Generating a Low-Density Primary Neuron Culture From Frozen Embryonic Neurons

Begin with a cryovial containing cryopreserved embryonic neurons and place it into a heating block for controlled thawing.

Next, transfer the thawed neurons into a tube and dilute them with a neurobasal medium dropwise to balance the osmotic pressure and prevent cell lysis.

This generates a homogeneous suspension with uniformly dispersed neurons.

Seed the diluted neural suspension onto a poly-L-lysine-coated multi-well plate containing a neurobasal medium enriched with nutrients and antibiotics.

Incubate to allow the positively charged poly-L-lysine to interact with the neurons via electrostatic interactions, facilitating their adherence.

Supply the wells with fresh neurobasal media.

Incubate the cells in the same media under humid conditions to prevent media evaporation.

The embryonic neurons utilize the nutrients and grow by developing neuronal processes and forming primary neurons.

The neurobasal medium maintains the primary neurons that appear widely spaced, generating a low-density primary culture with neuronal connections.

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 4 degrees Celsius.

To start cell seeding, add 50 microliters of the culture medium to each well in the coated plate. 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 cryovial from the liquid nitrogen tank and place it in a 37 degrees Celsius heat block to partially thaw its contents. Using a 1-milliliter pipette with a wide-bore tip, slowly transfer the vial's contents dropwise to a sterile 50-milliliter tube. Rinse the empty cryovial with 1 milliliter of room-temperature culture medium. Transfer the rinse solution to the 50-milliliter tube containing the cell suspension.

Next, add 9 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 multichannel pipette with wide-bore tips, dispense the cell suspension with a final count of 1.0 x 10⁴ cells per well into the coated 96-well plate.

Incubate the neurons for 1 to 2 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.