Cor.4U in CardioExcyte 96 assays

Transcription

1 Cor.4U in CardioExcyte 96 assays Application protocol Last update: July 2017 Europe Nattermannallee 1/S Cologne, Germany North America 600 W Germantown Pike, STE 110 Plymouth Meeting, PA 19462, USA

2 TABLE OF CONTENTS GENERAL INFORMATION 4 SAFETY INFORMATION 4 MATERIAL 4 Cells and media provided by Axiogenesis 4 Storage conditions 6 Required consumables 6 4. Required equipment 7 5. Components of the CardioExcyte 96 instrument 7 4. INSTRUMENT SETUP 8 4. Editing the instrument software 8 5. PREPARATIONS 8 5. Background measurement 8 5. Coating 9 6. CELL CULTURE 9 6. Thawing 9 6. Cell counting and viability determination 9 6. Seeding Maintenance ASSAY Compound treatment Short term compound treatment (24h) Washout of compounds Long term compound treatment REFERENCES RELATED DOCUMENTS TROUBLESHOOTING GUIDE 14!2

3 1 AXIOGENESIS LIMITED LABEL USE LICENSE 16 1 APPENDIX 17 1Recommended seeding densities 17 1Example of a test plate layout 17 1Example of compound test results 17!3

4 GENERAL INFORMATION This protocol covers thawing, seeding, maintenance, and compound addition when using Axiogenesis Cor.4U cardiomyocytes in a CardioExcyte 96 instrument (Nanion Technologies, Munich, Germany). The CardioExcyte 96 instrument is used for labelfree, real-time detection of the beating behavior of cardiomyocytes. It is recommended to use the cells earliest at day 3 post thaw, as the cells develop a syncytium and are beating synchronously at this time. Please read the entire protocol and the Nanion CardioExcyte 96 user manual before starting the experiment. The protocol is set up according to the state of purchased cryopreserved Cor.4U. When pre-seeded Cor.4U in CardioExcyte 96 Sensor Plates (NSP 96 plates) were purchased please see step 2 and 6.4. Cor.4U are produced by in vitro differentiation of transgenic human induced pluripotent stem cells (ipsc) and puromycin selection of the resulting cardiomyocytes. The ipsc line is generated by introducing defined transcription factors, described by Yamanaka (1) in a human skin fibroblast, using a non -viral system (2). The highly pure cardiomyocytes express cardiac-specific proteins, e.g. cardiac alpha-actinin and the gap junction protein connexin-43, a prerequisite for electric coupling of these cells. Patch clamp analyses, as well as micro-electrode array (MEA) recordings, demonstrate the normal electrophysiological properties of these cells. Cor.4U are particularly useful for cell-based in vitro assays in pharmacology, safety pharmacology, and toxicology. These cells are ideal for electrophysiological applications as well as for high content and high throughput screening applications. SAFETY INFORMATION Cor.4U cardiomyocytes are intended for in vitro research use only. The cells are not intended for diagnostics, therapeutic or clinical use and are not approved for human in vivo applications. Cor.4U cardiomyocytes are genetically modified human cells and should be handled according to local directives (Biosafety level 1, US-CDC, or S1, GenTSV, Germany). Cor.4U cardiomyocytes can be inactivated by autoclaving at 121 C for 20 minutes. Cor.4U cardiomyocytes should be cultured in a sterile environment. It is highly recommended that gloves and lab coats be worn when handling all reagents as some reagents contain chemicals that may be harmful. Please consult the Certificate of Analysis (CoA) and Material Safety Data Sheets (MSDS) for additional safety instructions where applicable. MATERIAL Cells and media provided by Axiogenesis!4

