General Instructions: Handling of Cor.4U human ipsc - derived Cardiomyocytes The different formats of Cor.4U human cardiomyocytes (i.e. 0.25x10 6, 1x10 6, and 5x10 6 ) are designed for specific applications and have been optimized with thawing and seeding protocols. These procedures are described in detail in the designated Cor.4U Application Protocols that can be downloaded from the Axiogenesis website www.axiogenesis.com. The following instruction is designed to give a general overview of the procedures to thaw, seed, and culture Axiogenesis' Cor.4U human cardiomyocytes. 1. Safety Instructions 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 via the Yamanaka protocols from a human skin fibroblast. For questions regarding the fibroblast donor, please email info@axiogenesis.com. The highly pure cardiomyocytes (> 99% 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. The kit is intended for in vitro Research Use Only. The kit is not intended for Diagnostic, Therapeutic or Clinical Use and is 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). Cor.4U cells can be inactivated by autoclaving at 121 C for 20 minutes. Cor.4U cardiomyocytes should be cultured in a sterile environment according to good cell culture and good laboratory practices. 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 CoA and Material Safety Data Sheets (MSDS) for additional safety instructions where applicable. 2. Material 2.1 Reagents Cor.4U cells (cat.-no. Ax-C-HC02-MP, Ax-C-HC02-1M; and Ax-C-HC02-5M). Upon receipt, transfer cells immediately to the vapor phase of liquid nitrogen! Do not keep the cells on dry ice; a prolonged contact with dry ice will damage the cells! 1
Cor.4U Thawing Medium (cat.-no. Ax-M-TMC-10) Cor.4U Culture Medium (cat.-no. Ax-M-HC250) Puromycin stock solution (10mg/ml) Store Cor.4U Culture Media and Puromycin solution at -20 C. Thaw media and Puromycin solution at 4 C overnight and avoid excess exposure to light. Once thawed, keep the reagents at 4 C for up to 2 weeks. 2.2 Requirements Cor.4U cardiomyocytes should be cultured in a sterile environment according to good cell culture and good laboratory practices 1. This requires certain instruments and materials that are not included in the kits; e.g.: Cell culture incubator set to 37 C, 95% humidity and 5% CO 2 Vertical laminar flow hood certified for Level I handling of biological materials Centrifuge with a swinging-bucket rotor for 50ml polypropylene tubes Water bath at 37 C Adjustable Micropipettes, e.g. Eppendorf P1000 and P100; and/or adjustable Multichannel pipettes, e.g. Eppendorf 8-channel P1200 Cell counter (e.g. Neubauer hemocytometer) Inverse microscope with phase contrast or bright field Sterile Tissue Culture flasks and dishes, e.g. T-25 cell culture flasks or multiwell (24w, 96w, 384w) cell culture plates. We highly recommend to use Falcon, Nunc, or Greiner plasticware. Sterile 50 ml polypropylene tubes Coating solutions, e.g. fibronectin (e.g. Sigma F1141) solution. Please refer to the designated Application Protocol for exact coating procedures! Sterile PBS with and w/o Ca ++ and Mg ++ 1x Trypsin/EDTA solution Trypan Blue solution for cell viability staining (e.g. Sigma T8154) 1 A general overview about Good Cell Culture Practice can be found here: http://www.atcc.org/en/documents/learning_center/resources_for_cell_biology.aspx http://www.researchgate.net%2fpublication%2f7583114_guidance_on_good_cell_culture_practice._a_report_of_the_second_ecv AM_task_force_on_good_cell_culture_practice%2Ffile%2F79e4150ec39af4fbaf.pdf&ei=nfouUtn5PMHX0QW5-4DYCA&usg=AFQjCNH0zXqkSpKwamzkTA8b0wZsA7YlzA&bvm=bv.51773540,d.d2k http://www.springerprotocols.com/abstract/doi/10.1007/978-1-61779-077-5_1 2
3. Method 3.1 Thawing and Seeding of Cor.4U Cardiomyocytes A. Coating of cell culture dishes (> 3h prior to thawing of the cells) Cor.4U cardiomyocytes have to be cultured on coated (e.g. Fibronectin) cell culture dishes, please note that coating has to be done at least 3h prior to thawing of the cells. Please refer to the designated protocols for detailed coating instructions. B: Thawing of 1 vial with 1x10 6 Cor.4U cardiomyocytes All formats of Cor.4U cardiomyocytes are thawed using the provided Cor.4U Thawing Medium. This medium is designed to adjust and compensate for the ingredients of freezing solution. Here, we describe the procedure for the standard vial format of 1x10 6 viable Cor.4U cardiomyocytes. Please refer to the designated protocols for detailed instructions on the thawing procedure, since this varies with the different formats! 1. Pipette 6 ml of Cor.4U Thawing Medium into a 50ml tube, add 3 µl of the puromycin stock solution, and warm to 37 C. Immediately before thawing the cells, pipette 3 ml of the warm Thawing Medium with puromycin into a fresh 50 ml tube. 2. Transfer the cells from the liquid nitrogen directly to the laboratory (using dry ice box), making sure that the cells are transferred as quickly as possible. Disinfect the outside of the vial and transfer it to the laminar flow hood. 3. Open the vial and immediately pipette 0.5 ml pre-warmed Cor.4U Thawing Medium from the first tube into the vial. 4. Tightly screw the vial, and soak it immediately into a 37 C waterbath until the frozen cell suspension detaches from the bottom of the vial (about 40 sec), dry and disinfect the vial and transfer it back into the laminar hood. 5. Transfer the cell suspension from the vial into the second 50 ml tube containing 3 ml prewarmed Cor.4U Thawing Medium using a P1000 pipette. Rinse the cell vial with 1ml Cor.4U Thawing Medium from the first tube and transfer to the second 50 ml tube containing the cell suspension; this now has a total volume of 5 ml. Do not centrifuge the cell suspension! 