iCell® Cardiomyocytes2
Application Protocol

Contractility Analysis of iCell Cardiomyocytes2:
Evaluating iCell Cardiomyocytes2 in co-culture with iCell Cardiac
Fibroblasts on the FLEXcyte 96 SystemIntroduction
Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes, specifically iCell® Cardiomyocytes2,
have become an important tool in the safety pharmacology space for monitoring the cardiotoxic effects ofcompounds. Numerous in vitro assays to measure the activity of these cardiac cells using multi-electrodearray (MEA) or calcium oscillations have been developed. Next-level approaches to monitor contractility,
however, have been limited by throughput and biological relevance. Furthermore, the electromechanicalfunction of cardiac muscle depends on the crosstalk of myocytes with other regenerative non-myocyte
cells, specifically cardiac fibroblasts. A major function of cardiac fibroblasts is to produce and secrete
growth factors and cytokines, which have specific effects on cardiomyocytes that can impact contractile
function.
To enable the isogenic co-culture of iCell Cardiomyocytes2 with human iPSC-derived cardiac fibroblasts,
FUJIFILM Cellular Dynamics has launched a new cell type with iCell Cardiac Fibroblasts. These cells aredifferentiated from the same 01434 female donor, are of high purity, have low lot-to-lot variability, andexpress cardiac fibroblasts specific markers such as POSTN, TE7, and Cx43.
In this application protocol, we provide recommendations for co-culturing iCell Cardiomyocytes2 with iCell
Cardiac Fibroblasts on the Nanion FLEXcyte 96 system and offer basic instructions for compound dosing,data acquisition, and analysis. This system uses a 96-well plate format with flexible PDMS membranes
which enables contractility measurements of cells cultured in a more physiological environment, thus
more closely reflecting the mechanical conditions of the native human heart versus overly stiff substrates
like glass or plastic. iCell Cardiomyocytes2 together with iCell Cardiac Fibroblasts can be co-cultured andmaintained on the FLEXcyte 96 system for extended durations allowing for the measurement of acuteand sub-acute drug-induced effects, thus offering an excellent platform for in vitro screening of compoundeffects on human cardiac contractility. This Application Protocol describes how to prepare and maintainthese cell types together in co-culture for use on the FLEXcyte 96 system.

Required Equipment, Consumables, and Software
The following equipment, consumables, and software are required in addition to the materials specified in
the iCell Cardiomyocytes2 User’s Guide and iCell Cardiac Fibroblasts Quick Guide.
ItemVendor(s)Catalog Numbers

Equipment
FLEXcyte 96Nanion Technologies GmbH

12-channel pipettor, 200 µl and 20 µl

Multiple Vendors

Consumables
iCell Cardiomyocytes2 Kit, 01434

FUJIFILM Cellular Dynamics, Inc. (FCDI)

R1017• iCell Cardiomyocytes2, 01434

FUJIFILM Cellular Dynamics, Inc. (FCDI)(included in kit)

• iCell Cardiomyocytes Plating Medium, 30 ml

FUJIFILM Cellular Dynamics, Inc. (FCDI)(included in kit)

• iCell Cardiomyocytes Maintenance Medium, 100 ml

FUJIFILM Cellular Dynamics, Inc. (FCDI)(included in kit)iCell Cardiac Fibroblasts Kit, 01434

FUJIFILM Cellular Dynamics, Inc. (FCDI)

R1257• iCell Cardiac Fibroblasts, 01434

FUJIFILM Cellular Dynamics, Inc. (FCDI)(included in kit)

• iCell Cardiomyocytes Maintenance Medium, 100 mlFUJIFILM Cellular Dynamics, Inc. (FCDI)(included in kit)

FLX-96 Plate

Nanion Technologies GmbH

201010Geltrex™ hESC-Qualified, Ready-To-Use, Reduced Growth
Factor Basement Membrane Matrix

ThermoFisher Scientific

A1569601

DPBS, no calcium, no magnesium

Multiple Vendors

15 ml Conical Tubes

Multiple Vendors

50 ml Conical Tubes

Multiple Vendors

200 μl and 20 μl Pipette Tips

Multiple Vendors

Sterile Reagent Reservoirs

Multiple Vendors

SoftwareCardioExcyte Control Software

Nanion Technologies GmbH

DataControl96 Software

Nanion Technologies GmbHWorkflow
It is recommended to assess the contractility of co-cultured iCell Cardiomyocytes2 (CM2) and iCellCardiac Fibroblasts (CF) on Day 6 to Day 9 of culture.
•Day -1: Coat the 96-well FLX-96 plate with 1:4 Geltrex:DPBS (-/-) overnight in a 37°C incubator.

