Cor.4U in microelectrode array (MEA) assays

Transcription

1 Cor.4U in microelectrode array (MEA) assays Application protocol Last update: Oct Europe Nattermannallee 1/S Cologne, Germany North America 600 W Germantown Pike, STE 110 Plymouth Meeting, PA 19462, USA

2 TABLE OF CONTENTS 1. GENERAL INFORMATION 3 2. SAFETY INFORMATION 3 3. MATERIAL Cells and media provided by Axiogenesis Storage conditions Required consumables Required equipment 6 4. PREPARATIONS Surfaces Coating 7 5. CELL CULTURE Thawing for pre-cultivation Cell counting and viability determination Detachment Seeding Maintenance MEA RECORDINGS REFERENCES RELATED DOCUMENTS TROUBLESHOOTING GUIDE AXIOGENESIS LIMITED LABEL USE LICENSE APPENDIX Example of Cor.4U activity in a MEA well Examples of typical activity maps from Maestro MEA measurements 18!2

3 1. GENERAL INFORMATION This protocol covers thawing, seeding and dissociation of Axiogenesis Cor.4U cardiomyocytes for use on Microelectrode Arrays (Multi Channel Systems, Reutlingen Germany; Axion BioSystems, Atlanta USA). The protocol is set up according to the condition of purchased Cor.4U (cryopreserved or pre-seeded) and the type of Microelectrode Arrays (1-well, 6-well, 9-well, 12-well, 48-well, 96-well MEAs). It is recommended to use the cells on day 3 post-thaw, as the cells develop a syncytium and are beating synchronously at this time. Please read the entire protocol before you start the experiment. Cor.4U cardiomyocytes are produced through 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 (~100 % purity) express cardiac-specific proteins, e.g. cardiac alpha-actinin and connexin-43, an indication of the ability for electric coupling of these cells. Patch clamp analyses, as well as multi-electrode array (MEA) recordings, demonstrate the normal electrophysiological properties of these cells. Cor.4U cardiomyocytes are particularly useful for cell-based in vitro assays in pharmacology, safety, and toxicology. These cells are ideal for electrophysiological applications as well as for high content and high throughput screening applications. 2. 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 therefore genetically modified organisms (GMO). They 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 at a gauge pressure of 1 bar. Cor.4U 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. 3. MATERIAL 3.1. Cells and media provided by Axiogenesis Cor.4U cardiomyocytes are available in different cryopreserved as well as precultured formats suitable for performing MEA assays (see table 1). The latter can be delivered either in culture flasks or ready-to-use in 96- Maestro MEA plates.!3

4 Cor.4U cardiomyocytes are offered with Cor.4U Culture Medium or 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. 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 survival and function is not compromised by long-term culture in presence of Ciprobay. Table 1: Overview of MEA suitable Cor.4U products Material Container Contents Storage Shelf life Cryopreserved Cor.4U (Ax-B-HC02-4M) Cryopreserved Cor.4U (Ax-B-HC02-1M) Pre-cultured Cor.4U (Ax-C-HC02-FR1) Pre-cultured Cor.4U (Ax-C-HC02-FR3) Pre-cultured Cor.4U (Ax-C-HC02-APL) Cor.4U Culture Medium (Ax-M-HC250) BMCC medium (serum-free) (Ax-M-BMCC250) Axio-Supplement (Ax-M-CB-5) Cryo vial 4 million cells Liquid nitrogen Cryo vial 1 million cells Liquid nitrogen Pre-cultured T25 flask Pre-cultured T75 flask Pre-cultured Maestro MEA 96-well Bottle Bottle 1 million cells Incubator 37 C 3 million cells Incubator 37 C Plate with cells and QC tested signal 250 ml medium 250 ml medium Incubator 37 C Frozen -20 C / Refrigerated 4 C Frozen -20 C / Refrigerated 4 C Cryo vial 250 µl Liquid RT, dark Max. 1.5 years from issue date on CoA Max. 1.5 years from issue date on CoA See expiry date on bottle label See expiry date on bottle label See expiry date on cryo vial label!4

