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Axol User Guide – Human iPSC-derived astrocytes

Product Information

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Cat. No. Product Name Format Storage on Arrival
ax0665 Human iPSC-derived Astrocytes > 1 million viable cells per vial Vapor phase nitrogen
ax0053 SureBond-XF (Coating Substrate) 1 mL 4oC
ax0047 FGF2 100 μg Reconstituted growth factor in 0.05% HSA can be stored in working aliquots at -80°C for up to 3 months

Required reagents from other suppliers

Cat. No. Product Name Supplier
12349015 NeurobasalTM- A ThermoFisher
A3160401 FBS, One Shot TM ThermoFisher
35050061 GlutaMAXTM ThermoFisher
AR008 N21-Max R&D Systems
A9731 Human Serum Albumin (HSA) Sigma-Aldrich
P7405 Poly-D-Lysine Sigma-Aldrich
100-03 Heregulin-b1 Peprotech

Preparation of Medium

Astrocyte Medium (100 mL) for mono-culture & co-culture with neurons

Components Volume Stock Conc. Final Conc.
NeurobasalTM-A Medium 95 mL N/A N/A
N21-Max2 mL50 X1 X 2ml 50 X 1 X
FBS, One ShotTM 2 mL N/A N/A
GlutaMAXTM1 mL100 X1 X 1ml 100 X 1 X
Heregulin-b1 100 μL 10 μg/mL 10 ng/mL

Preparation of Growth Factor Stock Solution

Preparation of Human Serum Albumin (HSA) D-PBS:

Human Serum Albumin (HSA, Sigma-Aldrich A9731)
D-PBS, no calcium, no magnesium (ThermoFisher, 14190-250)

  • Prepare a 0.05% stock concentration of HSA D-PBS by diluting 50 μg HSA in 100 ml D-PBS.
  • Aliquot into appropriate working volumes (recommended at 10 ml).
  • Aliquots can be stored long term at -80°C for up to 1 year. Preparation of Heregulin-b1 and FGF2:
  • Make a working stock at 10 μg/mL by reconstituting 10 μg of the growth factor in 1 mL HSA D-PBS
  • Aliquot into appropriate working volumes (recommended at 50 ~ 100 μL).
  • Aliquots should be made into sterile pre-labelled tubes.
  • Aliquots can be stored at -80°C for up to 3 months.

Preparation of Heregulin-b1 and FGF2:

  • Make a working stock at 10 μg/mL by reconstituting 10 μg of the growth factor in 1 mL HSA D-PBS
  • Aliquot into appropriate working volumes (recommended at 50 ~ 100 μL).
  • Aliquots should be made into sterile pre-labelled tubes.
  • Aliquots can be stored at -80°C for up to 3 months.

Coating culture vessels with substrates

Poly-D-Lysine

  • Add 50 mL of sterile tissue culture grade water to 5 mg of Poly-D-Lysine (Sigma-Aldrich, P7405), and aseptically coat the surface with 300 μL per cm2 of the solution. Incubate 5 minutes at room temperature. Remove the solution through aspiration and thoroughly rinse the surface twice with sterile water. Let dry for two hours at room temperature under sterile condition 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 cm 2 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.

Important! Axol Neural Cell Culture Media
DOES NOT contain antibiotics or antifungal agents. Axol 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

Thawing and plating ISPC-derived astrocytes

  • • Coat culture vessels with substrates as described above.
  • On the day of thawing the cells prepare Astrocyte Medium.
  • Aliquot the 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 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 2 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.
  • Aspirate the spent SureBond-XF solution from the pre-coated culture vessel and then immediately transfer the cells into the culture vessel.

Recommended cell seeding densities: 25,000 ~ 48,000 / cm 2 for monoculture or 10,000 / cm2 for neuronal co-culture experiments.

  • • 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).
  • Replace the culture medium on Day 2 with fresh, pre-warmed medium. The medium addition should be done gently by pointing the pipette tip  toward the wall of the cell culture vessel to avoid flushing the cells directly with medium.
  • Medium changes can then be performed every 2 days with fresh, pre-warmed medium.

Note: Axol human iPSC-derived astrocytes are assay-ready 2 day post thawing.
Prolonged culture of these cells is not necessary for functional maturation.

Co-culture

  • Axol’s human iPSC-derived astrocytes have been successfully grown in a variety of co-culture experiments with other neural cell types. These include iPSC-derived cerebral cortical excitatory (ax0025) & inhibitory neurons (ax0667) and iPSC-derived microglia (ax0666). In general, we recommend astrocyte : neuron ratio from 1:9 to 1:3 depending on your assay requirements (imaging or multi-electroarray assays).
  • For co-culture of multiple cell types, Axol can help with the development as we have devised a number of culture conditions to support a variety of complex culture environment.