Isolation and Culture of Primary Embryonic Mouse Midbrain Dopamine Neurons

Take midbrain floor tissue isolated from the ventral region of the midbrain of mouse embryos.

The tissue is rich in developing dopamine neurons, which release the neurotransmitter dopamine.

Add an enzyme solution to break down the tissue matrix, thereby loosening the cells.

Add a serum solution containing DNase I and mechanically dissociate the digested tissue to generate a cell suspension. The serum proteins inhibit the enzymes, while DNase I degrades extracellular DNA released during cell lysis, preventing cell clumping.

Let the tissue debris settle and transfer the suspended cells to a fresh tube.

Centrifuge and remove the supernatant, then resuspend the cells in a dopamine neuron medium, or DPM.

Take a microplate coated with a cell culture substrate at the center of the wells, creating micro-islands.

Transfer the cells to the middle of the micro-islands to culture them at a high density.

Incubate, allowing the cells to adhere to the micro-islands.

Fill the wells with DPM and incubate, facilitating the growth of dopamine neurons.

To set up primary embryonic midbrain cultures from mouse embryonic day 13.5 mouse embryos, pool all of the harvested mid-brain floors into the same 1.5-milliliter tube, and wash the samples three times, with 500 microliters of calcium and magnesium-free HBSS per wash. After the last wash, replace the HBSS with 0.5% trypsin for a 30-minute incubation at 37 degrees Celsius.

At the end of the incubation, add 500 microliters of a freshly prepared DNase I in FBS solution to the partially digested tissue and use a siliconized glass pipette with a fire-polished tip to triturate the tissue. When only tiny, barely visible particles can be observed, allow these tissue fragments to settle to the bottom of the microcentrifuge tube, and transfer the supernatant into an empty 15-milliliter conical polypropylene tube.

Add one milliliter of HBSS to the tube containing DNase I in FBS, and mix several times by pipetting. Transfer one milliliter of this solution to the remaining tissue particles, and triturate the samples again. Then, pool the digested cell suspension with the tube of supernatant without transferring any remaining tissue fragments. After triturating the tissue sample one more time with the remaining DNase I in FBS in HBS, sediment the collected cells by centrifugation, and aspirate the supernatant without disturbing the pellet.

Wash the cells two times in two milliliters of warmed dopamine neuron medium per wash, and resuspend the cells at 3 x 10⁴ cells per six microliters of fresh, warm medium concentration in a microcentrifuge tube. Next, remove the medium from each previously prepared micro-island and mix the cells with gentle pipetting before using a 10-microliter pipette to add six microliters of cells to each micro-island.

When all of the cells have been added, fill the empty wells at the edges of the plate with 150 microliters of water or PBS, and place the plate in the cell culture incubator for one hour. At the end of the incubation, add 100 microliters of fresh dopamine neuron medium to each well, and return the plate to the incubator.