Cor.4U Cardiomyocytes. Human ipsc-derived Cardiomyocytes

Transcription

1 Cor.4U Cardiomyocytes Human ipsc-derived Cardiomyocytes User Manual Updated January 2018

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3 Contents 1. General Information 2 2. Safety Information 2 3. Material Cells and media provided by Ncardia Storage conditions Required consumables Required equipment 5 4. Preparations Surfaces Coating 6 5. Cell Culture Thawing for pre-cultivation Counting of cells and determination of viability Detachment Seeding into multi-well plates Puromycin treatment Maintenance References Related Documents Troubleshooting Guide Ncardia Limted Label Use License Appendix 17

4 Getting Started Please make sure to read the entire User Guide carefully before you start thawing and culturing Cardiomyocytes. Cardiomyocytes are for in vitro life science research use only. A Material Safety Data Sheet (MSDS) for Cardiomyocytes is available on our website. Technical support and training Our scientists are ready to help you with any questions you may have regarding this user guide or our Cardiomyocytes. In addition, in-lab training is available upon request. For further information please visit our website or contact us directly by (support@ncardia.com) User Guide - Cardiomyocytes Ncardia.com 1

5 1. General Information This protocol covers general thawing, seeding and dissociation of Ncardia cardiomyocytes required for various assays and methods. It is recommended to use the cells earliest on day 3 post thaw, when a fully formed syncytium has been formed and the spontaneous beating is synchronized and stable. Please read the entire protocol and the appropriate assay-specific handling guide before you start your experiment. cardiomyocytes 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 of the ability 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. cardiomyocytes 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. Please note: This document describes general handling procedures for cardiomyocytes in a sequence how it is most commonly performed by Ncardia customers. However, the specific workflow (e.g., whether cells are pre-cultured prior to seeding into assay specific plates or not) very much depends on the downstream assay performed with the cells. Therefore, for procedures related to specific applications please also refer to the respective application protocols (listed in Section 7. Related Documents). 2. Safety Information 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. cardiomyocytes are genetically modified human cells and therefore genetically modified organisms (GMO). They should be handled according to local directives (Biosafety level 1, US-CDC, or S1, GenTSV, Germany). cardiomyocytes can be inactivated by autoclaving at 121 C for 20 minutes. cardiomyocytes should be cultured in a sterile environment. It is highly recommended that gloves and lab coats are 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. User Guide - Cardiomyocytes Ncardia.com 2

6 3. Material 3.1 Cells and media provided by Ncardia are available in different cryopreserved as well as pre-cultured formats (see Tables 1 and 2). The latter can be delivered either in culture flasks or ready-to-use in standard 96- or 384-well culture plates or in assay-specific plates. Cryopreserved are available as vials with 0.25, 1 or 4 million cells per vial. are offered as a bundle including cells, medium ( Culture Medium), and puromycin for optional use. The bundle contains enough culture medium to fit the needs for standard cultivation of the cells. Bundle ingredients can also be ordered separately (see Table 1 for respective order numbers). Bundles of pre-cultured cells also contain 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. Culture Medium is the standard culture medium used for thawing and seeding of. If required, serum-free BMCC medium can be purchased separately. BMCC medium avoids potential protein-binding of test compounds to serum components. cells in culture can be easily adopted to BMCC medium but potentially show a slightly reduced beating rate when cultured for a longer time period (gradual decrease over various days). Material Container Content Storage Shelf life Cryopreserved Cryo vial 0.25, 1 or 4 million Liquid Max. 1.5 years cells nitrogen from issue date Pre-cultured Culture Medium (Ax-M-HC250) BMCC medium (serum free) (Ax-M-BMCC250) Puromyin (optional) (Ax-M-PO-05) Axio-Supplement (Ax-M-CB-5) Cell culture Various Incubator flasks or culture plates 37 C Bottle 250 ml Frozen -20 C Bottle 250 ml Frozen -20 C Cryo vial 50 μl Frozen -20 C Cryo vial 250 μl Liquid RT, dark on CoA - See expiry date on bottle label See expiry date on bottle label See expiry date on vial label See expiry date on cryo vial label Table 1: Overview of available and related products User Guide - Cardiomyocytes Ncardia.com 3

