User Guide
Quick-Neuron™ Precursor - Human iPSC-derived Neural Precursor Cells
Catalog Numbers:

NP-mRNA-CW50065, NP-mRNA-CW10149, NP-mRNA-CW20300, NP-mRNA-CW50023,
NP-mRNA-CW70067, NP-mRNA-CW50025, NP-mRNA-CW50113, NP-mRNA-CW50114,
NP-mRNA-CW50115, NP-mRNA-CW50137, NP-mRNA-CW50147, NP-mRNA-CW60130,
NP-mRNA-CW60231, NP-mRNA-CW60236, NP-mRNA-CW20026, NP-mRNA-CW20090, orNP-mRNA-CW10130

Introduction
Elixirgen Scientific’s proprietary transcription factor-based stem cell differentiation method uses synthetic mRNAs toproduce highly pure populations of neurons without a genetic footprint. Quick-Neuron™ Precursor - Human iPSC-derived
Neural Precursor Cells continue to proliferate and express a variety of NPC markers such as nestin (NES), vimentin (VIM),
Hes Family BHLH Transcription Factor 5 (HES5), SRY-Box Transcription Factor 1 (SOX1), and SRY-Box Transcription
Factor 2 (SOX2). When handled and maintained according to the instructions in this user guide, NPCs are viable long-term
and are suitable for further characterization and differentiation.

Scale:

Quick-Neuron™ Precursor - Human iPSC-derived Neural Precursor Cells are available in twosizes: (Small) 1 million viable cryopreserved cells and (Large) 5 x 1 million viable cryopreserved
cells. The instructions outlined in this user guide are for seeding 1 million viable cells at
approximately 5 x 104 cells/cm2 into 2 wells of a 6-well plate (5 x 105 cells/well), 10 wells of a 24-wellplate (1 x 105 cells/well), or 62 wells of a 96-well plate (1.6 x 104 cells/well).

Related Products:Quick-Neuron™ Precursor - mRNA Kit, Catalog Number: NP-mRNA
Quick-Neuron™ Precursor - Maintenance Medium, Catalog Number: NP-MMContents
Upon receipt, immediately store the items at the indicated temperatures. Be especially careful to keep the frozen cells on
dry ice until placing them in liquid nitrogen and avoid any temperature fluctuation and slight thawing.
Contents

Amount (Small Size)

Amount (Large Size)

StorageThaw

Cryopreserved cells>1 million viable cells,(1 vial, 500 μl)

5 x >1 million viable cells,(5 vials, 5 x 500 μl)

Liquid nitrogen

37°C

Condition of UseThis product is for research use only. It is not approved for use in humans or for therapeutic or diagnostic use.

Technical Support
For technical support please refer to the FAQ on our website.
You may also contact us at cs@elixirgensci.com or call +1 (443) 869-5420 (M-F 9am-5pm EST).

Last revised: September 14, 2023

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1Required Consumables
ItemVendor

Catalog Number

6-well tissue-culture-treated polystyrene plate (e.g., Corning
Costar Flat Bottom Cell Culture Plates)

Fisher Scientific07-200-80

DMEM/F12

ThermoFisher

21331020Neurobasal Medium

ThermoFisher

21103049

Gibco™ B-27™ Supplement (50X), minus vitamin A

Fisher Scientific12-587-010

Gibco™ N-2 Supplement (100X)

Fisher Scientific17-502-001

Glutamax (100x)

ThermoFisher

35050061

Penicillin-Streptomycin

ThermoFisher

15140122

Recombinant Human FGF-basic (154 a.a.) (bFGF)

PeproTech

100-18B

Recombinant Human HB-EGF

PeproTech

100-47LDN 193189 dihydrochloride

R&D Systems

6053

SB 431542

R&D Systems

1614

Bovine Serum Albumin solution, 30% in DPBS

Sigma-Aldrich

A9576-50ML

TrypLE Select Enzyme (1X)

ThermoFisher

12563011

0.02% EDTA in DPBS

Sigma-Aldrich

E8008-100ML

0.01% Poly-L-Ornithine

Sigma-Aldrich

P4957-50ML

Extracellular Matrix such as
Laminin Mouse Protein, Natural, or
Geltrex Basement Membrane Matrix

ThermoFisher

23017015 orA15696-01

Phosphate-buffered saline (without Ca++ Mg++)*

ThermoFisher

20012050

Dimethyl sulfoxide (DMSO)

Sigma-Aldrich

D2650

AMSBIO

11890(Optional) STEM-CELLBANKER*** PBS should be used at room temperature unless otherwise specified.
** This is only required if you intend to cryopreserve the cells after differentiation.

