Growth and Differentiation of Magnetic Nano Particle-Loaded Neurons on Magnetic Platforms

1. Cellular magnetic nanoparticle (MNP) uptake

1. Prepare basic growth medium for PC12 cell culture by adding 10% horse serum (HS), 5% fetal bovine serum (FBS), 1% L-glutamine, 1% penicillin/streptomycin, and 0.2% amphotericin to Roswell Park Medical Institute (RPMI) medium, and filter using a 0.22 µm nylon filter.
2. Add 1% horse serum (HS), 1% L-glutamine, 1% penicillin/streptomycin, and 0.2% amphotericin to RPMI medium to prepare PC12 differentiation medium and filter using a 0.22 µm nylon filter.
3. Grow cells in a non-treated culture flask with 10 mL of basic growth medium; add 10 mL of basic growth medium to the flask every 2-3 days, and sub-culture the cells after 8 days.
4. For cellular uptake, centrifuge the cell suspension in a centrifuge tube for 8 min at 200 × g and room temperature, and discard the supernatant.
5. Resuspend the cells in 3 mL of fresh basic growth medium. Again, centrifuge the cell suspension for 5 min at 200 × g and room temperature, and discard the supernatant. Resuspend the cells in 3 mL of fresh differentiation medium.
6. Aspirate the cells 10x using a syringe and a needle to break up cell clusters. Count the cells using a hemocytometer, and seed 10⁶ cells in a regular uncoated 35 mm dish.
7. Add to the dish the calculated volume of MNP suspension and volume of differentiation medium to achieve the desired MNP concentration and total volume. Mix the cells, MNPs, and differentiation medium; incubate the dish in a 5% CO₂ humidified incubator at 37 °C for 24 h.
8. Centrifuge the cell suspension for 5 min at 200 × g at room temperature, and discard the supernatant. Resuspend the cells in 1 mL of fresh differentiation medium, and count the cells using a hemocytometer.

2. Cell differentiation and growth on magnetic platform

1. Clean the patterned substrate with 70% v/v/ ethanol and place the substrate in a 35 mm culture dish in the hood. Place a large magnet (~1500 Oersted) below the patterned substrate for 1 min and remove the magnet by first moving the dish up and away from the magnet, and then take the magnet out of the hood. Turn on the ultraviolet light for 15 min.
2. Coat the substrate with collagen type 1 by diluting collagen type 1 (solution from rat tail) 1:50 in 30% v/v ethanol. For coating a 35 mm dish, add 20 µL of collagen to 1 mL of 30% ethanol.
3. Cover the dish with the solution, and wait until all the solution evaporates, leaving the dish uncovered for a few hours. Wash 3x in sterile 1x PBS; the glass slide is ready for cell seeding.
4. Suspend the cells after cellular MNP uptake, seed 10⁵ cells in a 35 mm culture dish, and add 2 mL of differentiation medium. Incubate the culture in a 5% CO2, humidified incubator at 37 °C.
5. After 24 h, add 1:100 fresh murine β-NGF (final concentration of 50 ng/mL). Renew the differentiation medium and add fresh murine β-NGF every 2 days.