7 Material Container Cell number / quality Example applications (Ax-B-HC02-MPC) Frozen cryo vial 0.25 million cells Patch clamp (Ax-B-HC02-1M) Frozen cryo vial 1 million cells Not specified Frozen cryo vial 4 million cells xcelligence RTCA Cardio (Ax-B-HC02-4M) or CardioECR, CardioExcyte 96, Maestro MEA, 96-well and 384-well plating (e.g., for (Ax-C-HC02-APC) (Ax-C-HC02-FR1) (Ax-B-HC02-FR3) (Ax-C-HC02-96) (Ax-C-HC02-384) (Ax-C-HC02-APL) Pre-cultured T25 flask Pre-cultured T25 flask Pre-cultured T75 flask Pre-cultured 96-well plate Pre-cultured 384-well plate Pre-cultured Maestro MEA calcium transient assays) 0.5 million cells Patch clamp 1 million cells Not specified 3 million cells Not specified Confluent Calcium transient culture Confluent Calcium transient culture QC qualified Maestro MEA signal 96-well plate (Ax-C-HC02-NCE) Pre-cultured 96-well plate for QC qualified signal CardioExcyte 96 CardioExcyte 96 (Ax-C-HC02-EPL) Pre-cultured 96-well plate QC qualified signal xcelligence RTCA Cardio (E-plate 96) for xcelligence Table 2: Overview of available cell product formats and respective example applications* * Table lists product formats required to ensure sufficient cell numbers for respective example applications 3.2 Storage conditions Cryopreserved cells: Upon receipt of cryopreserved, 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. Cells pre-cultured in flasks: Upon receipt, transfer flasks into a sterile hood, aspirate the medium and add 7 ml medium (T25 flask) or 20 ml medium (T75 flask) of fresh pre-warmed Culture Medium. Replace the lid on the flask with the new sterile filter lid provided, and transfer the flasks immediately to a humidified incubator (37 C, 5 % CO2). Fresh cells are by default delivered on a fibronectin coated surface. User Guide - Cardiomyocytes Ncardia.com 4

8 Cells pre-cultured on multi-well plates: Transfer the plate to a sterile hood and carefully remove the sealing mat. Aspirate the medium and add 200 µl (96-well plate) or 50 µl per well (384-well plate) of pre-warmed Culture Medium. Replace the lid with the new sterile lid provided and transfer the plates immediately to a humidified incubator (37 C, 5 % CO2). Medium: Store frozen 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 is not frozen and can be stored at 4 C for up to 4 weeks. 3.3 Required consumables Consumables Vendor Cat. No. Tissue culture flask or multi-well plates Various - Sterile 50 ml polypropylene tubes Various - Sterile reservoir (optional) Various - Fibronectin Sigma-Aldrich F1141 Gelatin solution 2 % in H2O Sigma-Aldrich G1393 (tissue culture grade) (optional) Geltrex hesc-qualified, Thermo Fischer A Scientific Ready-to-use (optional) DPBS, sterile with Ca 2+ and Mg 2+ Sigma-Aldrich D8662 DPBS, sterile with Ca 2+ and Mg 2+ Sigma-Aldrich D8537 Accumax TM solution (optional) Sigma-Aldrich A7089 Trypan blue solution 0.4 % Sigma-Aldrich T8154 Table 3: Overview of required consumables 3.4 Required equipment Item Vendor 37 C water bath Various Laminar flow hood Cell culture incubator (37 C, 95 % humidity, 5 % CO 2 ) Neubauer hemocytometer Centrifuge (swinging bucket rotor) Inverse microscope Liquid nitrogen storage Various Various Various Various Various Various User Guide - Cardiomyocytes Ncardia.com 5