•

Day 0: Thaw iCell Cardiomyocytes2 and iCell Cardiac Fibroblasts according to their User’s Guideor Quick Guide using the online Certificate of Analysis (CoA) viable cell count. The cells are
combined in a 10:1 (CM2:CF) ratio and seeded on the FLX-96 plates in iCell Cardiomyocytes
Plating Medium (iCPM).

•Day 2: After 48hrs of plating in iCell Cardiomyocytes Plating Medium, gently vacuum aspirate
media and replace with 200 μl of iCell Cardiomyocytes Maintenance Medium (iCMM).

•Day 4 and Day 5: Perform a complete media change with 200 μl of iCell Cardiomyocytes
Maintenance Medium. It is important to change the media 24 hours before assay. Do NOTchange the media on the day of assay. EX: If you plan to assay on Day 6, perform a complete
media change on Day 5. If you plan to assay on Day 7, perform an additional complete media
change on Day 6.

•Day 3 to Day 6: Read the baseline signal of the wells every day on the FLEXcyte 96 System.
(This is optional but recommended for tracking the activity of the plate).

•Day 6 (day of assay): Read the baseline of the plate on the FLEXcyte 96 System. Dose withdesired compounds at 10X final concentration in 22 μl while the plate is on the instrument. Readplate on the FLEXcyte 96 after desired amount of time. FCDI recommends assaying the plate on
between Day 6-9.

Figure 1: Schematic diagram of the workflow for assaying iCell Cardiomyocytes2 (CM2) and iCell Cardiac
Fibroblasts (CF) on the FLEXcyte 96 System.

Tips Before Starting
1. Refer to the iCell Cardiomyocytes2 and iCell Cardiac Fibroblasts User’s Guide and Quick Guide for
information on storage and handling of the cells and media.
2. Read this entire Application Protocol before handling the cells to become familiar with the assay
workflow.
3. Obtain the Certificate of Analysis (CoA) for each cell type here: www.fujifilmcdi.com/coa-lookup/.
Based on the guaranteed number of cells provided per vial (≥5 x 106 for iCell Cardiomyocytes2 and≥0.5 x 106 for iCell Cardiac Fibroblasts), there are enough cells to seed at least 50 wells of the FLX96 plate. Two vials of iCell Cardiomyocytes2 and iCell Cardiac Fibroblasts are required to seed all 96
wells in a FLX-96 plate.
4. Thaw both bottles of media required for this assay, including iCell Cardiomyocytes Plating Medium(30 ml) and iCell Cardiomyocytes Maintenance Medium (100 ml), overnight at 4°C the day prior tothawing and plating cells.
5. Overnight coating with the Geltrex solution is imperative to assay performance.
6. Cells are thawed and seeded in iCell Cardiomyocytes Plating Medium. Wait 48 hours to perform amedia change.
7. The PDMS membrane on a FLX-96 plate is very thin (10 µm) and is easily ruptured. It is imperative toleave the plate in its protective receiver plate. Do NOT touch the pipette tips to the bottom of the welland carefully rest the tips on the side of the well when dispensing media.
8. A full media change is required 24 hours before assay. Do NOT change the media on day of assay.

