Cortical excitatory neurons user guide
This User Guide is for the following NSC products
| Catalog. No. | Product Name | Format | Storage on Arrival |
| ax0015, ax0016, ax0018, ax0111, ax0112, ax0113, ax0114, ax0211, | axoCells Human iPSC-derived Neural Stem Cells | ≥1.5 million cells/vial | Vapor phase LN storage |
Media Components – Axol
| Catalog. No | Product Name | Format | Stock Conc. | Storage on Arrival | Thawing Instructions |
| ax0031 | Neural Maintenance Medium | 500ml | 1x | Aliquot & store at -80°C for up to 6 months. Keep in the dark | Overnight at 4°C and store at 4°C for 2 weeks |
| ax0033 | Neural Plating Medium | 30ml | 1x | Store at -80°C for up to 6 months. Keep in the dark. | Overnight at 4°C |
| ax0674 | NeurOne Cortical Neuron supplement | ||||
| ax0674a | NeurOne Sup. A | 1 mL | 50x | Store at -80°C for up to 1 months. Keep in the dark. | Thaw in 37°C water bath |
| ax0674b | NeurOne Sup. B | 1 mL | 50x | Store at -80°C for up to 1 months. Keep in the dark. | Thaw in 37°C water bath |
| ax139800 (10 ug) | Recombinant Human Brain-Derived Neurotrophic Factor (BDNF) | 10 ug Lyophilized Powder | N/A | -20ºC | Reconstituted protein should be used immediately or stored in working aliquots at -80°C |
| ax0053 | SureBond-XF | 1ml | 200x | Refer to C of A | |
Media Components – Required
| Product Name | Supplier | Product Code |
| B-27 supplement | ThermoFisher Scientific | 17504044 |
| Neurobasal™-A Medium | ThermoFisher Scientific | 12349015 |
| GlutaMAX™ | ThermoFisher Scientific | 35050061 |
| 2-Mercaptoethanol (50 mM) | ThermoFisher Scientific | 31350010 |
| Poly-D-Lysine (PDL) | ThermoFisher Scientific | A3890401 |
| Dibutyryl-cAMP | Merck-Sigma | D0260 |
| Ascorbic Acid | Merck-Sigma | A4403 |
| Y-27632 dihydrochloride (ROCK inhibitor) | Focus Biomolecules | 10-2301 |
Process Overview
| Stages | Thawing & Plating | Neuronal Differentiation | Neuronal Maturation | Assay Ready | Long Term Culture |
| Day 0 | Day 1-6 | Day 7-14 | Day 15-27 | >Day 28 | |
| Basal Media | Neural Plating Media | Neural Maintenance Media | Neural Maturation Basal Media | Neural Maturation Basal Media | Neural Maturation Basal Media |
| Supplement | Y-27632.2HCl
SureBond-XF |
NeurOne Supplement A
SureBond-XF |
NeurOne Supplement B,
BDNF, cAMP, Ascorbic Acid, SureBond-XF |
BDNF, cAMP, Ascorbic Acid | BDNF, cAMP, Ascorbic Acid |
| Final Media | Complete Neural Plating Media | Differentiation Media | Complete Maturation Media | Long Term Culture Media | Long Term Culture Media |
| Coating | PDL & SureBond-XF | PDL & SureBond-XF | PDL & SureBond-XF | PDL & SureBond-XF | PDL & SureBond-XF |
Preparation of Media
Neural Plating Media
• Upon receipt, Neural Plating Media should be stored at -80°C until the given expiry date in the dark.
• When ready to use, thaw Neural Plating Media overnight at 4°C in the dark.
• Once thawed Neural Plating Media can be stored at 4°C for 2 weeks, protected from light.
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 light. Frozen Neural Maintenance Media can be stored at -80°C until the given expiry date.
• When ready to use, thaw an aliquot of Neural Maintenance Media overnight at 4°C in the dark.
• Once thawed Neural Maintenance Media can be stored at 4°C for 2 weeks, protected from light.
Neural Maturation Basal Media
Neural Maturation Basal Media is made according to the recipe below:
| Components | Volume |
| B-27 supplement | 10 mL |
| Neurobasal™-A Medium | 500 mL |
| GlutaMAX™ | 5 mL |
| 2-Mercaptoethanol (50 mM) | 250 µL |
• Prepare the above chemicals and media in a class II biosafety cabinet under sterile conditions.
• Perform sterile filtration of the media using 0.22μM PES filters.
• Store both media at 4°C and use within two weeks.