5 Table 2: Overview of recommended Cor.4U products for different formats of MEA plates MEA formate Amount Cor.4U product 96-well 2 1 million cryovials 1 4 million cryovials (enough for 2 x 96-well MEA plates) 1 T75 flask 3 million cells 48-well 1 1 million cryovials 1 T25 flask 1 million cells 12-well, 6-well, 1-well 1 1 million cryovials (enough for 48 wells) 1 T25 flask 1 million cells (enough for 48 wells) 3.2. 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. 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 Cor.4U 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 % CO 2 ). Fresh cells are by default delivered on a fibronectin coated surface. Cor.4U in flasks are ready for detachment once a stable beating can be observed, normally the day after delivery. Cells pre-cultured on a Maestro MEA 96-well plate: Directly after arrival please make a measurement of the plate in the Maestro MEA instrument without removing the silicon lid. This is to control that the plate was not damaged during delivery. Then transfer the plate to a sterile hood and carefully remove the sealing mat. Aspirate the medium and add 300 µl of pre-warmed Cor.4U Culture Medium /well. Replace the lid with the new sterile lid provided and transfer the plates immediately to a humidified incubator (37 C, 5 % CO 2 ). Cor.4U in plates are ready for compound treatment once a stable beating can be observed, normally the day after delivery. 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.!5

6 3.3. Required consumables Table 3: Overview of required consumables Item Vendor Cat. No. T25 or T75 tissue culture flask Various - Sterile 50 ml polypropylene tubes Various - Geltrex hesc-qualified, Ready-to-use (when ordered frozen or pre-cultured cells in flasks) Thermo Fischer Scientific A Fibronectin (when ordered frozen cells) Sigma Aldrich F1141 Accumax TM solution (when ordered frozen or pre-cultured cells in flasks) Sterile 10 cm petridish (when using 1-well MEA) Sigma Aldrich Various - A7089 DPBS, sterile with Ca 2+ and Mg 2+ Sigma-Aldrich D8662 DPBS, sterile without Ca 2+ and Mg 2+ Sigma-Aldrich D8537 Trypan blue solution 0.4 % Sigma-Aldrich T Required equipment Table 4: Overview of required equipment Item Vendor 37 C water bath Various Laminar flow hood Various Cell culture incubator (37 C, 95 % humidity, 5 % CO 2 ) Various Neubauer hemocytometer Various Centrifuge (swinging bucket rotor) Various Inverse microscope Various µl single channel electronical multistep pipette E.g. Eppendorf 300 µl 8-multichannel-pipette (for 96-well MEA) E.g. Eppendorf Microelectrode Array MCS, Axion BioSystems Liquid nitrogen storage Various!6

7 Cor.4U Human ipsc-derived cardiomyocytes 4. PREPARATIONS We highly recommend to pre-culture the cryopreserved Cor.4U in a cell culture flask before seeding cells on the MEAs. This procedure allows removal of dead cells prior to seeding on a MEA assay plate and will result in better assay performance and overall data quality Surfaces When pre-culturing Cor.4U cardiomyocytes they should be plated on fibronectin coated cell culture treated plastic flasks. The surfaces on MEAs can vary. Most common is cell culture plastic but also glass is used as surface, for example 1-well MEA (Multichannel system). Some MEAs have been plasma treated to get a more hydrophilic surface, this has shown less advantages for Cor.4U since it leads to a larger spread of the cells over the surface. A highly hydrophobic surface helps concentrate the cells on a small area on the electrodes and there is less risk that coating or cells are drying when applied in a small drop Coating Coating of culture flasks 1. Use a T25 flask for 1 x 106 Cor.4U or a T75 flask for 4 x 106 Cor.4U. Dilute the fibronectin (1 mg / ml) stock 1:100 in DPBS with Mg2+ and Ca2+ to a final concentration of 10 µg / ml. Coating should completely cover the surface, we recommend using 5 ml for a T25 and 10 ml for a T75 flask. 2. Add coating solution to the flask and incubate for 1.5 to 3 h at 37 C or over night at 4 C. Coating of MEA plates MEA coating should be performed on the day of dissociation and plating on MEAs. It is not possible to coat MEA plates a day in advance, due to the low volume of coating, which might dry on plate if coated longer then 3 h. We recommend to incubate coating for 30 minutes. 1. (For 1-well MEA, which has no lid; Place a sterile MEA in a sterile 10 cm petri dish under laminar flow hood. The petri dish act as sterile lid.) 2. For plates; position your plate in an approximately 45 angel by leaning it on for example a pipette box. 3. Transfer an appropriate amount of ice cold Geltrex ready-to-use solution in a pre-cold sterile 50 ml tube on ice. Please refer to table 1 for Geltrex volumes. 4. Place a drop of Geltrex directly onto the electrode array area of the MEA. For plates we recommend to use an electronic multistep pipette to improve working speed. The drop should not spread out, then there is a risk for coating to drying out. 5. Incubate the coated MEA for 30 min at 37 C in humidified chamber inside the incubator. Do not let the bottom of the MEA plate get in direct contact with the moist area, by placing it on a multi well lid in the humidified chamber. 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. ATTENTION: Keep the tube with Geltrex on ice throughout the whole procedure to avoid polymerization! ATTENTION: Avoid touching the electrical field surface of the MEAs with the pipette tip, this might damage the electrodes! ATTENTION: Handle the coated MEAs with care to avoid drifting of the Geltrex drop. ATTENTION: Avoid direkt contact between the bottom of the MEA plate and wet or moist surfaces since there is a risk of corrosion of the contactors.!7

