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Astrocytes user guide

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Updates to protocol:

Part Change Rationale Previous version New version
Product ax0665 to be replaced by ax0704 New product code ax0704 added New starting line needed (CENSOi004E) From alternative line (NCRM5) Includes ax0704 Astrocytes
Coating cell culture ware Included option to use hESC-qualified Matrigel Expands utility for users PDL + Surebond-XF only Includes additional reference to hESC Matrigel
Thawing Astrocytes Changed to add cell suspension dropwise to Align methodology with other cell Media was added dropwise to cell suspension Changed to add cell suspension dropwise to
media types media

Product Information

Catalog.

No.

 Product Name Format Stock Conc. Storage on Arrival Storage Once Thawed
ax0665 ax0704 axoCells Human iPSC-Derived Astrocytes Frozen Vial, >1 x

106 cells

 N/A -150°C / liquid nitrogen 37°C
ax0047 FGF2 100mg N/A -20°C Reconstituted growth factor in 0.05% HSA can be stored in working aliquots at -20°C for

up to 3 months
ax0053 Surebond-XF 1mL 200x 4°C N/A
Additional Reagents Required
Product Name Supplier Product Code
N21-Max R&D Systems AR008
FBS, One ShotTM  

 

ThermoFisher

 A3160401
Neurobasal™-A Media 12349015
GlutaMAX™ 35050061
Poly-D-Lysine (PDL) A3890401
hESC-qualified Matrigel 354277
Heregulin Peprotech 100-03
Human Serum Albumin (HSA) Sigma-Aldrich A9731

Lot-specific information such as specifications and quality control details are stated in the Certificate of Analysis. Expiry dates for Axol- supplied components are stated on the label. Consult the manufacturer’s guidelines for expiry dates of any additional reagents.

IMPORTANT!

Axol 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.

Preparation of Media

Astrocyte Media (100mL)

Components Volume Stock Concentration Final Concentration
Neurobasal™-A Medium 95mL N/A N/A
N21-Max 2mL 50 X 1 X
FBS, One ShotTM 2mL N/A N/A
GlutaMAX™ 1mL 100 X 1 X
Heregulin- β1 100µL 10µg/mL 10ng/mL
FGF2 80µL 10µg/mL 8ng/mL

Prepare all above chemicals and growth factor in a class II biosafety cabinet under sterile conditions.

Preparation of 0.05% Human Serum Albumin in D-PBS

  • Make a 05% stock concentration of HSA D-PBS by diluting 5mg HSA in 10mL D-PBS.
  • Aliquot into appropriate working
  • Aliquots can be stored long term at -80oC for up to 1 year and 2 months at 4oC.

Preparation of Heregulin-β1

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

Preparation of FGF2

  • Make a working stock at 10µg/mL by reconstituting 100µg of the growth factor in 10mL HSA D-PBS.
  • Aliquot into appropriate working volumes (recommended at 50 ~ 100µL).
  • Aliquots should be made into sterile pre-labelled
  • Aliquots can be stored at -20°C for up to 3

Coating Cell Culture Ware

A variety of extra cellular matrices can be used with the cells, i.e. PEI, Polyornithine, Poly-D-lysine, Laminin, Matrigel. We would recommend using either PDL + SureBond-XF or hESC-qualified Matrigel for axoCells™ astrocytes.

PDL

  • Dilute the liquid poly-D-lysine 1:1 in sterile water to create a 05mg/mL working solution (0.5x).
  • Add 300mL per cm2 of 0.5x PDL solution (05mg/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

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
  • Incubate for at least 4 hours at 37°C.
  • Remove the SureBond-XF from the culture vessel prior to plating of Do not wash the culture vessel after coating with SureBond-XF.
  • Do not let the SureBond-XF coating dry out before plating the

Matrigel

  • Thaw hESC-qualified Matrigel overnight at 2–8°C and keep on cold metal beads at all times during preparation and handling.
  • Dilute the hESC-qualified Matrigel in cold DMEM/F-12 to the required working concentration according to the manufacturer’s instructions.
  • Coat the culture surface with sufficient diluted Matrigel solution to completely cover the growth area (e.g., 100µL per well of a 96-well plate).
  • Incubate the coated culture vessel for at least 45 minutes at 37°C or overnight at 2–8°C.
  • Remove excess Matrigel solution immediately prior to cell
  • Rinse residual Matrigel from the wells with PBS before seeding

Thawing and Plating axoCells Human iPSC-Derived Astrocytes

  1. Coat culture vessels with the substrates as described
  2. On the day of thawing the cells prepare Astrocyte Media as per page
  3. Aliquot the media into a 50mL sterile conical tube and pre-warm to 37°C. Store the remaining medium at 4°C.
  4. Prepare a 15mL sterile conical tube with 8mL warm Astrocyte Media.
  5. To thaw the cells, transfer the vial of cells from nitrogen storage with the vial buried in dry ice.
  6. 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.
  7. Do not shake the vial during
  8. Spray the vial with 70% ethanol and wipe it down with a sterile paper towel before placing it in the cell culture hood.
  9. Once thawed completely, use a P1000 pipette to transfer the cells into the 15mL sterile conical tube dropwise. Gently wash the cryovial with 1mL of the medium and transfer this to the 15mL sterile conical tube.

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

  1. Centrifuge cells at 200 x g for 5 minutes at room
  2. Aspirate the media carefully and gently resuspend the cell pellet with 2mL of the

Astrocyte Media until they become mostly a single cell suspension.

  1. Perform a cell count and dilute the cell suspension to the required density. We recommend a cell seeding density of 25,000 to 48,000 cells / cm2 for monoculture or 10,000 / cm2 for co-culture experiments.
  2. Aspirate the spent SureBond-XF or Matrigel solution from the pre-coated culture vessel and then immediately transfer the cells into the culture vessel.
  3. Maintain the cells at 37°C, 5% CO2 in a humidified The day of seeding the cells is Day 0. Monitor the cell survival and attachment the following day (Day 1).
  4. Replace the culture medium on Day 2 with fresh, pre-warmed medium. The media 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.
  5. Medium changes can then be performed every 2 days with fresh, pre-warmed

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