Product Information

Product Information

Catalog. No. Product Name Format Conc. Stock Conc. Storage on Arrival Thawing Storage Once Thawed
ax0078 Human iPSC-Derived Motor Neuron Progenitors (Healthy) ≥2 million cells/vial N/A Liquid Nitrogen Follow
protocol
N/A
ax0073 Human iPSC-Derived Motor Neuron Progenitors (Healthy – sibling (C9ORF72 extension)) ≥2 million cells/vial N/A Liquid Nitrogen Follow
protocol
N/A
ax0074 Human iPSC-Derived Motor Neuron Progenitors (ALS (C9ORF72 extension)) ≥2 million cells/vial N/A Liquid Nitrogen Follow
protocol
N/A
ax0071 Motor Neuron Recovery Medium 50 mL 1x -80ºC Overnight at
4ºC
Once thawed, aliquot and store at 4ºC for up to 1 week or for long term store at -80ºC.
ax0072 Motor Neuron Maintenance
Medium
200 mL 1x -80ºC Overnight at
4ºC
Once thawed, aliquot and store at 4ºC for up to 1 week or for long term store at -80ºC.
ax0041 SureBond 3 x 120 μL 1 mg/mL -80ºC Overnight at
4ºC
Store at 4ºC for up to 1 weeks
ax0053 SureBond-XF 1 mL 200x 4ºC N/A Refer to CoA
ax0044 Unlock (Passaging reagent) 25 mL 1x Aliquot and store at -80ºC for 6 months Overnight at 4ºC Once thawed, store aliquot at 4ºC for up to 1 week
ax139888 (5 μg) Recombinant Human Ciliary-Neurotrophic Factor (CNTF) 5 μg Lyophilized Powder N/A -20ºC N/A Reconstituted protein should be used immediately or stored in working aliquots at -20°C
ax139800 (10 μg) Recombinant Human Brain-Derived Neurotrophic Factor (BDNF) 10 μg Lyophilized Powder N/A -20ºC N/A Reconstituted protein should be used immediately or stored in working aliquots at -20°C

Additional Reagents

Product Name Supplier Product Code
Retinoic acid Sigma-Aldrich R2625
Y-27632 2HCl (ROCK inhibitor) Focus Biomolecules 10-2301
Poly-D-Lysine Sigma-Aldrich P7405
Compound E Abcam ab142164

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

Preparation of Media and Coating Reagents

Motor Neuron Recovery Medium

  • Upon receipt, aliquot and store Motor Neuron Recovery Medium at -80°C protected from light.
  • When ready to use, thaw an aliquot of Motor Neuron Recovery Medium overnight at 4°C in the dark.
  • Motor Neuron Recovery Medium requires supplementing with retinoic acid before use.
  • Prepare the retinoic acid by creating a stock concentration of 1 mM in DMSO.
  • Prepare Motor Neuron Recovery Medium by adding retinoic acid to create a final concentration of 0.1 μM e.g. 5 μL in 50 mL.
  • During thawing and passaging Motor Neuron Recovery Medium should be supplemented with Y-27632 2HCl to a final concentration of 10 μM.

Motor Neuron Maintenance Medium

  • Upon receipt, aliquot and store Motor Neuron Maintenance Medium at -80°C protected from light.
  • When ready to use, thaw an aliquot of Motor Neuron Maintenance Medium overnight at 4°C in the dark.
  • Motor Neuron Maintenance Medium requires supplementing with Compound E once thawed.
    • Add Compound E (ab142164) to create a final concentration of 0.2 μM
Growth factor Stock concentration Final concentration Volume to add in 50 mL
Recombinant Human Ciliary-Derived Neurotrophic Factor (CNTF) (ax139888) 5 μg/mL 10 ng/mL 100 μL
Recombinant Human Brain-Derived Neurotrophic Factor (BDNF) (ax139800) 10 μg/mL 5 ng/mL 25 μL
Retinoic acid 1 mM 0.5 μM 25 μL

Choosing a Coating Solution

For cells destined for passaging or enzymatic dissociation we recommend using SureBond with poly-d-lysine (PDL). For cells destined for end-use assay or long-term culture without enzymatic dissociation, we recommend using SureBond-XF with PDL.

SureBond + PDL

  • Calculate the total surface area that requires coating.
  • Add 120 μL per cm² of 1x PDL solution (0.1 mg/mL) to the culture vessel. Incubate for 1 hour at 37°C, remove the solution through aspiration, and thoroughly rinse the surface twice with sterile water. Allow the culture vessel to dry out for at least 2 hours at 37°C, 5% CO2 before proceeding onto SureBond coating.
  • Upon receipt, store SureBond at -80°C or below.
  • Thaw the SureBond coating solution overnight at 4°C.
  • Dilute the SureBond stock solution (1 mg/ml) in D-PBS (without calcium or magnesium) to a final concentration of 20 μg/mL e.g. 120 μL in 6 mL.
  • Coat the surface of your culture vessel with the SureBond 1X working solution. We recommend coating at a volume of 200 μL per cm 2.
  • Incubate overnight at 37 oC.
  • Remove the SureBond from the culture vessel prior to seeding. Do not wash the culture vessel after coating with SureBond.
  • Do not let the SureBond coating dry out before seeding the cells.