5 Cor.4U Cor.4U are available cryopreserved as well as pre-cultured in CardioExcyte 96 Sensor Plate (NSP-96), suitable for performing impedance assays with the CardioExcyte 96 instrument (see table 1). Cor.4U is offered with Cor.4U Culture Medium. If required, serum-free BMCC medium can be purchased separately (see table 1). The Cor.4U Culture Medium is the standard culture medium used for thawing and seeding of Cor.4U. The BMCC medium is a serum-free culture medium and avoids potential proteinbinding of test compounds to serum components. Cor.4U can be easily adopted to the BMCC medium but potentially show a slightly reduced beating rate when cultured for a longer time period. Do not directly seed freshly thawed or dissociated Cor.4U in BMCC medium because the Cor.4U require the serum containing Cor. 4U Culture Medium for the attachment to the culture surface. Pre-cultured cells also comes with Axio-Supplement. Axio-Supplement contains the antibiotic Ciprobay (2 mg/ml) and may be added to the culture medium where the use of antibiotics is desired. Note that the use is optional; importantly, cell growth and function is not compromised by long-term culture in presence of Ciprobay. Table 1: Overview of CardioExcyte96 suitable Cor.4U products Material Container Contents Storage Shelf life Cryopreserved Cor.4U (Ax-B-HC02-4M) Cryo vial 4 million cells Liquid nitrogen See label (max. 5 years) Pre-cultured Cor.4U cardiomyocytes (Ax-C-HC02-NCE) Pre-cultured in NSP-96 plate Plate with Incubator cells and 37 C QC tested signal Cor.4U Culture Medium (Ax-M-HC250) Bottle 250 ml medium Frozen See expiry date -20 C / on bottle label Refrigerated 4 C BMCC medium (serum-free) (Ax-M-BMCC250) Bottle 250 ml medium Frozen See expiry date -20 C / on bottle label Refrigerated 4 C Axio-Supplement (Ax-M-CB-5) Cryo vial 250 µl Liquid RT, dark - ATTENTION: No puromycin is required for the use of Cor.4U cardiomyocytes in the CardioExcyte 96 instrument See expiry date on cryo vial label!5

6 Cor.4U Storage conditions Cryopreserved cells: Upon receipt of cryopreserved Cor.4U, transfer the vials directly to the vapor phase of liquid nitrogen for further storage. Do not expose the vials to room temperature and do not store cells at -80 C, as recrystallization will harm the cells. ATTENTION: Do not expose the vials longer than one minute to room temperature as recrystallization processes might harm the cells. Cells pre-cultured on a CardioExcyte 96 (NSP-96) plate: Directly after arrival please make a measurement of the plate in the CardioExcyte96 instrument without removing the silicon lid. This is to confirm that the plate was not damaged during delivery. Then transfer the plate to a laminar flow hood and carefully remove the sealing mat. Change medium upon arrival by removing 50 µl of the medium and replace with 100 µl of fresh, warm Cor.4U Culture Medium. Thereafter remove 100 µl medium and replace again with 100 µl fresh, warm Cor. 4U Culture Medium. Repeat this step 3 times to achieve a complete medium change. Replace the lid with the new sterile lid provided and transfer the plates immediately to a humidified incubator (37 C, 5 % CO2). Cor.4U in plates are ready for compound treatment once a stable beating can be observed, normally the day after delivery. Continue with instructions in 6.4 Maintenance. Medium: Store frozen Cor.4U Culture Medium and BMCC medium at -20 C upon receipt. Thaw medium overnight at 4 C ; avoid excessive exposure to light. Once thawed, medium can be kept at 4 C up to 4 weeks. The media delivered with cultured Cor.4U is not frozen and can be stored at 4 C for up to 4 weeks. Required consumables Table 2: Overview of required consumables Item Vendor Cat. No. CardioExcyte 96 Sensor Plate (NSP-96) Nanion Technologies DPBS, sterile with Ca2+ and Mg2+ Sigma-Aldrich D8662 DPBS, sterile without Ca2+ and Mg2+ Sigma-Aldrich D8537 Fibronectin Sigma-Aldrich F1141 Trypan blue solution 0.4 % Sigma-Aldrich T8154 Sterile reservoir Various - 96-deep well with U-shaped bottom Various - Sterile 50 ml polypropylene tubes Various -!6

