Protocol Guide: Midbrain Dopaminergic Neuron Monoculture

What are Midbrain Dopaminergic Neurons?

Dopaminergic neurons are a subtype of neurons that release dopamine as their primary neurotransmitter. Important for various neurological functions including motor coordination and executive function, dopaminergic neurons are a critical part of the central nervous system (CNS). Dopaminergic neurons can be found in the midbrain, and have been a crucial model for the study of regenerative medicine and the development of novel therapeutics for and understand of Parkinson’s disease, Huntington’s disease, ADHD, addiction, and epilepsy.

We provide, through partnership with BrainXell, dopaminergic neurons of a midbrain lineage that are fully differentiated, express MAP2/TH at DIV 14, and release dopamine as their primary neurotransmitter.  Here we describe a protocol for seeding and culturing midbrain dopaminergic neurons in vitro.

Section Overview:

• Dopaminergic Neuron Culture Materials
• Dopaminergic Neuron Culture Protocol

Dopaminergic Neuron Culture Materials

Dopaminergic cryovials should be immediately transferred to a liquid or vapor nitrogen storage system. To maintain BrainFast supplement stability, store at -20˚°C for 6 months or -80˚°C for 18 months; return the cryovial to -20˚°C between each use.

• BrainXell Human Midbrain Dopaminergic Neurons (BX-0200-30-1M)
• BrainFast Dopa (BX-2200-100uL)
• DMEM/F-12 Medium, +L-glutamine +HEPES (D0697)
• Neurobasal Medium (SCM003)
• B-27 Supplement (Thermo Fisher Scientific #17504044)
• N-2 Supplement (Thermo Fisher Scientific #17502048)
• dbcAMP (D0627)
• GlutaMAX Supplement (Thermo Fisher Scientific #35050061)
• Cultrex (R&D Systems #3432-001-01)
• Ascorbic Acid (A8960)
• PDL-coated 96-Well Plate (M0812, LPDL001)
• BDNF (SRP3014)
• GDNF (SRP3200)
• TGF-β1 (SRP0300)

Dopaminergic Neuron Culture Protocol 

Day 0: Seeding Preparation

2.2-3.3 million live cells are needed to seed a full 96-well plate, but additional cells may be used depending on experimental needs. Refer to the Certificate of Analysis (CoA) for further viability and seeding information.

1. Combine the Basal Medium components listed below into a sterile container in a sterile container. After combined, equilibrate the Basal Medium at room temperature for 15 minutes without placing the medium in a 37°C water bath to warm. Store Basal Medium at 4°C for up to 3 weeks.

2. Prepare a pre-diluted Cultrex solution by adding 495µl of cold DMEM/F12 medium to a 55µl aliquot of frozen Cultrex (straight from the -80°C freezer). The Cultrex solution can be stored at 4°C until experimentally necessary.
3. For cell counting, prepare a microcentrifuge tube with 25µl of Trypan Blue solution (see below). Prepare a separate 50ml conical tube with 3ml of Basal Medium for cell seeding.

Day 0: Seeding the Dopaminergic Neurons

1. Remove dopaminergic neuron cryovial from storage. Thaw the vial in a 37°C water bath but do not submerge the cap, as this can cause contamination.
2. Remove the cryovial from the water bath immediately as the last ice melts; this should take ~75-90 seconds. Disinfect the dopaminergic cryovial with 70% ethanol and transfer it to the cell culture hood.
3. Using a P1000 pipette, add 500µl Basal Medium from the prepared conical tube to the dopaminergic neuron cryovial slowly (~2-3 drops/sec). This process should take about 10 seconds.
4. Transfer the cryovial contents to the prepared 50ml conical tube that contains the remaining Basal Medium.
5. Centrifuge the conical tube of dopaminergic neurons at 465xg for 5 minutes.
6.  Carefully aspirate the supernatant from the dopaminergic neuron pellet. Pipette with a P1000 pipette up and down to resuspend the pellet in 950µl fresh Basal Medium.
7. Resuspend the 1ml neuron pellet with Basal Medium to a concentration of 1.0 x 10⁶ live cells/ml based on the number of viable cells/vial found in the Certificate of Analysis (CoA).
   a. For example: Dilute 5.3 million viable cells/vial to 5.3ml final volume.
8. Suspend the cells in the Basal Medium by swirling the conical tube and pipetting up and down. Transfer 25µl of the dopaminergic neuron suspension to the previously prepared Trypan Blue microcentrifuge tube (Step 3); pipette to evenly mix. Count the number of dead and live cells using a hemocytometer and determine the viability and live cell concentration (live cells/ml).
9.  Calculate the volume for the Dopa seeding suspension. The typical seeding density for a 96-well plate is 20,000-30,000 viable cells/100µl/well (~62,5000-93,750 viable cells/cm²). The seeding density recommendation may vary; refer to the CoA for lot-specific seeding information. Dilute the dopaminergic neurons to the desired seeding concentration based on the Trypan Blue calculation.

10. In a new sterile container, combine the above calculated volumes for the dopaminergic neuron suspension and Basal Medium.
11. Prepare the Dopaminergic Seeding Medium:

12. Completely mix the Dopaminergic Seeding Medium. Transfer 100µl of the medium to each of the wells in the PDL-coated 96-well plate using a liquid handler or multi-channel pipette. Do not agitate or move the plate during the seeding process, movement can lead to uneven cell attachment.
13. Let the plate to rest for at least 10 minutes after seeding to allow cells to settle at the bottom of the wells. After 10 minutes, gently move the seeded plate to a humidified incubator set at 37°C with 5% CO₂; this is designated as Day 0.

This dopaminergic thawing and plating protocol should not exceed 1 hour. The post-thaw health and viability of the cells can be severely impacted and lead to unsuccessful cultures if the protocol takes too long.

Day 1: Dopaminergic Neuron Culture Medium Replacement

1. Prepare the Maintenance Medium 24 hours after seeding:

2. Remove 100µl of the seeding medium from each well and replace with 100µl of fresh Day 1 Medium per well. Complete one column or row at a time to ensure wells do not dry out during medium replacement.

Day 4: Medium Addition

1. On Day 4 (96 hours after seeding), make fresh Maintenance Medium (see above).
2. Do not remove the Maintenance Medium from the wells. Add fresh 100µl/well Maintenance Medium to the entire plate; the total volume in each well is 200µl/well.

Day 7 and Beyond Medium Changes

1. Remove 100µl from each well and replace 100µl/well weekly (Day 7, 14, 21, etc.) with freshly prepared Maintenance Medium.

Dopaminergic neurons require approximately 14 days to mature. They can be maintained viable and adherent under the above conditions for at least 3 weeks after seeding.

*These products are available in only the US and UK.