Co-culture of human iPSC-derived Glutamatergic Neurons and Microglia

Co-culture of human iPSC-derived Glutamatergic Neurons and Microglia

Protocol overview

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. bit.bio’s 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.

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

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.

Cells

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.

Materials, equipment, and associated protocols

Protocol

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

The following protocol recommends general guidelines. We encourage users to optimise the critical steps according to their experimental conditions.

Read the entire protocol and prepare all the necessary materials and reagents required before starting your experiment.


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 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.

If using D1 ioMicroglia, remove the stabilisation media and replace with maintenance media 1-2 hours before seeding onto D10 ioGlutamatergic Neurons.

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 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.

This is a 50% medium change, please note that the concentrations of some compounds have been doubled to account for the other 50% of the media.

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.

Carry out culture with special care as neuronal cells are prone to mechanical stress which may cause detachment.

Perform medium aspiration and addition slowly and on the side of the well, using micropipettes instead of serological pipettes.

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.

Appendix 1 - Preparation of co-culture media

Table 1: Preparation of basal co-culture medium.

Reagent

Cat no

Supplier

Stock conc.

Final conc.

Volume per 250 mL

Advanced DMEM/F12

12634010

Thermo Fisher

-

-

Make up to 250 mL

Glutamax

35050061

Thermo Fisher

100X

1X

2.5 mL

Pen/Strep (optional)

15140122

Thermo Fisher

100X

1X

2.5 mL

N2 Supplement

17502001

Thermo Fisher

100X

1X

2.5 mL

B27 Supplement

17504044

Thermo Fisher

50X

1X

5 mL

2-Mercaptoethanol 

31350010

Thermo Fisher

50 mM

50 µM

250 µL

 

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

Reagents for 2x complete co-culture medium

Stock Concentration

Final Concentration

Volume per 50 mL

 Basal co-culture medium

-

-

Make up to 50 mL

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

 

Technical 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

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