7 4. Required equipment Table 3: Overview of required equipment Item Vendor Cat. No. CardioExcyte 96 instrument Nanion Technologies Inverse microscope Various - 37 C water bath Various - Laminar flow hood Various - Cell culture incubator (37 C, 95 % humidity, 5 % CO 2 ) Various - Liquid nitrogen storage Various - Freezer (-20 C) refrigerator (+4 C) Various - 10 µl, 100 µl, 1000 µl pipette Various µl 8-channel pipette Various - Neubauer hemocytometer Various - Pipettor for serological pipettes Various - 5. Components of the CardioExcyte 96 instrument The CardioExcyte 96 instrument is composed of the following components: CardioExcyte 96 instrument CardioExcyte 96 control software CardioExcyte 96 Sensor Plate (NSP-96) Fig CardioExcyte 96 instrument!7

8 Cor.4U 4. INSTRUMENT SETUP 4. Editing the instrument software Open the CardioExcyte software and click on the EDIT icon to select the sweep duration, number of sweeps and repetition interval. Choose the impedance mode for Cor.4U cardiomyocyte impedance measurements or the EFP mode for extracellular field potential recordings. Click on the EDIT COMPOUNDS icon and generate your compound and Solution-layout. For freshly seeded cells; before coating and seeding cells we recommend a background base impedance measurement of the CardioExcyte 96 Sensor Plate for normalization see 5. Click on the START MEASUREMENT icon. A little window will pop up to define the chip number of the CardioExcyte 96 Sensor Plate and the name of your experiment. ATTENTION: Please read the CardioExcyte 96 manual regarding settings for the measurement. ATTENTION: Cor.4U use in the CardioExcyte 96 instrument is standardized for an instrument calibrated to 470 Ohm. For pre-seeded cells in NSP-96 plate; background measurement data for normalization and base impedance measurements of the plated cells are present on the USB stick. First load measurements on the provided USB stick and use these as basis for further measurements. 5. PREPARATIONS 5. Background measurement Before coating and seeding, we recommend to perform a base impedance background measurement of the CardioExcyte 96 Sensor Plate for normalization, to compensate for possible plate and well variations. Transfer a minimum of 40 ml of sterile DPBS without Ca2+ and Mg2+ into a sterile reservoir suitable for an 8-channel pipette. Add 200 µl DPBS to each well. Incubate the CardioExcyte 96 Sensor Plate for 10 minutes. 4. Aspirate DPBS and repeat the addition of 200 µl DPBS to each well. 5. Place the CardioExcyte 96 Sensor Plate into the CardioExcyte 96 instrument inside the incubator. Before you start the background measurement, incubate the CardioExcyte 96 Sensor Plate for at least 5 min. 6. Perform the background measurement by clicking on the START MEASUREMENT icon. Use background basal impedance for normalization of the base impedance data. An acceptable starting point for compound measurement is 120 Ohm over background.!8