Methods
ECM Coating with Geltrex Solution (Day -1)
1. Prepare 1:4 Geltrex:DPBS by diluting 3 ml of Geltrex with 9 ml of DPBS without Ca2+ and Mg2+ in asolution reagent reservoir.
2. Dispense 100 μl of the Geltrex working solution into each well of a FLX-96 plate with a 200 µl multichannel pipettor.
3. Incubate the Geltrex-coated FLX-96 plate in a cell culture incubator at 37°C, 5% CO2 overnight.
Thawing and Plating iCell Cardiomyocytes2 and iCell Cardiac Fibroblasts for the FLEXcyte 96
SystemThese steps outline the requirements for seeding a full FLX-96 plate with iCell Cardiomyocytes2 and iCellCardiac Fibroblasts at a 10:1 ratio.
1. Obtain the number of viable cells per vial from the Certificate of Analysis (CoA) for the specific lot ofiCell Cardiomyocytes2 and iCell Cardiac Fibroblasts.
2. Calculate the final volume of iCell Cardiomyocytes Plating Medium needed to obtain a viable celldensity of 1 x 106 cells/ml, which yields 100,000 viable cells per 100 µl for iCell Cardiomyocytes2.
Example: If the number of viable iCell Cardiomyocytes2 cells/vial is 5.6 x 106, the final solution shouldbe 5.6 ml. Multiply the final volume of iCell Cardiomyocytes Plating Medium for the iCell
Cardiomyocytes2 by 2, because two vials will be thawed.
3. Warm the 30 ml bottle of iCell Cardiomyocytes Plating Medium to 37°C in a water bath prior to use.
4. Thaw the two vials of iCell Cardiomyocytes2 into a single sterile 50 ml centrifuge tube according to theiCell Cardiomyocytes2 User’s Guide. Bring the volume of the cell suspension up to the calculated
volume from step 2.
Note: The iCell Cardiomyocytes2 User’s Guide recommends thawing the vial of cells by transferring
1 ml of cell solution to the 50 ml conical tube, rinsing the vial with 1 ml of iCell Cardiomyocytes Plating
Medium and transferring slowly to the cell solution. After this is complete, add additional iCell
Cardiomyocytes Plating Medium to the conical tube to reach the calculated volume from step 2.
Expect this volume to be greater than or equal to 5 ml for each vial of iCell Cardiomyocytes2 .
5. Calculate the final volume of iCell Cardiomyocytes Plating Medium needed to obtain a viable celldensity of 100,000 cells/ml, which yields 10,000 viable cells per 100 µl for iCell Cardiac Fibroblasts.
Example: If the number of viable iCell Cardiac Fibroblasts cells/vial is 0.7 x 106, the final solution
should be 7 ml. Multiply the final volume of iCell Cardiomyocytes Plating Medium for the iCell Cardiac
Fibroblasts by 2, because two vials will be thawed.
6. Thaw the two vials of iCell Cardiac Fibroblasts into a single sterile 50 ml centrifuge tube according to
the iCell Cardiac Fibroblasts Quick Guide. Bring the volume of the cell suspension up to thecalculated volume from step 5.
Note: The iCell Cardiac Fibroblasts Quick Guide recommends thawing the vial of cells by transferring
0.5 ml of cell solution to the 50 ml conical tube, rinsing the vial with 1 ml of iCell Cardiomyocytes
Plating Medium and transferring slowly to the cell solution, then add an additional 0.5 ml of iCell
Cardiomyocytes Plating Medium dropwise to the conical tube (the extra 0.5 ml is to make up forcryopreservation solution provided at 0.5 ml per vial). After this is complete, add additional iCell
Cardiomyocytes Plating Medium to the conical tube to reach the calculated volume from step 2.
Expect this volume to be greater than or equal to 5 ml for each vial of iCell Cardiac Fibroblasts.

