axion Protocol Cell Culture on Microelectrode Arrays Cell Type: Axiogenesis - Cor.4U Human ipsc-derived Cardiomyocytes BioSystems v. 1.

Transcription

1 axion BioSystems Cell Culture on Microelectrode Arrays Cell Type: Axiogenesis - Cor.4U Human ipsc-derived Cardiomyocytes Protocol v. 1.1

2 Cor.4U Human ipsc-derived Cardiomyocytes from Axiogenesis axion BioSystems Introduction Axiogenesis Cor.4U Cardiomyocytes are human induced pluripotent stem cell-derived (ipsc) cardiomyocytes that exhibit typical biochemical, electrophysiological, mechanical, and pathophysiological characteristics of native human cardiac myocytes. Due to their human origin, high-purity, functional relevance, and ease of use, Cor.4U Cardiomyocytes represent an optimal test system for interrogating cardiomyocyte biology in basic research and many areas of drug development. The Maestro multielectrode array (MEA) system from Axion Biosystems is a non-invasive, label-free platform that measures local field potentials of electrically active cells, representing summed activity of underlying ion channels. Cor.4U Cardiomyocytes can be cultured on MEAs to form an electrically and mechanically active stabile syncytium amenable to electrophysiological interrogation. Together, Cor.4U Cardiomyocytes and Axion s MEA technology form an excellent, non-invasive platform for in vitro screening of compound effects on human cardiomyocyte physiology. This culture protocol describes how to handle Cor.4U Cardiomyocytes for use on the Maestro MEA system and provides basic instructions for compound treatments, data acquisition, and analysis. The cell culture protocol begins on page 3, example platings can be found on page 6, and a complete list of required materials can be found on page 7. Workflow A 1M vial of Cor.4U Cardiomyocytes are initially thawed onto standard T-25 tissue culture flasks coated with fibronectin. On day 2 post-thaw, the cells are dissociated and plated into Axion BioSystems MEA plates with fibronectin. After 5 days on the MEA, cells are ready for experimentation, and can maintain stable beating for at least 2 additional weeks with daily media changes. Handling of human ipsc cardiomyocytes during MEA plating procedures requires stringent sterile technique. To prevent bacterial contamination during handling-intensive steps, antibiotics may be added during the initial pre-plating and/or for the media addition directly following the seeding of cells to the MEA surface. Antibiotics should be removed for all other steps (e.g. routine culture steps/media changes, drug treatments, etc). We recommend the use of Ciprofloxacin or biosimilar due to its relatively low affinity for herg (~ 1 mm IC50). A working concentration of 2-10 µg/ml is suggested. Coat T-25 Tissue-Culture Flask and Thaw Cells Dissociate and Transfer to Axion MEA Plates Begin Compound Screening Days in Culture Replace Medium Daily Axion BioSystems Axiogenesis Cor.4U Human ipsc-derived Cardiomyocytes on Microelectrode Arrays 2

3 Notes: Methods Culturing Cryopreserved Cor.4U Cardiomyocytes 1. Coat one T-25 tissue culture flask with fibronectin (1:20 in PBS, final concentration of 50 µg/ml) for 3 hours according to the Cor.4U Cardiomyocytes User s Guide. Reconstitute fibronectin in sterile water at 1 mg/ml according to the manufacturer s instructions. Aliquot and store at -20 C, and dilute to 50 µg/ml the day of use. 2. Thaw 1 vial of Cor.4U cardiomyocytes according to the user guide. 3. Aspirate the fibronectin solution. Without centrifugation, gently (using a pipette) evenly distribute the entire cell suspension into the T-25 flask. 4. Incubate the cells for 3 hours at 37 C, 5% CO 2 and 95% humidity in Thawing Medium. 5. Warm Cor.4U Culture Medium to 37 C. 6. Inspect the cells under a microscope. The majority of the cells should have attached to the surface and formed a monolayer; some floating (dead) cells are normal and will be removed with the subsequent medium change. 7. For each T-25 flask (do not perform these steps simultaneously), carefully aspirate and replace the medium from the T-25 flask with the Cor.4U cells. Do not touch the surface of the flask Make sure to aspirate the supernatant only from the surface of the liquid at slowest speed. 8. Add 5 ml of warm Cor.4U culture medium to the flask at slowest speed and avoid pipetting over the culture area of the flask Culture the cells overnight at 37 C, 5% CO 2, and 95% humidity. 9. Maintain the cardiomyocytes according to the User s Guide for 2 days, replacing the medium every day. Preparing the MEA Plate 10. Place an 8 μl droplet of fibronectin over the recording electrode area of each well in the MEA plate. See Figure 1 on page 5 for appropriate drop placement. 11. Incubate the fibronectin-coated MEA plate in a cell culture incubator at 37 C, 5% CO 2 for at least 60 minutes. Axion BioSystems Axiogenesis Cor.4U Human ipsc-derived Cardiomyocytes on Microelectrode Arrays 3