8 Cor.4U Human ipsc-derived cardiomyocytes Table 5: Volume of Geltrex Microelectrode Array Volume of Geltrex (µl / well) 1-well (MCS or Axion BioSystems) 10 6-well (MCS) 5 9-well (MCS) 5 12-well (Axion BioSystems) 5 48-well (Axion BioSystems) 5 96-well (Axion Biosystems) 6 5. CELL CULTURE We recommend an overnight pre-culture of cryopreserved Cor.4U prior to performing assays. This procedure allows removal of dead cells prior to seeding on MEAs and will result in better assay performance and overall data quality Thawing for pre-cultivation Day 1 The procedure below applies for thawing of vials containing 1 or 4 million viable Cor. 4U and pre-cultivation in flasks. 1. Coat a T25 flask for 1 million vial Cor.4U or a T75 flask for 4 million vial Cor.4U with fibronectin, see Transfer 6.5 ml (for 1 million Cor.4U ) or 4 ml (for 4 million Cor.4U ) Cor.4U Culture Medium into a 50 ml tube and warm to 37 C. 3. (Optional: Prepare a reaction tube with 20 µl trypan blue solution for cell.number determination before seeding). 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.45 sec and 2 min, for 1 and 4 million vials respectively). 5. Disinfect and transfer the vial to the laminar flow hood, carefully tap the cryo vial to loosen the cell pellet. For 4 million cells, continue to step, For a 1 million vial, take 0.5 ml of Cor.4U Culture Medium from the 6.5 ml prewarmed media in the 50 ml tube and transfer into the cryo vial. (Optional: For determination of cell number and viability, transfer 20 µl of the cell suspension in the cryo vial into a reaction tube containing 20 µl trypan blue solution and mix gently). Then immediately continue to step million cells; Carefully transfer the cells with a 1000 µl pipette to the prewarmed medium in the 50 ml tube. The total volume is now 7 ml. Gently agitate the 50 ml tube to mix the cells million cells; plate the 7 ml cell suspension on a T25 flask see step 12. 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 lead to cell loss. ATTENTION: Do not force the cells to detach by pipetting. This harsh procedure will harm and stress the cells.!8

9 Cor.4U Human ipsc-derived cardiomyocytes 9. 4 million cells; Carefully transfer the cells with a 1000 µl pipette to the prewarmed medium in the 50 ml tube. The total volume is now 5 ml. Gently agitate the 50 ml tube to mix the cells million cells (Optional); withdraw 20 µl of the 5 ml cell suspension and transfer into the reaction tube containing the 20 µl trypan blue solution and mix gently for the determination of cell numbers and viability.) million cells; Add 15 ml of Cor.4U Culture Media to the cell suspension in the 50 ml tube and plate the 20 ml cell suspension on a T75 flask see step Remove the supernatant of the coating solution from the T75 (4 million cells) / T25 (1 million cells) flask by aspiration and immediately seed the cells into the flask. Move the flask back and forth for even distribution of the cells. 13. Immediately transfer the flask with the cells into the incubator. 14. (Optional: Follow the procedure in chapter 5.2 to determine the number and the viability of the cells.) 15. After 12 to 20 h post seeding, conduct a complete media change (the earlier the better, due to the DMSO content still present in the media). Aspirate the media in your flask and replace with 5 ml (T25) or 20 ml (T75) respectively fresh prewarmed Cor.4U Culture Media. 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. 16. Cells can be dissociated and re-plated to the desired MEA format after over night cultivation and 2 h after media change ; please refer to the detachment protocol for this procedure (see 5.3). For seeding densities see table Cell counting and viability determination 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 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. 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 1. Neubauer hemocytometer 4. Calculate the viability as follows: Number of viable cells / number of dead cells + viable cells x 100 = % viability!9