9 Cor.4U 5. Coating Day 0: Coating of the CardioExcyte 96 Sensor Plate (NSP-96) Prepare 6 ml fibronectin coating solution by diluting 60 µl fibronectin stock solution (1 mg/ml) in 6 ml of DPBS with Ca2+ and Mg2+. The final concentration of the coating solution is now 10 µg / ml fibronectin (Dilution 1:100). Mix the solution carefully, by pipetting with a 5 or 10 ml pipet. Transfer the coating solution into a sterile reservoir suitable for an 8-channel pipette. Pipette 50 µl of the coating solution into each well of the NSP-96 using an 8-channel pipette and incubate for 3 h at 37 C. Alternatively, incubate at 4 C overnight. 6. CELL CULTURE 6. Thawing Transfer 4 ml Cor.4U Culture Medium into a 50 ml tube and warm to 37 C. Prepare a reaction tube with 20 µl trypan blue solution for cell number determination. Quickly transfer the cells from liquid nitrogen directly to a 37 C water bath. Thaw the vial until the frozen cell suspension detaches from the bottom of the vial and only a small ice clump is visible (approx. 2 min). 4. Disinfect and transfer the vial to the laminar flow hood, carefully tap the cryo vial to loosen the cell pellet. 5. Carefully transfer the cells with a 1000 µl pipette to the pre-warmed medium in the 50 ml tube. The total volume is now 5 ml. 6. For determination of cell number and viability, gently agitate the tube to mix the cell suspension, transfer 20 µl of the cell suspension into a reaction tube containing 20 µl trypan blue solution and mix gently. 6. Cell counting and viability determination Apply 10 μl of the 1:1 mixture of cell suspension with trypan blue solution to a Neubauer hemocytometer and count viable (clear), dead (blue) and total cells. Count the number of cells in each of the four outer squares highlighted in red in figure Calculate the mean number of cells per red square. Calculate the number of trypan blue negative cells corrected by chamber factor (1 x 104), dilution factor (2), and total volume (5 ml). ATTENTION: Fibronectin is very susceptible to shear stress. Avoid harsh pipetting and do not vortex or spin the solution. Do not let coating dry. Do not freeze the fibronectin solutions. Be careful not to touch the bottom of the wells to avoid damage of the coating. ATTENTION: For transport of frozen vials from a liquid nitrogen storage tank to the cell culture room, it is recommended to use a dewar filled with liquid nitrogen. Don t use dry ice for the transport because this might affect the viability of the cells. ATTENTION: Do not spin down the cell suspension! Centrifugation directly after thawing will damage the cells and leads to cell loss. ATTENTION: When using an automated cell counter, please compare determined cell numbers with manual counting in a Neubauer hemocytometer. Many automated cell counters have difficulties determine the correct cell number of Cor.4U cell suspensions.!9

10 Cor.4U Calculation example E.g.: Mean number of viable cells per square = x 10,000 x 2 x 5 = 4,000,000 4 million living cells in the cell suspension Fig Neubauer hemocytometer 6. Seeding The recommended standard seeding density of Cor.4U cardiomyocytes in the CardioExcyte 96 plates (NSP-96) is 3 x 104 cells per well in 200 µl of medium. Prepare a cell suspension for 110 wells by diluting 3 x 106 viable cells in 22 ml of Cor.4U Culture Medium. Mix the cell suspension carefully, and transfer it into a sterile reagent reservoir. Aspirate the fibronectin from the wells of the NSP-96 plate and immediately add 200 µl of the cell suspension to each well of the plate. Mix cells in the reservoir every 3 rows by moving the container back and forth in order to avoid the cells settling down to the bottom of the reagent reservoir. 4. After adding the cell suspension, do not move the plate. Let it stand in the laminar flow hood at room temperature for 30 min. 5. Thereafter transfer the NSP-96 plate into the CardioExcyte 96 instrument in the incubator. 6. Start measurement Maintenance Day 1 - Day 2 Exchange the entire culture medium twice, approx.18 h (morning) and 26 h (evening) after seeding. When cultivating the Cor.4U cardiomyocytes in Cor.4U Culture Medium, a medium change twice per day over the complete culturing time is necessary. Warm at least 42 ml of Cor.4U Culture Medium to 37 C. Immediately before use, transfer the warm medium into a sterile reagent reservoir. Pause the CardioExcyte 96 instrument. Choose your Solution- Layout (e.g plate A for medium exchange with Cor.4U Culture Medium) Disengage the NSP-96 from the CardioExcyte 96 Station and transfer it to the laminar flow hood. 4. Perform a 100 % medium replacement in four steps. Remove 100 µl of the medium from every well and add the same amount of fresh medium to each well. Repeat this step 3 times. 5. Place the NSP-96 plate back into the CardioExcyte 96 Station as quickly as ATTENTION: Refer to the appendix of this protocol for suggestions regarding seeding densities. ATTENTION: Do not move the plate directly after seeding to avoid uneven distribution of the cells. ATTENTION: It is important to perform the stepwise medium change since it reduces unwanted side effects of the medium change to a minimum. Cor. 4U cardiomyocytes are very sensitive and should not be left without medium at any time. Whilst changing the medium, the cells may stop beating for a moment, but will recover quickly when returned into the incubator. ATTENTION: Remove medium carefully from the top, avoid touching the bottom of the plate with the pipette tips. Use slow speed when pipetting up and down.!10

