Cor.At. Cardiomyocytes derived from Mouse Embryonic Stem Cells. Protocol. Cor.At Cardiomyocytes Transfection

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1 Use our Discoveries to Help Advance Yours Cor.At Cardiomyocytes derived from Mouse Embryonic Stem Cells Protocol Cor.At Cardiomyocytes Transfection Using the Nucleofector Technology (Amaxa/Lonza Cologne)

2 1 Introduction Cor.At cells are cardiomyocytes derived from transgenic mouse embryonic stem cells. These cells are puromycin resistant and have the green fluorescent protein (GFP) reporter gene driven by a cardiac-specific promoter (MHC promoter). 1 Cor.At cells are produced through in vitro differentiation of mouse embryonic stem (ES) cells and puromycin selection of cardiomyocytes. Through BrdU incorporation assays, Cor.At cells were determined to have limited proliferative capacity similar to primary cells. These highly pure cells (>99% purity) express cardiac-specific Connexin-43, an indication of the ability for electric coupling of these cells, as shown in immunostaining. Patch clamp analysis, as well as multi-electrode array (MEA) recordings, demonstrate the normal electrophysiological properties of these cells. 1.1 Cor.At Cardiomyocytes for Pharmacological and Toxicological Screening Cor.At cardiomyocytes are particularly useful for cell-based in vitro assays in pharmacology and toxicology. These cells are ideal for high content screening, as well as, for medium to high throughput screening. Cor.At cells can be used for: Safety pharmacology Toxicological analysis of compounds (cardiac-specific cytotoxicity) Screening for pharmacological effects Drug development Molecular and cell biology Research & development 1.2 Cor.At Cardiomyocytes Compared to Cell Lines and Primary Cells Cor.At cardiomyocytes have many advantages over primary cells and cell lines. These cells are highly standardised and are 99.9% pure. The culture and maintenance of Cor.At cardiomyocytes require minimal laboratory time when compared to the culture and maintenance of rat neonatal cells. Reproducible results can be expected for every assay. These homogeneous cells are: Standardised from lot-to-lot 99.9% pure and have fully functional cardiac phenotype Frozen, stored and thawed with complete recovery of functionality An entire in vitro-based system Cor.At cells can be purchased frozen in cryo vials (Catalog # 06101). Cells are cultured for 48 hours to allow them to recover post thaw and should be cultured on BD BioCoat Fibronectincoated dishes (Catalog # for 35 mm dishes or Catalog # for 60 mm dishes). For coating plates and dishes of alternate sizes, we recommend using Sigma Fibronectin from bovine plasma (1 mg/ml solution), Catalog # F Page 2 of 7

3 1.3 The principle of the Nucleofector technology Non-viral Transfection Method The Nucleofector technology is a novel transfection technology especially designed for the needs of primary cells and difficult-to-transfect cell lines. It is a non-viral method which is based on a unique combination of electrical parameters and cell-type specific solutions. Ideal for DNA and RNA Optimal nucleofection conditions depend upon the individual cell type, not on the substrate being transfected. This means that identical conditions are used for the nucleofection of DNA and RNA. Consequently switching between substrates or performing co-transfections with DNA and RNA is straightforward. DNA Delivery Straight into the Nucleus Although Nucleofector technology is non-viral, it allows transfected DNA to directly enter the nucleus. In contrast, other commonly used non-viral transfection methods rely on cell division for the transfer of DNA into the nucleus. Thus, nucleofection provides the ability to transfect even nondividing cells, such as neurons and resting blood cells. High Throughput Transfection Two Nucleofector devices are available: The Nucleofector, working with single cuvettes for standard applications and the New Nucleofector 96-well Shuttle System, designed for high throughput transfection. 2 Materials and Reagents Please read the entire technical manual before beginning the culture of mouse atrial-like cardiomyocytes. 2.1 Product Specification Catalog # Product Description Unit / Format Storage Conditions Liquid nitrogen (6 months) * AX-001a-1M AX-001aCL AX-CCM250 N/A Invitrogen*** Lonza*** AAD-1001 or AAM-1001 Lonza*** Solution 114A3 Cor.At Cells & Cor.At CL Cells (Vials) Cor.At Culture Media 1x (0,05%) Trypsin-EDTA solution Lonza Nucleofector Device Transfection Solution 1 million viable Cor.At cardiomyocytes > 99.9% pure Culture medium for Cor.At cardiomyocytes Puromycin stock solution (10 mg/ml) Trypsin-EDTA solution for dissociation of Cor.At cardiomyocytes from fibronectin-coated dishes Single Cuvette Nucleofector 96 well shuttle Nucleofector Optimal solution for Cor.At cardiomyocytes Cryo vial 250 ml bottle -80 C (4 weeks) ** -20 C (6 months)* 4 C (2 weeks)** 1 ml vial see above 100 ml bottle ml bottle -20 C according to manufacturer's instructions according to manufacturer's instructions Page 3 of 7

