Neurosight -SUser GuideNeurosight® -S User Guide

Contents
1.

Product Information................................................................................................................... 2
Unpacking & Handling ........................................................................................................... 2
Components & Description ................................................................................................... 3
Safety Precaution & User Notice ........................................................................................... 52.

Introduction ................................................................................................................................ 63.

Preparing for Cell Culture .......................................................................................................... 7
Required Equipment and Consumables (Not Provided) .................................................... 7
Preparing Neurosight® -S Media ........................................................................................... 8
Preparing Cell Culture Surfaces ............................................................................................ 94.

Neurosight® -S Thawing and Plating ....................................................................................... 10
Thawing ................................................................................................................................. 10
Plating ................................................................................................................................... 12

5.

Neurosight® -S Maintenance .................................................................................................... 13

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1. Product Information
Unpacking & HandlingUpon receiving the shipment of Neurosight® -S, check whether all temperaturesensitive components are correctly frozen. If this is not the case, please contact our
support team immediately.
Immediately transfer each of the components to the appropriate storage conditions.
Please check the catalog number, lot number, and expiry date. The basal media
expiration date is the shortest (date of expiration on label) so experiments should beplanned accordingly.
The Neurosight® -S should be handled by technically qualified individuals complying
with good laboratory practices, applicable laboratory regulations, and the MSDS.
Following the User Guide herein is recommended for best results.
The Neurosight® -S is intended for research use only, not intended for any type of use inanimals or humans.

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Components & Description
COMPONENTSCAT#STORAGE ON ARRIVAL

Neurosight® -S Neurons
Cryopreserved, frozen vial
> 2 million cells> 4 million cells

N-001
N-002

Neurosight® -S Maintenance Media
Maintenance medium
50 ml
100 ml

NM-001
NM-002

Neurosight® -S MaintenanceSupplement

NS-001
NS-002NS-013NS-014

0.5 ml
1.0 ml0.15 ml0.25 ml

Liquid Nitrogen-20 °C-20 °C

Neurosight® -S EP MediaMEA* maintenance medium
50 ml
100 ml

NM-003
NM-004

Neurosight® -S EP Media
Supplement (50X)

NS-003NS-004

1.0 ml2.0 ml-20 °C-20 °C

Neurosight® -S Plating MediaMEA* Plating medium
20 ml

NM-010-20 °C

Neurosight® -S User Guide1
Certificate of Analysis (CoA)MSDS2
1 Also available online at www.nexel.co.kr
2 Enclosed with shipping documents.

Should any of the above components be missing from your shipment, please contact
us at NEXEL Co., Ltd. or the distributor in your country upon which our support teamwill provide the necessary assistance.

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Neurosight® -S Neurons
Cell Type

Human Induced Pluripotent Stem Cell (hiPSC) derived Neurons

Cell Line of Origin

hiPSC cell line reprogrammed from commercially available normal
donor fibroblast cell line

Quality Control

Please refer to the CoA for lot-specific information.
Virus clearance & STR analysis data is available upon request.

Neurosight® -S Media & Media Supplements
The Neurosight® -S Media & Supplements need to be combined to make the
Neurosight® -S Maintenance Media, after which it should be used within 1 month. DO
NOT FREEZE the Neurosight® -S Maintenance Media, but aliquot into smaller quantities
for best results.
The above also applies for Neurosight® -S EP Media and Supplements, and Plating
Media and Supplements.
The Neurosight® -S Maintenance Media are serum-free. For additional information on
the composition, please contact our technical support team.
The Neurosight® -S Maintenance Media are antibiotic and antifungal free as they are
not necessary if proper conditions are kept. NEXEL does not recommend the use ofsuch agents for accurate results, but they should be used if aseptic cell culture
conditions are not possible.

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Safety Precaution & User Notice

Biosafety Level: 1
For research use only, not intended for any type of use in animal or humans. Appropriate
safety procedures should always be used with this material. Please refer to the MSDS for
detailed instructions.