9 8-channel pipette (20 to 300 µl) (optional) Various Table 4: Overview of required equipment 4. Preparations 4.1 Surfaces can be cultured on various surfaces. Plastic adhere best on cell culture-treated plastic surfaces. We recommend to use plastic ware from Nunc (Nunclon Delta Surface) or Greiner (Greiner Bio-One). Glass The attachment of the on glass surfaces (e.g., cover slips for patch clamp or immunostaining) is not as tight as on plastic ware. For additional information on how to improve attachment rates please contact 4.2 Coating Different coating reagents are suitable for culturing. Due to specific assay requirements, a proper choice of coating reagent is recommended (see table 5). Coating agent Assay Comment Fibronectin Standard cultivation Susceptible to shear stress Gelatin Calcium transients CardioExcyte 96 xcelligence RTCA Manual Patch Clamp, Do not vortex and avoid harsh pipetting Store at 4 C; do NOT freeze Not suitable for hypertrophy assays since it induces hypertrophy Cells easily detached for replating using Accumax hypertrophy assays Ensure the gelatin solution is sterile (e.g., autoclave at 121 C at 1 bar for 20 min) Geltrex MEA Results in a very tight cell attachment Detachment is difficult Table 5: Suggested coating agents ATTENTION! Do not directly seed freshly thawed or dissociated in BMCC medium. The cells require the serum contained in Culture Medium in order to attach to the culture surface. Do not spin down the cells! Centrifugation directly after thawing will damage the cells and will lead to cell loss. Make sure that the flasks stand even during seeding to allow for an even distribution of cells. User Guide - Cardiomyocytes Ncardia.com 6

10 Coating with fibronectin 1. Prepare fibronectin coating solution by diluting fibronectin stock solution (1 mg / ml) in DPBS with Ca 2+ and Mg 2+ to a final concentration of 10 µg / ml fibronectin (1:100 dilution). Mix the solution carefully. See Table 6 or corresponding assay-specific handling guides for recommended volumes. 2. Pipette the coating solution into each flask or well of a culture plate and incubate in an incubator at 37 C for 3 h. Alternatively, incubate the coating solution at 4 C overnight. MEA coating with Geltrex MEA coating is not trivial. Please contact support@ncardia.com for detailed instructions. 1. Place sterile MEAs under laminar flow. 2. Transfer an appropriate amount of cold Geltrex Ready-to-use solution in a sterile 50 ml tube on ice. 3. Place a 6 µl drop of Geltrex directly onto the electrode array area of the MEA. 4. Handle the coated MEAs with care to avoid drifting of the Geltrex drop. Incubate the coated MEA for at least 30 min at 37 C in a humidified chamber. Coating with Gelatin 1. Dilute sterile 2 % gelatin solution to 0.1 % in DPBS with Ca 2+ and Mg Place the coverslips or petri dishes in the designated container. 3. Pipette a drop of gelatin solution (< 20 µl) onto the coverslips or petri dishes to cover the center of the slides. For seeding on 96-well or 384-well plates, coat the whole well by adding 50 µl gelatin solution per well. 4. Incubate the coverslips in a humid chamber or well plates in an incubator at 37 C for at least 1 h. Format T75 flask Coating volume 10 ml T25 flask 5 ml T12.5 flask 3 ml 96-well plates 50 μl / well 384-well plates 50 μl / well Table 6: Recommended fibronectin and gelatin coating volumes User Guide - Cardiomyocytes Ncardia.com 7

11 5. Cell Culture Different procedures for culture of cryopreserved prior to performing assays are possible. We recommend an overnight pre-cultivation on fibronectin coated flasks (described in Chapter 5.1.). This procedure allows removal of dead cells prior to seeding on assay-specific plates (e.g., for calcium transient and MEA assays; Chapter 5.4.). This will result in better assay performance and overall data quality. However, if required / preferred, it is possible to seed cells directly onto assay plates after thawing (see Chapter 5.5). 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. Do not use dry ice for the transport because this might affect the viability of the cells. 5.1 Thawing for pre-cultivation The procedure below applies for thawing of vials containing 4 million and pre-cultivation in flasks. 1. Coat a T75 flask with fibronectin (see 4.2.). 2. Transfer 4 ml Culture Medium into a 50 ml tube and pre-warm to 37 C. 3. Prepare a reaction tube with 20 μl trypan blue solution. 4. 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). 5. Disinfect, transfer the vial to the laminar flow hood. Carefully tap the vial to loosen the cell pellet. 6. 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. Gently agitate the tube to mix the cells. During the freezing process it is possible that cardiomyocytes may accumulate at the bottom of the vial. In doing so, these cells may appear as a pellet. This occurrence does not impair the viablility of the cells and it is important NOT to roughly triturate these sedimented cells (it is NOT a dense pellet as is typical upon centrifugation). Harsh trituration of the cells during this procedure may be harmful and will lead to loss of viable cells. The cells typically remain as single cells and are not truly clustered. To bring these cells back into suspension follow these steps: 7. Remove a small volume of media/cell suspension (roughly 1/4 of the volume) from the top of the vial, using a 1000 µl pipette. 8. At the bottom of the vial (near the sedimented cells), eject the media/cell suspension slowly out of the pipette under" the cell pellet you are trying to disperse. 9. The cells should gently lift and disperse and be brought back into suspension. 10. Use the same pipette tip to transfer the cell suspension into the Falcon tube. User Guide - Cardiomyocytes Ncardia.com 8