4 Lonza*** VHCA well Nucleofector Kit SE 1x96 reactions Kit containing a GFP vector for Cor.At CL cardiomyocytes only! according to manufacturer's instructions *Expiry date as stated on label (i.e. 6 months from date of Manufacture) **from date of delivery of product(s) ***Refer to respective company for more information 2.2 Additional Materials and Reagents Required Vertical laminar flow hood certified for Level I handling of biological materials Incubator with humidity and gas control to maintain 37 C and 95% humidity in an atmosphere of 5% CO 2 in air 37 C water bath Inverted fluorescence microscope with appropriate filter sets (e.g. Green Fluorescent Protein (GFP) or FITC filters; Texas Red or Cy3 filters) Neubauer hemocytermeter Sterile 50 ml an 15 ml polypropylene (PP) tubes Micropipettes (e.g. Eppendorf "Research") Sterile pipette tips PBS with Ca 2+ and Mg 2+ (at 4 C) PBS w/o Ca 2+ and Mg 2+ (at room temperature) Ethanol p.a. (99.9% pure) 3 Safety Instructions 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.At cells are genetically modified mouse cells and should be handled according to local directives (Biosafety level 1). Cor.At cells can be inactivated by autoclaving at 121 C for 20 minutes. Cor.At cardiomyocytes should be cultured in a sterile environment according to good cell culture and good laboratory practices. It is highly recommended that gloves and labcoats be worn when handling all reagents as some reagents contain chemicals that may be harmful. Please consult the PIS and Material Safety Data Sheets (MSDS) for additional safety instructions where applicable. Page 4 of 7

5 4. Procedure 7.5 million Cor.At cardiomyocytes are thawn and cultured on a fibronectin-coated 10 cm dish according to the Technical Manual. Cor.At cardiomyocytes should be cultured at least for 24 h to ensure optimal conditions. If longer pre-culture is desired, media should be changed every 24h. Prepare a sufficient amount of vector DNA to be transfected. Per 10 5 Cor.At cardiomyocytes, at least 0.4 µg DNA are needed. To culture the Cor.At cells after transfection, coat a 96 well TC plate with fibronectin according to the Technical Manual. The protocol was optimized using the 96 well shuttle Nucleofector system. Slightly different results may be obtained if the single cuvette Nucleofector is used. 4.1 Preparation of the transfection solution 1) Equilibrate a 50 ml tube with the amount of Cor.At medium w/o Puromycin suitable for the transfection (180 µl for each well of the 96 well plate) in the incubator to 37 C and 5% CO 2. Unscrew the lid a little bit to allow for gas exchange. 2) For each 96-well to be transfected, prepare 0.4 µg DNA in the transfection solution 114A3 with a toal volume of 20 µl. Example: If the concentration of vector is 1 µg/ml, you need 0.4 µl vector plus 19.6 µl transfection solution per well of the 96 well shuttle system. 4.2 Preparation of the Cor.At cell suspension 1) Prepare a 50 ml tube with 8 ml of Cor.At media and prewarm to 37 C in the waterbath. 2) Prepare a 15 ml tube with 2 ml 1x Trypsin solution and prewarm to 37 C in the waterbath. 3) Prepare a 50 ml tube with 25 ml PBS with Ca 2+ and Mg 2+ cool it down to 4 C. 4) Take the 10 cm dish with the Cor.At cells out of the incubator and gently remove the medium using a slow-running suction pump or a pipette. Hold plates in a 45 angle and place the pipette tips in the inner rim to prevent the tips from touching the cell layer. 5) Carefully rinse the dish with 10 ml 4 C cold PBS with Ca 2+/ Mg 2+. Let the PBS flow down the inner rim of the plate. Do not pipette directly onto the cell layer. 6) Gently remove the PBS using a slow-running suction pump. Hold plates in a 45 angle and place the pipette tips in the inner rim to prevent the tips from touching the cell layer 7) Add 10 ml 4 C cold PBS with Ca 2+ /Mg 2+ to the dish and incubate at 4 C for 10 min. Let the PBS flow down the inner rim of the plate. Do not pipette directly onto the cell layer 8) After incubation at 4 C, gently remove the PBS using a slow-running suction pump or pipette. Hold plates in a 45 angle and place the pipette tips in the inner rim to prevent the tips from touching the cell layer 9) Carefully rinse the cells on the plate once with 10 ml of PBS w/o Ca 2+ /Mg ) Add 2 ml 1x Trypsin solution and incubate for 5 min at 37 C in the incubator. Page 5 of 7

