ioGlutamatergic Neurons and ioAstrocytes are human induced pluripotent stem cell (iPSC)-derived glutamatergic neurons and astrocytes, deterministically programmed using opti-ox™ technology. This protocol describes a method for co-culturing ioGlutamatergic Neurons with ioAstrocytes to facilitate research into complex neuroglial interactions and improve neuronal network activity in multi-electrode array (MEA) systems.  

See data on how bit.bio’s ioAstrocytes support the network formation and synchronicity of ioGlutamatergic Neurons in a co-culture >

• Cryopreserved ioAstrocytes (ioEA1093)

• Cryopreserved ioGlutamatergic Neurons (io1001)
  

• Poly-D-Lysine (PDL)-hydrobromide (Sigma, P6407)

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

• Geltrex (Reduced GF) (ThermoFisher, A1413202)

• Advanced DMEM/F12 (12634010, Thermo Fisher)

• Glutamax (35050061, Thermo Fisher)

• N2 Supplement (17502001, Thermo Fisher)

• B27 Supplement (17504044, Thermo Fisher)

• 2-Mercaptoethanol (31350010, Thermo Fisher)

• Fibroblast Growth Factor (FGF) (AF-100-18B, Peprotech)

• Ciliary neurotrophic factor (CNTF) (257-NT, Biotechne)

• Heparin-binding EGF-like growth factor (E4643, Sigma)

• Bone morphogenetic protein 4 (BMP4) (120-05ET, Peprotech)

• dbcAMP (sc-201567A, Santa Cruz Biotechnology)

• N-acetyl-cysteine (A9165, Sigma)

• Recombinant human Neurotrophin-3 rh-NT3 (267-N3-025, Biotechne)

• Brain-derived neurotrophic factor (BDNF) (248-BDB-050, Biotechne)

• DAPT (2634/10, Biotechne)

• Doxycycline hyclate (D9891, Sigma)

• 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 three steps:
Step 1: Thaw and culture ioAstrocytes
Step 2: Thaw ioGlutamatergic Neurons and seed on ioAstrocyte cultures
Step 3: Establish ioGlutamatergic Neurons and ioAstrocyte co-culture

Notes

1. Thaw and culture ioAstrocytes

For co-culture with ioGlutamatergic Neurons, culture the ioAstrocytes up to day 9 according to the ioAstrocytes protocol outlined below.

Before starting:

Note: Each vial of ioAstrocytes contains ≥1 x 106 viable cells. bit.bio recommends a minimum seeding density of 30,000 cells/cm2.

• Prepare enough tissue culture vessel(s) with PDL-Geltrex coating prior to thawing the cryovial(s) (Appendix 4.5).
• Prepare and allow DMEM/F12 medium supplemented with ROCK Inhibitor (DMEM/F12 + Ri) to reach room temperature prior to thawing of cells (Appendix 4.4.1)
• Prepare and warm up astrocyte stabilisation medium supplemented with ROCK Inhibitor (Ri) (stab:Astro + Ri) to 37°C for revival (Appendix 4.4.3).
• Prepare the cell viability marker and slides for counting the cells (step 1.1.11) before beginning. This will ensure there is minimal time for the cells to settle before counting.
• Warm-up the water bath to 37°C.
• Remove the vials from liquid nitrogen immediately before step 1.1.1 and keep at -80°C or on dry ice. Do not store the cells at -80°C or on dry ice long term. 

Revival and culture procedures:

1.1. Cell thawing

1.1.1. Remove the cryovial from dry ice and immediately immerse into a 37°C water bath. Avoid submerging the cap. Use of a floating microcentrifuge tube rack is recommended.

1.1.2. Remove the cryovial from the water bath when only a very small fraction of ice is left visible (this should take approximately 1 minute and 50 seconds).

1.1.3. Spray the cryovial with 70% ethanol and take it to a biological safety cabinet.

1.1.4. Transfer the cells from each vial (1 mL)  into a single empty falcon tube of the appropriate size (15 or 50 mL tube).

1.1.5. Carefully wash the cryovial with 1 mL DMEM/F12 + Ri using a 1 mL micropipette, and add it to the tube in a dropwise manner, agitating the tube occasionally ensuring the cell suspension is mixed with DMEM/F12 + Ri.