4 Notes: Tip Longer incubation times are acceptable; however, the droplet of fibronectin should not be allowed to evaporate to avoid impacting proper cell attachment. Water may be added to the area outside of the wells to prevent the droplet from drying out. Collecting Cor.4U Cardiomyocytes from the T-25 Tissue Culture Flask 12. Warm Cor.4U Cardiomyocytes Culture Medium in a 37 C water bath. 13. Dilute 0.5% trypsin solution in PBS to a final concentration of 0.25%. Warm the 0.25% trypsin solution in a 37 C water bath for 10 minutes before use. 14. Gently aspirate the Culture Medium from each well containing Cor.4U cardiomyocytes. 15. Rinse the cardiomyocytes once with 5 ml of room temperature PBS without Ca 2+ or Mg 2+, discard the PBS, and then add another 5 ml of PBS with 2 mm EDTA and incubate in a cell culture incubator at 37 C, 5% CO 2 for 5 minutes. 16. Gently aspirate the PBS, and add 3 ml of 0.25% trypsin to the flask. Incubate in a cell culture incubator at 37 C, 5% CO 2 for 2 minutes. To ensure a consistent dissociation, it is recommended to dispense a stock of 0.5% trypsin solution into singleuse aliquots and store frozen until use. Thaw the 0.5% trypsin aliquot(s) at 4 C overnight. Always use freshly prepared 0.25% trypsin and do not warm trypsin in a 37 C water bath for prolonged periods of time. 17. Quickly wash the cardiomyocytes from the surface using a 1 ml pipettor by tilting the flask and repeatedly aspirating and dispensing the trypsin solution over the surface 2-3 times. 18. Rotate the plate 180 and repeat the previous step. Take care that the incubation with 0.25% trypsin solution is as short as possible (2 min or less is recommended). Incubation for longer than 5 min is not recommended and will cause loss of viable cells. 19. Transfer the cell suspension into a 15 ml centrifuge tube containing 3 ml of warm Culture Medium. 20. Rinse the plate with 2 ml of Culture Medium using a 5 ml serological pipette and add this to the 15 ml centrifuge tube suspension. Avoid over Axion BioSystems Axiogenesis Cor.4U Human ipsc-derived Cardiomyocytes on Microelectrode Arrays 4

5 Notes: A. trituration and introduction of air bubbles. The main goal is to detach the cardiomyocytes from the bottom of the flask before the rinsing step. Do not attempt to individualize the cardiomyocytes and avoid introducing air bubbles during the repeated rinses. 21. Centrifuge the cardiomyocyte suspension at 200 x g for 3 minutes. B. 22. Aspirate the supernatant, being careful not to disturb the cardiomyocyte pellet. 23. Resuspend the cardiomyocyte pellet in 300µL of Culture Medium and mix gently. 24. Count the cardiomyocytes using a hemocytometer. C. 25. Dilute the cardiomyocytes with Culture Medium to 2,500 viable cardiomyocytes/μl. Be mindful of cardiomyocyte viability after dissociation. Low viability (<70%) after dissociation could lead to excess debris and poor performance of the cardiomyocyte monolayer on the MEA Plating Cor.4U Cardiomyocytes onto the MEA Figure 1: Drop Placement Diagram The layouts above represent the bottom surfaces of wells in (A) a 12-well MEA, (B) a 48-well MEA, and (C) a 96-well MEA. The number of electrodes per well is different across the plate formats, however the drop placement is the same, with the drop (red circle) centered on the recording electrodes and staying within the ground electrodes. 26. Aspirate the fibronectin solution from the MEA. 27. Place an 8 µl droplet of Cor.4U Cardiomyocytes suspension over the recording electrode area of each well of the MEA. See Figure 1 on page 5 for appropriate drop placement. Timing is critical in this step. Cardiomyocyte attachment is compromised if the fibronectin is allowed to dry. Under typical conditions, the well will begin to dry within a few minutes after aspiration of the excess fibronectin solution. At this point the residual fibronectin in the well will begin to crystallize, turn white, and the well should then be ignored as cardiomyocyte attachment will be suboptimal. The cardiomyocytes will begin to settle quickly, requiring that the centrifuge tube be mixed frequently and gently throughout this step to ensure an even distribution. Axion BioSystems Axiogenesis Cor.4U Human ipsc-derived Cardiomyocytes on Microelectrode Arrays 5

