Neural Co-culture

Neural co-culture in 6 well Cytoview MEA plate with ibidi insert
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The brain is a collection of individual, but interconnected functional circuits.  Additionally, circuits within the nervous system interact with various other biological systems in the human body. Compartmentalized in vitro models allow two spatially distinct neural circuits or, more generally, two cell populations to develop functional connections and mimic key biological interactions.

Using Axion BioSystems' Maestro Pro and Edge systems, any scientist can now track functional network formation between neural co-cultures in the same well. Together, neural co-cultures on MEA plates create an ideal model for studying the functional connection between two networks, and the effects of synaptic blockade.

 

Tracking functional network formation between two neural populations

 

 

A two compartment well divider insert composed of silicone (ibidi, cat. 80209) was used to culture two cortical neuron populations within the same well of a CytoView MEA 6-well plate (M384-tMEA-6W). At 7 days in vitro, the well divider insert was removed, such that neurites could cross the cell-free gap and establish functional connections between the two distinct cortical networks. Experimental results from an optical image and activity map confirmed that a consistent, bi-directional functional connection between the two networks was formed within 10 days of insert removal.

CytoView MEA well with ibidi co-culture insert
10 days post insert removal the neurite growths can begin to be seen
Multielectrode activity map showing activity from the two neurite populations
Raster plot of network activity 17 days post insert removal

A) Two compartment silicone well divider insert. B) Neurites project across the cell-free gap within 10 days of removing the insert. C) Activity map illustrating synchronous activity between the two spatially separated networks. D) Raster plot at 17 days in vitro illustrating independent network activity from Network 1 and Network 2, followed by a whole-well network event with Network 1 driving the activity in Network 2.

Neural co-culture assay protocol steps

Getting started with Maestro Pro and Edge couldn't be easier. Place the well divider insert (ibidi, cat. 80209) into the well of a CytoView MEA 6 well plate (M384-tMEA-6W). Culture your neurons in the desired compartments of the well divider insert (Day 0). Remove the well divider insert after the cells have attached to the surface of the MEA plate (~1-7 days). Load the MEA plate into the Maestro MEA system and allow the environmental chamber to automatically equilibrate. Analyze the neural activity in the MEA plate label-free and in real-time with AxIS Navigator Neural Module software (Day 2+).

Download Protocol

 

Maestro MEA user

 

The advantages of measuring neural activity from neural-co-cultures on the Maestro MEA platform:

  • Measure what matters – The Maestro Pro and Edge MEA systems directly measure neuronal action potentials. Indirect measurements like calcium imaging are unable to capture important but subtle changes to neural network signaling while gene and protein expression are insufficient to characterize function. The Maestro MEA platforms measure activity in real-time, enabling you track functional network formation between neural co-cultures in the same well.

  • Analyze cell activity label-free – The Maestro MEA system performs noninvasive electrical measurements from the cultured neural population, circumventing the use of dyes/reporters that can perturb your cell model and confound results. Track activity over hours, weeks, and months from the same population of cells.

  • Precise assay environment – The smart environmental chamber finely controls temperature and CO₂ while rejecting electrical noise and mechanical vibrations.

  • See your cells – Sometimes you just want to look at your neural co-cultures under a microscope. CytoView MEA plates have a thin, transparent plate bottom for culture visualization and assay multiplexing.

  • 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 cells in an MEA plate, load your plate into the Maestro MEA system, and record your co-culture activity data. Axion's data analysis tools will do the rest, even generating the publication-ready graphs you need.

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.