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HomeStem Cell CultureProtocol Guide: Functional Tri-Culture of iPSC-Derived Human Neurons, Astrocytes, and Microglia

Protocol Guide: Functional Tri-Culture of iPSC-Derived Human Neurons, Astrocytes, and Microglia

Neuron, Astrocyte, and Microglia Tri-Culture

More often researchers have seen the need for more physiologically relevant models for the central nervous system (CNS), but it can be difficult to obtain cell or tissue samples. The number of cells that are needed to form a functional network can also make it difficult to recapitulate the system in vitro.

Co-culture of cortical neurons and cortical astrocytes or spinal motor neurons and spinal astrocytes has previously been established, but tri-culture with microglia presents additional complexity. Here we describe a protocol for, and the reagents involved with, establishing a tri-culture of iPSC-derived human neurons, astrocytes, and microglia in vitro. Neurons and astrocytes were cultured together and microglia were added after 7 days of co-culture. All cells were provided through partnership with BrainXell.

Section Overview

Neuron, Astrocyte, and Microglia Tri-Culture Materials

Immediately transfer the neuron cryovials to a liquid or vape nitrogen storage system. The BrainFast supplements should be stored at -20°C for 6 months or -80°C for 18 months; vial should be returned to -20°C between each use to maintain stability.

If culturing spheroids, we recommend either PrimeSurface 3D Culture Spheroid (ULA) plates (S-Bio #MS-9096WZ) or Nunclon Sphera 96-Well U-Shaped-Bottom Microplate (Thermo Fisher Scientific #174925). Successful cultures have resulted when seeding between 100 and 10,000 cells/well.

Neuron, Astrocyte, and Microglia Tri-Culture Protocol

Day 0: Seeding Preparation

  1. Combine all components listed below in the cell culture hood to make the Basal Medium for culture in a sterile container. Allow the Basal Medium to equilibrate at room temperature for 15 minutes, but do not place medium in a 37°C water bath to warm. Basal Medium can be stored at 4°C for up to 2 weeks.
    1. Growth factors are optional for Basal Medium; the recommended final concentrations are: 10ng/ml GDNF and 10ng/ml BDNF.
  1. Prepare a pre-diluted Cultrex solution by mixing 495µl of cold DMEM/F12 medium with a 55µl aliquot of frozen Cultrex. Store at 4°C until experimentally necessary.
  2. For cell counting, prepare a separate microcentrifuge tube with 25µl of Trypan Blue solution, as well as a 50ml conical tube with 3ml of Basal Medium for each cell type.

Day 0: Seeding the Neurons and Astrocytes

The recommended seeding ratio of neurons to astrocytes is 2:1 to 6:1; however, other ratios can be used depending on individual research needs.

  1. Remove neuron and astrocyte cryovials from liquid nitrogen storage and thaw in a 37°C water bath. Do NOT submerge the cap.
  2. After ~75-90 seconds or as the last of the ice melts, remove the vials from the water bath. Disinfect with 70% ethanol and transfer the vials to the cell culture hood.
  3. To the first cryovial, slowly add 500µl Basal Medium (at a rate of ~2-3 drops/sec) using a P1000 pipette. This process should take about 10 seconds.
  4. Transfer the contents from the neuron cryovial to the 50ml conical tube. Rinse the vial with an additional 1ml Basal Medium to collect the residual cells and transfer to the same 50ml conical tube. Repeat with the thawed astrocyte cryovials using a second prepared 50ml conical tube (Steps 2 and 3).
  5. Centrifuge the tubes of neurons and astrocytes at 200xg for 5 minutes.
  6. Aspirate the supernatant from the first pellet and resuspend the pellet in 950µl fresh Basal Medium by gently pipetting with a P1000 pipette. Repeat for the second tube (be sure to use a clean pipette tip).
  7. Based on the number of viable cells/vial found in the Certificate of Analysis, resuspend the cell pellets to a concentration of 1.0 x 106 live cells/ml using Basal Medium to the existing 1ml in each tube.
    1. For example, 6.7 million viable cells/vial is diluted to 6.7ml total volume.
  8. Count the resuspended cells. Evenly suspend the cells in the medium through swirling the conical tube. Transfer 25µl of the cell suspension to the prepared Trypan Blue solution and pipette up and down to mix. Count the number of live and dead cells using a hemocytometer and use that to determine the live cell concentration (live cells/ml) and the viability for each of the cell suspensions.
  9. Calculate the volume needed to make the final mixed seeding suspension for each cell type. For a 96-well plate, the initial combined seeding density is 30,000-40,000 viable cells/well is recommended (~95,000-125,000 viable cells/cm2). The recommended seeding density may vary, but a general recommended seeding ration for neurons to astrocytes is 2:1 to 6:1.

