This article describes a method for magnetizing neuronal cells using fluorescent magnetic nanoparticles (MNPs) to facilitate their orientation and attachment on a patterned substrate. The process involves cell culture, substrate preparation, and the application of growth factors to promote cell extension.
Begin by adding neuronal cells to a suitable medium in a culture dish.
Add fluorescent magnetic nanoparticles, or MNPs, containing a magnetic core tagged with a fluorescent dye.
During incubation, the cell takes up the MNP through endocytosis and becomes magnetized.
Place a clean magnetized patterned substrate in a culture dish. This glass substrate has embedded geometrical patterns with magnetic properties.
Sterilize the substrate by exposing it to ultraviolet light.
Coat the substrate with collagen to promote cell attachment.
Add the MNP-loaded cells to the substrate and incubate.
The external magnetic field of the substrate attracts the MNP-loaded cells, enabling their attachment.
Add a medium containing a specific growth factor at regular intervals.
The growth factor assists cells in forming extensions, while the magnetic pattern of the substrate directs the orientation of these extensions.
Seed 1 million cells in a regular, uncoated 35-millimeter dish. Add the calculated volume of MNP suspension and differentiation medium to the dish to achieve the desired MNP concentration. Mix the cells, MNPs, and differentiation medium, then incubate the dish in a 5% carbon dioxide humidified incubator at 37 degrees Celsius for 24 hours.
Clean the patterned substrate with 70% ethanol, and place it in a 35-millimeter culture dish in the hood. Place a large magnet below the patterned substrate for one minute, then remove it by moving the dish up and away, and taking the magnet out of the hood. Turn on the ultraviolet light for 15 minutes.
To prepare a collagen-coated glass substrate, dilute collagen type I at a 1-to-50 ratio in 30% ethanol. Cover the glass with the solution. Leave the dish uncovered in the hood for four hours until all the solution evaporates. Then, wash it three times in sterile PBS. Remove the cells from the incubator. Centrifuge the cell suspension for 5 minutes at 200 g and discard the supernatant. Resuspend the cells in 1 milliliter of fresh differentiation medium, and count the cells using a hemocytometer.
Seed 105 cells atop the substrate in a 35-millimeter culture dish, and add 2 milliliters of differentiation medium. Incubate the culture in a 5% carbon dioxide humidified incubator at 37 degrees Celsius. After 24 hours, add 1 to 100 fresh Murine beta-NGF to the cells. Renew the differentiation medium and add fresh beta-NGF every two days.
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