User Notice & Restrictions:User may use the Product (Neurosight® -S) for internal research including but notlimited to screening potential drug compounds for efficacy and safety, and for theprovision of such services to third parties. No other right is granted to User whether
expressly, by implication, by estoppel or otherwise. In particular, the purchase of the
Product does not include nor carry any right or license to use, develop or otherwise
exploit the Product commercially, and no rights are conveyed to User to use theProduct for any other purpose.
User agrees to use the Product in compliance with all applicable statutes andregulations, but not to use the Product for any administration or application tohumans. Moreover, User agrees not to use the Product in human subjects for humanclinical use for therapeutic, diagnostic or prophylactic purposes, or in animals forveterinary use for therapeutic, diagnostic or prophylactic purposes, including but notlimited to clinical applications, cell therapy, transplantation, and/or regenerative
medicine without an appropriate license.
In the case that User transfers Product to a third party, User shall convey the User
Restrictions set forth herein to such third party.

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2. Introduction
NEXEL Co., Ltd. strives to provide high quality human neurons derived from induced
pluripotent stem (iPS) cells using optimized proprietary protocols. The Neurosight® -S is ahighly pure and electrophysiologically active population of cells, suitable for all types ofexperiments in the field of neuron research. As such, they are the perfect choice to test theadvance of science in tissue-specific research, toxicity screening, efficacy testing, and drug
discovery.
This User Guide will help you seed the Neurosight® -S at the appropriate densities to create
mature neuronal networks comprised of functional neurons appropriate for a variety of
applications related to the electrophysiological behavior such as calcium fluxes or MEA assays
and neurite outgrowth or degeneration. However, please keep in mind that the best individual
results will be obtained by close observation, care, and optimization from the user. Example
morphology of the Neurosight® -S can be found below as a reference (Figure 1).

Figure 1. Morphology of Neurosight® -S at day 14 of culture. Image on the left was taken
at 100X magnification, whereas the image on the right was taken at 200X magnification.

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3. Preparing for Cell Culture
Required Equipment and Consumables (Not Provided)
ITEM
Coating Material
Poly-D-Lysine
LamininCAT#

VENDOR

A3890401
354232

Gibco
Corning

Typical Cell Culture Equipment
Liquid Nitrogen Storage Tank
37 °C Water Bath
Tabletop Centrifuge
Biological Safety Cabinet with UV Lamp
Hemocytometer or Automated Cell Counter
Phase Contrast Microscope
Pipettes
Cell Culture Incubator

Typical Cell Culture Consumables
Centrifuge Tubes
Cell Culture Plates
Pipette Tips
Trypan Blue
Phosphate Buffered Saline (PBS)

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Preparing Neurosight® -S Media
1. Thaw the Neurosight® -S Media and Supplements by placing them at 4°C 24 hours prior touse.
2. In a biosafety cabinet, thaw and add the Neurosight® -S Maintenance Supplement to thethawed medium to make corresponding Media. Store at 4°C for up to 1 month after
addition of the supplement. DO NOT FREEZE Neurosight® -S Maintenance Media.
To avoid oxidation of the media due to air contact and repeated warming/opening,
it is recommended to aliquot the media into quantities enough for 2~3 media
changes.

MEDIA TYPE
Neurosight® -S Maintenance Media
Neurosight® -S EP Media
Neurosight® -S Plating MediaCOMPONENTS
Neurosight® -S Maintenance media
Neurosight® -S Maintenance Supplement
Neurosight® -S EP media
Neurosight® -S EP Supplement
Neurosight® -S Plating media

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Preparing Cell Culture Surfaces
1. Calculate the amount of coating media required using the following table as a reference.

