Tri-Culture of Cerebral Cortical Excitatory Neurons, Inhibitory Interneurons and Astrocytes User Guide

Preparation of Media

Neural Plating Media

• Neural Plating Media is created by supplementing Complete Neural Plating Media with Y-27632 2HCl to a final concentration of 10μM before use.
• Once made the Complete Neural Plating Media must be used and cannot be refrozen.

Neural Maintenance Media

• Upon receipt, aliquot the Neural Maintenance Media according to your requirements.
• Store the aliquoted Neural Maintenance Media at or below -80°C protected from Frozen Neural Maintenance Media can be stored at -80°C for 6 months.
• When ready to use, thaw an aliquot of Neural Maintenance Media overnight at 4°C in the
• Once thawed Neural Maintenance Media can be stored at 4°C for 2 weeks, protected from

Neural Maturation Media

Neural Maturation Media is made according to the recipe below:

Components	Volume
Neurobasal™- A Media	500 mL
B-27 supplement	10 mL
GlutaMAX™	5 mL
2-Mercaptoethanol (50 mM)	500 µL

 

• Prepare the above media in a Class II Biosafety Cabinet.
• Perform sterile filtration of the media using 0.22 µM filter bottles.
• Store both media at 4^oC and use within three weeks.

Preparation of Stock Solutions

 

	Stock concentration	Reconstitution	Storage after reconstitution
Y-27632	10 mM (1000x)	Sterile tissue culture grade water	3 months, -20 to -80 °C
BDNF	20 µg/mL (1000x)
cAMP	50 mM (100x)
Ascorbic Acid	20 mM (100x)
PDL	0.5 mg / mL (1x)

• Prepare all above chemicals and growth factor in a Class II Biosafety Cabinet under sterile conditions.

Preparation of NeurOne Supplement A (ax0674a)

• Each supplement should be aliquoted, depending on the customer’s requirements (each supplement requires a 1 in 50 dilution of the stock) g. 1ml of NeurOne Supplement A (ax0674a) will supplement 50 ml of Neural Maintenance Medium (ax0031) to create Neural Differentiation Medium. Three full medium changes of Neural Differentiation Medium will be required in total.
• Once each 1ml supplement is thawed, aspirate the contents to ensure appropriate resuspension of the contents using a 1ml Gilson, before aliquoting.
• Prepare supplements in a Class II Biosafety Cabinet under sterile conditions.

Preparation of NeurOne Supplement B (ax0674b)

• The NeurOne Supplement B (ax0674b) should be thawed and aliquoted, depending on the customers’ requirements (each supplement requires a 1 in 50 dilution of the stock) e.g. 1ml of NeurOne Supplement B will supplement 50 ml of Neurobasal-A Media to create Neural Maturation Medium. Four full medium changes of Neural Maturation Medium will be required in
• After the initial supplement is thawed, aspirate the contents to ensure appropriate resuspension of the contents using a 1ml Gilson, before
• Prepare supplements in a Class II Biosafety Cabinet under sterile conditions.

Coating Cell Culture Vessel

A variety of extra cellular matrices can be used for the cells, such as PEI, Polyornithine, Poly-D-lysine, Laminin or Matrigel.

We would recommend the following ECM combination for culturing Cortical Inhibitory Interneuron Progenitors in either monoculture or co-culture with Axol’s Cortical Excitatory Neuron Progenitors.

PDL

• Add 10ml sterile tissue grade water to 5 mg of poly-D-lysine to achieve desired concentration. Recap and leave for 24 hours at RT to ensure full dissolution.
• Add 300 mL per cm² of 1x PDL solution (0.5 mg/mL) to the culture vessel. Incubate for 60 mins at 37^oC, remove the solution through aspiration and thoroughly rinse the surface twice with sterile water.
• Remove water and allow the surface to dry for at least two hours before proceeding onto SureBond-XF

SureBond-XF

• Dilute the SureBond-XF stock solution (200x) in D-PBS (without calcium or magnesium) to make 1x working solution, e.g. 30 μL in 6 mL.
• Coat the culture surface with 200 μL per cm² of the SureBond-XF 1x working solution.
• Incubate for at least 4 hours at 37°C.
• Remove the SureBond-XF from the culture vessel prior to plating of cells. Do not wash the culture vessel after coating with SureBond-XF.
• Do not let the SureBond-XF coating dry out before plating the cells.

Thawing and Plating the Neural Stem Cells and Interneurons (Day 0) 

1. Add 10 µM Y-27632 (1 in 1000 dilution of the stock solution) to the Complete Neural Plating Media.
2. Pre-warm the Complete Neural Plating Media to 37°C.

