This article describes a microfluidic device designed for the culture and differentiation of neural stem cells (NSCs) into neurons. The device features separate compartments for somatic and axonal growth, facilitating the study of neuronal development.
Take a microfluidic device consisting of reservoir wells connected to two compartments, a somatic and an axonal, separated by a microgroove barrier.
The compartments and microgrooves are coated with a cell culture substrate to facilitate cell adhesion.
Add a neural stem cell medium to each well and incubate to precondition the device for cell culture.
Discard the media. Add neural stem cells, or NSCs, to the wells of the somatic compartment and allow the cells to adhere.
Add the NSC medium to each well and incubate in a humidified chamber to prevent media evaporation. During incubation, the NSCs proliferate.
Replace the NSC medium with a neural differentiation medium and incubate.
The small molecules in the medium induce the differentiation of NSCs into neurons within the somatic compartment, with their axons extending through the microgrooves into the axonal compartment.
The differentiated neurons with extended axons are ready for further assays.
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 4 degrees Celsius overnight. If using a Petri dish, wrap it with parafilm and place it at 4 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 a 37 degrees Celsius incubator with 5% CO2 to precondition the chip.
To seed human neural stem cells into the multicompartment chip, first, count the cell concentration using a hemocytometer. Then, aspirate the media from the wells. Pipette 5 microliters of the cell solution to the upper right well, followed by 5 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 degrees Celsius incubator within a humidifier tray.
繁體中文
