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  • Cardiomyocytes in FLPR 384-well plate format
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Citations Cellartis hiPS-CM citation list
Home › Learning centers › Stem cell research › Application protocols › Cardiomyocytes in FLPR 384-well plate format

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Citations Cellartis hiPS-CM citation list
STEM CELL APPLICATION PROTOCOL

Cellartis cardiomyocytes in FLPR 384-well plate format

Cellartis cardiomyocytes are derived from human induced pluripotent stem cells and provide a promising physiologically-relevant, human model for preclinical safety evaluation and drug screening. The FLIPR Tetra High-Throughput Cellular Screening System is a real-time kinetic cellular assay screening system that allows for detection of intracellular Ca2+ flux and cardiac beating in a high-throughput format. Cellartis cardiomyocytes used in combination with this technology demonstrate the potential to predict cardiotoxic responses accurately and to screen compound efficacy.

Materials required Protocol

Materials required  

  • Cellartis Cardiomyocytes (from ChiPSC22) Kit (Takara Bio, Cat. # Y10075)
    • Cellartis Cardiomyocytes (from ChiPSC22)
    • Cellartis CM Thawing Base
    • Cellartis CM Culture Base
  • Fetal Bovine Serum (FBS) (Fisher Scientific, Cat. # 16140)
  • Y-27632
  • Fibronectin (Sigma-Aldrich, Cat. # F0895)
  • 384-well plate (Corning Costar, Cat. # 3712)
  • MicroClime lid (Labcyte, Cat.# LLS-0310)
  • PBS Dulbecco's with Ca2+ & Mg2+ (D-PBS +/+)
  • FLIPR Calcium 5 Assay Kit (Molecular Devices, Cat # R8185, R8186, or R8187)
  • FLIPR Tetra High-Throughput Cellular Screening System (Molecular Devices)
  • General cell culture equipment used in cell culture laboratory

Protocol  

NOTE: These procedures should be performed under aseptic conditions as much as possible.

A. Coating of the 384-well plate

  1. Dilute the required volume of Fibronectin in D-PBS +/+ to a final concentration of 50 µg/ml.
  2. Add the diluted Fibronectin solution into each well to be used. Use 18 µl/well.

    NOTE: To minimize edge effects, seed cells in the central 308 wells only. Fill the outside wells with the PBS and cover the plates with a MicroClime lid filled with PBS for optimal recordings.

  3. Incubate at 37°C for a minimum of 2.5 hr.
  4. Aspirate the Fibronectin solution from the cell culture plate just before use. 

B. Medium Preparation 

Preparing Cellartis CM Thawing Medium

  1. Thaw Cellartis CM Thawing Base.
  2. Decontaminate the external surface of all bottles with an appropriate disinfectant and place into the biological safety cabinet.
  3. Add 8 ml FBS per 32 ml Cellartis CM Thawing Base to make Cellartis CM Thawing Medium.
    • Cellartis CM Thawing Medium should be stored at 4°C and expires one month after the date of preparation.
    • Always discard any leftover warmed Cellartis CM Thawing Medium.

Preparing Cellartis CM Thawing Medium with Y-27632

  1. On the day of use, prepare Cellartis CM Thawing Medium with Y-27632 by adding Y-27632 to a final concentration of 10 μM to Cellartis CM Thawing Medium.
    • Cellartis CM Thawing Medium with Y-27632 should be used on the day of preparation.

Preparing Cellartis CM Culture Medium

  1. Thaw Cellartis CM Culture Base.
  2. Decontaminate the external surface of supplement and medium bottle with appropriate disinfectant and place into the biological safety cabinet.
  3. Add 10 ml FBS per 90 ml Cellartis CM Culture Base to make Cellartis CM Culture Medium.
    • Cellartis CM Culture Medium should be stored at 4°C and expires one month after the date of preparation.
    • Always discard any leftover warmed Cellartis CM Culture Medium.