1.1.6. Add a further 4 mL of DMEM/F12 + Ri per vial of cells thawed in a dropwise manner, agitating the tube occasionally.

1.1.7. Gently pipette up and down once using the serological pipette to ensure the even distribution of DMSO. 

1.1.8. Centrifuge the cells at 200 x g for 5 minutes at room temperature.

1.1.9. Return cells to the biological safety cabinet and carefully remove the supernatant, without disturbing the cell pellet, by tilting the tube to a 45-degree angle and aspirating all the medium from the meniscus up to the point where the tube starts to taper down towards the conical base.

1.1.10. Add 1 mL of stab:Astro + Ri to the cell pellet and resuspend the cells gently by pipetting up and down 4 times with a 1 mL micropipette.

1.1.11. Count the cells using a haemocytometer or automatic cell counter, including a cell viability marker such as Trypan Blue or AOPI. The typical recovery from one cryovial is >1 x 10⁶ viable cells.

1.2. Cell Seeding (Day 0)

1.2.1 Day 0: Dilute the cell suspension to the required cell concentration (Table 1 below for reference) using stab:Astro + Ri to achieve the optimal seeding density for your desired experimental conditions (for all other plate formats, contact us at technical@bit.bio). A minimum seeding density of 30,000 cells/cm² is routinely used at bit.bio. We do not expect compatibility issues with 384 well microplates, but these have not been tested at bit.bio.

Table 1: Recommended cell concentrations and medium volumes based on plate formats tested by bit.bio (*) or calculated using the recommended seeding density/cm² for Corning^® plates. 

1.2.2. Aspirate the Geltrex coating solution from the culture vessel(s).

1.2.3. Directly add the required volume of cell suspension to the coated culture vessel(s).

1.2.4. Immediately transfer the culture vessel(s) to a standard normoxic tissue culture humidified incubator at 37°C, 5% CO₂.

1.2.5. To ensure an even cell distribution, gently cross-shake the plate once on the incubator shelf (back and forth, side to side, 4 to 5 times).

1.3. Cell Stabilisation (Day 1 to Day 3) 

1.3.1. Day 1: 24 hours post thawing, gently aspirate 100% of the spent medium and replace it with fresh pre-warmed stab:Astro medium (no ROCK Inhibitor). 

1.3.2. Day 2: No medium change required.

1.4. Establishment of ioAstrocytes Mono-culture (Day 3 to Day 9)

1.4.1. Day 3: Gently aspirate 50% of the spent medium and replace it with fresh pre-warmed cult:Astro + D1µg/mL medium.

1.4.2. Day 4: No medium change required.

1.4.3. Day 5: Gently aspirate 50% of the spent medium and replace it with fresh pre-warmed cult:Astro + D1µg/mL medium.

1.4.4. Day 6: No medium change required.

1.4.5. Day 7: Gently aspirate 50% of the spent medium and replace it with fresh pre-warmed cult:Astro + D1µg/mL medium.

1.4.6. Day 8: No medium change required.

2. Thaw ioGlutamatergic Neurons and seed onto Day 9 ioAstrocyte mono-cultures

Before starting:

Note: Each ioGlutamatergic Neurons vial contains either ≥1.0 x 10⁶ (small vial) or ≥5.0 x 10⁶ (large vial) viable cells. bit.bio recommends a minimum seeding density of 30,000 cells/cm².

• Prepare basal glutamatergic neuron medium (b:GN) (Appendix 4.4.6).
• Prepare complete glutamatergic neuron medium (comp:GN) (Appendix 4.4.7).
• Prepare astrocyte culture medium (cult:Astro) (Appendix 4.4.4).
• Prepare co-culture pre-maintenance medium (pm:CC) (Appendix 4.4.8).
• Allow basal glutamatergic neuron medium (b:GN) and co-culture pre-maintenance medium (pm:CC) to reach room temperature prior to thawing the cells.
• Prepare the cell viability marker and slides for counting the cells (step 2.1.11) before beginning. This will ensure there is minimal time for the cells to settle before counting.
• Warm-up the water bath to 37°C.
• Remove the vials from liquid nitrogen immediately before step 2.1.1 and keep at -80°C or on dry ice. Do not store the cells at -80°C or on dry ice long term. 