Preparation of Stock Solutions
| Component | Stock concentration | Reconstitution | Storage after reconstitution |
| Y-27632 dihydrochloride (ROCK inhibitor) | 10 mM (1000x) | Sterile tissue culture-grade water | 3 months -20 to -80°C |
| Ascorbic Acid | 20mM (100x) | Sterile tissue culture-grade water | 3 months -20 to -80°C |
| BDNF | 20 µg/mL (1000x) | 0.05% HSA in D-PBS | 12 months -20 to -80°C |
| cAMP | 50mM (100x) | 0.05% HSA in D-PBS | 6 months -80°C |
• Prepare all above chemicals and growth factors in a Class II Biosafety Cabinet under sterile conditions.
• cAMP should be filter sterilized through a 0.22μM PES filter prior to use.
Preparation of BDNF Growth Factor (ax139800)
• Prepare a 0.05% solution of HSA in Dulbecco’s Phosphate-Buffered Saline (D-PBS) (without calcium or magnesium). If necessary, filter sterilize the HSA prior to use (0.2μm).
• Prepare a stock solution of BDNF by resuspending the lyophilized powder in 0.05% HSA. The recommended concentrations and dilution factors are outlined in the table above.
• We do not recommend repeated freeze-thaws of reconstituted proteins. We would recommend dilution to stock concentration and aliquoting into single-use aliquots of appropriate volumes e.g. BDNF at 50 μL of 20μg/mL for 50 mL media. The growth factors can be aliquoted and stored at -80°C or -20°C for up to 6 months.
Preparation of NeurOne Supplement A & B
• Each supplement should be aliquoted, depending on the customer’s requirements (each supplement requires a 1 in 50 dilution of the stock).
• Thaw each supplement at 37°C to ensure optimal performance.
• Once each 1ml supplement is thawed, aspirate the contents to ensure appropriate resuspension of the contents using a 1ml pipette, before aliquoting.
• Prepare supplements in a class II biosafety cabinet under sterile conditions.
Coating Cell Culture Vessel
A variety of extracellular 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 Neural Stem Cells to Cortical Excitatory Neurons:
PDL
• Dilute the liquid poly-D-lysine 1:1 in sterile water to create a 0.05mg/ml working solution (0.5x).
• Add 300 μL per cm2 of 0.5x PDL working solution (0.05 mg/mL) to the culture vessel. Incubate for 60 mins at 37oC, 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 coating.
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 immediately 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 axoCells Neural Stem Cells (Day 0)
1. Add 10 μM Y-27632.2HCl (1 in 1000 dilution of the stock solution) and SureBond-XF at a 1:400 dilution to Neural Plating Media to make Complete Neural Plating Media.
2. Pre-warm the Complete Neural Plating Media to 37°C.
3. To thaw the axoCells neural stem cells, transfer the vial of cells 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 Complete Neural Plating Media and transfer this to the 15 mL sterile conical tube.
9. 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.
10. Centrifuge cells at 200 x g for 5 minutes at room temperature.
11. Aspirate the media carefully and gently resuspend the cell pellet in 2 mL of Complete Neural Plating Media until they become mostly a single-cell suspension.
12. Perform a cell count. We recommend seeding axoCells Cortical Excitatory Neurons at a density range between 100,000 – 200,000 cells per cm2 for mono-culture.
13. 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 dry out during the process.
14. Plate the resuspended cells dropwise and evenly.
15. Gently rock the culture vessel back and forth to ensure an even seeding density.
16. Incubate the cells at 37°C, 5% CO2.
Differentiation of the axoCells Neural Stem Cells (Day 1-6)
1. Add 1x NeurOne Supplement A (1 in 50 dilution of the stock solution) and SureBond-XF (1 in 400 dilution) to Neural Maintenance Media to make Differentiation Media.
2. Pre-warm the Differentiation Media to 37°C.
3. Replace the spent plating media with warm 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 Neural Maturation Basal Media to make Complete Maturation Media.
2. Pre-warm the Complete Maturation Media to 37°C.
3. Replace the spent Differentiation Media with warm Complete Maturation Media containing SureBond-XF at a 1:400 dilution.
4. Perform half media changes with pre-warmed Complete Maturation Media every other day on Day 9, 11 and 13.
Long term culture of axoCells Cortical Excitatory Neurons (after Day 14)
1. On Day 15, add 20 ng/mL BDNF, 0.5 mM cAMP and 0.2 mM ascorbic acid to Neural Maturation Basal Media to make Long Term Culture Media.
2. Pre-warm the media to 37°C.
3. Replace the spent maturation media with pre-warmed Long Term Culture Media.
4. Perform half media changes with the pre-warmed Long Term Culture Media every other day from Day 15.
5. The neurons are assay-ready post-Day 20 with an assay window of at least 14 more days.