Workflow* From Day 7, users may maintain NPCs in NPC Medium to repeat this cycle or cryopreserve cells for future use.

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2Media Preparation
0.002% Poly-L-Ornithine solution (ornithine)
1. Take 1 ml 0.01% Poly-L-Ornithine solution and mix it with 4 ml PBS.
2. The 0.002% Poly-L-Ornithine solution, hereafter referred to as ornithine, can be stored at 4°C for up to 2 weeks.
1 mg/ml laminin stock solution (laminin)
1. Thaw Laminin Mouse Protein, Natural and chill PBS at 4°C or on ice.
2. Mix the Laminin Mouse Protein, Natural and PBS to make the 1 mg/ml stock solution, hereafter referred to aslaminin.
○ Laminin concentration varies by lot, so use the number specified on the vial or CoA when making yourcalculations.
3. Make aliquots of a convenient volume (e.g., 35 μl) and store at -20°C.
0.5X TrypLE Select with EDTA (Solution D1)
1. Mix 1.5 ml TrypLE Select Enzyme (1X) with 1.5 ml 0.02% EDTA in DPBS.
2. This mixture, hereafter referred to as Solution D1, can be stored at 4°C for 2 weeks.
10 μg/ml bFGF stock solution
1.
2.
3.
4.

Prepare 1 mg/ml bFGF by following the vendor’s instructions.
Further dilute with sterile 0.1% BSA prepared with PBS to 10 μg/ml bFGF stock solution.
Make aliquots of a convenient volume (e.g., 100 μl).
This solution, hereafter referred to as 10 μg/ml bFGF, can be stored at -80°C.

100 μg/ml HB-EGF stock solution
1.
2.
3.
4.

Prepare 1 mg/ml EGF by following the vendor’s instructions.
Further dilute with sterile 0.1% BSA prepared with PBS to 100 μg/ml EGF stock solution.
Make aliquots of a convenient volume (e.g., 20 μl).
This solution, hereafter referred to as 100 μg/ml HB-EGF, can be stored at -80°C.

10 mM LDN-193189 stock solution
1. Dissolve 10 mg LDN-193189 in 1.99 ml DMSO.
2. Make aliquots of a convenient volume (e.g., 100 μl).
3. This solution, hereafter referred to as 10mM LDN-193189, can be stored at -20°C.
1 mM LDN-193189 stock solution
1. Dilute 10 μl 10mM LDN-193189 in 90 μl DMSO.
2. Make aliquots of a convenient volume (e.g., 10 μl).
3. This solution, hereafter referred to as 1 mM LDN-193189, can be stored at -20°C.
10 mM SB 431542 (1,000x) stock solution
1. Dissolve 10 mg SB 431542 in 2.38 ml DMSO.
2. Make aliquots of a convenient volume (e.g., 100 μl).
3. This solution, hereafter referred to as 10mM SB 431542, can be stored at -20°C.
NPC Medium
1.

Prepare NPC Medium (A) using the reagents listed in the table below. The volume below is sufficient for use fromthawing cells to a day before first passaging at Day 7.

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○○
○
○

Thaw N2 supplement (100x), B-27 Supplement (50x), 10 µg/ml bFGF, and 100 µg/ml HB-EGF on ice for
20-30 minutes.
Warm all other reagents at room temperature for 20-30 minutes.
Briefly spin down all tubes before use.
Store NPC Medium for up to 2 weeks at 4°C. The leftover reagents can be saved for other uses.
Reagents

VolumeDMEM/F1217.3 ml

Neurobasal

17.3 ml

200 mM GlutaMAX

180 μlPenicillin-Streptomycin (10000 units/ml; 100x)

360 μlN2 supplement (100x)

360 μl

B-27 Supplement (50X)

720 μl

10 µg/ml bFGF

72 μl

100 µg/ml HB-EGF

7.2 μl

1 mM LDN-193189

2.2 μl10 mM SB431542 (1,000x)

22 μl

NPC Medium (A)1.

