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Sensory Neuron Progenitor User Guide

Product Information

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Catalog. No. Product Name Format Stock Conc. Storage on Arrival Thawing Instructions Storage Once Thawed
ax0055 axoCells Human iPSC-derived Sensory Neuron Progenitors ≥0.5 million cells/vial N/A Vapor Phase Nitrogen Follow protocol N/A
ax0033 Neural Plating medium 30 mL 1 X -80ºC Overnight at 4oC Must be used immediately once thawed
ax0060 Sensory Neuron Maintenance Medium 250 mL 1 X Aliquot & store at -80ºC for up to 6 months Overnight at 4oC Once thawed store aliquot at 4ºC for up to 1 week
ax0058 Sensory Maturation Maximizer Supplement 1 mL 100 X Aliquot & store at – 80ºC (See Vial for date of expiry) Thaw at room temperature in biosafety cabinet After re-freezing the aliquots must be used immediately once thawed
ax0053 SureBond-XF 1 mL 200 X 4°C N/A Stable at 4ºC for up to 6 months
ax139855 (10 μg) Recombinant Human Glial-Derived Neurotrophic Factor (GDNF) 10 μg Lyophilized Powder N/A -20ºC N/A Reconstituted protein should be used immediately or stored in working aliquots at -80°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 -80°C
ax139789 (20 μg) Recombinant Human Nerve Growth Factor (NGF) 20 μg Lyophilized Powder N/A -20ºC N/A Reconstituted protein should be used immediately or stored in working aliquots at -80°C
ax139811 (10 μg) Recombinant Human Neurotrophin-3 (NT-3) 10 μg Lyophilized Powder N/A -20ºC N/A Reconstituted protein should be used immediately or stored in working aliquots at -80°C

Additional Reagents

Product Name Supplier Product Code
Mitomycin C Sigma-Aldrich or Bio-Techne M4287 / 3258
Poly-D-Lysine Thermo-Fisher A3890401
Y-27632 2HCl (ROCK inhibitor) Focus Biomolecules 10-230
0.05% human or bovine serum albumin (HSA or BSA) Customer’s choice Customer’s choice

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 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 techniques are adopted.

Preparation of Media and Coating Reagents

Neural Plating Medium

  • Upon receipt, store Neural Plating Medium at -80°C.
  • When ready to use, thaw Neural Plating Medium overnight at 4°C.
  • Once thawed, Neural Plating Medium must be used and cannot be refrozen.
  • Neural Plating Medium requires supplementing with Y-27632 dihydrochloride (ROCK Inhibitor) to a final concentration of 10μM before use.
  • To aid cell adherence, we recommend adding SureBond-XF at a 1:400 dilution to Neural Plating Media. Once made the supplemented Neural Plating Media must be used immediately and cannot be refrozen.

Supplement Sensory Neuron Maintenance Medium with Growth Factors and Sensory Maturation Maximizer 

  • Upon receipt, aliquot and store Sensory Neuron Maintenance Medium at -80°C protected from light.
  • When ready to use, thaw an aliquot of Sensory Neuron Maintenance Medium overnight at 4°C in the dark.
  • Prepare a 0.05% solution of HSA or BSA in Dulbecco’s Phosphate-Buffered Saline (D-PBS) (without calcium or magnesium). If necessary, filter sterilize the HSA or BSA prior to use (0.2µm).
  • Prepare 10 µg/mL stock solutions of each growth factor by resuspending the lyophilized powder in 0.05% human or bovine serum albumin (HSA or BSA) solution. The growth factors can be aliquoted and stored at -80°C.
  • Thaw an aliquot of the Sensory Maturation Maximizer Supplement (Maximizer) at room temperature.
  • Prepare supplemented Sensory Neuron Maintenance Medium by adding the following:
Growth factor Stock concentration Final concentration Volume to add in 20 mL
Sensory Maturation Maximizer Supplement (ax0058) 100 X 1 X 200 μL
Recombinant Human Glial-Derived Neurotrophic Factor (GDNF) (ax139855) 10 μg/mL 25 ng/mL 50 μL
Recombinant Human Beta Nerve Growth Factor (β-NGF) (ax139789) 10 μg/mL 25 ng/mL 50 μL
Recombinant Human Brain-Derived Neurotrophic Factor (BDNF) (ax139800) 10 μg/mL 10 ng/mL 20 μL
Recombinant Human Neurotrophin-3 (NT-3) (ax139811) 10 μg/mL 10 ng/mL 20 μL

The growth factors and the Maximizer should be added fresh each time an aliquot of Sensory Neuron Maintenance Media is thawed. Do not refreeze supplemented Media.

N.B: For one full media change of a 96 well plate, approximately 10 mL of supplemented Sensory Neuron Maintenance Media will be required.