CELL CULTURE
PLATE

6-well(9.6 cm2)

12-well(3.8 cm2)

24-well(1.9 cm2)

48-well(1.0 cm2)

96-well(0.33 cm2)

COATING VOLUME

1 ml

500 µ l300 µ l100 µ l50 µ l

MEA
48 well
Plate
50 µ l

2. Pipette appropriate amount of Poly-D-Lysine solution to each well.

※ Do not swirl the MEA plate
3. Incubate the plate at 37°C CO2 incubator overnight.
4. Next day, remove the Poly-D-Lysine solution in each well.
5. Rinse two times with sterile PBS. As for the MEA 48-well plate, let it completely dry in thebiosafety cabinet (over 2 hours).
6. Prepare the laminin coating solution to working concentrations immediately before use as
described in the following table. Both coating solutions can be kept at 4°C for a shortperiod of time but this is not recommended.

COATING TYPESTOCK CONCENTRATION

WORKING
CONCENTRATION

Laminin

Varies by bottle. Checklabel for concentration.

5 µ g/ml

7. Pipette the correct amount of coating solution to each well you intend to use. For MEA
plates, we recommend pipetting 5 µ l of laminin solution on top of the electrodes.
8. Gently swirl the plate and check whether all wells are completely covered.

※ For MEA plates, DO NOT swirl the plate.
9. Incubate at 37°C for at least an hour.

※ For MEA plates, incubating the laminin coating for 1 hour is critical. Please refer tothe “Neurosight® -S Application Protocol for the Axion Maestro MEA” for moredetail.

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4. Neurosight® -S Thawing and Plating
Thawing
The Neurosight® -S can be thawed using typical cell culture thawing protocols. Here, we
present NEXEL’s optimized protocol and recommend our users to follow the instructions tomaximize results. We strongly recommend thawing 1 vial at a time to minimize cell exposure
to liquid DMSO.

1. Calculate the amount of Neurosight® -S Maintenance Media required. For each vial, 10 mlof Maintenance Media is required for resuspending the vial. The amount of MaintenanceMedia required can be calculated by the number of wells; recommendations for different
cell culture plates can be found below.

CELL CULTURE
PLATE

6-well(9.6 cm2)

12-well(3.8 cm2)

24-well(1.9 cm2)

48-well(1.0 cm2)

96-well(0.33 cm2)

PLATING VOLUME

2 ml

1 ml

500 µ l300 µ l200 µ lMEA
48-wellplate
300 µ l2. Warm the Maintenance Media at Room Temperature (RT, 25 °C) for at least 30 mins. For
each vial to thaw, aliquot 8 ml of Maintenance Media in a 15 ml centrifuge tube.
3. Retrieve the Neurosight® -S vial(s) from the liquid nitrogen storage tank.
4. Submerge the vial(s) 2/3 in a 37 °C water bath so that the mouth of the vial does notcontact with the water. Constantly check how much has thawed and once ~20 % remain(~3 mins), spray the vial(s) with 70 % Et-OH, wipe and place it in your biosafety cabinet.
Ideally, the vial(s) should have completely thawed exactly when you start step 5.
5. Open the vial(s) and transfer the contents (~1 ml) using a 1 ml pipette to the aliquoted 8
ml of Maintenance Media dropwise while gently swirling the tube.
Dropwise pipetting while gently swirling the tube minimizes osmotic shock andmaximizes mixing, which ensures high viability. Drops will remain on the surface for~1 second and then drop towards the bottom of the tube (visible due to the DMSO
content). For dropwise pipetting, simply pipette slowly into the air ~1 cm above themedia surface. It should take approximately 1 minute per 1 ml.
6. Use 1 ml of Maintenance Media to gently rinse the emptied vial and transfer dropwise to
the centrifuge tube containing the cells from step 5 while gently swirling the tube.
7. Centrifuge the suspended cells at 180 x g for 3 minutes at room temperature.
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8. Carefully discard the supernatant. Be careful not to disturb the cell pellet at bottom of the
tube to avoid any cell loss.
9. Resuspend the cells gently using 1 ml of Maintenance Media and check the cellconcentration using a hematocytometer or cell counter and Trypan Blue. Immediately
move on to the Plating section.
Avoid rigorous pipetting of the cells to maximize viability. Single cell resuspensionof the Neurosight® -S during thawing should easily be achieved by gently pipetting 3~4
times.

