in health and disease.Through advanced bioelectronics and neural technology, we'll focus on neuroplasticity, olfactory coding, developing AI, and memory-enhancing strategies for novel therapeutics and brain machine interfaces.

Watch this Scientific Journal Video about In-Vitro Culture of Human-Induced Pluripotent Stem Cells iPSC on High-Density Microelectrode Array HD-MEA at JoVE.com

In-Vitro Culture of Human-Induced Pluripotent Stem Cells (iPSC) on High-Density Microelectrode Array (HD-MEA)

in-vitro-culture-human-induced-pluripotent-stem-cells-ipsc-on-high

Research

JoVE Journal

Neuroscience

326 次观看.  German Center for Neurodegenerative Diseases (DZNE). 首先，以 HD-MEA 芯片为例。用用 96% 乙醇润湿的纸巾擦拭玻璃环的芯片。将每个装置放入引擎盖下的无菌培养皿中，并用70%乙醇填充MEA储液器。吸入乙醇后，用无菌、过滤、双蒸水清洗储液罐三次。加入一毫升预处理培养基并孵育过夜。第二天，从设备中取出预处理介质。在每个设备的有效区域涂上一毫升 PDLO。第二天，从设备中吸出 PDLO。用双蒸水清洗设备三次，?...

视频: 在高密度微电极阵列 （HD-MEA） 上人诱导的多能干细胞 （iPSC） 的体外培养 

recording and analyzing multimodal large-scale neuronal ensemble dynamics on cmos-integrated high-density microelectrode array

Here, we employ HD-MEA to delve into computational dynamics of large-scale neuronal ensembles, particularly in hippocampal, olfactory bulb circuits, and human neuronal networks. Capturing spatiotemporal activity, combined with computational tools, provides insights into neuronal ensemble complexity. The method enhances understanding of brain functions, potentially identifying biomarkers and treatments for neurological...

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preparation of human ipsc recording workspace and network-wide recordings on high-density microelectrode array (hd-mea)

Preparation of Human iPSC Recording Workspace and Network-Wide Recordings on High-Density Microelectrode Array (HD-MEA)

ex-vivo circuit-wide recordings from hippocampal-cortical and olfactory bulb slices using high-density microelectrode array (hd-mea)

Ex-Vivo Circuit-Wide Recordings from Hippocampal-Cortical and Olfactory Bulb Slices Using High-Density Microelectrode Array (HD-MEA)

technical applications of microelectrode array and patch clamp recordings on human induced pluripotent stem cell-derived cardiomyocytes

Technical Applications of Microelectrode Array and Patch Clamp Recordings on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as a promising in vitro model for drug-induced cardiotoxicity screening and disease modeling. Here, we detail a protocol for measuring the contractility and electrophysiology of...

in vitro neuromuscular junction induced from human induced pluripotent stem cells

In vitro Neuromuscular Junction Induced from Human Induced Pluripotent Stem Cells

Here we provide a protocol to generate in vitro NMJs from human induced pluripotent stem cells (iPSCs). This method can induce NMJs with mature morphology and function in 1 month in a single well. The resulting NMJs could potentially be used to model related diseases, to study pathological mechanisms or to screen drug compounds for...

high-quality seizure-like activity from acute brain slices using a complementary metal-oxide-semiconductor high-density microelectrode array system

Author Spotlight: Unraveling Seizure Dynamics and Novel Therapeutics for Status Epilepticus Using CMOS High-Density Microelectrode Array Systems

Here, we outline a protocol for using complementary metal-oxide-semiconductor high-density microelectrode array systems (CMOS-HD-MEAs)&#160;to record seizure-like activity from ex vivo brain...

Consistent Induction and Recording of Seizure-Like Activity Using CMOS-HD-MEAs

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in vitro generation of somite derivatives from human induced pluripotent stem cells

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic...

in vitro induction of neuromuscular junctions from engineered human-induced pluripotent stem cells

This experiment details a method for generating neuromuscular junctions from human-induced pluripotent stem cells. It uses a staged differentiation process to promote myotube and motor neuron development and synapse formation, yielding functional neuromuscular...

In Vitro Induction of Neuromuscular Junctions from Engineered Human-Induced Pluripotent Stem Cells

an automated culture system for maintaining and differentiating human-induced pluripotent stem cells

Author Spotlight: Automating iPSC Culture for Enhanced Reproducibility 

Here, we present a protocol for an automated cell culture system. This automated culture system reduces labor and benefits the users, including researchers unfamiliar with handling induced pluripotent stem (iPS) cells, from the maintenance of iPS cells to differentiation into various types of...