Mitomycin C

  • Prepare a 0.5 mg/mL stock concentration of mitomycin C by solubilizing 2 mg in 4mL of sterile water. Diluted mitomycin C in water is not stable and should be made fresh each time for use.  You can revert to the previous method and make up mitomycin C in DMSO (which can be stored for up to 4 weeks at -80°C) but performance in Axol’s hands is better when made up in water.

Supplement Sensory Neuron Maintenance Medium with Mitomycin C

  • Prepare medium containing 1.5 μg/mL of mitomycin C by adding 60 μL of the 0.5 mg/mL mitomycin C stock to 20 mL of growth factor and Maximizer supplemented Sensory Neuron Maintenance Medium.
  • This medium should then be filter sterilized with a 0.22 μM filter prior to use.
  • Prepare and use on the day required, do not store.

Coating Cell Culture Ware

Poly-D-Lysine (PDL) Coating Solution

  • Dilute the liquid poly-D-lysine 1:1 in sterile water to create a 0.05mg/ml working solution.
  • Add 312 µL per cm2 of working PDL solution (0.05 mg/mL) to the culture vessel. Incubate for 60 mins at 37°C, remove the solution through aspiration and thoroughly rinse the surface twice with D-PBS.
  • Remove D-PBS and allow the surface to dry for at least two hours before proceeding onto SureBond-XF coating.

SureBond-XF Coating Solution

This coating is used for differentiating the sensory neuron progenitors on plastic surface.

  • 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 312.5 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.

Differentiation of axoCells Sensory Neuron Progenitors

Thawing and Plating

  • Prepare coating of culture vessels as described above.
  • Thaw Neural Plating Medium overnight at 4°C.
  • Pre-warm Neural Plating Medium and culture vessels to 37°C before use.
  • 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 warm Neural Plating Medium and transfer this to the 15 mL sterile conical tube.
  • Add 8 mL of Neural Plating 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 in 2 mL of the plating medium until they become mostly a single cell suspension.
  • Perform a cell count. We recommend seeding the sensory neuron progenitors at 100,000 – 150,000 cells/cm2.
  • Remove 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.
  • 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% CO2.
  • The day after plating (Day 1), replace the medium with fresh pre-warmed Sensory Neuron Maintenance Medium supplemented with the growth factors and the Maximizer.

Growth Arrest and Purification

  • Two days after the medium change (Day 3), replace the spent medium with mitomycin C supplemented Sensory Neuron Maintenance Medium (prepared as described above).
  • Incubate the cells for 2 hours at 37ºC, 5% CO2.
  • After the incubation period, remove all the medium from the culture vessel and gently rinse the cells twice with warm Sensory Neuron Maintenance Media.
  • Add pre-warmed, Sensory Neuron Maintenance Medium supplemented with the growth factors and the Maximizer. To aid cell adherence, we recommend adding SureBond-XF at a 1:400 dilution to the Sensory Neuron Maintenance Media supplemented with the growth factors and the Maximizer.

Please note: The effect of mitomycin C treatment is not immediate. Non-neuronal cell elimination will not occur until 4 days post-treatment. The full effect will be apparent after 7 days.

Maturation of axoCells Sensory Neurons

  • On Day 5, perform a half media change with fresh, pre-warmed Sensory Neuron Maintenance Medium supplemented with the growth factors, Maximizer and SureBond-XF.
  • To aid cell adherence, we recommend adding SureBond-XF at a 1:400 dilution to the Sensory Neuron Maintenance Media supplemented with the growth factors and the Maximizer, for maturation up to Day 14.
  • To maintain a healthy culture, replace half the volume of the medium with fresh pre-warmed, supplemented Sensory Neuron Maintenance Media (with growth factors, Maximizer and SureBond-XF)  every 2 to 3 days ae.g. feed on Monday, Wednesday and Friday.

After 21 days of culturing, TrpV1 and Nav1.7 and Nav1.8 sodium channels should be expressed by the sensory neurons, and the cells are assay ready. We suggest that you carry out your experiments with the cells between day 21 and day 29.

Outline of the Cell Culture Preparation

DAY INSTRUCTION MEDIA SUPPLEMENTS
0 Thaw and plate the cells Neural Plating Medium ROCKi to 10µM final concentration and SureBond-XF
1 Full medium change Sensory Neuron Maintenance Medium Growth Factors, Maximizer and SureBond-XF                                                     
3 Two-hour treatment with mitomycin C at 37°C Sensory Neuron Maintenance Medium Growth Factors, Maximizer and mitomycin C (no SureBond-XF)   
3 Rinse Sensory Neuron Maintenance Medium                 
3 Full media change Sensory Neuron Maintenance Medium Growth Factors, Maximizer and mitomycin C and SureBond-XF                             
5 (then every 2 -3 days) Half media change Sensory Neuron Maintenance Medium Growth Factors, Maximizer and mitomycin C and SureBond-XF   

Assay time window: Day 21 ~ 29

For each vial of cells, approximately 10 mL of supplemented Sensory Neuron Maintenance Medium will be required for one full media change of a 96 well plate.