6 Notes: 28. Incubate the MEA plate with the seeded cardiomyocytes in a cell culture incubator at 37 C, 5% CO 2 for 3 hours. Tip Place the seeded MEA plate in an extra-humidified box within a cell culture incubator at 37 C, 5% CO 2 for 3 hours. Prolonged incubation without increased humidification may cause evaporation of the droplet and loss of cell viability. 29. Gently add 1/2 of the final volume of Culture Medium to each well of the MEA. Adding the medium too quickly will dislodge the adhered cardiomyocytes. Recommended well volumes for each plate type are: 12-well = 500µL, 48-well = 300µL, 96 well = 200µL. Using a pipettor, add medium first in a semi-circle along the outer edge of the flat bottom area. Progressively add medium so it fills evenly towards the center, stopping before contact is made with the droplet in the center. Gently bridge the gap with additional medium. The goal is to prevent a rush of medium in either direction that might dislodge the cardiomyocytes. 30. Repeat the step 25 a second time to reach the final recommended volume of Culture Medium. 31. Incubate in a cell culture incubator at 37 C, 5% CO For optimal cell health, be sure to exchange medium daily. Though cardiomyocyte beats may be detectable within 24 hours, optimal Na+ spike and T-wave amplitudes are typically achieved after 5 days in culture. Axion BioSystems Axiogenesis Cor.4U Human ipsc-derived Cardiomyocytes on Microelectrode Arrays 6

7 Visualization of Typical Cardiomyocyte Seeding Results Figure 2: Cor.4U Cardiomyocyte Morphology Human ipsc-derived Cardiomyocytes (20,000) at day 4 in vitro in a 12-well MEA, 10x magnification. Notice that cardiomyocyte morphology is easily recognizable. Figure 3: Cor.4U Cardiomyocyte Morphology and Placement Human ipsc-derived Cardiomyocytes (20,000 in a 8 µl dot) at day 4 in vitro in a 12-well MEA, 4x magnification. Notice the dotting method described above confines cells to the area indicated by the dotted blue line surrounding the grid of circular electrodes. *Axion can provide transparent 48-Well blank plates with no electrodes to confirm cellular adhesion. Axion BioSystems Axiogenesis Cor.4U Human ipsc-derived Cardiomyocytes on Microelectrode Arrays 7

8 Required Materials Consumables Item Vendor Catalog Number Cor.4U Cardiomyocytes Axiogenesis AG Ax-B-HC02-1M (or 4M) Cor.4U Thawing Medium Axiogenesis AG Ax-M-TMC-10 Cor.4U Culture Medium Axiogenesis AG Ax-M-HC250 Puromycin Axiogenesis AG EDTA 0.5M Life Technologies PBS without Ca 2+ and Mg 2+ Life Technologies Fibronectin Roche % Trypsin Life Technologies Ciprofloxacin or similar (optional) Sigma KimWipes Pipettes and Pipettors 15 ml and 50 ml Centrifuge Tubes Pipette Aid and Sterile Pipettes Equipment Item Vendor Catalog Number Maestro MEA System Axion BioSystems 12-Well MEA Axion BioSystems M768-GLx 48-Well MEA Axion BioSystems M768-KAP Axion Integrated Studio (AxIS) Axion BioSystems 37 C Water Bath Cell Culture Incubator Hemocytometer or Automated Cell Counter Biological Safety Cabinet Tabletop Centrifuge Phase Contrast Microscope Liquid Nitrogen Storage Links to Resources Axion BioSystems Technical Support Applications Axion BioSystems Axiogenesis Cor.4U Human ipsc-derived Cardiomyocytes on Microelectrode Arrays 8

9 Trademarks Axion BioSystems, Inc. and the logo are trademarks of Axion BioSystems, and may not be used without the express written permission of Axion BioSystems, Inc. All other brands, product names, company names, trademarks and service marks are the properties of their respective owners. Restrictions and Liabilities This document is provided as is and Axion BioSystems will assume no responsibility for any typographical, technical or other inaccuracies in this document. Axion BioSystems does not make any commitment to provide any changes, updates, enhancements, or other additions to this document to you within any time frame or at all. This document might contain references to third-party sources of information, hardware or software, products or services and/or third-party websites (collectively the Third-Party Information ). Axion BioSystems has no control over and is not responsible for any Third-Party Information, including, without limitation the content, accuracy, copyright compliance, compatibility, performance, trustworthiness, legality, decency, links or any other aspect of Third-Party Information. The inclusion of Third-Party Information in this document does not imply endorsement with your use of Axion BioSystems technology. Conditions of Use You are responsible for understanding and performing the protocols that are described within. Axion BioSystems makes no guarantee for any results you may achieve. These protocols are provided as a recommendation by Axion BioSystems based on its use and experience. Origin Axion BioSystems Microelectrode Arrays are manufactured in the United States of America. Copyright Notice 2014 Axion BioSystems, Inc. All rights reserved. This document may not be reproduced, distributed, modified or publicly displayed without the express written permission of Axion BioSystems. Acknowledgement axion BioSystems Axion BioSystems would like to thank Axiogenesis AG for providing their experience and resources toward the creation of this application note. Axion BioSystems Axiogenesis Cor.4U Human ipsc-derived Cardiomyocytes on Microelectrode Arrays 9