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Enriched Cerebral Cortical Neuron Protocol

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The User Guide is for the following NSC products

Catalog. No. Product Name Format Storage on Arrival
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 SThawing Instructions
ax0031a Neural Maintenance Medium Supplement 7.5ml 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
ax0031b Neural Maintenance Basal Media 500ml 1x Aliquot & store at 2-8°C for up to 6 months. Keep in the dark.
ax0033 Neural Plating Medium 30ml 30ml 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
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
Recombinant Human BDNF R&D Systems 248-BDB
Dibutyryl-cAMP Merck-Sigma D0260
Ascorbic Acid Merck-Sigma A4403
Y-27632 dihydrochloride (ROCK inhibitor) Focus Biomolecules 10-2301
Poly-D-Lysine (PDL) Sigma-Aldrich P7405

Process Overview

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Preparation of medium 

NEURAL PLATING MEDIUM (ax0033)

  • Upon receipt, store Neural Plating Medium (ax0033) at or below -80°C protected from light.
  • When ready to use, thaw Neural Plating Medium overnight at 4°C in the dark.
  • Once thawed, Neural Plating Medium must be used and cannot be refrozen.
  • Neural Plating Medium requires supplementing with Y-27632 dihydrochloride to a final concentration of 10μM before use

NEURAL MAINTENANCE MEDIUM SUPPLEMENT (ax0031a)

  • Upon receipt, thaw Neural Maintenance Medium Supplement (ax0031a) overnight at 4°C protected from light.
  • Aliquot the Neural Maintenance Medium Supplement according to your requirements. E.g. 750μl of Neural Maintenance Medium Supplement (ax0031a) will supplement 50ml of Neural Maintenance Medium (ax0031b) to create Supplemented Neural Maintenance Medium (ax0031).
  • Store the aliquoted Neural Maintenance Medium Supplement at or below -80°C protected from light. Frozen Neural Maintenance Medium Supplement can be stored at -80°C for 6 months.
  • When ready to use, thaw an aliquot of Neural Maintenance Medium Supplement overnight at 4°C in the dark.
  • Once thawed Neural Maintenance Medium Supplement can be stored at 4°C for 2 weeks, protected from light.

NEURAL MATURATION BASAL MEDIUM

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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 4OC AND USE WITHIN THREE WEEKS.

PREPARATION OF STOCK SOLUTIONS

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Prepare all above chemicals and growth factor 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 50ml of Supplemented Neural Maturation Basal Media to create Neural Maturation
  • Medium. Four full medium changes of Neural Maturation Medium will be required in total.
  • After the initial 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.

COATING CELL CULTURE VESSEL

  • A variety of extra cellular matrices can be used the cells, i.e. PEI, polyornithine, Poly-D-lysine, Laminin.
  • We would recommend the following ECM combination for developing cortical neurons.

PDL

  • Add 50mls of sterile tissue grade water to 5 mg of poly-D-lysine. 300 μL per cm 2 of 1x PDL solution (0.1 mg/mL) to the culture vessel. Incubate for 10 mins at 37o C, remove the solution through aspiration and thoroughly rinse the surface twice with sterile water.
  • Allow the surface to completely dry for at least two hours before proceeding onto SureBond-XF coating.
  • Derivation of Cerebral Cortical Neurons from Axol iPSC-Derived NSCs 8

SureBond-XF

  • Dilute the SureBond-XF stock solution (200x) in D -PBS (without calcium or magnesium) to make 1x working solution, e.g. 30μL in 6mL.
  • Coat the culture surface with 200μL 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 (DAY 0)

  1.  Add 10 mM Y-27632 (1 in 1000 dilution of the stock solution) to Neural Plating Medium (ax0033).
  2. Pre-warm the plating medium to 37°C.
  3. To thaw the NSCs, transfer the vial of NSCs 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 plating medium and transfer this to the 15 mL sterile conical tube.
  9. Add 8 mL of plating medium dropwise to the cell suspension in the conical tube. Gently mix the cells with the medium with a 10 mL serological pipette.
  10. Important! Do not mix the cells vigorously. Avoid generating bubbles while pipetting.
  11. Centrifuge cells at 200 x g for 5 minutes at room temperature.
  12. Aspirate the medium carefully and gently resuspend the cell pellet in 2 mL of the plating medium until they become mostly a single cell suspension.
  13. Perform a cell count. We recommend seeding the NSCs at a density range between 100’000 – 200’000 cells per cm 2.
  14. Remove SureBond-XF coating solution from the culture vessel. Add an appropriate volume of the plating medium to the culture vessel. Do not let the coating to dry out during the process.
  15. Plate the resuspended cells dropwise and evenly.
  16. Gently rock the culture vessel back and forth to ensure an even seeding density.
  17. Incubate the cells at 37°C, 5% CO 2 .

DIFFERENTIATION OF THE NEURAL STEM CELLS (DAY 1-6)

  1. On Day 1, add 1x NeurOne Supplement A (1 in 50 dilution of the stock solution) to Supplemented Neural Maintenance Medium (ax0031) to make Neural Differentiation Medium.
  2. Pre-warm the Neural Differentiation Medium to 37°C.
  3. Replace the spent Neural Plating Medium with warm Neural Differentiation Medium.
  4. Perform a full medium changes every other day on Day 3 and 5.
  5. There will be three full medium changes with Neural Differentiation Medium in total.

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 Medium to make Complete Neural Maturation Medium.
  2. Pre-warm the maturation medium to 37°C.
  3. Replace the spent Neural Differentiation Medium with pre-warmed Complete Neural Maturation Medium.
  4. Perform a full medium change with pre-warmed Complete Neural Maturation Medium every other day on Day 9, 11 and 13.
  5. There will be four full medium changes with Complete Neural Maturation Medium in total.

LONG TERM CULTURE OF CEREBRAL CORTICAL 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 Medium to make long-term Neuronal Culture Medium.
  2. Pre-warm the medium to 37°C.
  3. Replace the spent Neural Maturation Medium with pre-warmed long-term Neuronal Culture Medium.
  4. Perform a 50% medium change with the pre-warmed medium every other day from Day 15.
  5. There will be at least three half-medium changes of Neuronal Culture Medium in total.
  6. The neurons are assay-ready post Day 20