Compartmentalization of Human Stem Cell-Derived Neurons within Pre-Assembled Plastic Microfluidic Chips

Compartmentalized microfluidic devices allow neurons to be studied in their highly polarized form. These devices are instrumental for both basic and translational neuroscience research. This protocol describes how to use cyclic olefin copolymer chips to compartmentalize neurons differentiated from human stem cells.

These COC chips produce healthier cultures of differentiated neurons over PDMS multi-compartment devices. In this protocol, we demonstrate the culture of neurons differentiated from the NIH-approved H9 stem cell line. Similar procedures can be used to differentiate neurons from mIPSCs.

The multi-compartment chips must be placed into secondary containment with extra humidification. The channels of the multi-compartment chips must be filled with a precoat solution, and then flushed with PBS. To begin, dissolve poly-L-ornithine in cell culture grade distilled water to make 600 microliters of solution per chip.

Aspirate the remaining PBS from the wells, making sure that the pipette tip is away from the channel opening. Load 150 microliters of poly-L-ornithine working solution in the upper right well. Wait for 90 seconds and add 150 microliters of the solution to the lower right well.

Wait five minutes and repeat the loading process with the solution for the left wells. Then, place the humidifier tray with chip at four degrees Celsius overnight. If using a Petri dish, wrap it with Parafilm and place it at four degrees Celsius overnight.

Next, aspirate the solution from the wells, ensuring that the pipette tip is placed away from the channel opening. And repeat loading the right and left wells with 150 microliters each of sterile water twice. Prepare the laminin working solution.

Load the right and left wells with 150 microliters each of the laminin working solution. Incubate the chip within the tray for two hours at 37 degrees Celsius. Rinse the chip with PBS without calcium and magnesium.

Load the right and the left wells with 150 microliters each of PBS. Wait five minutes. Aspirate the PBS from the wells and rinse the chip with NSC media.

Load the right and the left wells with 150 microliters each of the NSC media. Incubate the chip in the tray overnight in the 37 degree Celsius incubator with 5%CO2 to pre-condition the chip. To seed human neural stem cells into the multi-compartment chip, first count the cell concentration using a hemocytometer.

Then, aspirate the media from the wells. Pipette five microliters of the cell solution to the upper right well, followed by five microliters to the lower right well. Use a microscope to check that cells have entered the channel.

Wait five minutes for cells to adhere to the bottom of the chip. Pipette approximately 150 microliters of NSC media to the right and left wells. Incubate at 5%CO2, 37 degrees Celsius within a suitable humidified container.

After 48 hours, aspirate NSC media from the wells and replace it with neural differentiation media by adding 150 microliters to each top well and each bottom well. Culture neurons within a 5%CO2, 37 degree Celsius incubator within a humidifier tray. Remove the remaining neural differentiation media from the axonal compartment and place it into a centrifuge tube.

Store at 37 degrees Celsius. Mix 50 microliters of the virus solution with 150 microliters of the warmed media. Pipette 100 microliters of the mixture to both wells of the axon compartment.

Incubate for two hours within a 37 degree Celsius incubator. Withdraw and dispose of virus-containing media. Gently pipette 75 microliters of the fresh media to one axon compartment well and let the media flow through the channel into other axon well.

Repeat this process once. Finally, fill the axon compartment with the saved media, and transfer the chip to the incubator. After one week in the chip with the differentiation media, human neural stem cells differentiated into neurons, attached and distributed evenly within the somatic compartment.

In comparison, neurons in PDMS devices aggregated as early as five days post addition of differentiation media, leading to compromised cell health. Visualization of labeled neurons and dendritic spines with mCherry fluorescent protein showed that NSC-derived neurons differentiated within the chips form mature synapses. Neurons were labeled for the excitatory synaptic marker, vGlut1.

Immunostaining results show that virally labeled neurons could co-localize with vGlut1, and neuron-specific marker, Beta-tubulin three. Virally transduced mCherry neurons extend projections into an axon-localized microenvironment established within the preassembled chip. A comparison between the images of mCherry labeled neurons before and immediately after axotomy showed completely severed axons.

It is critical to make sure that the channels remain filled with fluid except when performing the axotomy procedure. COC-compartmentalized chips are easy to use and can open the door to numerous investigations related to neuronal injury, synaptogenesis, synaptic plasticity, and pathophysiology of disease.