The human brain is a complex and heterogeneous environment, containing multiple types of neurons and immune cells. Co-culture of neuronal and glial cells help researchers to model the complexities of the human central nervous system, 

Microglia are the resident immune cells in the brain, and work in close proximity with neurons to undertake their job of clearing pathogens, cell debris, redundant synapses, and protein aggregates. bit.bio’s ioMicroglia are capable of forming a stable co-culture with ioGlutamatergic Neurons, while retaining the ability to selectively phagocytose pathogenic particles.

See the co-culture and phagocytosis data on the product page >

In this protocol, we describe two methods of co-culturing ioMicroglia with ioGlutamatergic Neurons.

Cryopreserved vial of ioGlutamatergic Neurons (io1001).
Please refer to the ioGlutamatergic Neurons user manual for the generation of ioGlutamatergic Neurons cells, recommended reagents, equipment and associated protocols.

Cryopreserved vial of ioMicroglia (io1021).
Please refer to the ioMicroglia user manual for the generation of ioMicroglia cells, recommended reagents, equipment and associated protocols.

This protocol was optimised using the wild type ioGlutamatergic Neurons and ioMicroglia and can also be used for the related ioCell products listed in appendix 3.

• ioMicroglia Cell detachment protocol for re-seeding Recommended reagents, equipment and protocol in the ioMicroglia Cell detachment protocol.

• Poly-D-Lysine (PDL)-hydrobromide (Sigma, P6407)
  Preparation described in appendix 1.

• Borate buffer (20X) (ThermoFisher, 28341)

• Geltrex (Reduced GF) (ThermoFisher, A1413202)

• Poly-L-Lysine Solution (0.01%) (Sigma, P4707-50ML)

• Advanced DMEM/F12 (12634010, Thermo Fisher)

• Glutamax (35050061, Thermo Fisher)

• Pen/Strep (15140122, Thermo Fisher) (optional)

• N2 Supplement (17502001, Thermo Fisher)

• B27 Supplement (17504044, Thermo Fisher)

• 2-Mercaptoethanol (31350010, Thermo Fisher)

• NT (267-N3-025, Biotechne)

• BDNF (248-BDB-005, Biotechne)

• Recombinant Human M-CSF (300-25, Peprotech)

• Recombinant Human IL-34 (200-34, Peprotech)

• Biological safety cabinet

• Standard tissue culture wares (pipettes, tips) 

• Normoxic cell culture incubator (37°C, 5% CO₂)

• Bench top centrifuge 

• Cell counter and associated equipment

This protocol is split into four steps:
Step 1: Thaw and culture ioGlutamatergic Neurons
Step 2: Thaw and culture ioMicroglia
Step 3: Detach the ioMicroglia
Step 4: Establish ioGlutamatergic Neurons and ioMicroglia co-culture

Notes

1. Thaw and culture ioGlutamatergic Neurons

1.1. Coat a 24-well plate(s) with PDL-Geltrex, according to the ioGlutamatergic Neurons user manual.

1.2. Thaw a vial of ioGlutamatergic Neurons (Small vial: >1 x 10⁶ viable cells, Large vial: >5 x 10⁶ viable cells) and seed at a density of 30,000 cells/cm², according to the ioGlutamatergic Neuron user manual.

1.3. Culture the ioGlutamatergic Neurons up to day 10, according to the ioGlutamatergic Neuron user manual.

2. Thaw and culture ioMicroglia

2.1. Coat a 24-well plate(s) with Poly-L-lysine, according to the ioMicroglia user manual.

2.2. Thaw a vial of ioMicroglia (Small vial: >1.5 x 10⁶ viable cells, Large vial: >5 x 10⁶ viable cells) and seed at a density of 39,500 cells/cm², according to the ioMicroglia user manual.

2.3. Culture ioMicroglia up to day 1 or day 10*, according to the ioMicroglia user manual.

*When culturing ioMicroglia to day 10 for detachment, you will see 50-70% cell loss that is inherently part of  the dissociation and re-seeding process. Seed a sufficient number of cells to account for this.

3. Detach the ioMicroglia

3.1. Prepare the required volume of basal co-culture medium (0.5 mL per well of a 24-well plate) according to Table 1 in Appendix 1, and mix well.

3.2. Prepare the required volume (250 µL per well of a 24-well plate) of 2x complete co-culture medium, according to Table 2 in appendix 1. The following additives are added to support glutamatergic (NT3 and BDNF) and microglial (IL-34 and M-CSF) functions.

3.3. Follow the ioMicroglia Cell detachment protocol for re-seeding experiments (using manual agitation and no centrifuge step)

4. Establish ioGlutamatergic Neurons and ioMicroglia co-culture.

4.1. Remove the supernatant and resuspend the ioMicroglia cell pellet in 200 µL of basal co-culture medium.

4.2. Perform a cell count and determine cell viability; >90% viability is required to proceed with the rest of the co-culture protocol.

4.3. Centrifuge ioMicroglia cells at 300 x g for 5 minutes.

4.4. Resuspend ioMicroglia cells at a density of 48,000 cells/mL in 2x complete co-culture medium, prepared as described in Table 2 in appendix 1.

4.5. Gently remove 50% (250 µL) of the medium from each well of the 24-well plate containing day 10 ioGlutamatergic Neurons.

Tip: We recommend a seeding density ratio of 1:5 ioMicroglia:ioGlutamatergic Neurons. Seed the ioMicroglia at a density of 6,000 cells/cm² onto the day 10 ioGlutamatergic Neurons.

4.6. Gently add 250 µL of ioMicroglia cells resuspended in the 2x complete co-culture medium directly to each well of the 24-well plate containing day 10 ioGlutamatergic Neurons.

4.7. Immediately transfer the 24-well plate(s) to a standard normoxic tissue culture humidified incubator at 37°C, 5% CO₂. To ensure an even cell distribution, gently cross-shake the plate once on the incubator shelf (back and forth, side to side, 2-3 times).

4.8. Once the co-culture has been established, perform a 50% media change every 3-4 days with the 2x complete co-culture medium.

Tip: We suggest maintaining the co-culture for up to 8 days, but this can be modified according to the downstream application. See the reference data below for details on how to perform ICC to visualise and validate this co-culture.

Table 1: Preparation of basal co-culture medium.

Table 2: Preparation of 2x complete co-culture medium.

Table 3 – ioCell products that may be used in this protocol.

If you have any questions or need assistance, please reach out to technical@bit.bio and we will do our best to support you.

Published March 2025, version 2