Protocol Guide: Neuron and Microglia Co-Culture Assay

Neuron and Microglia Co-Culture

The central nervous system (CNS) includes multiple cell types, but two of the most common are neuron and microglia. Studying how these cell types interact is important for understanding the interplay between the immune system and the CNS, as well as how microglia mediate neurotoxicity. Co-culture of neurons and microglia is a growing tool for scientists to model these interactions.

Through our partnership with BrainXell, we provide a detailed protocol for the co-culture of iPSC-derived human neurons and microglia. 

Section Overview

• Neuron and Microglia Co-Culture Materials
• Neuron and Microglia Co-Culture Protocol
• Related Materials

Neuron and Microglia Co-Culture Materials

Neuron cryovials should be immediately transfers to a liquid or vape nitrogen storage system. To maintain stability, BrainFast supplements should be stored at -20°C for 6 months or -80°C for 18 months; return cryovial to -20°C between each use.

• BrainXell Human Neurons – multiple options (BX-0100-30-5M or BX-0100-30-1M or BX-0300-30-5M or BX-0300-30-1M or BX-0400-30-1M or BX-0700-30-1M)
• BrainXell Human Microglia (BX-0900-30-1M or BX-0900-32-1M)
• BrainFast supplements (neuron type specific):  Motor (BX-2100-100uL) or Glut (BX-2300-100uL) or GABA (BX-2400-100uL)
• BrainFast D4 (BX-2040-100uL)
• BrainFast SK (BX-2020-100uL)
• 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)
• GlutaMAX Supplement (Thermo Fisher Scientific #35050061)
• Cultrex (R&D Systems #3432-001-01)
• Chemically Defined Lipid Concentrate (Thermo Fisher Scientific #11905031)
• M-CSF (SRP3110)
• IL-34 (SRP3287)
• Ascorbic Acid (A8960)
• PDL-coated 96-Well Plate (M0812, LPDL001)
• BDNF (SRP3014)
• GDNF (SRP3200)
• TGF-β1 (SRP0300)

Neuron and Microglia Co-Culture Protocol

Day 0: Seeding Preparation

1. Gather the components listed below in the cell culture hood and combine to make the Basal Medium for culture in a sterile container. Equilibrate Basal Medium at room temperature for 15 minutes, but do not place medium in a 37°C water bath to warm. Store Basal Medium at 4°C for up to 2 weeks.
   1. The recommended final concentrations for growth factors are 2µg/ml TGF-β1, 10 µg/ml GDNF and 10 µg/ml BDNF. Growth factors are optional for Basal Medium. 

*To make the Ascorbic Acid solution, click here.

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

Day 0: Seeding the Neurons

1. Remove neuron cryovial from storage and let thaw in a 37°C water bath. Do NOT submerge the cap – this can cause contamination.
2. As soon as the ice melts (or after ~75-90 seconds) remove the cryovial from the water bath. Disinfect the cryovial with 70% ethanol and transfer to the cell culture hood.
3. Add 500µl Basal Medium from the prepared conical tube to the cryovial slowly, i.e. at a rate of ~2-3 drops/sec, using a P1000 pipette. Add Basal Medium should take about 10 seconds.
4. Transfer the 1ml cell solution to the prepared 50ml conical tube.
5. Centrifuge the conical tube of neurons at 465xg for 5 minutes.
6. Aspirate the supernatant from the cell pellet carefully and gently pipette with a P1000 pipette to resuspend the pellet in 950µl fresh Basal Medium.
7. Based on the number of viable cells/vial, found in the Certificate of Analysis (CoA), resuspend the 1ml cell pellet with the Basal Medium to a concentration of 1.0 x 10⁶ live cells/ml.
   1. Example: Dilute 5.3 million viable cells/vial to 5.3ml final volume.
8. Gently swirl to suspend the neurons in the Basal Medium and transfer 25µl of the cell suspension to the prepared Trypan Blue microcentrifuge tube. Pipette up and down to evenly mix. Using a hemocytometer, count the number of live and dead cells; determine the viability and live cell concentration (live cells/ml).
9. Calculate the volume for the motor neuron (MN) seeding suspension. For a 96-well plate, the recommended seeding density is 20,000-30,000 viable cells/100µl/well (~62,5000-93,750 viable cells/cm²). The recommended seeding density may vary based on lot – refer to the CoA for lot-specific seeding information. Dilute the cells to the desired seeding concentration based on the Trypan Blue calculation.