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Plating
NEXEL recommends seeding the Neurosight® -S at a density of ~100,000 cells/cm2 for moststandard applications. Application specific protocols are available upon request. The best
results are obtained by the User’s own optimization, for which NEXEL will try to provide as
much assistance as possible.
1. Calculate the volume of Maintenance Media and cells required to match the correct
density for the culture platform of choice. Below is a table with cell numbers.

CELL CULTURE
PLATE

6-well(9.6 cm2)

12-well(3.8 cm2)

24-well(1.9 cm2)

48-well(1.0 cm2)

96-well(0.33 cm2)

MEA
48-well
Plate

PLATING VOLUME

2 ml

1 ml

500 µ l300 µ l200 µ l300 µ lCELL NUMBER

960,000

380,000

190,000

100,000

33,000100,000

Well area (cm2) can vary between different vendors, please check with yourproviders for exact calculations.

2. Combine the volumes as calculated above. Add laminin to a final concentration of 1 µ g/ml.
3. Remove the coating solution in the cell culture plates. Avoid drying out the coated wells asmuch as possible.
4. Gently mix by pipetting and evenly distribute the appropriate volumes of cells with
Maintenance Media.
5. Move the cell culture plate to the incubator, shake the plate in perpendicular directions toevenly distribute the cells for attachment.
6. The day after, perform the first media change with Maintenance Media (Neurosight® -S
Maintenance Step 1).

*For MEA plates, follow the plating procedure below.
1.
2.
3.
4.
5.

Calculate the number of cells required for seeding.
Centrifuge and dissolve in media, to a final concentration of 1.0 x 105 cells per 5 µ l
Carefully remove the laminin coating solution and immediately add 5 µ l of the cellsuspension to the coated area.
Incubate for at least 30 min at RT in the biosafety cabinet.
After incubation, carefully add 300 µ l of Maintenance Media.
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5. Neurosight® -S MaintenanceStarting 24 hours after plating the cells, the media needs to be changed every two to threedays. Ideally, the media should be changed at 48-hour intervals. When performing
electrophysiological assays on the cells, we recommend changing the media on the morningof the experiment to ensure there are enough nutrients for the cells and to deliver the desired
drug concentration.
1. Warm the correct volume of Neurosight® -S Maintenance Media volume (half of thevolume for plating) at room temperature for at least 30 mins.
2. Immediately before media change, add Laminin media to a final concentration of 1 µ g/ml.
3. Perform a half-media change with the newly warmed media in a biosafety cabinet. Pipette
softly onto the cell culture plate walls to avoid any damage to the cell culture.
Performing half-media changes is crucial for neuronal cultures. First, it preventseven the shortest contact with air which can damage neuronal cultures. If the cell is
exposed to air, cell death will be visible within a few days. Second, it prevents removalof cytokines secreted by the cells, which results in higher quality cell cultures. An
example of half-media change is: for 1 ml of media in the well, remove ~400 μl andadd 500 μl.
While performing half-media changes, it is critical to keep the plate flat on asurface. Tilting the plate during half-media changes can cause the cells to shift
position and potentially induce aggregation towards the tilted direction.
For MEA plates, replace a full volume of Plating Media with EP Media 24 hours after
plating. To remove the spent media, aspirate the media using a pipette without tilting
the plate. Leave a small amount of media so that the cells do not come in contact withair. Then gently add appropriate amount of pre-warmed EP Media. After the initialfull-media change, perform half-media changes on 48-hour interval.
4. Place the plate back in the incubator.
5. Repeat 1 to 3 every 2~3 days.

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We recommend performing any planned assays with the Neurosight® -S from Day 14 onwards.
Long-term culture over 28 days can lead to aggregation or cell detachment, we recommend
careful and constant observation of the cell culture. Aggregation on its own is not always
detrimental to experiment results, particularly in electrophysiological assays. We do notrecommend subculture/passaging of the neuronal cell culture as mature neurons are veryfragile and neurite destruction may lead to cell death.

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