12 11. To determine cell numbers and viability, transfer 20 µl of the cell suspension into a reaction tube containing 20 µl trypan blue solution and mix gently. 12. Add 15 ml of media to the cell suspension in the 50 ml tube. 13. Remove the supernatant of the coating solution from the T75 flask by aspiration and immediately seed the cells into the flask. Move the flask back and forth for even distribution of the cells in the flask. 14. Immediately transfer the flask with the cells to the incubator. 15. Follow the procedure in chapter 5.2 to determine the number and the viability of the cells. 16. After 12 to 20 h post seeding, conduct a complete media change (the earlier the better). Aspirate the media in the flask and replace with 20 ml fresh pre-warmed Cor.4U Culture Media. Note: Before medium change, it is normal that a larger amount of dead cells are visible in the culture medium. These dead cells will be almost completely removed by the media change. 17. Cells can be dissociated and re-plated to the desired formate after over night cultivation and 2 h after media change (see Chapters 5.3. and 5.4.). 5.2 Counting of cells and determination of viability 1. 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. 2. Count the number of cells in each of the four outer squares highlighted in red in figure 1. Calculate the mean number of cells per red square. 3. Calculate the number of trypan blue negative cells corrected by chamber factor (1 x 104), dilution factor(2), and total volume (e.g., 5 ml). 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 Figure 1. Neubauer hemocytometer 4. Calculate the viability as follows: Number of viable cells / number of dead cells + viable cells x 100 = % viability User Guide - Cardiomyocytes Ncardia.com 9

13 5.3 Detachment After an overnight culture and 2 hours after medium exchange, pre-cultured are ready for detachment. Pre-culture of may be extended up to 3 days without loss of efficiency of the dissociation and cell quality. A longer pre-culture will impair the dissociation and will lead to cell loss and an impaired viability. 1. For one T75 flask, equilibrate 5 ml Accumax to room temperature before use, do not warm to 37 C (activity of the enzyme decreases rapidly at 37 C). 2. Prepare a reaction tube with 20 µl trypan blue solution. 3. Wash the cells with DPBS without Ca 2+ and Mg Pipette 5 ml Accumax into the flask and incubate the cells for 5 to 10 min at 37 C in the incubator. 5. Check the detachment process of the cells: After 5 min the cells usually start to detach, but tend to stay clustered. It is beneficial to incubate them for additional 3 to 5 min at 37 C to achieve a single cell suspension. Do not incubate longer than 10 min. 6. Once a single cell suspension is achieved, or at the latest after 10 min, add 5 ml Culture Medium to the T75 flask to stop the dissociation process and transfer the cell suspension to a 50 ml tube. 7. Carefully detach remaining cells from the flask surface by rinsing the surface of the flask with additional 5 ml Culture Medium and transfer the solution to the 50 ml tube containing the cell suspension. 8. Centrifuge the cell suspension for 3 min at 180 x g, and aspirate the supernatant. 9. Carefully resuspend the cell pellet in 1 ml Culture Medium and transfer 20 µl of the cell suspension into the reaction tube with trypan blue solution. 10. Determine the cell number and viability according to Chapter 5.2. The cell viability should be 90 %. 11. Adjust the the cell density with Culture Medium according to the requirements of the assay plate format (see table 7). ATTENTION! Do not force the cells to detach by pipetting. This harsh procedure will harm and stress the cells. 5.4 Seeding into multi-well plate Proceed with seeding of according to assay-specific protocols or see general seeding instructions for standard multi-well formats as described below. Please note that the measures of the wells of specific assay plates like the xcelligence E-plate vary from standard cell culture plates and there- User Guide - Cardiomyocytes Ncardia.com 10

