Culturing and Maintaining Dopaminergic Neurons from Mouse Embryonic Brain Tissue

Abstract

Source: Weinert, M. et al., Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains. J. Vis. Exp. (2015)

This video outlines a method for cultivating dopaminergic neurons from mouse embryonic brain tissue. The process includes treating the midbrain with a proteolytic enzyme, detaching the cells, and culturing them on polymer-coated coverslips in a nutrient-rich medium with antibiotics for their growth and long-term viability.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Dissociation of Ventral Midbrain Cells

1. Transfer the pieces of the ventral midbrain under a laminar flow hood. Remove the Hanks' balanced salt solution (HBSS) and add 1 ml pre-warmed (37 ºC) 0.05% trypsin-ethylenediaminetetraacetic acid (trypsin-EDTA) into the conical tube (volumes enough for up to 12 pieces of ventral midbrain). Incubate the tissue at 37 ºC for 5-10 min.
2. Remove trypsin-EDTA under the hood and add 1 ml de-activation medium (the serum will deactivate trypsin) to the tissue.
3. Remove the deactivation medium and wash the tissue in 1 ml of the complete medium twice.
   NOTE: To prevent discarding the dissociated cells, avoid removing all the medium from the tube and pipetting the tissue.
4. Add 1 ml of complete medium and triturate the tissue with a fire-polished glass pipette. Avoid bubble formation and continue triturating until a single-cell suspension is achieved.
   NOTE: Usually, 8-10 passes through the restricted tip are required for complete dissociation.
5. Centrifuge the cells at 400 x g for 5 min at room temperature (RT) and remove the medium.
6. Re-suspend the pellet in 1 ml complete medium by slowly pipetting up and down up to four times.

2. Plating Ventral Midbrain Cells

1. Mix 10 µl of the cell suspension with 90 µl Trypan blue solution and count the number of cells with a hemocytometer.
   NOTE: The viability of the cells can also be checked at this stage.
2. Place sterilized microcentrifuge tube caps in 100 mm Petri dishes and transfer the Poly-L-ornithine/Laminin coated coverslips, using forceps, one at a time on top of the caps.
   NOTE: Do not wash the coverslips and avoid drying the laminin, as this may cause uneven cultures and decrease the cells' survivability.
3. Adjust the volume to 1,500 cells per 1 µl by adding a complete medium and 100 µl (total of 150,000 cells) of the cell suspension on top of each coverslip (the volume is optimal to cover the surface of the coverslip without spillage). Close the Petri dishes and incubate them for 1 hr in a humidified tissue culture incubator (37 °C, 5% CO2).
   NOTE: CRITICAL STEP: This step is required to enhance the attachment and viability of the cells and to increase the number of coverslips per embryo. Alternatively, the cells can be transferred directly into the wells (containing coverslips), but the number of cells should be increased to 350,000 per well (instead of 150,000). In other words, using this method, 8-10 coverslips can be acquired, while direct transfer yields about 3-4 coverslips because of the cells that attach to the plates (beside or underneath the coverslips). The average viability of the cultures using this method was around 90%.

3. Culture Growth and Maintenance

1. After 1 hr of incubation, carefully transfer the coverslips, including the medium, into 24-well plates containing 400 µl pre-warmed (to 37 ºC) complete medium (total volume: 500 µl). Incubate the cells overnight (O/N) at 37 ºC.
2. Within 24 hr after incubation of the wells, gently add 500 µl complete medium into each well.
   NOTE: The first few hours are the most critical stage for the survival of the dopaminergic neurons, and the number of surviving cells does not change significantly beyond this point.
3. Do not add additional media to the cultures for the first two weeks or until the medium becomes yellow. If culturing for more than two weeks or the medium color changes to yellow, exchange half of the media (500 µl) with fresh, complete pre-warmed media (typically every two weeks).
   NOTE: Dopaminergic neurons within the cultures would survive for more than six weeks under these conditions.

Materials

Dulbecco's modified Eagle medium nutrient mixture F-12	Invitrogen	11330
Hanks' Balanced Salt Solution (HBSS) (1X), liquid	Invitrogen	24020-117
Fetal bovine serum, heatinactivated (FBS)	Invitrogen	16140
N2 Supplement (100X), liquid	Invitrogen	17502-048
D-(+)-Glucose solution (45% (wt/ vol) in water	Sigma	G8769
Bovine serum albumin BSA	Sigma	A9430
Laminin from Engelbreth-HolmSwarm murine sarcoma basement membrane	Sigma	L2020
Penicillin/streptomycin	Invitrogen	15070
Trypsin (0.05% (wt/vol)	Invitrogen	25300
Bovine serum albumin (BSA) cell culture tested	Sigma	A9418
Phosphate-buffered saline (PBS)	Sigma	P3813
Poly-L-ornithine, 0.01% solution	Sigma	P4957
Trypan blue solution (0.4% (wt/vol))	Biowhittaker	17-942E