7. Transfer 10 ml of each cell suspension to a separate 50 ml conical tube to make a combined master
cell suspension. Gently pipette up and down to mix the cells.
8. Transfer the mixed master cell suspension to a reagent reservoir.
9. Remove the Geltrex-coated FLX-96 plate from the cell culture incubator and work in a biologicalsafety cabinet.
10. Aspirate the Geltrex solution from the plate quickly with a glass pipette fitted to a vacuum line fromthe first four rows. Do NOT touch the bottom of the well with the pipette; it is recommended to havethe pipette touching the wall while aspirating.
11. Gently pipette the combined master cell suspension of iCell Cardiomyocytes2 and iCell Cardiac
Fibroblasts up and down 1-2 times in the reservoir to evenly resuspend cells. Pipette 200 µl of themaster cell suspension into each well of the FLX-96 plate using a multichannel pipette. Mix the cells
in the reagent reservoir by gently pipetting up and down between each row.
12. Repeat steps 9 and 10 to seed the last four rows.
Culturing of iCell Cardiomyocytes2 and iCell Cardiac Fibroblasts in the FLEXcyte 96 System
1. Equilibrate the iCell Cardiomyocytes Maintenance Medium to 37°C in a water bath prior to use.
Note: Aliquots of iCell Cardiomyocytes Maintenance Medium (~20 ml) may be removed from thestock bottle and warmed up separately.
2. On Day 2 (or 48 hours) post-plating, perform a complete media change by vacuum aspirating iCell
Cardiomyocytes Plating Medium from the plate 2 rows at a time. Add 200 μl of iCell Cardiomyocytes
Maintenance Medium to each well using a multichannel pipette.
3. Incubate the microplate in a cell culture incubator at 37°C, 5% CO2 after changing the medium.
4. Maintain the co-culture of iCell Cardiomyocytes2 and iCell Cardiac Fibroblasts by replacing 100% of
the spent medium with fresh iCell Cardiomyocytes Maintenance Medium every 2 days.
5. On Day 5 (24 hours before planned assay), perform a complete media change.

Data Acquisition and Analysis
Data Acquisition
Please refer to the CardioExcyte Control and DataControl96 Software manuals for complete instructions
on how to acquire data on the instrument.
Compound Application
1. The day before compound addition, replace 100% of the spent medium with 37°C iCell
Cardiomyocytes Maintenance Medium, iCell Cardiomyocytes Serum-Free Medium or iCell CardioTox
Assay Medium.
Note: Changing the medium about 18 hours before compound treatment ensures that thecardiomyocytes have stabilized after medium replacement and that the medium volumes are uniform
across the FLX-96 plate.
2. Prepare stock solutions of test compounds in fresh medium at a concentration of 10X the final
concentration in a regular 96-well cell culture plate.
Note: Final DMSO concentrations above 0.1% should be used with caution. Therefore, if test
compounds are dissolved in DMSO, the 10X compound solutions should not exceed 1% DMSO.
3. Equilibrate the cell culture plate containing the 10X compound solutions (covered with a lid) in a cellculture incubator at 37°C, 5% CO2.
4. To begin the assay, quickly transfer 20 µl of the 10X compound solutions from the cell culture plate to
the FLX-96 plate. Gently mix by pipetting 2-3 times.
Note: Beating rate and amplitude are temperature dependent. It is recommended to add thecompounds while the FLX-96 plate is placed on the FLEXcyte 96 system. If this is not possible, theFLX-96 plate should not be kept outside the incubator for more than 2 minutes while compounds areadded.
Data Acquisition and Analysis
The CardioExcyte Control Software enables automatic beating detection of cardiomyocytes cultured on aFLX-96 plate placed onto the FLEXcyte 96 system. DataControl96 Software allows automated analysis ofdifferent contractility parameters before and after compound addition. Furthermore, DataControl96
Software enables the calculation of IC50 and EC50 of test compounds. See the CardioExcyte Control andDataControl96 Software manuals to discover all their features.

Representative DataFigure 1. Comparison of iCell Cardiomyocytes2 in mono-culture vs. in co-culture with iCell Cardiac
Fibroblasts A) Co-culture of iCell Cardiomyocytes2 (100K) and iCell Cardiac Fibroblasts (10K) results in a higher
signal amplitude of contractility on the FLEXcyte 96 (representative data from Day 6 shown here). Contractility can betracked over time in culture and B) the signal amplitude of cells together in co-culture is greater than that of iCell
Cardiomyocytes2 alone at every time point measured, while C) the beat rate of mono- vs. co-culture remains
relatively similar over time.