10 5.3. Detachment Day 2 After an overnight culture and 2 hours after medium exchange, pre-cultured Cor.4U are ready for detachment. Pre-culture of Cor.4U 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. Perform a complete medium change. Aspirate media and replace with 7 ml (T25) or 20 ml (T75) fresh Cor.4U Culture Medium. Incubate for 2 h before detachment of cells. 2. Coat a MEA plate with Geltrex (see chapter 4.3). 3. Equilibrate aliquots of Accumax (1 ml / T25 and 3 ml / T75) to 20 C at room temperature before use, do not warm to 37 C (activity of the enzyme decreases rapidly at 37 C). 4. Prepare two reaction tubes with each 20 µl trypan blue solution for cell number estimation (Tube 1) and (Tube 2). 5. Wash the cells with pre-warmed (37 C) DPBS without Ca 2+ and Mg 2+ (5 ml / T25 and 15 ml / T75). 6. Pipette 20 C tempered Accumax (use 1 ml per T25 flask and 3 ml per T75 flask) into the flask and incubate the cells for 5 to 10 min at 37 C in the incubator. 7. Check the detaching process of the cells: After 5 min the cells normally start to detach, but tend to cluster. Tap on the side of the flask to detach cells, check under the microscope. It is beneficial to incubate them a little longer at 37 C to get single isolated cells. Do not incubate longer than 10 min. 8. Once a single cell suspension is achieved, or at the latest after 10 min, add 1 ml (T25 flask) or 3 ml (T75 flask) Cor.4U Culture Medium to the flask to stop the dissociation process and transfer the cell suspension to a 50 ml tube. 9. Take an 20 µl aliquot for cell counting estimation and add to the reaction tube with trypan blue (Tube 1). 10. Carefully detach remaining cells from the flask surface by rinsing the surface of the flask with additional 1 ml (T25 flask) or 3 ml (T75 flask) Cor.4U Culture Medium and transfer the solution to the 50 ml tube containing the cell suspension. 11. Centrifuge the cell suspension for 3 min at 180 x g, and aspirate the supernatant. 12. While centrifugating cells, estimate the number of living cells by counting cells in Tube1 according to 5.2. (Total volume is 2 ml (for T25) or 6 ml (for T75), and dilution factor 2). 13. Determine the volume you need for dilution of cells to reach a concentration of 2 x 10 6 /ml. Multiply this volume with This is the volume of media in which the cells are resuspended after centrifugation. 14. Carefully resuspend the cell pellet in the volume of Cor.4U Culture Medium calculated in step 13. and transfer 20 µl of the cell suspension into the reaction tube with trypan blue solution (Tube 2, which is then a 1:2 dilution).!10

