The overall goal of this procedure is to initiate new neurites, rapidly extend them, and precisely connect them to target cells. This procedure is only possible by using cells grown in a controlled and reproducible environment. This method allows us to answer key questions in neuroscience.
What limits the growth rate of neurons? How fast can they grow? Do mechanical clues play a role?
We can also study how synapses are formed, how do neurons find a target cell? How do synapses age, mature? And thirdly, we can start building systematically neural networks, study how neurons compute.
The main advantages of this technique are that it enables easy manipulation, experimental reproducibility, and ultimately a high degree of control at the single-cell level. To begin this procedure, clean and prepare the desired number of sterile cover slips. Next, coat each cover slip with 0.5 to 1 milliliters of 100 microgram per milliliter PDL for at least two hours at room temperature.
After that, wash the cover slips twice with water and remove all the liquid. Let them dry in a sterile environment for 5 to 10 minutes or until the surface is completely dry. Then, place the microfluidic devices with the patterns facing up under the UV light in a biosafety cabinet for 10 minutes.
After 10 minutes, place a microfluidic device with the pattern facing down in contact with the clean cover slip. Use the tweezers to softly press the device so it adheres to the glass. To fill the single population device with medium, add 50 microliters of complete cell medium to the right upper well.
And then add another 50 microliters of complete cell medium to the well diagonal to it. To fill the multiple populations device with medium, add 30 microliters of complete NBM to one row of wells. Make sure the medium flows between the wells.
Then, add 50 microliters of medium to each of the remaining wells. Place the devices in a bigger plate with an open dish of autoclaved water, and place the dish in the incubator for one to two hours while preparing the cell culture. Now, 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.
Afterwards, 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 micro channels.
Add 20 microliters of MBM 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 device's removal, add two 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 cover slips leaving a patterned configuration of neurons. In this procedure, and 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 one milliliter syringe, then perfuse the sample with physiological saline solution. Next, under the microscope, check the gap between the two isolated neuronal populations to insure 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 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 two micrometers, then pause for five minutes to allow neurite elongation.
After successful initiation and neurite extension for the first five 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 cover slip, 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 bead as well. Afterward, carefully place the sample back in the incubator to strengthen the neuronal connection for future experiments. You can use whole cell patch clamp recordings to investigate the electrical functionality of the micromanipulated connection.
Shown here are the isolated neuronal populations separated by a 100 micrometer gap in PDMS microdevices. Pair to patch clamp recordings were performed in whole-cell configuration, and the mechanically induced connections were analyzed, and compared with those in naturally connected neuronal populations, and non-connected populations. The electrical responses after pre-synaptic action potential recorded from the neurons connected naturally and by micromanipulation are significantly higher and temporarily correlate with the pre-synaptic activity.
Once mastered, this technique can be performed in eight hours if done properly, and while attempting this procedure, it is important to remember to have healthy and well-patterned long term cell cultures to guarantee fast and reproducible results. Following this procedure different methods can be used to verify the electrical functionality of the micromanipulated connection. After watching this video, you should have a good understanding of how to grow cell populations in microfluidic chambers, and use pipette manipulations to initiate, extend, and connect neurites.