14 fore require adjustment of the seeding cell densities and the culture medium volumes. 1. Adjust the cell suspension (i.e., viable cells per ml) adequately with Culture Medium depending on the suggested assay conditions and mix the suspension carefully (see Table 7 for recommended seeding densities). 2. Transfer the coated plates to the laminar flow hood. 3. Mix the cell suspension by gently agitating the 50 ml tube; transfer the cell suspension to a sterile reservoir. 4. Remove coating by aspiration (using a 8-channel aspirator for 96- and 384 wells) immediately prior to seeding. Do not let the coating dry. 5. Plate cells using a manual or electrical 8 channel pipette (for 96- and 384-wells) and a one channel pipette for 24- and 48- wells. Move reservoir back and forth to mix cells every 4 rows. Make sure that the plate is in a horizontal position on the bench when seeding, otherwise you run the risk of having an uneven distribution of cells. Keep the tips of the pipette close to the bottom of the plate when seeding; this reduces the risk of air bubbles. 6. After seeding, let plates stand for 30 minutes under the hood to achieve an even distribution of the cells within the wells to avoid edge effects. 7. After 30 minutes, transfer plates carefully to the incubator. Volume of cell suspension / Cell Cell density Cell Cell density Format well number 1 (10 6 cells / ml) 1 number 2 (10 6 cells / ml) 2 24-well 1000 μl 120, , well 500 μl 60, , well 200 μl 20, , well 50 μl 8, , Seeding pre-cultured cells (Chapter 5.4) 2 Direct seeding (Chapter 5.5) Table 7: Overview of recommended seeding densities in different standard formats to obtain confluent monolayers Thawing of a cryo vial with 4, 1 or 0.25 million cells 1. Coat a flask or multi-well plate of desired format with fibronectin or other desired coating (see 4.2.). 2. Transfer 4 ml Culture Medium to a 50 ml tube and warm to 37 C. 3. Prepare a reaction tube with 20 µl trypan blue solution. 4. Quickly transfer the cells from liquid nitrogen directly into a 37 C water bath. Thaw the vial until the frozen cell suspension detaches from the bottom of the vial and only a small clump of ice is visible (approx. 45 sec to 2 min, dependent on vial size and frozen cell volume). 5. Disinfect and transfer the vial to the laminar flow hood, and carefully tap the cryo vial to loosen the cell pellet. User Guide - Cardiomyocytes Ncardia.com 11

15 6. For vials with 1 or 0.25 million cells (skip this step for vials with 4 million cells): Add 0.5 ml of Culture Medium to the vial, and mix the cell suspension very gently. For cell counting, remove 20 µl cell of the suspension and transfer it to a tube containing 20 µl trypan blue solution; mix gently. For vials with 4 million cells (skip this step for vials with 1 or 0.25 million cells): Carefully transfer the cells with a 1000 µl pipette to the 50 ml tube with pre-warmed medium. The total volume is now 5 ml. Mix the cell suspension by gently agitating the 50 ml tube. For cell counting, remove 20 µl cell of the suspension and transfer it to a tube containing 20 µl trypan blue solution; mix gently. 7. Follow the procedure in Chapter 5.2. to determine the number and viability of the cells. 8. Dilute cells with Culture Media to the desired seeding density. (See Table 8 for seeding into cell culture flasks; see Table 7 for seeding into multi-well plates; also see specific application protocols - Chapter 7. Related documents). 9. For seeding of into multi-well plates proceed to Chapter For seeding into flasks aspirate coating and immediately add your diluted cell suspension. Transfer flasks immediately to the incubator (37 C, 5 % CO2). Vial size Plating format Cell suspension Culture medium volume volume 4 x 106 cells T75 flask 20 ml 20 ml 1 x 106 cells T25 flask 7 ml 7 ml 0.25 x 106 cells T12.5 flask 5 ml 5 ml Table 8: Suggested plating formats (flasks) 5.5 Puromycin treatment are puromycin resistant and survive exposure of 2 µg / ml puromycin for up to 24 h (longer exposure maybe be toxic). The puromycin selection step prevents the outgrowth of fibroblasts which can occur during long-term experiments. Fibroblast growth is slow especially in confluent monolayers and does not interfere with short-term experiments. When planning long-term experiments exceeding two weeks (especially when cultured in low density), a puromycin treatment can be carried out to avoid fibroblasts outgrowth. The puromycin treatment should be started earliest after cells have started regular beating (24 to 48 h after seeding) and is only performed in flasks (i.e., not in 96- or 384-well plates). Prophylactic puromycin treatment is only effective if done within the first 72 h of initial culture. 1. Warm the necessary amount of Culture Medium to 37 C and add puromycin to a final concentration of 2 µg / ml. 2. Aspirate the medium carefully and add the medium with puromycin; avoid pipetting directly User Guide - Cardiomyocytes Ncardia.com 12