6. If necessary, perform a live cell counting as described in the designated Cor.4U Application Protocols. Adjust the cell suspension to the desired density using Cor.4U Culture Medium. C: Seeding of Cor.4U cardiomyocytes Cor.4U cardiomyocytes can be seeded either directly in the desired format (e.g. a multiwell plate); or pre-cultured in a TC flask or dish for a few days before used in the desired application. The latter is a necessity with applications like automated Patch Clamp, where no direct use is possible, but also helpful if the desired format or application does not allow for a stable culture over several days, e.g. Micro Electrode Arrays. Please refer to the designated protocols for detailed instructions on the seeding densities! 3
For direct seeding of the Cor.4U cardiomyocytes, it has to be decided whether the cells should be confluent, or sub-confluent. This is determined by the application: if single cells or clusters of cardiomyocytes are desired, the seeding density should be sub-confluent; if a synchroneously beating monolayer of cardiomyocytes is desired, a confluent culture has to be seeded. It is important to know that only those cells that are connected via gap junctions will beat synchroneously within a culture! For any pre-culture that requires the enzymatic detachment of the Cor.4U cardiomyocytes, a sub-confluent culture should be used. Please note that preculture and detachment of Cor.4U cardiomyocytes will always result in a loss of cells. Dish / Multiwell Plate Surface Cor.4U cells per Volume of area (cm 2 ) unit (x 10 6 )* Medium (ml) 35 mm dish 11.78 1 3 60 mm dish 21.29 2 5 1 well of a 6 well plate 9.62 1 3-4 1 well of a 12 well plate 3.80 0.35 2 1 well of a 24 well plate 2.00 0.2 1 1 well of a 48 well plate 0.75 0.075 0.5 1 well of a 96 well plate 0.32 0.02 0.2-0.25 3.2 Maintenance of Cor.4U Cardiomyocytes Cor.4U cardiomyocytes can be used for most types of experiments within the first 4 days after thawing, but can be kept in culture for several weeks, if desired. To keep the cells in a metabolically stable and physiologically normal state during the culture time, the culture medium has to be replaced at certain intervals. Please refer to the designated protocols for detailed instructions on the (pre)-culture times as well as medium exchange intervals! The first medium change is always done at the day after thawing; this will remove the last residues of the freezing solution, as well as remaining dead cells and cell debris from the thawing procedure. It is most important not to touch the cell layer when aspirating the old medium, and to avoid pipetting directly onto the culture surface, because this will result in damage of the cell layer, and possible loss of cells. It is highly recommended to check all Cor.4U cardiomyocyte cultures on a daily basis using a microscope, and to take pictures to keep record of the cultures. Synchroneous beating of Cor.4U monolayers appears usually around day 2 post thaw. It is normal to have floating dead cells and cell debris within the cultures prior to the first medium change; these cells are removed with the subsequent media changes. 4
3.3 Detachment of Cor.4U Cardiomyocytes The detachment of Cor.4U cardiomyocytes after a preculture period is described in the designated Application Protocols, e.g. for seeding onto Micro Electrode Arrays (MEA), or for the use in automated Patch Clamp Instruments. In general, depending on the experimental set-up and aims, it is possible to dissociate the Cor.4U cardiomyocytes between day 3 and 7 after thawing. It is not recommended to dissociate the cells prior day 3 or after day 7! 4. Associated Cor.4U Application Protocols All Cor.4U Application Protocols can be downloaded from the Axiogenesis Website www.axiogenesis.com Cor4U-protocol_ManualPatchclamp.pdf Cor4U-protocol_Preculture-Dissociation-PatchLiner.pdf Cor4U-protocol_MEA.pdf Cor4U-protocol_RTCA.pdf Cor4U-protocol_Seeding384wMTP.pdf Please refer to the designated Cor.4U Application Protocol for detailed instructions on coating, seeding densities, and maintenance of the cardiomyocytes. 5
4. AXIOGENESIS Label License A. AXIOGENESIS Intellectual Property Rights This product is covered by patent families including, but not limited to, EP1348019; EP1002080; EP1745144; EP1644485; JP4904153; JP4159358; JP3956154; JP4814875; DE10136702 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 nonexclusive, nontransferable right to use the purchased amount of Cor.4U cells and the associated AXIOGENESIS Intellectual Property for (i) notfor - profit internal research conducted by the buyer and (ii) certain forprofit activities, including lead discovery, testing and/or research and development of other products. The forprofit use in disease models and tissue models is expressly excluded. 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 EP1970446 and US8048999 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, noncommercial 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 and Cor.4U are registered trademarks of AXIOGENESIS AG, Cologne, Germany. Mel.Cor is a trademark of AXIOGENESIS AG, Cologne, Germany. MitoXpressTM HS is a trademark of Luxcel Biosciences, Cork, Ireland, registered in the European Union. TurboGFPTM and TurboRFPTM are trademarks of Evrogen, Moscow, Russia. For information on the patents, patent applications, and licenses associated with the product contact the AXIOGENESIS Business Development Department, AXIOGENESIS AG at: info@axiogenesis.com. 6