Co-culture of hiPSC-derived ioGlutamatergic Neurons with ioAstrocytes

Co-culture of hiPSC-derived ioGlutamatergic Neurons with ioAstrocytes

Protocol overview

ProtocolGraphic

Introduction

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 >

Protocol

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

Alert 1

Alert 2

Alert 3

Alert 4


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:

Alert 5

Alert 6

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.

Alert 7

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.

Alert 8

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.

Alert 9

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. 

Plate format

Surface (cm2)

mL/well

Cells/well

  6 well*

9.60

2.000

288,000

12 well

3.80

1.000

114,000

24 well*

1.90

0.500

57,000

48 well

0.95

0.250

28,500

96 well

0.32

0.125

9,600

 

Alert 10

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)

Alert 11

Alert 12

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.

Alert 13

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.

Alert 14

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.

Alert 15

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.

Plate format

Surface (cm2)

mL/well

Cells/well

  6 well

9.60

2.000

288,000

12 well

3.80

1.000

114,000

24 well*

1.90

0.500

57,000

48 well

0.95

0.250

28,500

96 well

0.32

0.125

9,600

 

Alert 16

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).

Alert 17

Alert 18

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. 

Supplementary information

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

Reagent

Supplier

Cat no.

Storage

Poly-D-Lysine hydrobromide

Sigma

P6407

-20°C to -80°C

Sterile water

Sigma

W3500

Room temperature

Geltrex LDEV-Free, hESC-Qualified, Reduced Growth Factor Basement Membrane Matrix

Gibco

A1413302

-20°C to -80°C

Doxycycline hyclate

Sigma

D9891

2°C to 8°C

ROCK Inhibitor (Y27632)

Abcam

Ab144494

-20°C to -80°C

DMEM F12

Gibco

31330-038

2°C to 8°C

Neurobasal

Gibco

21103-049

2°C to 8°C

Foetal Bovine Serum

Gibco

10100-147

-20°C to -80°C

N2 Supplement (100)

Gibco

17502-001

-20°C to -80°C

GlutaMAX (100x)

Gibco

35050-038

Room temperature

2-Mercaptoethanol (50 mM)

Gibco

31350-010

2°C to 8°C

B27 Supplement (50x)

Gibco

17504-001

-20°C to -80°C

MEM Non-Essential Amino Acids (100x)

Gibco

11140-035

2°C to 8°C

FGF

Peprotech

AF-100-18B

-20°C to -80°C

CNTF

Biotechne

257-NT

-20°C to -80°C

BMP4

Peprotech

120-05ET

-20°C to -80°C

Sodium Pyruvate

Gibco

11360-070

2°C to 8°C

HB-EGF

Sigma

E4643

-20°C to -80°C

cAMP

Santa Cruz Biotech

SC-201567A

-20°C to -80°C

N-Acetyl-L-cysteine

Sigma

A8199

2°C to 8°C

NT3

Biotechne R&D Systems

267-N3-025

-20°C to -80°C

BDNF

Biotechne R&D Systems

248-BDB-005

-20°C to -80°C

DAPT

Biotechne R&D Systems

2634

2°C to 8°C

 

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.

Reagent

Stock solution

Working concentration

Storage

Doxycycline

2 mg/mL for cult:Astro + D1µg/mL and pm:CC


To prepare, reconstitute 20 mg in 10 mL of sterile water

1 μg/mL for cult:Astro + D1µg/mL and pm:CC


0.5 μL of 2 mg/mL stock solution
per 1 mL of medium

-20°C to -80°C




10 μg/mL for m:CC

To prepare, add 5 μL of 2 mg/mL stock to 1 mL of Neurobasal medium

10 ng/mL for m:CC 

1 μL of 10 µg/mL stock solution
per 1 mL of medium

-20°C to -80°C

ROCK Inhibitor

10 mM (1000x)

To prepare, reconstitute in 2 mL of sterile water

10 μM 

1 μL of stock solution per 1 mL of medium

-20°C to -80°C

HB-EGF

25 μg/mL

To prepare reconstitute 50 μg in 2 mL of 0.1% BSA/PBS

5 ng/mL

0.2 μL of stock solution per 1 mL of medium

-20°C to -80°C

N-Acetyl-L-cysteine

50 mg/mL

To prepare, weigh 50 mg of the powder then reconstitute in 1 mL of sterile water

Filter sterilise before use

5 μg/mL

0.1 μL of stock solution per 1 mL of medium

2°C to 8°C

FGF

20 μg/mL

To prepare, reconstitute in 1 mL of sterile water before further diluting with 4 mL of 0.1% BSA/PBS

8 ng/mL

0.4 μL of stock solution per 1mL of medium

-20°C to -80°C

CNTF

25 μg/mL

To prepare reconstitute 50 μg in 2 mL of 0.1% BSA/PBS

5 ng/mL for stab:Astro

0.2 μL of stock solution per 1 mL of medium

-20°C to -80°C




10 ng/mL for cult:Astro 

0.4 μL of stock solution per 1 mL of medium

-20°C to -80°C

BMP4

25 μg/mL

To prepare reconstitute 250 μg in 10 mL of 5 mM HCL

10 ng/mL

0.4 μL of stock solution per 1 mL of medium

-20°C to -80°C

cAMP

50 mg/mL

To prepare reconstitute 500 mg in 10 mL of DMSO

500 μg/mL

10 μL of stock solution per 1 mL of medium

-20°C to -80°C

NT3

50 μg/mL (5000x solution)