11 Cor.4U possible and resume the measurement. 6. Change medium twice per day to secure regular beating performance. Day 3: Medium change to serum free media Optional; many compounds bind serum, therefor it is often desirable to use serum free medium for compound testing. A change from Cor.4U Culture Medium to serum-free BMCC medium can be performed one day prior compound treatment. Warm at least 42 ml BMCC medium to 37 C. Perform a 100 % medium change in 4 steps as described in Exchange the medium twice on day 1, once in the morning and once in the evening. Repeat the 100 % medium change in 4 steps as described in every day until you perform a compound treatment. 4. As soon as a stable baseline beating can be monitored (a stable and regular beating rate and Amplitude) you can start with the compound treatment. 7. ATTENTION: Due to the different medium composition, a drop in the total base impedance value will occur after changing to serum-free BMCC medium. ATTENTION: When Cor.4U are cultivated in BMCC media,media change once per day is usually sufficient. This is due to a reduced metabolism of the cells in this medium. ASSAY 7. Compound treatment The first contraction signal of Cor.4U cardiomyocytes can be detected approx. 24 h after plating. The beating frequency might be high due to incomplete coupling of the Cor.4U cardiomyocytes. As soon as a stable baseline beating can be monitored (approx. 72 h after plating), the cells can be treated with test compounds. We recommend checking the following quality criteria before starting a compound treatment ( Basal Impedance = Absolute basal impedance - well background impedance): ATTENTION: The final concentration of solvent (e.g. DMSO) must not exceed 0.1 %. We recommend using at least triplicates for each compound concentration. Table 3: Quality criteria before starting a compound treatment: Cell type Basal Impedance Beating Rate Amplitude Cor.4U > 120 Ohm bpm > 2 Ohm cardiomyocytes!11

12 Cor.4U 7. Short term compound treatment (24h) Day 4 Perform a 100 % medium change as described in approx. 2 h before adding the compounds. Prepare at least 120 µl per well of double-concentrated compound dilutions in the designated medium. Prepare the compound solutions in a 96-deep well plate (preferentially with an U-shaped bottom following exactly the defined layout). An example for a plate layout can be found in the appendix. Equilibrate all compound solutions in the cell culture incubator for at least 30 min to adjust temperature and ph. 4. Perform a reference measurement before you add the compounds. Choose your preferred interval, e.g. every 2 min for 10 min and resume your measurement. 5. Pause the CardioExcyte 96 instrument, choose your compound - Layout and disengage the NSP-96 plate from the station and transfer it to the laminar flow hood, together with the equilibrated compound plate. 6. Remove 100 µl of the medium from the top of each well using an 8-channel pipette and add 100 µl of the equilibrated double-concentrated stock solution, following the layout design. 7. Transfer the NSP-96 plate back into the station as quickly as possible and immediately resume the measurement e.g with a preferred interval of every 2 min for 60 min. ATTENTION: It is recommended to use a multichannel pipette at a slow speed in order to add the compounds as quickly as possible, but without disturbing the cells. 7. Washout of compounds (Optional) Warm 42 ml of the designated medium without compounds to 37 C. Pause the CardioExcyte 96 instrument, disengage the NSP-96 plate from the station and transfer it to the laminar flow hood. Remove 100 µl from every well and add 100 µl of fresh medium to each well using an 8-channel pipette. Repeat this medium exchange 4 times. 4. Return the NSP-96 plate back into the CardioExcyte 96 station as quickly as possible and resume the measurement for additional 2 h to 12 h. ATTENTION: Some compounds are sticky and requires additional medium changes to achieve a complete washout Long term compound treatment Day 5 Exchange the compound solution with fresh, 1 x concentrated compound solution once per day. The first medium change should be performed after the 24 h measurement. Prepare 1 x concentrated compound dilutions in the designated medium and equilibrate all compound solutions in the cell culture incubator for at least 30 min to adjust temperature and ph. Every 24 hours, pause the CardioExcyte 96 instrument, disengage the NSP-96 ATTENTION: For compounds with an anticipated long-term effect, the treatment protocol can be extended to at least 72 hours.!12