2.1. Cell thawing

2.1.1. Day 9: Remove the cryovial from dry ice and immediately immerse into a 37°C water bath. Avoid submerging the cap. Use of a floating microcentrifuge tube rack is recommended.

2.1.2. Remove the cryovial from the water bath when only a very small fraction of ice is left visible (this should take approximately 2 minutes).

2.1.3. Spray the cryovial with 70% ethanol and take it to a biological safety cabinet.

2.1.4. Transfer the cells from each vial into a single empty tube of the appropriate size (15 mL or 50 mL tube).

2.1.5. Carefully wash the cryovial with 1 mL of b:GN and add the media to the tube in a dropwise manner, agitating the tube occasionally.

2.1.6. Add a further 4 mL of b:GN per thawed vial in a dropwise manner, agitating the tube occasionally.

2.1.7. Gently pipette up and down once using the serological pipette to ensure the even distribution of DMSO.

2.1.8. Centrifuge the cells at 200 x g for 3 minutes at room temperature.

2.1.9. Return cells to the biological safety cabinet and carefully remove the supernatant, without disturbing the cell pellet, by tilting the tube to a 45-degree angle and aspirating all the medium from the meniscus down to the point where the tube starts to taper down towards the conical base.

2.1.10. Add 1 mL (small vial) or 5 mL (large vial) of pm:CC to the cell pellet and gently resuspend the cells by slowly pipetting up and down 4 to 5 times with a 1 mL micropipette.

2.1.11. Count the cells including a cell viability marker. The typical recovery from one cryovial is ≥1.0 x 10⁶ viable cells (small vial) or ≥5.0 x 10⁶ viable cells (large vial); >85% viability is required to proceed with the rest of the co-culture protocol.

2.2. Cell seeding

2.2.1. Dilute the ioGlutamatergic Neurons cell suspension to the required cell concentration (Table 2 below for reference) using pm:CC to achieve the optimal seeding density for your desired experimental conditions (for all other plate formats, contact us at  technical@bit.bio).

We recommend a minimum seeding density of 30,000 cells/cm² ioGlutamatergic Neurons onto the Day 9 ioAstrocytes at a ratio of 1:1 ioAstrocytes: ioGlutamatergic Neurons. We do not expect compatibility issues with 384 well microplates, but these have not been tested at bit.bio. 

Table 2: Recommended cell concentrations and medium volumes based on plate formats tested by bit.bio (*) or calculated using the recommended seeding density/cm² for Corning^® plates.

2.2.2. Immediately transfer the plate(s) to a standard normoxic tissue culture humidified incubator at 37°C, 5% CO2.

2.2.3. To ensure an even cell distribution, gently cross-shake the plate once on the incubator shelf (back and forth, side to side, 2 to 3 times).

3. Establish ioGlutamatergic Neurons and ioAstrocytes co-culture

3.1. Day 10: No medium change required.

3.2. Day 11: Gently aspirate 100% of the medium and replace it with fresh pm:CC + DAPT (Appendix 4.4.9).

3.3. Day 12: No medium change required.

3.4. Day 13: Gently aspirate 100% of the medium and replace it with fresh m:CC (Appendix 4.4.10).

3.5. Day 14: No medium change required.

3.6. Day 15: The co-culture is established. For optimal co-culture maintenance, bit.bio recommends half-medium change every 48 hours, i.e., replacing 50% of the medium with fresh m:CC.

Tip: At bit.bio we have maintained the co-culture for 2 weeks, but this can be modified according to the downstream application. 

4. Appendices

4.1 Equipment required

• Biological safety cabinet

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

• 37°C water bath  

• Haemocytometer or calibrated automatic cell counter

• Liquid nitrogen storage unit

• Standard tissue culture wares (pipettes, tips, culture plates)

• Bench top centrifuge

• Integra Voyager automated pipette (optional)

• Corning^® tissue culture plates (Cat nos. 3516, 3513, 3526, 3548, 3596)

4.2 Recommended reagents

4.3. Preparation of stock solutions for media preparation

Note: to avoid freeze-thaw cycles, aliquot the stock solutions as appropriate for future use. The full media formulations are available in section 4.4.