SureBond-XF + PDL

  • Calculate the total surface area that requires coating.
  • Add 120 μL per cm² of 1x PDL solution (0.1 mg/mL) to the culture vessel. Incubate for 1 hour at 37°C, remove the solution through aspiration, and thoroughly rinse the surface twice with sterile water. Allow the culture vessel to dry out for at least 2 hours at 37°C, 5% CO2 before proceeding onto SureBond-XF coating.
  • Upon receipt store SureBond-XF at 4°C.
  • Calculate the total surface area that requires coating.
  • Dilute the SureBond-XF stock solution (200x) in D-PBS (1x) (without calcium or magnesium) to make 1x working solution e.g. 30 μL in 6 mL.
  • Coat the surface of your culture vessel with 200 μL per cm² of SureBond-XF 1x working solution.
  • Incubate for at least 4 hours at 37°C.
  • Do not wash the culture vessel after coating with SureBond-XF.
  • Do not let the SureBond-XF coating dry out before plating the cells.

Culturing Human iPSC-derived Motor Neuron Progenitors

Thawing and Plating

  • Prepare culture vessels by coating with SureBond overnight prior to thawing cells. T-25 flasks or 60 mm dishes are recommended for initial plating of the cells.
  • Thaw an aliquot of Motor Neuron Recovery Medium overnight at 4°C. On the day of thawing the progenitors, prepare Motor Neuron Recovery Medium by adding Y-27632 2HCl to a final concentration of 10 μM and retinoic acid to a final concentration of 0.1 μM.
  • Pre-warm the medium and culture vessels to 37°C before use.
  • Transfer the vial of cells from storage by transporting the vial buried in dry ice.
  • Remove the vial from dry ice and transfer it to a 37°C water bath.
  • Quickly thaw the vial of cells in 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.
  • Take the vial of cells to a biological safety cabinet, spraying the vial and working surface of the cabinet thoroughly with 70% ethanol and wiping with a sterile paper towel before placing the vial in the cabinet.
  • Using a P1000 pipette, gently add the cell suspension dropwise into a 15 mL sterile conical tube containing 8 mL Motor Neuron Recovery Medium. Gently rinse the cryovial with 1 mL of fresh Motor Neuron Recovery Medium and transfer the medium with remaining cells into the 15 mL sterile conical tube.
  • Centrifuge cells at 200 x g for 5 minutes at room temperature.
  • Carefully aspirate and discard the supernatant using a 10 mL pipette.
  • Using a P1000 pipette, gently resuspend the cell pellet in 1 mL of Motor Neuron Recovery Medium until they are in a single cell suspension.
  • Perform a cell count. We recommend seeding the progenitors at densities ranging from 100,000-150,000 cells/cm 2.
  • Remove the SureBond coating solution and add an appropriate volume of medium to the culture vessel. Do not let the coating to dry out during the process.
  • Plate the resuspended cells dropwise and evenly.
  • Gently rock the culture vessel back and forth to ensure an even seeding density.
  • Incubate the cells at 37°C, 5% CO 2.
  • The day after plating, replace all of the medium with fresh pre-warmed, 37°C, Motor Neuron Recovery Medium (without Y-27632 2HCl).
  • Every 2 days replace all of the medium with fresh pre-warmed Motor Neuron Recovery Medium (without Y-27632 2HCl). Passage the motor neuron progenitors at day 5-7, depending on confluency.

Passaging and Differentiation

  • When the culture becomes 70% confluent, it is ready to be passaged.
  • The day before passaging, thaw an aliquot of Unlock and Motor Neuron Maintenance Medium overnight at 4ºC. Pre-coat culture vessels with SureBond and PDL as describe above.
  • **Note – At this stage, if the cells are destined for end-use assay and will not require further passaging, we recommend coating in SureBond-XF as outlined above. If the cells will require further passaging do NOT useSureBond-XF as it will prevent enzymatic dissociation.
  • Prepare Motor Neuron Maintenance Medium by adding Y-27632 2HCl to a final concentration of 10 μM, retinoic acid to a final concentration of 0.5 μM, BDNF to a final concentration of 5 ng/mL and CNTF to a final concentration of 10 ng/mL.
  • Pre-warm the medium and culture vessels to 37ºC before use.
  • Remove all spent medium from cell culture vessels.
  • Gently rinse the surface of the cell layer once with the D-PBS (without calcium or magnesium). We recommend using 2 mL D -PBS per 10 cm 2 of culture surface area.
  • Use Unlock to detach the cells: add 1 mL per 10cm 2 of culture surface area of 4oC Unlock. Evenly distribute it over the entire cell layer. Incubate the cells for 5 minutes at 37°C.
  • Use a P1000 pipette to transfer the cells dropwise into a 15 mL sterile conical tube and then gently add four volumes of pre-warmed Motor Neuron Maintenance Medium. (e.g. if 1 mL of Unlock is used, then add 4 mL of the medium to stop the
    reaction). Gently pipette up and down a few times to disperse the medium.
  • Centrifuge cells at 200 x g for 5 minutes at room temperature.
  • Carefully aspirate and discard the supernatant using a pipette.
  • Using a P1000 pipette, gently resuspend the cell pellet in 2 mL of Motor Neuron Maintenance Medium until they are in a single cell suspension.
  • Perform a cell count to ensure optimal seeding density.
  • Remove the coating solution and add an appropriate volume of medium to the culture vessel. Do not let the culture vessel dry out.
  • Plate the resuspended cells dropwise and evenly at densities ranging from 100,000-150,000 cells/cm 2.
  • The day after plating, replace the spent medium with fresh pre-warmed Motor Neuron Maintenance Medium (without Y-27632 2HCl).

Maturation and Maintenance of Human iPSC-derived Motor Neurons

  • To maintain a healthy culture, replace half the volume of medium every other day with fresh pre-warmed Motor Neuron Maintenance Medium  supplemented with CNTF (10 ng/mL), BDNF (5 ng/mL) and retinoic acid (0.5 μM).
  • The motor neurons should be maintained in the above medium for a minimum of two weeks to achieve strong expression of HB9, ChAT and ISL-1.
  • Repetitive firing of action potentials can be observed from week 6.
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