Example dilution calculation at a combined 40,000 cells/well with a 3:1 ratio of neurons to astrocytes:

  1. In a new sterile 50ml conical tube, add the above calculated volumes for each cell type and Basal Medium to reach 11ml of mixed cell suspension.
  2. Prepare the Seeding Medium:
  1. Using a 5ml serological pipette, mix the suspension slowly to ensure a homogeneous suspension. Seed 100µl/well of the Seeding Medium onto a PDL-coated 96-well plate using a multi-channel pipette or liquid handler. Do not move or agitate the plate during the seeding process, as this can lead to uneven cell attachment.
  2. Allow cells to settle to the bottom of the wells by resting the plate for 10 minutes after seeding. Gently transfer the seeded plate to a humidified incubator set at 37°C with 5% CO2; this is considered Day 0.

The post-thaw health and viability of the cells can be severely impacted and can lead to an unsuccessful culture if the thawing and plating protocol should exceed 1 hour or go on too long.

Day 1: Medium Replacement

  1. Prepare the Day 1 Medium fresh:
  1. Remove 100µl medium/well and replace with 100µl/well Day 1 Medium; complete one column or row at a time to ensure the wells do not dry out.

Day 4: Medium Addition

  1. Prepare the Day 4 Medium 96 hours after seeding:
  1. Add 100µl/well Day 4 Medium to the entire plate; the total concentration of each well is 200µl/well.

Day 7: Microglia Seeding

  1. Mix the components listed below to make the Day 7 MG Seeding Medium in a sterile container. The medium should equilibrate at room temperature for at least 15 minutes. Do NOT warm in a 37°C water bath.
  1. Prepare one 50ml conical tube with 3ml of Day 7 MG Seeding Medium. Prepare a separate microcentrifuge tube with 25µl pf Trypan Blue solution.
  2. Remove the microglia cryovial from liquid storage and place in a 37°C water bath. Do NOT submerge the caps to avoid contamination.
  3. Remove the cryovial from the water bath when the last of the ice melts (~75-90 seconds). Disinfect the cryovial with 70% ethanol and transfer to the cell culture hood.
  4. Add 500µl of the Day 7 MG Seeding Medium slowly at a rate of ~2-3 drops/second using a P1000 pipette to the cryovial. This should take 10 seconds.
  5. Transfer the 1ml cryovial contents to the prepared 50ml conical tube; rinse the cryovial with an additional 1ml of the Day 7 MG Seeding Medium to collect residual cells. Transfer this to the 50ml conical tube.
  6. Centrifuge the microglia tube at 200xg for 5 minutes.
  7. Aspirate off the supernatant, being sure to leave the cell pellet, and resuspend the cells in 950µl of fresh Day 7 MG Seeding Medium. Pipette up and down with a P1000 to mix gently.
  8. Based on the Certificate of Analysis value (viable cells/vial), resuspend the cells to a concentration of 1.0 x 106 live cells/ml using Day 7 MG Seeding Medium.
    1. For example, 2.3 million live cells per CoA will be resuspended to a total final volume of 2.3ml.
  9. Swirl the conical tube and pipette up and down to evenly suspend the cells in the medium. Transfer 25µl microglia suspension to the microcentrifuge tube that is prefilled with 25µl Trypan Blue. Pipette to mix. Count the number of viable and dead cells using a hemocytometer; determine the live cell concentration and viability.
  10. Calculate the volume needed to make the final microglia seeding suspension. General recommendation is to use a seeding density equal to the astrocyte density, but the final ratios may vary depending on experimental needs. Tri-culture totals > 50,000 live cells/well can appear clustered due to higher density.
    1. Dilute the cells in Day 7 MG Seeding Medium to the desired seeding concentration using the calculation table below as an example.
  1. Remove 100µl/well of medium from each well and add 100µl/well of the microglia seeding suspension.
  2. Do not immediately transfer the plate to the incubator after seeding; allow the cells to settle to the bottom of the well for 15 minutes. Transfer the plate to a humidified incubator set at 37°C with 5% CO2.

Day 9 and Onward Medium Changes

  1. Prepare Maintenance Medium using the components listed below:
  1. Change 100µl/well twice weekly, i.e. on Day 9, 12, 15, etc., and replace with freshly prepared Maintenance Medium.

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

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