Cardiosight -SUser Guide(Advanced media)

Cardiosight® -S User Guide(Advanced media)

Contents
1. Product Information............................................................................................................. 2
Unpacking & Handling ..................................................................................................... 2
Components & Description ............................................................................................. 3
Safety Precaution & User Notice ..................................................................................... 5
2. Introduction .......................................................................................................................... 6
3. Preparing for Cell Culture .................................................................................................... 7
Required Equipment and Consumables (Not Provided) .............................................. 7
Preparing Cardiosight® -S Advanced Media ................................................................... 8
Preparing Cell Culture Surfaces ...................................................................................... 94. Cardiosight® -S Thawing and Plating ................................................................................. 10Thawing ........................................................................................................................... 10Plating .............................................................................................................................. 12
5. Cardiosight® -S Maintenance ............................................................................................. 13

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Cardiosight® -S User Guide(Advanced media)

1. Product Information
Unpacking & HandlingUpon receiving the shipment of Cardiosight® -S, check whether all temperaturesensitive components are correctly frozen. If this is not the case, please contact oursupport 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 Cardiosight® -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 Cardiosight® -S is intended for research use only, not intended for any type of use
in animals or humans.

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Cardiosight® -S User Guide(Advanced media)
Components & Description
COMPONENTS

CAT#STORAGE ON ARRIVAL

Cardiosight® -S Cardiomyocytes
Cryopreserved, frozen vial
>2.5 million cells>5 million cells

C-001
C-002

Cardiosight® -S Advanced MediaAdvanced Plating medium
30 ml
45 ml
Advanced Maintenance medium
100 ml200 ml

CM-010A
CM-020A

Liquid Nitrogen

4°C

CM-001A
CM-002A

Cardiosight® -S Advanced PlatingSupplement (50X)
0.6 ml0.9 ml

CS-010A
CS-020A

1 ml
2 ml

CS-001A
CS-002A

Cardiosight® -S Advanced MaintenanceSupplement (100X)

-20°C

-20°C

Cardiosight® -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.

Cardiosight® -S Cardiomyocytes
Cell Type

Human Induced Pluripotent Stem Cell (hiPSC) derived
cardiomyocytes

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.

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Cardiosight® -S Advanced Media & Media Supplements
The Cardiosight® -S Advanced Medium & Supplements need to be combined to makethe Cardiosight® -S Advanced Plating or Maintenance Media, after which it should be
used within 1 month. DO NOT FREEZE the Cardiosight® -S Advanced Plating or
Maintenance Media, aliquot into smaller quantities for best results.
The Cardiosight® -S Advanced Plating or Maintenance Media are serum-free. For
additional information on the composition, please contact our technical support team.
The Cardiosight® -S Advanced Plating or Maintenance Media are antibiotic andantifungal free as they are not necessary if proper conditions are kept. NEXEL does notrecommend the use of such 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 (Cardiosight® -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 theProduct 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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Cardiosight® -S User Guide(Advanced media)

2. Introduction
NEXEL Co., Ltd. strives to provide high quality human cardiomyocytes derived from inducedpluripotent stem (iPS) cells using optimized proprietary protocols. The Cardiosight® -S is ahighly pure and electrophysiologically active population of cells, suitable for all types ofexperiments in the field of cardiomyocytes. Advanced Media is newly designed to enhance theelectrophysiological profiles of Cardiosight® -S, which adequately supports a robust maturation
as well as excitation-contraction coupling. Together with Advanced Media, Cardiosight® -S havebeen validated on multiple electrophysiological platforms, resulting in synchronous beatingwith adequate FPD, higher calcium influx, and stronger contraction. As such, they are theperfect choice to test the advance of science in tissue-specific research, toxicity screening,efficacy testing, and drug discovery.
This User Guide will help you seed the Cardiosight® -S at the appropriate densities to create
synchronous layers of cardiomyocytes appropriate for a variety of applications related to theelectrophysiological behavior assays. However, please keep in mind that the best individual
results will be obtained by close observation, care and optimization from the user.