6 11) After incubation, resuspend the Cor.At cells in 2 ml of the prewarmed Cor.At media and transfer the total cell suspension (4 ml) into the tube with the remaining 6 ml of media. Mix carefully by inverting the tube a few times / turning the tube upside down a few times. 12) Take an aliquot of the cell suspension and count the cells as described in the Technical Manual using the Neubauer Hemocytometer. Calculate the amount of viable cells. 4.3 Transfection of Cor.At cells 1) For each well of the 96 well shuttle system, 10 5 viable Cor.At cells are needed. Lable one tube for each well to be transfected and transfer the appropriate volume of cell suspension to the tube. Example: If a total amount of 5x10 6 viable cells in a total volume of 10 ml was prepared, add 200 µl of cell suspension to each tube. 2) Adjust the volume in every tube to at least 1 ml and spin the Cor.At cardiomyocytes at 200 x g for 5 min. 3) After centrifugation, discard the supernatant using a slow-running suction pump or a pipette. Flip against the tube to loosen the cell pellet. 4) Add 20 µl of the transfection solution to each tube. Do NOT resuspend the cells in the transfection solution! 5) Transfer the suspension from each tube into one well of the 96 well Nucleofector plate. 6) Seal the plate with the lid and tap the plate onto a surface to discard air bubbles in the solution. 7) Transfer the plate into the 96 well shuttle Nucleofector, choose programm 96-CD-150, and start the program. 8) After transfection is finished, take the plate out of the Nucleofector and transfer it to the laminar flow hood again. 9) Add 80 µl of equilibrated Cor.At media (prepared in 4.1, step 1) to each well and transfer the suspension (100 µl total volume) into the corresponding well of the fibronectin-coated 96 well plate. 10) Rinse eache well of the Nucleofector 96 well plate with additional 100 µl of the equilibrated Cor.At media and transfer the media into the corresponding well of the fibronectin-coated 96 well plate. 11) Culture the transfected cells at 37 C, 95% humidity and 5% CO 2 for 24 h before analysing them using a fluorescence microscope. Page 6 of 7

7 6 Literature 1. Kolossov E, Lu Z, Drobinskaya I, et al. Identification and characterization of embryonic stem cell-derived pacemaker and atrialcardiomyocytes. FASEB. J. 2005;19(6): Limited Use Label License Cor.At cells, including its derivatives or modifications, are produced in part with technology that is the subject of pending German, U.S. and foreign patent applications and/or patents owned or licensed by Axiogenesis AG (hereinafter "Axiogenesis IP"). The purchase of this product conveys to the buyer the non-exclusive, non-transferable right to use the purchased amount of Cor.At cells and the associated Axiogenesis IP for (i) not-for-profit internal research conducted by the buyer (whether the buyer is an academic or for-profit entity), where "Research" means non-commercial uses or activities which (or the results of which) do not generate revenue, and (ii) certain for-profit activities, including lead discovery, testing and/or research and development of products or potential products that may or may not be, or become, commercially available, quality control of commercial products, and provision of services consisting of these applications to third parties. Axiogenesis AG hereby waives its right to any compensation beyond the price paid by purchaser for Cor.At cells when Cor.At are used for the activities permitted by this license, including the right to any intellectual property or any profits generated by purchaser through purchasers use, directly or indirectly, of Cor.At cells. Accordingly, Axiogenesis AG will not assert a claim against the buyer of infringement of its relevant patents based upon the manufacture, use or sale of a product (including a therapeutic, clinical diagnostic, vaccine, prophylactic or research product) developed in research by the buyer in which Cor.At cells were employed, provided that neither Cor.At cells nor any of its components were used directly in the manufacture of such product. This limited use license does NOT include the right to resell the product, use Cor.At cells directly in the manufacture of a product or as a component of a product, reverse engineer Cor.At cells or Axiogenesis IP, or to use Axiogenesis IP in any way that is separated from intact Cor.At cells (including the isolation and/or use of integrated genetic constructs that represent, in whole or in part, Axiogenesis IP). For information on the foregoing patents or patent applications or on purchasing a license to use Axiogenesis IP for purposes other than those permitted above, contact Business Department, Axiogenesis AG, Nattermannalle 1 / S 20, Cologne, Germany. info@axiogenesis.com tel: fax: Page 7 of 7