Prepare NPC Medium (A) using the reagents listed in the table below.
○ Warm/thaw all reagents at room temperature for 20-30 minutes.
○ Briefly spin down all tubes before use.
○ IMPORTANT! Prepare NPC Medium (A) the day of thawing and plating the cells.
Reagents

VolumeNPC Medium

18 ml

1 mM LDN-193189

1.8 μl10 mM SB431542 (1,000x)

18 μl

Day 0

5-6 hours

Plate Preparation
IMPORTANT! Cells can be plated in 6-well, 24-well, and 96-well plates depending on the desired format. Refer to the table
at the bottom of this page for the recommended volumes per well.
1.
2.
3.
4.

Vortex ornithine briefly and centrifuge it at a maximum speed for 1 minute.
Add ornithine to each well in the volume specified in the table.
Incubate the plate at 37°C, 5% CO2 for at least 2 hours (or at 4°C overnight one day before plating).
Thaw laminin and chill PBS on ice for 20-30 minutes in the volumes calculated in the table. Add 1/100 volume oflaminin to chilled PBS. Mix well.
○ All PBS washes should be done dropwise and with room temperature PBS. Chilled PBS is only for thecoating step.
5. Aspirate the supernatant from each well and add PBS in the volume specified in the table.
6. Repeat Step 5.
7. Aspirate PBS from each well and add diluted laminin in the volume specified in the table.
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8. Incubate the plate at 37°C, 5% CO2 for at least 2 hours.
9. While the plate is incubating, prepare Medium iN(S1P) using the volume of Medium N(S1P) indicated in the tablecalculated for the number of wells in use plus 1.1 ml for resuspension (i.e., 2 wells of a 6-well plate needs 3.3 ml
Medium N(S1P) (1.1 x 2 + 1.1), 10 wells of a 24-well plate needs 4.4 ml Medium N(S1P) (0.33 x 10 + 1.1) for thisstep).
○ Thaw iROCK at room temperature for 20-30 minutes.
○ Add 1/1000 of iRock to Medium N(S1P).
○ Keep the rest of Medium N(S1P) at 4°C for later use.
10. After the laminin incubation, aspirate most, but not all, of the supernatant and add PBS in the volume specified inthe table. Add the PBS dropwise to each well.
11. Pipet out most of the PBS, but not all, from each well.
12. Repeat Steps 10-11.
13. Add Medium iN(S1P) to each well in the volume specified in the table.
14. Incubate the plate at 37°C, 5% CO2 until cells are ready for plating.
Recommended volumes per well for different plate formats.
Recommended volume per wellReagents

Corresponding
steps

6-well plate24-well plate

96-well plate

Ornithine

1, 2

1.5 ml300 µl

50 µl

PBS for laminin dilution

41.65 ml

330 µl55 µl

5, 6, 10, 112 ml

500 µl100 µl

Diluted laminin

71.5 ml

300 µl

50 µl

Medium N(S1P) for Medium iN(S1P)

91.1 ml

330 µl55 µl

Medium iN(S1P)

13500 µl

200 µl33 µl

PBSThawing Cells
1. Warm Medium N at room temperature for 20-30 minutes.
2. Take out the vial of frozen cells from the liquid nitrogen storage tank.
3. Incubate the cryovial in a water bath set at 37°C (do not submerge the cap) until most of the content is thawed but asmall ice crystal remains (~2 min).
4. Wipe the vial with a dry paper towel. Spray the vial with 70% ethanol and place it inside a biosafety cabinet.
5. Transfer 4.5 ml room temperature Medium N to a new 15 ml conical tube.
6. Set a P1000 pipette to 1 ml but take approximately 500 μl Medium N from the 15 ml conical and add it to the cryovial
dropwise at 1 drop per 1-2 seconds.
○ IMPORTANT! Use the same pipette tip for Steps 6-10.
7. Gently pipet the cell suspension up and down once.
8. Gently transfer all of the cell suspension to the 15 ml conical tube prepared in Step 5.
9. Take 1 ml of the cell suspension from the conical tube and add it to the original cryovial and pipet up and down 2-3
times and then transfer the whole contents back to the same 15 ml conical tube.
10. Mix the contents in the conical tube by gently pipetting cell suspension up and down 3 times.
11. Centrifuge the cell suspension in the 15 ml conical tube at 200 x g for 4 minutes.
12. Use an aspirator to remove most of the supernatant from the conical tube, leaving a small volume of thesupernatant (<50 μl) to cover the pellet.
13. Tap the side of the conical tube up to 10 times to break up the cell pellet.
14. Add 1 ml room temperature Medium iN(S1P) to the conical tube using a P1000 pipettor and pipet up and down nomore than 2-3 times.

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5Plating Cells
1.