An Automated Culture System for Maintaining and Differentiating Human-Induced Pluripotent Stem Cells

transfecting and nucleofecting human induced pluripotent stem cells

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process.  To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human...

high efficiency, site-specific transfection of adherent cells with sirna using microelectrode arrays (mea)

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA

The article details the protocol for site-specific transfection of scrambled sequence of siRNA in an adherent mammalian cell culture using a microelectrode array...

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays (MEA)

application of live mitochondria staining in cell-sorting to purify hepatocytes derived from human induced pluripotent stem cells

Author Spotlight: Advancing Hepatocyte Purification from Human Induced Pluripotent Stem Cells for Regenerative Medicine

Hepatocytes derived from pluripotent stem cells can be purified through cell sorting, using a combination of mitochondrial and activated leukocyte cell adhesion molecule (ALCAM, also known as CD166)...

Purification of Human iPSC-Derived Hepatocytes by Mitochondrial-ALCAM Sorting

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in vitro differentiation of human pluripotent stem cells into trophoblastic cells

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

High-Quality Seizure-Like Activity from Acute Brain Slices Using a Complementary Metal-Oxide-Semiconductor High-Density Microelectrode Array System

differentiating human induced pluripotent stem cells into neurons on micro-electrode arrays

The video demonstrates a method for generating excitatory cortical neurons from human-induced pluripotent stem cells (hiPSCs). Upon doxycycline induction, the hiPSCs overexpress neurogenin-2, a neural-specific transcription factor. By using specific media and co-culturing with astrocytes, this process facilitates the hiPSCs&#39; differentiation into neurons with synaptic...

Differentiating Human Induced Pluripotent Stem Cells into Neurons on Micro-Electrode Arrays

differentiation of human-induced pluripotent stem cells into macrophages

This video describes an in vitro method for generating macrophages from human induced pluripotent stem cells (iPSCs). Macrophage generation from iPSCs occurs in three stages: (i) aggregation of iPSCs in suspension to form three-dimensional embryoid bodies (EBs) that differentiate into mesodermal cells, (ii) formation of macrophage precursor cells from the differentiated EBs, and (iii) differentiation of the macrophage precursor cells into mature, functional...

Differentiation of Human-Induced Pluripotent Stem Cells into Macrophages

differentiation and characterization of osteoclasts from human induced pluripotent stem cells

Differentiation and Characterization of Osteoclasts from Human Induced Pluripotent Stem Cells

This protocol presents the differentiation of human osteoclasts from induced pluripotent stem cells (iPSCs) and describes methods for the characterization of osteoclasts and osteoclast precursors.

efficient generation human induced pluripotent stem cells from human somatic cells with sendai-virus

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced...

Large-scale neuronal networks and their complex distributed microcircuits are essential to generate perception, cognition, and behavior that emerge from patterns of spatiotemporal neuronal activity. These dynamic patterns emerging from functional groups of interconnected neuronal ensembles facilitate precise computations for processing and coding multiscale neural information, thereby driving higher brain functions. To probe the computational principles of neural dynamics underlying this complexity and investigate the multiscale impact of biological processes in health and disease, large-scale simultaneous recordings have become instrumental. Here, a high-density microelectrode array (HD-MEA) is employed to study two modalities of neural dynamics - hippocampal and olfactory bulb circuits from ex-vivo mouse brain slices and neuronal networks from in-vitro cell cultures of human induced pluripotent stem cells (iPSCs). The HD-MEA platform, with 4096 microelectrodes, enables non-invasive, multi-site, label-free recordings of extracellular firing patterns from thousands of neuronal ensembles simultaneously at high spatiotemporal resolution. This approach allows the characterization of several electrophysiological network-wide features, including single/-multi-unit spiking activity patterns and local field potential oscillations. To scrutinize these multidimensional neural data, we have developed several computational tools incorporating machine learning algorithms, automatic event detection and classification, graph theory, and other advanced analyses. By supplementing these computational pipelines with this platform, we provide a methodology for studying the large, multiscale, and multimodal dynamics from cell assemblies to networks. This can potentially advance our understanding of complex brain functions and cognitive processes in health and disease. Commitment to open science and insights into large-scale computational neural dynamics could enhance brain-inspired modeling, neuromorphic computing, and neural learning algorithms. Furthermore, understanding the underlying mechanisms of impaired large-scale neural computations and their interconnected microcircuit dynamics could lead to the identification of specific biomarkers, paving the way for more accurate diagnostic tools and targeted therapies for neurological disorders.

Here, we employ HD-MEA to delve into computational dynamics of large-scale neuronal ensembles, particularly in hippocampal, olfactory bulb circuits, and human neuronal networks. Capturing spatiotemporal activity, combined with computational tools, provides insights into neuronal ensemble complexity. The method enhances understanding of brain functions, potentially identifying biomarkers and treatments for neurological disorders.

Large-scale neuronal networks and their complex distributed microcircuits are essential to generate perception, cognition, and behavior that emerge from ...