To prepare, reconstitute 25 μg
in 500 μL of PBS containing 0.1% BSA

10 ng/mL


0.2 μL of stock solution
per 1 mL of medium

-20°C to -80°C

BDNF

10 μg/mL (2000x solution)


To prepare, reconstitute 5 μg
in 500 μL of PBS containing 0.1% BSA

5 ng/mL


0.5 μL of stock solution
per 1 mL of medium

-20°C to -80°C

DAPT

20 mM (2000x solution)


To prepare, reconstitute 10 mg in 1156 µL of DMSO according to

the manufacturer’s protocol

10 µM


0.5 µL of stock solution per 1 mL of medium

-20°C to -80°C

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:

Media 1

DMEM/F12 + Ri

Section 4.4.1

Media 2

stab:Astro

Section 4.4.2

Media 3

stab:Astro + Ri

Section 4.4.3

Media 4

cult:Astro 

Section 4.4.4

Media 5

cult:Astro + D1μg/mL

Section 4.4.5

Media 6 

b:GN

Section 4.4.6

Media 7

comp:GN

Section 4.4.7

Media 8

pm:CC

Section 4.4.8

Media 9

pm:CC + DAPT

Section 4.4.9

Media 10

m:CC

Section 4.4.10

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)

Reagent/ Media

For 10 mL

For 100 mL

DMEM/F12 

10 mL

100 mL

ROCK Inhibitor

10 μL

100 μL

4.4.2 stab:Astro: astrocyte stabilisation medium

Reagent/ Media

For 100 mL

For 500 mL

Neurobasal 

94.80 mL

474.00 mL

MEM Non-Essential Amino Acids (100x)

1 mL

5 mL

GlutaMAX (100x)

1 mL

5 mL

Foetal Bovine Serum 

1 mL

5 mL

2-Mercaptoethanol 

100 μL

500 μL

B27 supplement (50x)

2 mL

10 mL

FGF

40 μL

200 μL

CNTF

20 μL

100 μL

BMP4

40 μL

200 μL

Doxycycline (2 mg/mL stock for final conc. 1 μg/mL)

50 μL

250 μL

 

Alert 19

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

Reagent/ Media

For 10 mL

For 100 mL

stab:Astro

10 mL

100 mL

ROCK Inhibitor

10 μL

100 μL

4.4.4 cult:Astro: astrocyte culture medium

Reagent/ Media

For 50 mL

For 100 mL

DMEM/F12

23.95 mL

47.90 mL

Neurobasal

23.95 mL

47.90 mL

GlutaMAX (100x)

500 μL

1 mL

2-Mercaptoethanol

50 μL

100 μL

Sodium Pyruvate

500 μL

1 mL

N2 Supplement (100x)

500 μL

1 mL

N-Acetyl-L-cysteine

5 μL

10 μL

HB-EGF

10 μL

20 μL

CNTF

20 μL

40 μL

BMP4

20 μL

40 μL

cAMP

500 μL

1 mL

 

Alert 20

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

Reagent/ Media

For 50 mL

For 100 mL

cult:Astro

50 mL

100 mL

Doxycycline (2 mg/mL stock for final conc. 1 μg/mL)

25 μL

50 μL

 

Alert 21

4.4.6 b:GN: basal glutamatergic neuron medium

Reagent/Media

For 200 mL

For 500 mL

Neurobasal

197.90 mL

494.75 mL

GlutaMAX (100x)

2 mL

5 mL

2-Mercaptoethanol 

100 μL

250 μL

 

Alert 22

4.4.7 comp:GN: complete glutamatergic neuron medium

Reagent/Media

For 50 mL

For 200 mL

b:GN

49.00 mL

195.86 mL

B27 (50x)

1 mL

4 mL

NT3 

10 μL

40 μL

BDNF

25 μL

100 μL

 

Alert 23

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

Reagent/Media

For 100 mL

For 200 mL

cult:Astro

50 mL

100 mL

comp:GN

50 mL

100 mL

Doxycycline (2 mg/mL stock for final conc. 1 μg/mL)

50 μL

100 μL

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

Reagent/Media

For 100 mL

For 200 mL

pm:CC

100 mL

200 mL

DAPT

50 μL

100 μL

4.4.10 m:CC: co-culture maintenance medium

Reagent/Media

For 100 mL

For 200 mL

pm:CC

100 mL

200 mL

DAPT

50 μL

100 μL

 

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. 

Alert 244.5.2. 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.

Coating solution

96 well

48 well

24 well

12 well

6 well

Poly-D-Lysine 

42 µL

125 µL

250 µL

500 µL

1 mL

 

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.

Alert 25

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.

Alert 26

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.

Coating solution

96 well

48 well

24 well

12 well

6 well

Geltrex:DMEM/F12 

42 µL

125 µL

250 µL

500 µL

1 mL

 

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.

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 June 2024, version 1

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