16 onto the cells. 3. Transfer the dish back into the incubator and incubate the cells over night, at maximum for 24 h. 4. Replace the medium with pre-warmed Culture Medium without puromycin. 5. Continue incubation at 37 C, 5 % CO2. Inspect the cells frequently and change the medium regularly. 5.6 Maintenance We recommend to perform a 100 % medium change 24 h after seeding to remove remaining DMSO from the freezing medium. The subsequent medium change procedure depends highly on the culture vessel and assay used. When are seeded into culture flasks, the medium should be changed every 2 to 3 days to maintain a healthy ratio of medium to cells. When are seeded into culture plates or specific assay plates, the medium should be changed on a daily basis due to a much lower ratio of medium to cells compared to flasks. Please also follow the instructions in the corresponding assay-specific protocols (available at 6. References 1. 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): Axiogenesis AG Patent WO A1, A non-viral system for generation of induced pluripotent stem (ips) cells. 7. Related Documents 1. in calcium transient assays - Application protocol 2. in microelectrode array (MEA) assays - Application protocol 3. in CardioExcyte 96 assays - Application protocol 4. in xcelligence RTCA Cardio assays - Application protocol 5. in xcelligence RTCA CardioECR assays - Application protocol 6. in manual patch clamp assays - Application protocol 7. in automated patch clamp assays - Application protocol User Guide - Cardiomyocytes Ncardia.com 13

17 8. Troubleshooting Guide Problem Not enough cells in vial after thawing Comment and suggestion Were all cells transferred to the 50 ml falcon? It is important to loosen the pellet in the vial before transferring the cells to the 50 ml tube; otherwise a large amount of cells may be retained in the cryo vial. Were the cell suspension properly mixed before withdrawing a sample for counting? Too many dead cells after thawing 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 are stored at higher temperatures (e.g., in a -80 C freezer). Storage or transportation on dry ice impairs the viability of. 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. What was the workflow during thawing? Work speedily. 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 centrifuged after thawing? This causes pronounced cell death. Too many dead cells after dissociation Which dissociation enzyme was used? How long was the incubation time? Trypsin / EDTA induces pronounced cell death after 5 min of incubation. Were the cells exposed to shear force, e.g., by harsh pipetting? User Guide - Cardiomyocytes Ncardia.com 14

18 Cells do not attach Which plastic material was used? Which coating material was used? 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. Geltrex polymerizes when stored and/or pipetted at room temperature. Were the cells stored properly after arrival? Were the cells thawed according to protocol? Wrong storage and wrong thawing procedure can influence the attachment capacity of the cells. Bubbles occur during coating and / or seeding of cells into 96- or 384-well plates Make sure to avoid any electrostatic charge. Take microplates out of the package at least 24 hours before seeding. 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 the cell layer may peal off the plate. Were plates or flasks with cell culture-treated plastic used? 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 ca. 30 minutes after return to the 37 C incubator. Was the medium changed regularly? A lack of nutrition will result in arrest of beating. User Guide - Cardiomyocytes Ncardia.com 15

19 Too many or too few cells in the culture dish/ plates/well You must use a manual hemocyotmeter: automated cell counters are not designed to analyze the smaller sized and often multinucleated cardiomyocytes. Do your cell counts make sense for how many cells should be found within a given vial (e.g. ~ 10+ million cells when there should be closer to 4)? A list of Frequently Asked Questions (FAQ) is available at 9. 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, non-transferable 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 Ncardia for a license for disease and tissue models at patent@ncardia.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. User Guide - Cardiomyocytes Ncardia.com 16

20 For information on commercial licensing, contact the Evrogen Licensing Department at: 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. xcelligence is a registered trademark of ACEA Biosciences, Inc. For information on the patents, patent applications, and licenses associated with the product contact the Ncardia Business Development Department at: patent@ncardia.com. 10. Appendix Cor.4U morphology in culture Figure 2. Cor.4U morphology one day after thawing. 4 million cells were plated on a fibronectin coated T75 flask. (Magnification 100x and 200x). User Guide - Cor.4U Cardiomyocytes Ncardia.com 17

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