Design, Surface Treatment, Cellular Plating, and Culturing of Modular Neuronal Networks Composed of Functionally Inter-connected Circuits

This manuscript describes a protocol to grow in vitro modular networks consisting of spatially confined, functionally inter-connected neuronal circuits. A polymeric mask is used to pattern a protein layer to promote cellular adhesion over the culturing substrate. Plated neurons grow on coated areas establishing spontaneous connections and exhibiting electrophysiological activity.

The overall goal of this procedure is to grow in vitro modular networks consisting of spatially confined, functional, interconnected neuronal circuits. This is accomplished by first preparing the PDMS stencils to pattern a protein layer to promote cellular adhesion over the culturing substrate. The second step is to clean the culturing substrates.

Specifically the surfaces of Petri dishes, cover slips and multi electrode arrays. Next, the PDMS stencils are deposited on the culturing substrate and the desired adhesive protein layer patterns are created. The final step is the plating of the neuronal glial cells.

Ultimately, multi electrode array recordings and calcium imaging are used to show the dynamics of the achieved modular neuronal networks. This method can help answer key open questions in the neuroscience field, such as investigating the dynamics of communication among neuronal assemblies, and there will counter experimental conditions. Polymethyl soane or PDMS is made by first mixing one part curing agent with 10 parts of base agent.

After five minutes of mixing, move the mixture to a vacuum chamber for 15 minutes. After 15 minutes, check for bubbles and return the solution to the chamber for 15 more minutes. Next, prepare a wafer on the spin coder.

Open the gas knobs for nitrogen gas. Put a wafer on the spinner and secure it in place with the vacuum. Then pour a thin layer of PDMS over the wafer.

Spin the wafer with PDMS for a minute at 1000 RPM to make a 100 micron thick PDMS coating on the wafer. Then transfer the wafer to a hot plate at 100 degrees Celsius. Let it bake there for 30 minutes.

After the PDMS has hardened, use a pipette to outline the stencils borders with more PDMS. Then bake the added PDMS onto the wafer. As before, when the PDMS borders have hardened, cut the stencil along the borders pae the silicone stencil off the wafer to begin RINs.

23 millimeter square glass cover slips with distilled water, followed by 70%ethanol, then acetone, then isopropanol, and finally distilled water. Again, dry the squares under a nitrogen gas stream. Next, gently press one pattern PDMS stencil onto each cover slip.

Put the cover slips with the stencils in a vacuum chamber for 15 minutes. Once vacuumed, drop a milliliter of PDL onto the stencils and return the assemblies to the vacuum chamber for 20 minutes. Repeat the 20 minute vacuum cycle once, and then let the PDL dry overnight the next day.

Prepare the petri dishes that will support the networking cells.First. Cover 3.5 centimeter dishes with a milliliter of PDL for two hours. At room temperature later, remove the PDL from the dish by aspiration.

Then wash the dishes with distilled water and leave them to dry. Once a dish is dried, add a small volume of silicone grease to the dish according to the four corners of the cover slip. Place the cover slips on the dish with the PDMS facing up and gently press them down to make certain they're attached.

Now gently tweezed A-P-D-M-S off the cover slips leaving the PDL pattern. Finally sterilize the dishes by exposing them to UV for seven minutes. First, clean the multi electrode array by washing it under tap water and then sonicate it in a concentrated enzymatic detergent.

Repeat these steps three times. Then sonicate the MEA in pure distilled water three times after which under a hood. Wash the MEA with distilled water before.

Sterilizing it with UV light for 30 minutes. Next, prepare the supporting network first. Pour PDMS into a 12 or 24 well plate.

Once the PDMS has hardened, remove it with a needle. Now cut holes at the center of the discs to make rings, which work as mold support. Now place a mold support at the center of the MEA and cover the external exposed surface of the MEA.

Let this settle for two hours at room. Then aspirate the PDL wash, the external exposed surface of the MEA with distilled water, and remove the mold support so that the MEA can be allowed to dry. Now attach a stencil to a prepared micro manipulator under an inverted microscope.

Inspect and align the patterned structure to match the electrodes of the MEA. Then use the micro manipulator to lower the stencil to the MEA surface. Once attached, raise the microm manipulator and if needed, use tweezers to apply a small amount of pressure to prevent the PDMS from detaching.

Now transfer the MEA to the vacuum chamber for 15 minutes. Then apply a one milliliter drop of PDL to the stencil and return to the vacuum chamber for two cycles of 20 minutes. Let the PDL dry overnight the next day.

Remove the P DM S stencils from the meas using tweezers. Lastly, UV sterilize the meas for seven minutes. They're then ready for the cells in preparation culture cells and prepare suspensions as in the text protocol.

After Resus suspending the required number of cells, plate them at the center of the patterned area on the MEA or cover slip. An MEA should be loaded with 100 microliter volumes and a cover slip. Accommodates 1000 microliters.

Incubate the plate cells for 40 minutes, then add plating medium to maintain the cells every four days. Add back fresh supplemented growth medium. After the sixth or seventh days, neuronal connections between the modules should start forming at that point.

Dilute the culture medium with an anti-mitotic agent to prevent glial overgrowth. After four days in vitro, it is possible to observe that neurons attached within the PD DL coded spots of 600 microns square after 14 days. In culture, neuron spontaneously established connection within the modules and among the modules forming active functional networks.

Neuronal circuits of 300 micron square were observed by having the PDMS feature size while keeping the same plating concentration. Calcium imaging was performed using a Forex objective to broadly activity in the cultured networks. Between the green and pink modules, a larger number of connections are seen while the blue and red modules are less connected to the other modules.

A roster plot of the calcium events onsets is shown for a movie acquired at 30 hertz with a 1 million pixel image. Green and pink modules were highly synchronized, whereas the blue and red modules were less. So.A cortical modular network composed of three distinct circuits was recorded at 21 days in vitro.

Using an MEA neuronal circuits about 600 microns apart were found to be interconnected. Their electrophysiological activity was reconstructed. Using a precise timing spike detection algorithm, a roster plot of five minutes of this data was made with color coded cluster data.

This provides a whole network and single cluster activity comparison and gives insight into intra cluster synchrony. After watching this video, you should have a good understanding of how to prepare PDM stencils and use them to pattern the adhesion of neuroglial cells leading to the establishment of functional modal network.