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3. Preparing for Cell Culture
Required Equipment and Consumables (Not Provided)
ITEM
Coating Material
Matrigel, Basal Membrane
Fibronectin, Human

CAT#VENDOR

356235
F0895

CorningSigma

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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Cardiosight® -S User Guide(Advanced media)
Preparing Cardiosight® -S Advanced Media
1. Thaw the Cardiosight® -S Advanced Media and Supplements by placing them at 4°C 24
hours prior to use.
2. In a biosafety cabinet, thaw and add the Cardiosight® -S Advanced Plating (50X) orMaintenance Supplement (100X) to the thawed medium to make to corresponding Media.
Store at 4°C for up to 1 month after addition of the supplement. DO NOT FREEZE
Cardiosight® -S Advanced Plating or 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 TYPECOMPONENTS

Cardiosight® -S Advanced Plating
Media
Cardiosight® -S Advanced MaintenanceMediaCardiosight® -S Advanced Plating medium
Cardiosight® -S Advanced Plating Supplement
Cardiosight® -S Advanced MaintenancemediumCardiosight® -S Advanced MaintenanceSupplement

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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
COATING VOLUME

6-well(9.6 cm2)
1 ml

12-well(3.8 cm2)
500 µ l24-well(1.9 cm2)
300 µ l48-well(1.0 cm2)
100 µ l96-well(0.33 cm2)
50 µ l2. Prepare the coating media to working concentrations immediately before use as describedin the following table. Both coating solutions can be kept at 4°C for a short period of time
but this is not recommended.

COATING TYPESTOCK CONCENTRATION

WORKING
CONCENTRATION

MatrigelNA, 100X

1X

Fibronectin

1 mg/ml

50 µ l (1:20 dilution)

Fibronectin > Lamin > Matrigel
Fibronectin consistently gives the best results for Cardiosight® -S culture, but it is also
the most expensive. Laminin (50 μg/ml) is a compromise between cost and quality.
Whereas Matrigel is the most economic option at the cost of attachment quality. For
long-term and any electrophysiological platform cultures, we strongly suggest using
Fibronectin for consistent results.
3. Pipette the correct amount of coating solution to each well you intend to use.
4. Gently swirl the plate and check whether all the wells are completely covered.

5. Incubate at 37°C for at least an hour.

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4. Cardiosight® -S Thawing and PlatingThawing
The Cardiosight® -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 Cardiosight® -S Advanced Plating Media required. For each vial, 10ml of Advanced plating Media is required for resuspending the vial. The amount ofAdvanced Plating Media required can be calculated by the number of wells;
recommendations for different cell culture plates can be found below.

CELL CULTURE PLATE
PLATING VOLUME

6-well(9.6 cm2)
2 ml

12-well(3.8 cm2)
1 ml

24-well(1.9 cm2)
500 µ l48-well(1.0 cm2)
300 µ l96-well(0.33 cm2)
200 µ l2. Warm the Advanced Plating Media at Room Temperature (RT, 25°C) for at least 30 mins.
For each vial to thaw, aliquot 8 ml of Advanced Plating Media in a 15 ml centrifuge tube.
3. Retrieve the Cardiosight® -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 not comein contact 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 8ml of Advanced Plating 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 DMSOcontent). For dropwise pipetting, simply pipette slowly into the air ~1 cm above themedia surface. It should take approximately 1 min per 1 ml.
6. Use 1 ml of Advanced Plating 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.
9. Resuspend the cells gently using 1 ml of Advanced Plating 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 Cardiosight® -S during thawing should easily be achieved by gently pipetting
3~4 times.

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Cardiosight® -S User Guide(Advanced media)
Plating
NEXEL recommends seeding the Cardiosight® -S at a density of ~150,000 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 Advanced Plating 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)

PLATING VOLUME

2 ml

1 ml

500 µ l300 µ l200 µ lCELL NUMBER

1,440,000

570,000

285,000

150,000

50,000Well area (cm2) can vary between different vendors, please check with yourproviders for exact calculations.

2. Combine the volumes as calculated above.
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 withAdvanced Plating 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 Advanced Maintenance Media(Cardiosight® -S Maintenance Step 1).

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5. Cardiosight® -S MaintenanceStarting 24 hours after plating the cells, the media needs to be changed every two days.
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 Cardiosight® -S Advanced Maintenance Media volume (half ofthe volume for plating) at 37°C in a water bath or room temperature for at least 20 mins.
2. Perform a 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.
24 hours after plating, replace a full volume of Plating Media with MaintenanceMedia. 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 300 μl/well of pre-warmed Maintenance Media.
After the initial full-media change, perform half-media changes on 48-hour interval.
Performing half-media changes is crucial for cardiomyocyte cultures. Contact with
air can damage cardiomyocyte cultures, which results in cell detachment from theplate, preventing long-term culture. An example of half-media change is: for 1 ml ofmedia in the well, remove ~500 μl and add 550 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.
3. Place the plate back in the incubator.
4. Repeat 1 to 3 every 2 days.
We recommend performing any planned assays with the Cardiosight® -S from Day 7 onwards.
Long-term culture over 14 days can lead to aggregation or cell detachment, we recommend
careful observation of borders and use of fibronectin coating for best results.

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