Count the cells to determine the volume of cell suspension needed for the chosen number of wells and include a
10% buffer for cell number and volume (e.g., for a 24-well plate scenario, a total of 1.1 x 106 cells to plate 1 x 105
cells in each of the 10 wells). If the volume of the cell suspension needs to be adjusted, centrifuge the required
volume of cell suspension at 200 x g for 4 minutes, remove the supernatant, and resuspend the pellet with MediumiN(S1P) to reach the multiplied volume of cell suspension with the number of wells.
2. Add cell suspension to the center of each well. Since each well already has Medium iN(S1P), the total volume of themedium in each well is indicated in the table below.
3. Incubate at 37°C, 5% CO2 overnight.
Recommended amounts
6-well plate24-well plate

96-well plate5 x 105 cells

1 x 105 cells1.6 x 104 cells

Required total volume of cell suspension● (Volume of cell suspension/well) + 10% buffer

550 µl110 µl19 µlVolume of cell suspension distributed/well

500 µl100 µl

17 µl

Total volume/well● Medium iN(S1P) + cell suspension

1 ml

300 µl

50 µl

Viable cells/well

Day 1< 1 hour

Maintenance
1. Warm Medium N(S1P) at room temperature for 30 minutes.
2. Pipet out the old medium from each well and add PBS to each well along its wall according to the table below.
○ IMPORTANT! To avoid lifting cells, leave a small volume of the old medium (i.e., just enough to cover thesurface of the well). PBS should be applied along the wall of the well very slowly.
Required volume per wellPBS

6-well plate24-well plate

96-well plate

2 ml

500 µl100 µl3. Pipet out the PBS from each well and add Medium N(S1P) to each well along its wall according to the table below.
Required volume per well
Medium N(S1P)

6-well plate24-well plate

96-well plate

2 ml

500 µl100 µl4. Incubate at 37°C, 5% CO2 for 2-3 days.

Day 3 or 4

< 1 hour

Maintenance
1. Warm Medium N(S1P) at room temperature for 20-30 minutes.
2. Pipet out half the old medium from each well and add Medium N(S1P) according to the table below.
Required volume per well

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6-well plate24-well plate

96-well plate6

Medium N(S1P)

1 ml

400 µl75 µl

3. Incubate at 37°C, 5% CO2 for 2-3 days.

Day 6< 1 hour

Assay or Continuous Maturation●●

Differentiated neurons can be observed on Day 1. For more mature neurons, we recommend culturing cells until
Day 6. From Day 6, users may maintain differentiated neurons in the medium best suited for their needs, though werecommend Quick-Neuron™ Sensory Maintenance Medium, Catalog Number: SS-MM
Differentiation into sensory neurons after using Quick-Neuron™ Sensory - Human iPSC-derived Neurons can beconfirmed with the sensory markers, PRPH, ISL1, and BRN3A.

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Appendix

Figure 1. Representative phase contrast images of Quick-Neuron™ Sensory - Human iPSC-derived Neurons cell cultures on days 1, 2, 6and 7 post-thaw (scale bar = 100 μm).

Figure 2. Immunofluorescent staining of Quick-Neuron™ Sensory - mRNA- CW Kit cell cultures shows typical neurite growth andexpression of PRPH, TUBB3, BRN3A, and ISL1 on day 10 post-differentiation (scale bar = 100 μm). Staining conditions: Peripherin
Rabbit polyclonal antibody (ThermoFisher, Catalog number: PA110018, 1:2000 dilution) was used in combination with a secondary
antibody (Invitrogen, Catalog number: A32731 Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus
488, 1:500 dilution). Anti-β-III tubulin monoclonal antibody (R&D Systems, Catalog Number: MAB1195, 1:250 dilution) in combination
with a secondary antibody (Invitrogen, Catalog Number: A11032 Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary
Antibody, Alexa Fluor 594, 1:500 dilution). BRN3A(14A6):mouse antibody (Santa Cruz Biotechnology, Catalog number:sc-8429 ,1:200dilution) in combination with a secondary antibody (Invitrogen, Catalog Number: A11032 Goat anti-Mouse IgG (H+L) HighlyCross-Adsorbed Secondary Antibody, Alexa Fluor 594, 1:500 dilution). ISL1 (DSHB, Catalog number: 39.4D5, 1:200 dilution) antibody
in combination with a secondary antibody (Invitrogen, Catalog Number: A11032 Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed
Secondary Antibody, Alexa Fluor 594, 1:500 dilution). Nuclei were counterstained with Hoechst 33342.

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