Introduction
The human brain is a complex and heterogeneous environment, containing multiple types of neurons and immune cells, each with a unique role to support the healthy function of the neuronal network as a whole. In order to make brain research as relevant as possible, it is important for researchers to create an in vivo environment, such as a co-culture of neuronal and glial cells, that more closely resembles the complex native environment in the human brain.
Microglia are the resident immune cells in the brain that contribute to brain development, maintenance of neuronal networks, and repair following infection or injury. They work within close proximity with neurons to undertake their job of clearing pathogens, cell debris, redundant synapses, and protein aggregates. ioMicroglia™️ are capable of forming a stable co-culture with ioGlutamatergic Neurons™️, while retaining the ability to selectively phagocytose pathogenic particles without disrupting neuronal networks.
ioMicroglia and ioGlutamatergic Neurons are human induced pluripotent stem cell (iPSC)-derived microglia and glutamatergic neurons, precision reprogrammed using opti-ox™ technology. In this protocol, we describe the two methods of co-culturing the ioMicroglia with the ioGlutamatergic Neurons in order to facilitate research into complex interactions between immune cells and neurons.
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Protocol overview
Materials, equipment, and associated protocols
-
ioGlutamatergic Neurons (cat no: io1001)
Please refer to the ioGlutamatergic Neurons user manual for the generation of ioGlutamatergic Neuronal cells, recommended reagents, equipment and associated protocols -
ioMicroglia (cat no: io1021)
Please refer to the ioMicroglia user manual for the generation of ioMicroglia cells, recommended reagents, equipment and associated protocols -
ioMicroglia Cell detachment protocol
Recommended reagents, equipment and protocol in the ioMicroglia Cell detachment protocol to detach ioMicroglia cells -
Poly-D-Lysine (PDL)-hydrobromide (Sigma, P6407) - preparation described in Appendix 1
-
Pen/Strep (15140122, Thermo Fisher) (optional)
-
Biological safety cabinet
-
Standard tissue culture wares (pipettes, tips)
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Normoxic cell culture incubator (37°C, 5% CO2)
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Bench top centrifuge
-
Cell counter and associated equipment
Protocol
This protocol is split into four sections:
1: Thaw and culture ioGlutamatergic Neurons
2: Thaw and culture ioMicroglia
3: Detach the ioMicroglia
4: Establish ioGlutamatergic Neurons and ioMicroglia co-culture
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 (>1 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 (>1.5 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.
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 below, and mix well.
Table 1: Preparation of basal co-culture medium.
|
Reagent |
Catalogue no. |
Brand |
Stock conc. |
Final conc. |
Volume per 250 mL |
Volume per 500 mL |
|
Advanced DMEM/F12 |
12634010 |
Thermo Fisher |
- |
- |
Make up to 250 mL |
Make up to 500 mL |
|
Glutamax |
35050061 |
Thermo Fisher |
100X |
1X |
2.5 mL |
5 mL |
|
Pen/Strep (optional) |
15140122 |
Thermo Fisher |
100X |
1X |
2.5 mL |
5 mL |
|
N2 Supplement |
17502001 |
Thermo Fisher |
100X |
1X |
2.5 mL |
5 mL |
|
B27 Supplement |
17504044 |
Thermo Fisher |
50X |
1X |
5 mL |
10 mL |
|
2-Mercaptoethanol |
31350010 |
Thermo Fisher |
50mM |
50µM |
250 µL |
500 µL |
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 below. The following additives are added to support glutamatergic (NT3 and BDNF) and microglial (IL-34 and M-CSF) functions.
Table 2: Preparation of 2x complete co-culture medium.
|
Reagent |
Stock Conc. |
Final Conc. |
Volume |
|
Basal co-culture medium |
- |
- |
Make up to |
|
NT3 |
50 µg/mL |
20 ng/mL |
20 µL |
|
BDNF |
10 µg/mL |
10 ng/mL |
50 µL |
|
IL-34 |
10 µg/mL |
200 ng/mL |
1 mL |
|
M-CSF |
10 µg/mL |
20 ng/mL |
100 µL |
3.3. Follow the ioMicroglia Cell detachment protocol up to step 7.
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 xg 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 above.
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.
4.8. 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).
4.9. 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.
Reference data - characterisation of the co-culture by immunocytochemistry
Characterisation of the ioGlutamatergic Neurons and ioMicroglia co-culture by immunocytochemistry
Immunocytochemistry (ICC) may be performed on the co-culture, using the primary antibodies detailed in Table 3 below, against IBA1 (microglia marker) and MAP2 (pan neuronal marker). Figure 1 shows example ICC images obtained with ioGlutamatergic Neurons and ioMicroglia co-cultures after 8 days.
Table 3: Recommended antibodies for co-culture ICC staining
|
Antibody |
Supplier |
Cat number |
Dilution |
Host |
|
Invitrogen |
PA5-27436 |
1:1000 |
Rabbit IgG |
|
|
Abcam |
ab5392 |
1:2000 |
Chicken IgY |
Figure 1: Example ICC image obtained at day 8 of the ioGlutamatergic Neurons and ioMicroglia co-culture. In this case, ioMicroglia were detached after 10 days of monoculture, and seeded onto day 10 ioGlutamatergic Neurons; this image was taken 8 days after the co-culture was established The co-cultures show expression of MAP2 (red; pan neuronal marker) and IBA1 (orange; microglia marker). DAPI counterstain (blue). Images taken at 100x magnification.
Application support
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 October 2023, version 1