C, Thawing and plating of Cellartis cardiomyocytes

NOTES:

  • It is recommended that not more than two to three vials are thawed at once.
  • For your protection, wear a protective face mask and protective gloves. Use forceps when handling a frozen vial. Never hold the vial in your hand as it may explode due to rapid temperature changes.
  1. Prepare the appropriate volume of Cellartis CM Thawing Medium with Y-27632 as described (see Section B above) and warm to room temperature (RT, 15–25°C).
  2. Transfer, as quickly as possible, the frozen vial from liquid nitrogen to a 37°C ± 1°C water bath using forceps.
  3. Thaw the cells by gently pushing the vial under the surface of the water without swirling the vial. Do not submerge the cap of the vial in the water bath as this could contaminate the cells.
  4. Take the vial out of the water bath as soon as the thawing is completed (approximately 3 min; the vial should still be cold on the outside).
  5. Wipe the vial with an appropriate disinfectant and place into the biological safety cabinet.
  6. As soon as possible, gently transfer the cell suspension into a sterile 50 ml tube using a pipette.
  7. Rinse the vial with 1 ml of Cellartis CM Thawing Medium with Y-27632 and carefully add it to the cell suspension dropwise.
  8. Add 8 ml of Cellartis CM Thawing Medium with Y-27632 dropwise. Gently swirl the tube a few times in between.
  9. Centrifuge the tube at 200g for 5 min at RT and remove the supernatant.
  10. Carefully resuspend the cell pellet with Cellartis CM Thawing Medium with Y-27632, using 6 ml medium per thawed vial.
  11. Count the cells and measure viability.
  12. Adjust the number of viable cells to 2.5 x 105 cells/ml with Cellartis CM Thawing Medium with Y-27632.

    NOTE: Aspirate the Fibronectin solution just before adding the cell suspension. Prepare one column at a time, since drying of the surface might result in crystallization of the Fibronectin and subsequent damage to the cells.

  13. Aspirate the Fibronectin solution from the first column.
  14. Carefully mix your cell suspension and pipet 36 μl per well (corresponding to 9 x 103 cells/well).
  15. Optional: If foaming of the medium occurs, spin the plate for 1 minute at 200g at room temperature. This will remove the majority of bubbles seen in the media. Spin another minute if bubbles remain.
  16. Place the plate in the incubator (37°C ± 1°C, 5% CO2, and >90% humidity).

D. Medium change day 1

It is recommended to wash the cells with fresh medium the day after thawing and plating

NOTE: Work very gently in order not to detach the cells.

Medium preparation

Prepare the appropriate volume of Cellartis CM Culture Medium as described in Section B above and warm to 37°C ± 1°C before use.

Medium change including washing the cells once

  1. Aspirate most of the medium per well column-by-column with a multichannel aspirator, taking care not to touch the bottom of the wells.
  2. Add 36 μl of fresh Cellartis CM Culture Medium.
  3. Aspirate the newly added medium as described in bullet 1 above.
  4. Add 50 μl of fresh Cellartis CM Culture Medium.
  5. Place the plate in the incubator (37°C ± 1°C, 5% CO2, and >90% humidity).

E. Medium change day 3 and onwards

It is recommended to perform medium changes every 2–3 day until analysis. Also, perform a medium change on the morning of the day of recording.

NOTE: Work very gently in order not to detach the cells.

Medium preparation

Prepare the appropriate volume of Cellartis CM Culture Medium as described in Section B above and warm to 37°C ± 1°C before use.

Medium change

  1. Aspirate most of the medium per well column-by-column with a multichannel aspirator, taking care not to touch the bottom of the well.
  2. Add 50 μl of fresh Cellartis CM Culture Medium.
  3. Place the plate in the incubator (37°C ± 1°C, 5% CO2, and >90% humidity).

NOTE: Cells are optimally recorded on day 10 post-thaw (in the afternoon to let cells recover after the morning medium change).

