Culturing and Maintaining Dopaminergic Neurons from Mouse Embryonic Brain Tissue
Transcript
Under a hood, remove the HBSS from the collection of ventral midbrain. Then, add a milliliter of warm 0.05% trypsin-EDTA, enough solution for 12 pieces of tissue. Incubate the tissue at 37 degrees Celsius for 5 to 10 minutes. Under the hood, replace the trypsin-EDTA with 1 milliliter of deactivation medium.
Gently swirl the mix, and then remove the deactivation medium, but leave enough medium behind so dissociated cells are not discarded. Next, wash the tissues with complete medium twice without losing the cells. Now, add 1 milliliter of complete medium. Then, triturate the tissue with the fire-polished pipette, without forming bubbles until a single-cell suspension is obtained. About 8 to 10 passes should suffice.
After the trituration, centrifuge the cells of 400 g's for 5 minutes. Remove the medium, and resuspend the cells in 1 milliliter of complete medium. Pipette the cells up to 4 times to achieve a suspension. Begin by counting and assessing the health of the cells in the suspension using trypan blue exclusion with a hemocytometer.
Now, adjust the cell suspension to 1,500 cells per microliter. Then, transfer the sterilized microcentrifuge tube caps to 100-millimeter dishes. Place the prepared coverslips onto the caps using forceps. Do not wash the coverslips, and try not to let them dry out. Load 100 microliters of suspension onto each coverslip.
This somewhat unusual method provides 90% culture viability, and is a critical step to success. Then, close the Petri dish, and transfer it to the incubator for an hour. After the incubation, carefully transfer the coverslips with the medium to 24-well plates, containing 400 microliters of complete medium warmed to 37 degrees Celsius in each well. Then, incubate the cells overnight.
The first few hours are the most critical to cell survival. Within 24 hours, gently add half a milliliter of complete medium to each well. Every 2 weeks, change half the media. If the culture turns yellow, then the half-media change can be performed sooner, but the change should still be infrequent. Dopaminergic neurons will survive for more than 6 weeks under these conditions.