13 plate from the CardioExcyte 96 Station and transfer it to the laminar flow hood. 4. Cells in BMCC medium: Remove 100 µl of the compound solution from the top of each well, and replace it with 100 µl of the freshly prepared compound solution. 5. Cells in Cor.4U Culture Medium: Remove 100 µl of the compound solution from the top of each well, and replace it with 100 µl of the freshly prepared compound solution, repeat this step 3 times. 6. Transfer the NSP-96 plate back into the station as quickly as possible and resume the measurement. 7. Repeat this step every 24 h. 8. After the last measurement is finished, perform a washout step as described above. 8. REFERENCES Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., Yamanaka, S. (2007) Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors. Cell Nov 30;131(5):861-7 Axiogenesis AG Patent WO A1, A non-viral system for generation of induced pluripotent stem (ips) cells. 9. RELATED DOCUMENTS Cor.4U Handling guide Nanion CardioExcyte 96 user manual (Nanion Technologies)!13

14 10. TROUBLESHOOTING GUIDE Problem Not enough cells in vial after thawing Too many dead cells after thawing No base impedance detected Comment and suggestion - Were all cells transferred to the 50 ml falcon? It is important to tip on the cell pellet in the cryo vial to loosen the pellet before transferring the cells to the 50 ml tube; otherwise a large amount of cells may be retained in the cryo vial. - Was the cell suspension properly mixed before withdrawing a sample for counting? - How were cells handled upon arrival? Cells should immediately be transferred to the vapor phase of liquid nitrogen upon arrival. - What were the storage conditions? Storage in liquid nitrogen is a prerequisite for good survival of frozen cells. Viability will be impaired if Cor.4U are stored at higher temperatures (e.g., in a -80 C freezer). Storage or transportation on dry ice impairs the viability of Cor.4U. - Was the transport ok? Were the cell frozen in liquid nitrogen upon arrival? Partial thawing and refreezing of cells will lead to complete loss of cells. - Were cells transported in liquid nitrogen prior to thawing? A slow thawing process will result in cell loss. - How was the workflow during thawing? Work speedy. Transfer cells to media immediately after thawing. Cells die when staying too long in freezing media after thawing due to the high DMSO concentration. Don t let cells stand during thawing. Pelleting of cells increases cell death. Avoid extensive pipetting of cells. Cells are very sensitive to shear force. - Were Cor.4U centrifuged after thawing? This causes pronounced cell death. - No or low base impedance is an indication of cells not attaching. This can have several reasons. Either cells died or could not attach to the surface. - Which coating material was used? Proper coating is a prerequisite for good attachment of cells. - How was the coating material stored? Fibronectin looses activity when frozen before use. Fibronectin is very susceptible to shear stress. Avoid harsh pipetting and do not vortex or spin the solution. Were the cells stored properly after arrival? Were the cells thawed according to protocol? Wrong storage and wrong thawing procedure can influence the attachment ability of the cells.!14