Figure 2. Cell Density Titration A) iCell Cardiomyocytes2 and B) iCell Cardiomyocytes2 and iCell Cardiac
Fibroblasts in Co-Culture. A) iCell Cardiomyocytes2 density was varied in co-culture with 10% iCell Cardiac
Fibroblasts. Example: 50K iCell Cardiomyocytes2 were cultured with 5K iCell Cardiac Fibroblasts, etc. Results
showed that the strongest signal amplitude were in cultures containing 100K and 125K iCell Cardiomyocytes2. It isrecommended to plate 100K Cardiomyocytes2 per well in the FLX-96 plate. B) 100K Cardiomyocytes2 were cultured
with varying iCell Cardiac Fibroblasts densities. Results showed the highest amplitude signal in wells containing 10%iCell Cardiac Fibroblasts. It is recommended to culture 100K iCell Cardiomyocytes2 and 10K iCell Cardiac Fibroblasts
per well of FLX-96 plate.

Amplitude

Beat Rate
70

Beats per MinuteSignal Amplitude54321060
5040

Geltrex30

Matrigel
Matrigel & FN20100Day 4Day 8

Day 10 Day 13 Day 15

Time in Culture

Day 4Day 8Day 10 Day 13 Day 15

Time in Culture

Figure 3. ECM optimization. The type of extracellular matrix coating on the FLX-96 plate had an effect on the cells’
contractility signal amplitude over time. Cells seeded on Geltrex (1:4 dilution Geltrex:DPBS(-/-)), 10ug/mL Matrigel,and 10ug/mL Matrigel with 10ug/mL Fibronectin coatings were tested. Results showed that Geltrex supported thehighest contractility signal amplitude over time. There was no effect on beat rate from different coatings. It isrecommended to plate 100K iCell Cardiomyocytes2 and 10K iCell Cardiac Fibroblasts on Geltrex-coated wells in theFLX-96 plate.

Figure 4. Pharmacology. Co-cultured iCell Cardiomyocytes2 and iCell Cardiac Fibroblasts were dosed withcompounds on day 7 in culture. Representative traces of the beat pattern when exposed to DMSO control (A),Epinephrine (B), and E-4031 (C). Expectedly, beta-adrenergic receptor antagonist, epinephrine, resulted in an
increased beat rate and contraction amplitude. Additionally, hERG channel blocker, E-4031 expectedly decreased thecontractility amplitude and disrupted and elongated the beating pattern.

SummaryThe details in this application protocol outline the steps to co-culture iCell Cardiomyocytes2 with iCell
Cardiac Fibroblasts on the FLX-96 plate. The FLEXcyte system measures the contractility force of the coculture, thereby enabling the user to study the pharmacological effects on contractility force. The data
within this protocol is a comprehensive look at representative data for the optimization of culture
conditions on the FLX-96 plate with additional pharmacological response to compounds. It has been
demonstrated that iCell Cardiomyocyctes2 co-cultured with isogenic iCell Cardiac Fibroblasts result in ahigher contractility force compared to iCell Cardiomyocytes2 in mono-culture. The co-culture of cells on
Geltrex resulted in the highest contractility force amplitude. Additionally, dosing the co-culture resulted in
the expected response to known cardiotoxic compounds.

For questions about culturing our iCell Cardiomyocytes2 and iCell Cardiac Fibroblasts, please contact
FCDI’s Technical Support at• fcdi-support@fujifilm.com• +1 (877) 310-6688 (US toll-free) or +1 (608) 310-5100
For inquiries about Nanion’s FLEXcyte platform, please contact Nanion’s Technical Support at• Nanion HQ for general enquiries: info@nanion.de• Nanion’s Technical Support: support.cellular.networks@nanion.de

Customer’s Responsibilities
FUJIFILM Cellular Dynamics, Inc. (FCDI), does not guarantee that you will obtain equivalent results from using iCell or MyCell products as described herein orthat such use will not infringe any intellectual property right(s) of any third party(ies). You are solely responsible for obtaining any licenses you may require foryour specific research use(s) of the iCell or MyCell products not expressly conveyed under FCDI’s terms and conditions of sale or other transfer of the iCell orMyCell products to you.
Conditions of Use
The cells are FOR RESEARCH USE ONLY and NOT FOR THERAPEUTIC USE. See www.fujifilmcdi.com/terms-and-conditions/ for USE RESTRICTIONS
applicable to the cells and other terms and conditions related to the cells and their use.
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Revision History
Version 1.0: September 2023
AP-CFCFLEXcyte_14SEP2023