Example dilution calculation:

10. Add the above calculated volumes for each cell type and Basal Medium to a new sterile conical tube.
11. Prepare the Neuron Seeding Medium:

*Depends on neuron type
**Cultrex should be added to the final seeding suspension immediately before seeding the plate.

12. Mix completely and add 100µl of the Neuron Seeding Medium to each well in the PDL-coated 96-well plate using a liquid handler or multi-channel pipette. Do not agitate or move the plate during this process - this can lead to uneven cell attachment.
13. Rest the plate for 10 minutes after seeding to allow cells to settle in the wells. Transfer the seeded plate gently to a humidified incubator set at 37°C with 5% CO₂. The day of plating is Day 0.

The thawing and plating protocol should not go longer than 1 hour, as the post-thaw health and viability of the neurons can be severely impacted and can lead to an unsuccessful culture if the protocol takes too long.

Day 1: Medium Replacement

1. Prepare the Day 1 Medium fresh:

2. Remove the 100µl medium from the well and replace with 100µl fresh Day 1 Medium per well. To ensure the cells do not dry out, complete one column or row at a time.

Day 4: Medium Addition

1. 96 hours after seeding, prepare the Day 4 Medium:

**Adding BrainFast SK is recommended in the Day 4 Medium for cultures that contain cortical GABAergic neurons. If culturing neurons for longer than 7 days, adding BrainFast SK is recommended.

2. Add 100µl/well Day 4 Medium to the entire plate, do not remove media from the wells. The total volume of each well is 200µl/well.

Day 7: Microglia Seeding

1. In a sterile container, mix together the components listed below to make the Day 7 MG Seeding Medium. Equilibrate the medium at room temperature for at least 15 minutes; do not warm the medium in a 37°C water bath.

2. Add 3ml of Day 7 MG Seeding Medium to a new 50ml conical tube. Prepare a microcentrifuge tube with 25µl Trypan Blue for cell counting.
3. Take the microglia cryovial from liquid storage and put in a 37°C water bath to thaw. Do not submerge the caps to minimize contamination.
4. When the last of the ice melts (~75-90 seconds), remove the cryovial from the 37°C water bath. Disinfect with 70% ethanol and transfer the cryovial to the cell culture hood.
5. Slowly add 500µl of the Day 7 MG Seeding Medium (~2-3 drops/second) using a P1000 pipette to the cryovial; this process should take ~10 seconds.
6. Transfer the microglia cryovial contents to the prepared 50ml conical tube from Step 2. Rinse the cryovial with an additional 1ml of the Day 7 MG Seeding Medium to collect residual cells and transfer to the 50ml conical.
7. Centrifuge the microglia tube at 200xg for 5 minutes.
8. Aspirate off the supernatant and using 950µl of fresh Day 7 MG Seeding Medium resuspend the cell pellet in. Mix by pipetting up and down with a P1000.
9. Resuspend the cells to a concentration of 1.0 x 10⁶ live cells/ml based on the Certificate of Analysis value (viable cells/vial) using Day 7 MG Seeding Medium to the existing 1ml in the tube.
   1. For example, 2.3 million live cells per CoA will be resuspended to a total final volume of 2.3ml.
10. Pipette up and down to evenly mix the cells in the Medium. Transfer the 25µl cell suspension to the prepared Trypan Blue microcentrifuge tube and pipette to mix. Using a hemocytometer, count the number of dead and viable cells; determine the live cell concentration and viability and remember to calculate in the dilution factor.
11. Calculate the volume needed to make the final seeding suspension. The general recommendation is to use a ratio of neuron:microglia of 2:1 to 4:1, but the final ratios will vary depending on experimental needs. Co-culture totals of greater than 45,000 live cells per well may appear clustered due to the higher density.
    1. Dilute the microglia to the desired seeding concentration in Day 7 MG Seeding Medium using the calculation table below as an example.

12. Remove 100µl of medium from each well and add 100µl to each well of the microglia seeding suspension.
13. Do not transfer the plate to the incubator immediately after seeding. Allow the cells to settle to the bottom of the wells with the plate in the hood for 15 minutes. Transfer the plate gently to a humidified incubator set at 37°C with 5% CO₂.

Day 9 and Onward Medium Changes

1. Prepare Day 9 Maintenance Medium using the components listed below:

*Room temperature

2. Change 100µl/well (half of the medium) twice weekly, i.e. on Day 9, 12, 15, etc. Replace with the freshly prepared Maintenance Medium.

Under the above conditions, co-cultures can be maintained adherent and viable in culture for at least 3 weeks post-seeding.

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