4.4. Preparation of media

The preparation of an ioGlutamatergic Neurons and ioAstrocytes co-culture requires the user to make 10 media formulations. Please see table below:

Note: 

• Media 2 (stab:Astro) and Media 3 (stab:Astro + Ri) share the same base formulation with the only difference being the addition of ROCK Inhibitor.

• Media 4 (cult:Astro) and Media 5 (cult:Astro + D1μg/mL) share the same base formulation with the only difference being the addition of doxycycline.

• Media 8 (pm:CC), 9 (pm:CC + DAPT) and 10 (m:CC) are all a 1:1 composition of astrocyte culture medium (cult:Astro) and complete glutamatergic neuron medium (comp:GN) with the only difference being the addition of DAPT and/or doxycycline.

4.4.1. DMEM/F12 + Ri: DMEM/F12 medium + ROCK Inhibitor (Ri)

4.4.2 stab:Astro: astrocyte stabilisation medium

4.4.3 stab:Astro + Ri: astrocyte stabilisation medium + ROCK Inhibitor (Ri)

4.4.4 cult:Astro: astrocyte culture medium

4.4.5 cult:Astro + D1µg/mL: complete astrocyte culture medium + 1 μg/mL doxycycline

4.4.6 b:GN: basal glutamatergic neuron medium

4.4.7 comp:GN: complete glutamatergic neuron medium

4.4.8 pm:CC: co-culture pre-maintenance medium

4.4.9 pm:CC + DAPT: co-culture pre-maintenance medium + 10 µM DAPT

4.4.10 m:CC: co-culture maintenance medium

4.5. Preparation of PDL-Geltrex coated vessels

To make PDL-Geltrex coated plates, treat culture vessels first with PDL solution and subsequently with Geltrex according to the protocol below. 

4.5.1. Preparation of PDL coating solution

Note:

• To avoid freeze-thaw cycles, aliquot the coating solution as appropriate for future use.

• The following coating solution is prepared using the recommended reagents in Appendix 4.2.

1. Make up 50 mL of 1x borate buffer by diluting 2.5 mL of the 20x stock with 47.5 mL of sterile water.
2. Resuspend a 5 mg vial of PDL in 50 mL of the 1x borate buffer for a working concentration of 100 μg/mL. 

4.5.2. PDL coating

1. Calculate the total surface area to be coated.
2. Coat the surface area of your culture vessel with the PDL solution ensuring the entire surface of the well is coated. We recommend the coating volumes shown in the table below.

3. Incubate the coated plates, overnight at 37°C or for at least 3 hours at 37°C.
4. Aspirate PDL solution and then wash 3 times with sterile water, 5 minutes per wash. For each wash, use the same volume used for coating.
5. Aspirate the water and allow coated surfaces to dry completely in a laminar flow hood (without lids). This typically requires 1 hour.

6. Proceed with the Geltrex coating as described below.

4.5.3 Preparation of Geltrex aliquots

For the preparation of Geltrex aliquots and PDL-Geltrex coated plates, please follow the manufacturer’s instructions. In brief:

1. Remove Geltrex stock from -80°C and thaw on ice in a 4°C fridge overnight. 
2. The next day, prepare aliquots according to foreseen use. Store at -80°C to minimise further freeze-thawing.

3. Depending on the volume, the smaller aliquots should take about 30 minutes to thaw while kept on ice.

4.5.4 Geltrex coating
1. Calculate the total surface area to be coated.
2. Dilute the Geltrex 1:100 in chilled DMEM/F-12 (e.g. 100 µL in 10 mL).
3. Coat the surface area of your culture vessel with the Geltrex:DMEM/F12 coating solution.

4. Incubate the coated plates at 37°C for a minimum of 60 minutes.
5. Carefully aspirate off the excess Geltrex, then immediately plate the cells.

4.6 ioCells products compatible for co-culture with ioAstrocytes

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 June 2024, version 1