Related Products

Cat. # Product Size Price License Quantity Details
Y10075 Cellartis® Cardiomyocytes (from ChiPSC22) Kit 1 Kit $853.00

License Statement

ID Number  
C005 This product is manufactured and sold by Takara Bio Europe AB based on a license to Cellectis SA. The use of this product is strictly limited to purchaser’s own internal research, including basic research, drug discovery research up to and including IND-enabling preclinical toxicological studies or equivalents thereof, excluding any use in the T-cell field. Purchaser cannot have any right to use this product or its components in humans for any purposes including but not limited to diagnostics and/or therapeutics, or otherwise clinical trials. Purchase does not include any right to resell or transfer this product to a third party regardless of whether or not compensation is received. Purchasers wishing to use this product for purposes other than internal research use should contact us. No express or implied license is granted to the purchaser.
L67 This product is covered by one or more claims of the issued patents and pending patent applications: U.S. Patent Nos. 8,048,999, 8,058,065, 8,129,187, 8,211,697, 8,257,941, 8,278,104, 8,530,238, 8,900,871, 8,927,277, 8,951,801, 9,213,999, 9,404,124, 9,499,797, 9,677,141 and 9,714,433, and foreign counterparts thereof, including their divisions, continuations, extensions, substitutions, and those claiming priority therefrom or those claiming the same priorities therein.

This Product is manufactured and sold by Takara Bio Inc. and/or its affiliate(s) based on a patent license from iPS Academia Japan, Inc. No express or implied license is granted to the purchaser.

"Product" means the differentiated cells manufactured and sold by Takara Bio Inc.

"Internal research purpose" means the use of this Product as a tool of internal research for the sole benefit of the purchaser or purchaser’s contract research customer, (including screening of compounds, antibodies, proteins, peptides and others in developing pharmaceuticals (including diagnostic agents) for human and/or animal use), provided, however, that the following activities using this Product are strictly prohibited and expressly excluded from Internal research purposes:
1) Administration of this Product to humans.
2) Use of this Product for the diagnostic testing, prevention, or treatment of human and/or animal diseases.
3) Use of this Product in studies involving administration to humans for cell therapy, regenerative medicine etc.

Purchaser may use this Product for its own Internal research purposes.

Purchaser shall not use the Products for its commercial purposes.

Notwithstanding the foregoing, the purchaser of this Product may provide third parties with contract research services by using this Product only for such third party’s Internal research purposes.

Cellartis Cardiomyocytes (from ChiPSC22) are human cardiomyocytes derived from a human induced pluripotent stem (iPS) cell line. The cells are provided frozen with thawing and maintenance media bases.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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Cellartis Cardiomyocytes (from ChiPSC22) respond in a dose-dependent manner to hERG blocker E4031 as measured by MEA (MED64 AlphaMED MEA system)

Cellartis Cardiomyocytes (from ChiPSC22) respond in a dose-dependent manner to hERG blocker E4031 as measured by MEA (MED64 AlphaMED MEA system)
Cellartis Cardiomyocytes (from ChiPSC22) respond in a dose-dependent manner to hERG blocker E4031 as measured by MEA (MED64 AlphaMED MEA system). BL = baseline and EAD = early after depolarization.

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Cellartis Cardiomyocytes (from ChiPSC22) express cardiomyocyte markers, including cTroponin (red)

Cellartis Cardiomyocytes (from ChiPSC22) express cardiomyocyte markers, including cTroponin (red)
Cellartis Cardiomyocytes (from ChiPSC22) express cardiomyocyte markers, including cTroponin (red). Nuclei are labelled with DAPI (blue). F-actin is labeled with Phalloidin (green).


Cellartis cardiomyocytes derived from human induced pluripotent stem cells

A highly homogeneous population of cardiomyocytes derived from human induced pluripotent stem cells.

Human stem cell-derived cardiomyocytes


Cellartis hiPS-CM citation list

Publications using Cellartis hiPS-CM.

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Takara Bio USA, Inc. (TBUSA, formerly known as Clontech Laboratories, Inc.) provides kits, reagents, instruments, and services that help researchers explore questions about gene discovery, regulation, and function. As a member of the Takara Bio Group, TBUSA is part of a company that holds a leadership position in the global market and is committed to improving the human condition through biotechnology. Our mission is to develop high-quality innovative tools and services to accelerate discovery.

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