11 Cor.4U Human ipsc-derived cardiomyocytes 15. Determine the cell number and viability according to chapter 5.2. The cell viability should be %. The concentration of viable cells should be >2 x 106 cells/ ml Seeding The procedure below applies for seeding on a MEA plate. 1. Adjust the the cell density with Cor.4U Culture Medium to 2 x 106 cells /ml (10k cells in 5 µl). Gently mix the cell suspension by carefully swinging the vial back and forth. 2. Aspirate the Geltrex coating solution from the MEAs. 3. Plate cells on the pre-coated MEA plates by adding a droplet (see table 6.) of the cell suspension in the very center of the well, directly on the electrode field of each well. Work quickly so the coating is not allowed to dry. 4. Place the MEA plate in a humidified box, but do not let the bottom of the MEA plate get in direct contact with the moist area by placing it on a multi well lid in the humidified chamber and incubate in an incubator at 37 C for 3 h to allow the cardiomyocytes to attach to the surface. 5. After 3h carefully add appropriate volume of Cor.4U Culture Medium to each well of the MEA plate (see table 6). 6. Culture the cells in an incubator at 37 C, 5 % CO2 in a humidified atmosphere. ATTENTION: If using multi well MEAs, aspirate Geltrex and seed cells well per well to avoid drying of the Geltrex coating, alternatively one row when using a multistep pipette! ATTENTION: Cell should be seeded as a drop on the coated microelectrode area of the MEA! Table 6: Cor.4U@ seeding densities and media volumes Microelectrode Array Cor.4U@ number Volume of cell / well suspension (µl) Volume of medium (ml) 1-well (MCS) 2 x well (MCS) 1 x (rounded well) 0.7 (triangular well) 9-well (MCS) 1 x well (Axion BioSystems) 1x well (Axion BioSystems) 1 x well (Axion Biosystems) 1 x ATTENTION: Synchroneous beating of the Cor.4U@ monolayer appears around day 2 after seeding. MEAs should be checked in the MEA analyzer once a day from day 2 onwards. Compounds can be added as soon as a stable baseline is obtained but it is recommended not to begin earlier than day 3 after seeding Maintenance Day 3 1. Perform a complete medium change every other day. 2. Pre-warm a required amount of Cor.4U Culture Medium to 37 C.!11

12 3. Inspect the cells under a microscope if using a transparent bottom MEA. The majority of the cells should have attached to the surface and formed a contracting cell layer. To achieve a good MEA signal it is important that the cells are positioned in the middle of the electrode field and covering the measuring electrode. 4. Carefully aspirate the medium from edge of the MEA-well and carefully add the required amount of warm Cor.4U Culture Medium on the side of the well. 6. MEA RECORDINGS Start recording not earlier then 1 h after the last media change. If serum free medium is required for compound testing, we recommend serum-free BMCC Medium. Medium change can be performed as described in chapter 5.5. Change from Cor. 4U Culture Medium to serum-free BMCC Medium is sufficient one hour before compound addition. Compounds should be diluted in the media of use. 1. Switch on the Middleman of the Maestro MEA system. 2. Start the AxIS software and switch on the temperature control, set to 37 C. 3. Wait until the device temperature has reached 37 C. 4. Before placing a MEA plate into the Maestro device, dry the bottom of the MEA plate with a smooth tissue. It is important that the plate is dry, when in contact with the measuring electrodes. Humidity on the electrodes will interfere with the measurement and result in a noisy background measurement. 5. Choose your settings for recording, as described in detail in the Maestro handling guide. 6. Before starting a recording, let the cardiomyocytes acclimatize for 2 min. 7. For standard analysis of Cor.4U, we recommend the following settings: Maestro configuration -> cardiac Filter Settings Fig 2. Configuration -> Cardiac Filter & Settings 8. Start data acquisition.!12

13 7. 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. 8. RELATED DOCUMENTS 1. Cor.4U Handling guide 2. AxISUserGuide2.1.pdf (Axion Biosystems) 9. 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 tip on the cryo vial to loosen up the pellet before transferring cells to the 50 ml falcon otherwise a large amount of cells might 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 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. - What 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.!13

14 Problem Too many dead cells after dissociation Cells do not attach Comment and suggestion - 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? - 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. 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 CO 2 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. MEA measurement shows a noisy signal in several wells - Remove plate from MEA device and dry the bottom of the plate with a soft tissue. Humidity on the electrodes disturbs the measurement.!14

15 Problem MEA signal was lost Comment and suggestion - The activity map showed very good signal from most electrodes, but the next day there was no signal or signal from less electrodes. - Was the cell number calculated correctly? When using a higher cell number then recommended on the MEA plate, this can result in a higher contraction force which in term can result in a movement of the cell drop on the plate. Check if the cells are still centered on the electrodes (when removing the media the position of the cells on the plate can be visualized).!15

16 10. 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 SKET.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 11. APPENDIX 11.1.Example of Cor.4U activity in a MEA well a) b) Fig 2.a) Typical signal from one well in a 96 well Maestro MEA plate with 8 electrodes on day 4 after plating. b) Magnification of the signal from one electrode (lower left electrode).!17

18 11.2.Examples of typical activity maps from Maestro MEA measurements a) b) Fig 3. Screenshots of activity maps of Cor.4U plated on a 96 -well Maestro plate a) 1 day after plating and b) Day 4 after plating. The spike amplitude is set at 300µV!18