Initiating Rapid Neurite Extension and Formation Functional Neuronal Connections

Remove the medium from the upper wells of the microfluidic devices without emptying the wells. Then, add 20 microliters of the concentrated cell solution into the top right well of the microfluidic device, and check in the microscope how cells flow inside the single population microfluidic device.

To plate cells in the multiple populations' device, add 20 microliters of the concentrated cell solution into each of the top wells. Under the microscope, check if the cells are inside the chambers. Afterward, place the devices in the incubator for 15 to 30 minutes to promote cell attachment to the substrate.

After the cells attach to the substrate, add 40 microliters of NBM to the two top wells of the single population device. This will help remove the air bubbles from the microchannels. Add 20 microliters of NBM to the top wells of the multiple populations device, the same wells where the cells were injected. The media should protrude slightly to form a positive meniscus, and give the wells a muffin-top aspect.

One to two days before the microfluidic devices removal, add 2 milliliters of pre-warmed NBM to each sample dish, and flood the chambers. Then, keep the devices in the incubator. Subsequently, use sterile tweezers and a pipette tip to remove the microfluidic devices from the coverslips, leaving a patterned configuration of neurons.

In this procedure, add 40 to 60 microliters of the prepared PDL-coated beads to the cell culture. Then, return the sample to the incubator for one hour to promote the formation of synaptic contacts. Subsequently, install the sample in an experimental setup such that the cells can be assessed from above by two micropipettes and also be accessed optically. In this configuration, a CCD camera is located on the side port of the microscope for image capture. Connect each pipette filled with saline to a 1-milliliter syringe. Then, perfuse the sample with physiological saline solution.

Next, under the microscope, check the gap between the two isolated neuronal populations to ensure that the two populations are not naturally connected. Select a PDL bead that is not attached to the neuron in the field of view. Align the bead with a micropipette tip by focusing on the bead and then on the micropipette.

Subsequently, bring the tip down as close as possible to the bead by monitoring it through the microscope. Then, apply negative pressure to the pipette to pick up the bead, and maintain the negative pressure throughout the experiment.

Now, select a PDL bead attached to a neuron in the field of view, and attach it to the second micropipette using suction. Pull the PDL bead-neuron complex by slowly moving either the micromanipulator or the sample stage at 0.5 micrometers per minute for 1 micrometer, and pause for five minutes to allow neurite initiation.

Pull the PDL bead-neuron complex by slowly moving it laterally at 0.5 micrometers per minute over 2 micrometers. Then, pause for five minutes to allow neurite elongation. After successful initiation and neurite extension for the first 5 micrometers, continuously pull the neurite at 20 micrometers per minute.

To connect the neurons, select a region rich in neurites. Use other beads to gauge the pipette tip height above the coverslip, and lower the PDL bead-neurite complex so that it physically contacts the neurites. Leave the PDL bead-neurite complex in contact with the target neurite while manipulating the second micropipette. Then, lower the second pipette with the second PDL bead on top of the newly formed neurite about 20 micrometers from the first bead, and use the second PDL bead to push the new neurite filament towards the target cell.

Next, hold both beads in place for at least one hour. Verify the absence of focal swelling, a thickening of the neurites contacting the bead with the microscope. During this time, slowly change the medium of the sample from physiological saline to pre-warmed carbon dioxide-equilibrated NBM.

Release the bead from the second pipette by releasing the suction. If the new neurite remains attached, release the first speed as well. Afterward, carefully place the sample back in the incubator to strengthen the neuronal connection for future experiments.