Protocol Guide: Motor Neuron Monoculture

What are Motor Neurons?

Located in the spinal cord or at the brain stem within the motor cortex, motor neurons indirectly or directly carry signals to muscles, organs, or glands from the brain. These neurons have one axon with multiple dendrites, and can be classified either as upper motor neurons, which signal between the brain and the spinal cord, or lower motor neurons, which carry signals to muscles from the spinal cord.

Through BrainXell we provide iPSC-derived motor neurons of a spinal cord lineage that is representative of lower motor neurons found in the ventral horn of the spinal cord. They are fully differentiated, express FOXP1 and MAP2 at DIV7 with spontaneous firing by DIV14, and release acetylcholine as the primary neurotransmitter. Here we describe how to culture motor neurons for use in assays developing novel therapeutics for amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), and spinal cord injury.

Section Overview

• Motor Neuron Culture Materials
• Motor Neuron Culture Protocol
• Related Materials

Motor Neuron Culture Materials

Immediately transfer the neuron cryovials to a liquid or vape nitrogen storage system. Store BrainFast supplements at -20°C for up to 6 months or -80°C for up to 18 months.

• BrainXell Human Motor Neurons (BX-0100-30-5M or BX-0100-30-1M)
• BrainFast Motor/Neuron Seeding Supplement (BX-2100-100uL)
• BrainFast D4/Day 4 Supplement (BX-2040-100uL)
• BrainFast SK/Supplement K (BX-2020-100uL)
• DMEM/F12 Medium, +L-glutamine, +HEPES (D0697)
• B-27 Supplement (Thermo Fisher Scientific #17504044)
• GlutaMAX Supplement (Thermo Fisher Scientific #35050061)
• Cultrex (R&D Systems #3432-001-01)
• Ascorbic Acid (A8960)
• PDL-coated 96-Well Plate (M0812, LPDL001)

Motor Neuron Culture Protocol

Day 0: Seeding Preparation

For a 96-well plate seed 2.2-3.3 million live cells, but additional cells can be used depending on experimental needs. For viability and seeding information see the Certificate of Analysis.

1. Make Basal Medium by combining all components in the cell culture hood or biological safety cabinet. Allow the medium to come to room temperature for 15 minutes. Be sure to not place medium in a 37°C water bath. This medium stock can be stored at 4°C for up to 3 weeks.
   1. The addition of growth factors is optional -- recommended final concentrations are: 2µg/ml TGF-β1, 10µg/ml GDNF, and 10µg/ml BDNF.

2. The 200mM ascorbic acid stock should be made with DMEM/F12, mixed only by inversion. The ascorbic acid must be added into the DMEM/F12 medium slowly. Ascorbic acid is temperature, light, and pH sensitive.
3. Preparation of 200mM Ascorbic Acid Solution:
   1. Aliquot 17.2ml of DMEM/F12 medium into a 50ml tube and gradually transfer 1.0g of ascorbic acid into the tube of DMEM/F12. 
      NOTE: Add no more than 0.2g at a time and wait 10 seconds between each addition.
   2. Add cap and invert to mix. Allow ascorbic acid to dissolve.
   3. Place solution in a 37°C water bath for 30 minutes. Invert the tube every 10 minutes to mix.
   4. If not fully dissolved after 30 minutes, invert tube to mix and incubate overnight at 4°C.
   5. Confirm ascorbic acid is completely dissolved after overnight storage.
   6. Filter the ascorbic acid solution using a Steriflip 0.22µm sterile filter.
   7. Aliquot into 1.5ml amber Eppendorf tubes at the recommended volumes of 10µl or 550µl. Freeze aliquots at -20°C for up to 6 months. Do not refreeze.

2. Prepare a pre-diluted Cultrex solution by adding 495µl of cold DMEM/F12 medium into a 55µl aliquot of frozen Cultrex – this should be taken directly from the -80°C freezer. Mix to dissolve and store at 4°C. This should be added to the final seeding suspension right before plate seeding.
3. Add 3ml of Basal Medium to a 50ml conical tube. Add 25µl of Trypan Blue solution to a microcentrifuge tube for cell counting.

Day 0: Seeding the Neurons

1. Place a cryovial of cells in a 37°C water bath to thaw – do not submerge the cap.
2. Remove the vial from the water bath immediately as the last of the ice melts. Disinfect the vial with 70% ethanol and transfer to the cell culture hood.
3. Transfer 500µl of the Basal Medium from the 50ml conical tube to the cell vial using a P1000 pipette at a rate of 2-3 drops/sec. This process should take about 10 seconds.
4. Transfer the 1ml contents in the cell cryovial to the 50ml conical tube.
5. Centrifuge the cells at 465xg for 5 minutes.
6. Aspirate off the supernatant and resuspend the cell pellet in 950µl fresh Basal Medium by gently pipetting up and down using a P1000 pipette.
7. Resuspend the cells to a concentration of 1.0 x 10⁶ live cells/ml based on the CoA value by slowly adding Basal Medium to the existing 1ml in the conical tube.
   1. For example, 5.3 million viable cells/vial is diluted to 5.3 total volume.
8. Count the cells using a Trypan Blue solution. Evenly suspend cells in the Basal Medium by pipetting up and down 3-5 times and transfer 25µl of the cell suspension to the prepared microcentrifuge tube filled with 25µl Trypan Blue solution. Pipette up and down to mix. Count the number of viable and dead cells using a hemocytometer; determine the live cell concentration and viability.
9. Calculate the volume needed to make the motor neuron seeding suspension. The typical seeding density is 20,000-30,000 viable cells/100µl/well for a 96-well plate (~62,500-93,750 viable cells/cm²), but the recommended seeding density may vary (refer to the CoA for lot-specific seeding information). Dilute cells to the desired seeding concentration based on the Trypan Blue cell count.
   

Example of dilution calculation: 

10. In a new 50ml conical tube, mix the calculated volumes of Basal Medium and cell suspension to make a 11ml final volume motor neuron seeding suspension solution.
11. Make the Seeding Medium:

12. Mix completely and transfer 100µl/well of the Seeding Medium to a PDL-coated 96-well plate.
    NOTE: Do not move the plate during seeding – this can lead to uneven cell attachment.
13. Rest plate for 10 minutes after seeding to allow the cells to settle to the bottom of the wells. Gently transfer plate to a humidified incubator at 37°C with 5% CO2.

This protocol should not exceed 1 hour – the post-thaw viability and health of the cells will be impacted if the process is too long.

Day 1: Medium Replacement 

1. Prepare fresh Day 1 Medium for 24 hours after seeding:
   

2. Aspirate the 100µl of medium in each well and gently replace with 100µl/well of the Day 1 Medium. Complete this process one row or column at a time to ensure that the wells do not dry out as you replace the media.

Day 4: Medium Addition

1. Prepare the Day 4 Medium fresh 96 hours after seeding:

*NOTE: The addition of BrainFast SK on Day 4 is optional for motor neurons if culturing for only 7 days, but it is recommended if culturing for longer than 7 days.

2. Add 100µl/well of Day 4 medium to the entire plate for a final volume of 200µl/well.

Day 7 and Onward Medium Changes 

1. Change half of the media weekly, i.e. on Day 7, 14, 21, etc., using the Basal Medium (Step 1). Aspirate 100µl/well and slowly add 100µl/well of Basal Medium to the entire plate.
   1. The addition of low concentration BrainFast SK (0.1-0.5X) in the medium may be helpful for long-term cultures.

These motor neurons mature rapidly and can be maintained in culture under the above conditions for at least 3 weeks post-seeding.

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