Using this protocol iPSC-Derived Neural Stem Cells are plated into the final assay format and then spontaneously differentiated, using Neural Maintenance-XF Medium, from iPSC-Derived Neural Stem Cells to produce a mixed population of neuronal and glial cells.
|Cat. No. Product Name||Format||Stock Conc.||Storage on Arrival||Thawing Instructions||Storage Once Thawed|
|Axol iPSC-derived NSCs||million cells/vial||N/A||Vapour phase Nitrogen||Follow protocol||N/A|
|Aliquot & store|
|ax0032-Neural||at -80°C for up||Once thawed,|
|500 Maintenance- XF Medium||500ml||1x||to 6 months. Keep in the||Overnight at 4°C||store aliquot at 4°C for up to 1|
|ax0033 Neural Plating Medium||30ml||1x||-80°C||Overnight at 4°C||Must be used once thawed immediately|
|ax0053 Surebond-XF||1ml||200x||4°C||N/A||Store at 4°C for up to 1 month|
|Product Name||Supplier||Product Code|
|Y-27632 2HCl (ROCK inhibitor)||Focus Biomolecules||10-2301|
Important! Axol Neural Cell Culture Media DOES NOT contain antibiotics or antifungal agents. Axol bioscience does not recommend the use of antimicrobial agents such as penicillin, streptomycin and amphotericin. Antimicrobial agents should not be necessary if proper aseptic technique is adopted.
Preparation of Reagents
Neural Plating Medium
- Upon receipt, store Neural Plating Medium 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 2HCl to a final concentration of 10 μM before use.
Neural Maintenance-XF Medium
- Upon receipt aliquot and store Neural Maintenance -XF Medium at or below – 80°C protected from light.
- When ready to use, thaw an aliquot of Neural Maintenance-XF Medium overnight at 4°C in the dark.
- A thawed aliquot of Neural Maintenance-XF Medium can be stored at 4°C for 1 week, protected from light.
- Neural Maintenance-XF Medium is fully supplemented and ready to use.
Preparation of Coating Reagents and Coating Culture Vessel
SureBond-XF +Poly-D-Lysine (PDL) Coating Solution
- Add 10mls of sterile tissue grade water to 5 mg of poly-D-lysine.
- Add 300 μL per cm2 of 1x PDL solution (0.5 mg/mL) to the culture vessel. Incubate for 10 mins at 37 o C, remove the solution through aspiration and thoroughly rinse the surface twice with sterile water.
- Allow the surface to dry for at least two hours before proceeding onto SureBond-XF coating.
- 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 iPSC-derived Neural Stem Cells
- Coat culture vessels with substrate(s) as described above.
- On the day of thawing the cells prepare Neural Plating-XF Medium as described above.
- Aliquot required amount of medium into a 50 mL sterile conical tube and pre-warm to 37°C.
- Store the remaining medium at 4°C.
- To thaw the cells, transfer the vial of cells from nitrogen storage with the vial buried in dry ice.
- 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.
- Do not shake the vial during thawing.
- Spray the vial with 70% ethanol and wipe it down with a sterile paper towel before placing it in the cell culture hood.
- Once thawed completely, use a P1000 pipette to transfer the cells into a 15 mL sterile conical tube. Gently wash the cryovial with 1 mL of the medium and transfer this dropwise to the 15 mL sterile conical tube.
- Add 8 mL of the medium 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.
- Centrifuge cells at 200 x g for 5 minutes at room temperature.
- Aspirate the medium carefully and gently resuspend the cell pellet with 1 mL of the medium until they become mostly a single cell suspension.
- Perform a cell count and dilute the cell suspension to the required density.
Recommended Seeding Density: 100,000 cells per cm2
|Format||Viable Cells Required per well||Seeding Volume per well||Culture Volume per well|
|6-well Plate||906,000||1 mL||2 mL|
|24-well Plate||200,000||0.5 mL||1 mL|
|96-well Plate||32,000||50 μL||100 μL|
- Aspirate the spent coating solution from the pre-coated culture vessel and then immediately transfer the cells into the culture vessel.
- Maintain the cells at 37°C, 5% CO2 in a humidified incubator. The day of seeding the cells is Day 0.
- Monitor the cell survival and attachment the following day (Day 1).
- On Day 1 (18-24 hours after plating) perform a full media change replacing the volume of media with fresh, pre-warmed to 37°C Neural Maintenance-XF Medium.
- This will allow the cells to recover after thawing.
- Perform media change by pipetting media gently down the side of the culture vessel to avoid flushing cells with the media.
- To maintain a healthy neuronal culture, replace half the volume of medium with fresh, pre-warmed 37°C Neural Maintenance-XF Medium every 2 days.
- It is important that the medium is pre-warmed and that the medium changes are conducted gently by pipetting to the side of the culture vessel.
- Terminally differentiated neurons can now be maintained in culture until ready for experimental assays.