Easy and Reproducible Low-Density Primary Culture using Frozen Stock of Embryonic Hippocampal Neurons

The frozen stocks of rat embryonic neurons are ready to use, and no advanced techniques are required for culturing the cells. This technique is very easy and you don't need any permission for animal experimentation, arranging timed pregnant animals, or dissecting embryos. The neurons can be cultured in other culture vessels and applied for other types of experiments.

For example, microelectrode array plates for electrophysiological experiments. Begin by coating a 96-well microplate with 100 microliters of poly-L-lysine per well. Next, incubate the plates overnight at 37 degrees Celsius.

At the end of the incubation, remove the poly-L-lysine solution. Wash the plates twice with sterilized water and once with supplement-free, fresh culture medium. Place the plates out to dry.

Dry plates can be wrapped and stored for up to one month at four degrees Celsius. To start cell seeding, add 50 microliters of the culture medium to each well in the coated plates. Fill the peripheral wells with 200 microliters of sterilized water and incubate the plate for one hour at 37 degrees Celsius with 5%carbon dioxide.

Remove the neuron cryo vial from the liquid nitrogen tank and place it in a 37 degree Celsius heat block to partially thaw its contents. Using a one-milliliter pipette with a wide bore tip, slowly transfer the vials contents dropwise to a sterile, 50-milliliter tube. Rinse the empty cryo vial with one milliliter of room temperature culture medium.

Transfer the rinse solution to the 50-milliliter tube containing the cell suspension. Next, add nine milliliters of the room temperature culture medium dropwise into the 50-milliliter tube, making up the volume to 11 milliliters. After counting the cells using a hemocytometer, transfer the cell suspension into a reservoir.

Now, using a multi-channel pipette with wide bore tips, dispense the cell suspension with a final count of 1.0 times 10 to the four cells per well into the coated 96-well plate. Incubate the neurons for one to two hours at 37 degrees Celsius under 5%carbon dioxide. Then, replace the culture medium with 100 microliters of preheated culture medium and incubate again at the same conditions.

After four days in vitro, add cytosine beta-D-arabinofuranoside at the final concentration of 0.2 micromolar per well to reduce the growth of glial cells. Place the plate back in the incubator. After 21 days in vitro, treat the cells with the drugs of interest.

To maintain a positive control, treat cells with 100 micromolar glutamate per well for 10 minutes at 37 degrees Celsius before fixation. Before performing immunocytochemical studies, fix the cells for 20 minutes by replacing the culture solution with 100 microliters of paraformaldehyde in each well. After fixation, wash the wells twice with 250 microliters of phosphate-buffered saline per well for five minutes each.

In the present study, the neurons were developed normally without exchanging the culture medium for three weeks. Immunohistochemistry revealed drebrin-positive dendritic spines along the MAP2-positive dendrites. The dose-dependent reduction of drebrin cluster densities against glutamate stimulation was observed.

The transplant of the cell suspension before it becomes too warm is very important. The dropwise addition of culture medium is also crucial to avoid sudden change in osmolarity.