Neural Characterization and Development

Neural Development Application
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Developing advanced electrically active cell models is challenging. Can you capture the complexity of your stem cell-derived neuronal function with just imaging or expression data? Track the emergence of neuronal activity and watch as complex synchronous patterns emerge. Quantify network properties and classify how your cells behave.

Over minutes or months, gain unprecedented access to neuronal activity with the Maestro MEA platform. Noninvasively monitor cells in culture as they mature and establish their unique phenotype. Measure dozens of endpoints with AxIS Navigator to fully classify and characterize your model's activity.

Example Data

 

Functionally optimize neural differentiation and culture conditions

Action potential firing and synaptic activity are fundamental properties of neurons in the brain. Bardy et al. [PNAS, 2015]  have recently reported that Neurobasal® Medium and DMEM/F-12 support neuron survival but suppress their synaptic activities in culture. Since it is desirable for human pluripotent stem cell (hPSC)-derived neurons to have spontaneous electrical activity BrainPhys Neuronal Medium, was developed. Here the effect of BrainPhys™ and DMEM/F-12 based media on the network activity of hPSC-derived neurons during 18 week in culture was tested.

Neuro differentiation protocol
Raster plot shoring the firing patterns of neurons across 64 electrodes in a well of a MEA plate
Network bursts were detected at week 6

(A) Raster plots showing the firing patterns of neurons across 64 electrodes after 18 weeks in culture. Each black line represents a detected spike. Blue lines represent single channel bursts - a collection of at least 5 spikes, each separated by an inter-spike interval (ISI) of no more than 100 ms. Network bursts are marked with purple boxes and are defined as a collection of at least 10 spikes from a minimum of 25% of participating electrodes across the entire well, each separated by an ISI of no more than 100 ms. Neurons cultured in BrainPhys had increased firing compared to DMEM/F-12/NB-A. (B) Network bursts were first detected at week 6 in BrainPhys™ and DMEM/F-12/NB-A conditions. The number of network bursts increased gradually over time, suggesting that both cultures became more synchronous as they matured. After 18 weeks in culture, the number of network bursts detected in a 10-minute recording in BrainPhys™ and DMEM/F-12/NB-A were 114 and 54, respectively. Data courtesy of STEMCELL Technologies, taken from Mak et al. 2016 presented at SfN2016.

 

Identify unique network activity patterns with cell cultures from specific brain regions

Differences in electrical activity patterns from primary murine neuronal cell cultures were compared on the Maestro MEA system. As a result of their phenotypic receptor and neuron type composition, primary neuronal cell cultures show very specific and complex activity patterns after four weeks in vitro. This complexity results from a high level of network organization in primary cultures.

Network spike train patterns of brain-region specific primary cell cutlures from embryonic murine tissue
Neuro characterization of brain region
Network spike train patterns of midbrain and frontal cortex specific primary cell cutlures from embryonic murine tissue

Raster plots of brain region-specific primary cell cultures derived from embryonic murine tissue of the frontal cortex, spinal cord (with dorsal root ganglia), hippocampus, and midbrain (co-cultured with frontal cortex) were compared. Plotted are 60 seconds of 25 neurons of spontaneous network activity at 28 days in vitro. Spontaneous activity plotted for 60 seconds shows distinguishable patterns based on the brain region of origin.  Data courtesy of Neuroproof GMBH, taken from Voss et al. 2014 presented at SfN2014.

Assay Steps

Neural characterization protocol

Protocol taken from Mak et al. 2016 presented at SfN2016.  Neural progenitor cells derived from hPSCs (XCL1-NPC) were cultured in STEMdiff™ Neuron Differentiation Medium on poly-L-ornithine (PLO)/laminin-coated 6-well plate for 5 days. On day 5, neural progenitor cells were dissociated and single cells were re-plated onto a PLO/laminin-coated CytoView MEA plates at 30,000 cells/cm2 in STEMdiff™ Neuron Differentiation Medium.  After one day, half of the medium was replaced with differentiation media (BrainPhys™ Neuronal Medium + supplements: 1% N2 Supplement-A, 2% NeuroCult™ SM1 Neuronal Supplement, 20 ng/mL GDNF, 20 ng/mL BDNF, 1 mM db-cAMP and 200 nM Ascorbic Acid). Half-medium changes were performed every 3 - 4 days throughout the culture period.  Spontaneous neuronal activity was acquired at 37°C under a 5% CO₂ atmosphere using the Maestro MEA system. A 15-minute recording was taken twice a week and analyzed with AxIS Navigator Neural Module software.