1. To thaw the cortical excitatory neurons/neural stem cells and interneurons, transfer the vial of cells from nitrogen storage with the vial buried in dry ice.
2. Remove the vial from dry ice and transfer it immediately to a 37°C water bath. Do not completely submerge the vial (only up to 2/3rd of the vial). Remove the vial before the last bit of ice has melted.
3. Do not shake the vial during thawing.
4. Spray the vial with 70% ethanol and wipe it down with a sterile paper towel before placing it in the cell culture hood.
5. Once thawed completely, use a P1000 pipette to transfer the cells into a 15 mL sterile conical tube.
6. Gently wash the cryovial with 1 mL of the warm Complete Neural Plating Media and transfer this to the 15 mL sterile conical tube.
7. Add 8 mL of Complete Neural Plating Media dropwise to the cell suspension in the conical tube. Gently mix the cells with the media with a 10 mL serological pipette.

Important! Do not mix the cells vigorously. Avoid generating bubbles while pipetting.

1. Centrifuge cells at 200 x g for 5 minutes at room temperature.
2. Aspirate the media carefully and gently resuspend the cell pellet in 2 mL of the Complete Neural Plating Media until it becomes mostly a single cell suspension.
3. Perform a cell count. We recommend seeding Cortical Neurons at a density range between 100,000 – 200,000 cells per cm² for mono-culture.
4. For Co-culture we recommend a ratio between 3:1-9:1 of Cortical Excitatory Neurons to Interneurons.
5. Remove SureBond-XF coating solution from the culture vessel. Add an appropriate volume of the Complete Neural Plating Media to the culture vessel. Do not let the coating to dry out during the process.
6. Plate the resuspended cells dropwise and evenly.
7. Gently rock the culture vessel back and forth to ensure an even seeding density.
8. Incubate the cells at 37°C, 5% CO₂.

Differentiation of the Neural Stem Cells and Interneurons (Day 1-6) 

1. Add 1x NeurOne Supplement A (1 in 50 dilution of the stock solution) to Neural Maintenance Media to make Neural Differentiation Media.
2. Pre-warm the Neural Differentiation Media to 37°C.
3. Replace the spent plating media with warm Neural Differentiation Media.
4. Perform a full media change every other day on Day 3 and 5.

Maturation of the young neurons (Day 7-14) 

1. On day 7, add 1x NeurOne Supplement B (1 in 50 dilution of the stock solution), 20 ng/mL BDNF, 0.5 mM cAMP and 0.2 mM ascorbic acid to Complete Neural Plating Media to make Neural Maturation Media.
2. Pre-warm the Neural Maturation Media to 37°C.
3. Replace the spent Neural Differentiation Media with warm Neural Maturation Media.
4. Perform a full media change with pre-warmed Neural Maturation Media every other day on Day 7, 9, 11 and 13.

Thawing and Plating the Astrocytes (Day 19)

1. Add 10ng/ml Heregulin (1 in 1000 dilution of the stock solution) to Neural Maintenance Media to make Co-Culture Media.
2. Pre-warm the Co-Culture Media to 37°C
3. To thaw the astrocytes, transfer the vial of astrocytes from nitrogen storage with the vial buried in dry ice.
4. Remove the vial from dry ice and transfer it immediately to a 37°C water bath. Do not completely submerge the vial (only up to 2/3rd of the vial). Remove the vial before the last bit of ice has melted.
5. Do not shake the vial during thawing.
6. Spray the vial with 70% ethanol and wipe it down with a sterile paper towel before placing it in the cell culture hood.
7. Once thawed completely, use a P1000 pipette to transfer the cells into a 15 mL sterile conical tube.
8. Gently wash the cryovial with 1 mL of the warm Co-Culture Media and transfer this to the 15 mL sterile conical tube.
9. Add 8 mL of Co-Culture Media dropwise to the cell suspension in the conical tube. Gently mix the cells with the medium with a 10 mL serological pipette.

Important! Do not mix the cells vigorously. Avoid generating bubbles while pipetting.

1. Centrifuge cells at 200 x g for 5 minutes at room temperature.
2. Aspirate the medium carefully and gently resuspend the cell pellet in 2 mL of the Co-Culture Media until they become mostly a single cell suspension.
3. Perform a cell count. We recommend adding the astrocytes at ratio of 1:9 to the original excitatory neuron/NSC seeding density (typically 20,000 – 30,000 cells per cm²).
4. Plate the resuspended cells dropwise and evenly.
5. Incubate the cells at 37°C, 5% CO₂.

Tri-culture of Cerebral Cortical Excitatory Neurons, Inhibitory Interneurons and Astrocytes (day 19-25)

1. On Day 21, pre-warm the Co-Culture Media to 37°C.
2. Replace half the spent Co-Culture Media with fresh pre-warmed Co-Culture Media.
3. Perform half-media changes with the pre-warmed Co-Culture Media on Day 23 and Day 25.
4. The co-culture is assay ready from Day 26 onwards. Spontaneous electrical activity should appear from around Day 21 and Synchronised Burst Firing should be well-established by around Day 30.