15 Problem Bubbles occur during coating and /or seeding of cells into plates Comment and suggestion - Make sure to avoid any electrostatic charge. Place an anti-static and grounded mat under the laminar flow hood. - Wear anti-static shoes and disposable gloves. Hold the pipet tips close to the bottom to minimize the number of air bubbles. - Make sure the coating is removed completely before seeding. Coating leftovers in the plate can increase the generation of air bubbles. Cells detach from plate - Were cells plated in a very high density? When the cell layer is very dense it may peal off the plate. - Was the correct coating used and properly prepared? - Does the incubator keep the correct CO2 percentage and temperature? Cells stopped beating - Did the cells get cold? When temperature decreases the beating rate decreases and eventually stops. Addition of cold media results in arrest of spontaneous beating. Usually cells start beating again 30 minutes after return to the 37 C incubator. - Was the medium changed regularly? A lack of nutrition will result in arrest of beating.!15

16 1 AXIOGENESIS LIMITED LABEL USE LICENSE A. AXIOGENESIS Intellectual Property Rights This product is covered by patent families including, but not limited to, EP ; EP ; EP ; EP ; JP ; JP ; JP ; JP ; DE and other families of patent applications ( AXIOGENESIS Intellectual Property ). Purchase of the product does not transfer any rights other than those outlined below. The purchase of this product conveys to the buyer the non-exclusive, nontransferable right to use the purchased amount of the product and the associated AXIOGENESIS Intellectual Property for (i) for non-profit internal research conducted by the buyer and (ii) certain for-profit activities, including lead discovery, testing and/ or research and development of other products. The use in disease modeling and tissue modeling is expressly excluded in this license. Please contact Axiogenesis for a license for disease and tissue models at patent@axiogenesis.com. B. Use restrictions This product is not suitable for any clinical, therapeutic (including cell therapy, transplantation, and regenerative medicine), or clinical diagnostic applications. The purchaser shall not use the product in any way that contravenes applicable laws or regulations. The product should be used according to the User Guide. Failure to comply with any provisions in section A, B, or C will make any warranty claims invalid. No rights are conveyed to modify, reproduce, or clone any part of this product or to use AXIOGENESIS Intellectual Property in any way that is separate from the purchased product. C. Other patents AXIOGENESIS products which were derived from ips cells are covered by patents in patent family EP and US licensed from ips Academia (Kyoto University). Additionally, GFP and RFP positive products are covered by patents owned by Evrogen. The GFP and RFP positive products are for internal, non-commercial research use only. The right to use a GFP positive product specifically excludes the right to validate or screen compounds. For information on commercial licensing, contact the Evrogen Licensing Department at: license@evrogen.com. Cor.At, Cor.4U, and Mel.Cor are registered trademarks of AXIOGENESIS AG, Cologne, Germany. Peri.4U, Dopa.4U, CNS.4U, and Astro.4U neural cells, as well as Skel.4U, are trademarks of AXIOGENESIS AG, Cologne, Germany. TurboGFP and TurboRFP are registered trademarks of Evrogen, Moscow, Russia. For information on the patents, patent applications, and licenses associated with the product contact the AXIOGENESIS Business Development Department at: patent@axiogenesis.com.!16

17 1 APPENDIX 1Recommended seeding densities The recommended standard seeding density of Cor.4U cardiomyocytes for the CardioExcyte 96 instrument is 3 x 10 4 cells per well in 200 µl of medium. However, depending on the experimental needs, the seeding density can be modified. 1Example of a test plate layout Fig Each color represents one test compound. Vehicle controls (e.g. DMSO) as well as standard controls (e.g. Isoproterenol and E-4031) are included. We recommend starting with compounds with well-known effects on cardiomyocytes, e.g. E-4031, Isoproterenol, Nifedipine, etc. 1Example of compound test results Fig 4. Compound test on Cor.4U in CardioExcyte 96 impedance assay. Shows different concentrations of Doxorubicin in a long term assay. Doxorubicin reduces impedance dose-dependently.!17