 

Key Features

 

Maestro MEA user

 

The advantage of neural differentiation and characterization experiments on the Maestro Pro and Edge systems:

  • Measure what matters – Indirect measures of cell quality are regularly used to track stem cell differentiation efficiency. However, expression levels of protein markers often poorly correlate with cell model performance. Maestro tracks neuronal excitability in real-time enabling you to measure what matters: do your hiPSC-neurons fire as expected?

  • Analyze cell activity label-free – Neurons exist as a functional network of inter-linked cells. The Maestro MEA platforms preserve the complex functionality of your neural models. Platforms that require single-cell suspensions (automated patch clamp, flow cytometry), require more sample handling and destroy the networks that define the functionality of these neural cultures.

  • Probe cell models in the same plate they were cultured in – Neurons exist as a functional network of inter-linked cells. The Maestro MEA platforms preserve the complex functionality of your neural models. Platforms that require single-cell suspensions (automated patch clamp, flow cytometry), require more sample handling and destroy the networks that define the functionality of these neural cultures.

  • It's easy – You don't have to be an electrophysiologist to use the Maestro MEA system. Just culture your neurons in an MEA plate, load your plate into the Maestro MEA system, and record your neural data. Axion's data analysis tools will do the rest, even generating the publication-ready graphs you need.

Technology

Neural MEA technology

Neural MEA

 

What is a microelectrode array (MEA)?

Microelectrode arrays (MEA), also known as multielectrode arrays, contain a grid of tightly spaced electrodes embedded in the culture surface of the well. Electrically active cells, such as neurons, are plated and cultured over the electrodes. When neurons fire action potentials, the electrodes measure the extracellular voltage on a microsecond timescale. As the neurons attach and network with one another, an MEA can simultaneously sample from many locations across the culture to detect propagation and synchronization of neural activity across the cell network.

That’s it, an electrode and your cells. Since the electrodes are extracellular, the recording is noninvasive and does not alter the electrophysiology of the cells - you can measure the activity of your culture for minutes, days, or even months!

CytoView well bottom

An MEA of 64 electrodes embedded in the substate at the bottom of a well.

Rendering of cells growing over the electrodes at the bottom of the well

Neurons attach to the array and form a network. The microelectrodes detect the action potentials fired as well as their propagation across the network.

 

 

 

Brain waves in a dish

Neurons communicate with other cells via electrochemical signals. Many neural cell types form cellular networks, and MEAs allow us to capture and record the electrical activity that propagates through these networks.

Neurons fire action potentials that are detected by adjacent electrodes as extracellular spikes. As the network matures, neurons often synchronize their electrical activity and may exhibit network bursts, where neurons repeatedly fire groups of spikes over a short period of time.

The MEA detects each cell's activity, as well as the propagation of the activity across the network, with spatial and temporal precision. Patterns as complex as EEG-like waveforms, or "brain waves in a dish", can be observed. Axion's MEA assay captures key features of neural network behavior as functional endpoints - activity, synchrony, and network oscillations.

Action potentials recorded from electrodes

Action potentials are the defining feature of neuron function. High values indicate frequent action potential firing and low values indicate the neurons may have impaired function.

Synchrony reflects the prevalence and strength of synaptic connections, and thus how likely neurons are to generate action potentials simultaneously

Synapses are functional connections between neurons. Synchrony reflects the prevalence and strength of synaptic connections, and thus how likely neurons are to generate action potentials simultaneously on millisecond time scales.

Network oscillations, or network bursting, are defined by alternating periods of high and low activity

Network oscillations, or network bursting, as defined by alternating periods of high and low activity, are a hallmark of functional networks with excitatory and inhibitory neurons. Oscillation is a measure of how the spikes from all of the neurons are organized in time.

 

Do more with multiwell

Axion BioSystems offers multiwell plates, ranging from 6 to 96 wells, with an MEA embedded in the bottom of each well. Multiwell MEA plates allow you to study complex neural biology in a dish, from a single cell firing to network activity, across many conditions and cell types at once.