The overall goal of this procedure is to culture, record and stimulate neuronal networks on micro electrode arrays. This is accomplished by first preparing reagents and micro electrode arrays for culturing. The second step of the procedure is to dissociate cortical neurons from a day 18 rat embryo.
The third step of the procedure is to plate neurons on microelectrode arrays and culture for two to three weeks. The final step of the procedure is to experiment with the neuronal networks via stimulating and recording from the microelectrode arrays. Ultimately, results can be obtained that show spontaneous and stimulus evoked activity in neuronal networks with recording and stimulation software through meticulous cell culturing on Microelectrode arrays.
Hello, I'm Professor Steve Potter. I'm the director of the Laboratory for Neuro Engineering in the culture Department of Biomedical engineering at Georgia Tech. That department is shared with Emory University School of Medicine.
And I'm Chad Hales, a postdoc in Dr.Potter's lab. I'm also a cognitive behavioral fellow in the Department of Neurology at the Emory University School of Medicine. Today we're gonna be showing you how to culture and care for neurons on multi electrode arrays.
Like these. We use them to study learning and memory in vitro and network information processing. So let's get started In a laminar flow hood, begin preparing the multichannel systems micro electrode array or MEA pipette 100 microliters of polyethylene amine into the center of a clean and sterile MEA resting in a 100 millimeter Petri dish to avoid damaging the electrodes.
Never touch solid objects to the MEA surface. Lightly covered the dish with a sterile lid fitted with a gas permeable Teflon membrane and incubate in the hood for 30 minutes. The Teflon membrane lid provides for gaseous exchange with minimal evaporation and the ability to culture in lower humidity environments.
The lower humidity allows the use of delicate electronics in the incubator and reduces contamination rates. Aspirate away the PEI rinse the MEA three times with one to two milliliters of sterile deionized water. Allow them to dry with the lid off in the hood for at least 30 minutes or until they are completely dry.
Complete an E 18 cortical neuron dissociation as described in the text portion of this protocol. Strain the cells after straining. Transfer the cell suspension to a 15 milliliter conical tube and bring the cell suspension volume to four milliliters with cell medium carefully.
Layer 500 microliters of a 5%BSA solution underneath the cell suspension centrifuge at 200 Gs for six minutes. While the cells are spinning, check that the MEA is completely dry. Carefully pipette 20 microliters of laminate solution into the center of the MEA.
Avoid introducing air bubbles and ensure that all the electrodes are covered. Gently place the Teflon lid onto the MEA to prevent evaporation of the laminin. The MEA should be on a completely flat surface when the lid is placed.
Otherwise, the pressure might crack the glass bottom of the array. Incubate the dish at 35 to 37 degrees Celsius, 5%carbon dioxide, 9%oxygen, and 65%humidity for 20 minutes. Now transfer the spun cells to the hood.
A cell pellet should be visible in the bottom of the tube. Remove and reserve the supernatant in case of incomplete pelleting. Gently resuspend the pellet.
Count the number of cells in 10 microliters of the suspension on a hemo cytometer and dilute the suspension to the desired concentration. Typically 1000 to 3000 cells per microliter with cell medium. When the laminate incubation of the array is complete, aspirate the droplet away immediately.
Pipette 15 to 20 microliters of the cell suspension over the laminate coated area of the MEA. Cover the dish with the Teflon lid and place in the incubator for 30 minutes. Under a phase contrast microscope, inspect the dish to ensure that the cells have adhered and cover all of the electrodes.
If coverage is incomplete, add more cells and re incubate. Slowly flood the dish with one milliliter of warmed equilibrating cell medium. Replace the Teflon lid and place the dish back in the incubator for 24 hours.
The next day. Under the phase contrast microscope, check that the cultured cells are adhered and that the amount of debris and cell death is limited with the proper care and feeding. As explained in the accompanying text, neurons cultured in this way can survive for more than a year.
After two to three weeks, cultures have reached a steady state of activity and can be recorded from this video will now demonstrate recording with the custom made neuro writer program. Transfer the dish into the recording incubator again kept at 35 to 37 degrees Celsius, 5%carbon dioxide, 9%oxygen, and 65%humidity. To maintain ideal temperature and pH, when moving the dish, carry it parallel to the ground and avoid quick movements that may dislodge the cells from the substrate.
Lift the MEA from the dish and place it into the multichannel systems. Recording preem amplifier, being sure to properly match up the internal ground with the Preem amplifier ground. The pre amplifier is sitting on a pelt device, which functions to dissipate heat as described in the accompanying text here, the pre amplifier has been moved out of the incubator.
Better show proper seating of the dish. Use a wipe moistened with 70%ethanol to clean the contacts around the perimeter of the array. Make sure the MEA is seated properly lower and latch the lid.
Use a cotton swab to align the contacts of the array if necessary. In neuro writer, choose settings appropriate for the experiment. The record switch must be flipped up.
To write the data to a file, click the start button to begin recording. The spike screen will now show running traces of activity and background noise with occasional action potentials and bursts of activity. If an electrode displays too much noise, there may be a misaligned pin on the pre amplifier lid, a small piece of trash, a drop of medium, a degraded electrode surface, or an overlapping clear membrane from the lid disrupting the contact.
Check the contact pin and adjust with a cotton swab soaked in 70%ethanol if necessary. Click on the spike waveform tab to change the screen view to spike detection mode where action potentials are displayed in static three millisecond long windows. To stimulate the neuronal network on the MEA train the salpa algorithm under the recording settings tab to limit the stimulation artifact, activate salpa in the online settings tab.
Once recording is started, click on the STEM tab to set up an appropriate stimulation paradigm to use the open loop program to stimulate one electrode and watch the responses in the dish. Choose the appropriate settings in the open loop section and then press start. Visualize responses on the main spikes tab and the spike wave forms tab.
This movie portrays developing in vitro day seven neurons and glial cells as they grow and form a connected network on an MEA for micro electrodes are visible. This is a raster plot of two minutes of spontaneous activity from a neuronal network. Each black dot represents an action potential.
Two bursts of activity are indicated with the blue arrows. The response over multiple electrodes is a function of the network connectivity. Here is a raster plot of 16 seconds of stimulus evoked activity.
Red stars represent stimuli. The red arrows show five stimuli that successfully induced bursts of synaptic activity across multiple electrodes. Occasionally a stimulus does not yield a burst.
Here at the blue arrow, We've just shown you how to culture record from and stimulate neuronal networks on micro electrode arrays. Probably the most important thing worry about is to prevent the cultures from getting infected and to reduce evaporation. If used lids with semi-permeable membranes like this, they're sealed and they prevent evaporation and infection, and you might be able to culture the cultures for months, perhaps even more than a year.
So that's it. Thanks for watching and good luck with your experiments.
