Compartmentalized Primary Murine Neuron Culture Using Plastic Microfluidic Chips

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparation and Coating of the Multicompartment Chips

1. In a bio-safety cabinet, place the chip into a Petri dish or other suitable sterile container.
2. Add 100 µL of pre-coating solution to the upper left well of the chip and allow it to flow through the main channel into the adjoining well.
   NOTE: The pre-coating solution is used to pre-coat the microfluidic channels to eliminate the potential for trapping air bubbles within the chip.
3. Fill the lower left well with 100 µL of pre-coating solution. Wait 5 min to allow the solution to flow through the microgrooves.
4. Add 100 µL of pre-coating solution to the upper right well and allow it to flow through the main channel into the adjoining well. Fill the lower right well with 100 µL of pre-coating solution.
5. Aspirate the solution from each well. Aspirate away from the main channels to avoid removing liquid from the main channels (Figure 1A). Immediately add 150 µL of phosphate-buffered saline (PBS) to the upper left well. Wait 1.5 min.
   CAUTION: Do not aspirate all liquid from the enclosed main channels.
6. Add 150 µL PBS to the lower left well. Wait 5 min to allow liquids to flow through the microgrooves. Add 150 µL PBS to the upper right well. Add 150 µL PBS to the lower right well. Wait 10 min.
7. Repeat steps 1.5-1.6 for a second PBS wash.
8. Check the chip under a tissue culture microscope for bubbles in the main channels. If bubbles are present, perform the procedures below. If no bubbles are present, skip to step 1.9.
   1. Aspirate PBS from the wells, angling the pipet tip away from the channel opening (Figure 1A).
   2. Dispense 100 µL of pre-coating solution into the upper well, angling the tip of the pipet near the channel opening (Figure 1B). The bubbles should move through the channel into the lower well. Wait 1.5 min.
   3. Repeat steps 1.3-1.8.
9. Aspirate PBS from the wells, angling the pipet tip away from the channel opening (Figure 1A).
10. Add 100 µL of 0.5 mg/mL poly d-lysine (PDL) to the upper left well of the chip. Wait 1.5 min. Fill the lower left well with 100 µL of PDL.
11. Add 100 µL of PDL to the upper right well of the chip. Wait 1.5 min. Add 100 µL to the lower right well.
12. Close the petri dish and place the chip in an incubator at 37 °C for 1 h.
13. Repeat PBS wash steps 1.5-1.6 twice to remove excess PDL.
14. Aspirate the PBS from the device.
15. Immediately add 100 µL of cell culture media to the upper left well of the chip. Wait 1.5 min. Add media to the lower left well. Add media to the upper right well. Wait 1.5 min. Add 100 µL medium to the lower right well of the chip.
16. Place the chip in the 37°C incubator until ready to plate cells.

2. Seeding Neurons into the Multicompartment Chips

1. Prepare cell suspension of dissociated rat hippocampal neurons according to established protocols to yield a density of ~12 × 10⁶ cells/ mL.
   NOTE: Use of cell suspension densities between 3 and 12 × 10⁶ cells/mL is possible. If a lower density is used, the volume of cell suspension to be added to the chip may be increased. The procedure described below is applicable for murine dissociated cortical or hippocampal neurons. Optimal cell densities for other neuron types may vary.
2. Remove the majority of media in each well of the chip, leaving approximately 5 µL in each well. Aspirate away from the main channels to avoid removing liquid from the main channels (Figure 1A).
   CAUTION: Do not aspirate liquid from the enclosed main channels. Air bubbles may become trapped in the chip if fluid is aspirated from the main channels.
3. Load 5 µL of cell suspension in the upper right well and another 5 µL of cell suspension in the lower right well (~120,000 cells total). Load the cells by dispensing them close to the main channel (Figure 1B). Check under a microscope to ensure the neurons are in the main channel. Wait for 5 min to allow the cells to attach.
   NOTE: Neurons can be loaded into either compartment. For explanation purposes, the somatic compartment is the main channel on the right side, but either compartment can be used as the somatic compartment. Use of lower cell densities down to 60,000 cells per chip is possible. Up to 10 µL of cell suspension may be added to each well of the somatic compartment in combination with a cell suspension with fewer cells than described above.
4. Add approximately 150 µL of neuronal culture media to each of the upper and lower right wells, and then add 150 µL of media to each upper and lower left well. Place the chip into the humidified tray in a 5% CO₂ 37 °C incubator.
5. After 24 h, perform a media change by removing media from the wells. Make sure the main channel remains filled. Add 150 µL of media to each top well, then fill the bottom wells.
6. Place the chip back in the incubator for the desired number of days.
   NOTE: Monitor the media every few days to ensure it remains light pink. If the media is yellowish, replace 50% of it with fresh media. If the fluid level is low, ensure adequate humidity and appropriate secondary containment of the chips to prevent evaporation. Minimizing or eliminating media changes is possible using secondary containment and/or covering the dish containing the chip with polytetrafluoroethylene (PTFE)-FEP film.