General Handling Guide: Cor.4U Human ipsc-derived Cardiomyocytes

Transcription

1 General Handling Guide: Cor.4U Human ipsc-derived Cardiomyocytes

2 1 of 13 TABLE OF CONTENTS 1. GENERAL INFORMATION SAFETY INFORMATION MATERIAL CELLS AND MEDIA STORAGE CONDITIONS REQUIREMENTS PREPARATION SURFACES COATING OF CULTURE SURFACES THAWING AND SEEDING OF COR.4U THAWING OF CRYOPRESERVED COR.4U (4 X 106 CELLS) THAWING OF CRYOPRESERVED COR.4U (1 X 106 CELLS) THAWING OF CRYOPRESERVED COR.4U (0.25 X 106 CELLS) COUNTING OF COR.4U IN A NEUBAUER HEMOCYTOMETER PUROMYCIN TREATMENT MAINTENANCE DETACHMENT AXIOGENESIS LIMITED LABEL USE LICENSE... 13

3 2 of General Information This protocol covers general thawing, seeding and dissociation of Axiogenesis Cor.4U 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 first before you start your experiment. Detailed handling guides for specific applications and assays are available and can be downloaded from our homepage: 2. Safety Information Cor.4U 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 via the Yamanaka protocol from a human skin fibroblast. 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 micro-electrode array (MEA) recordings, demonstrate the normal electrophysiological properties of these cells. Cor.4U 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. Cor.4U 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 are genetically modified human cells and therefore genetically modified organisms (GMO). They should be handled according to local directives (Biosafety level 1). Cor.4U can be inactivated by autoclaving at 121 C for 20 minutes. Cor.4U 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.

4 3 of Material 3.1 Cells and Media Cor.4U is offered with Cor.4U Culture Medium (see table 1). If required, serumfree BMCC medium can be purchased separately. 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. 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. Cor.4U are available in different cryopreserved as well as pre-cultured formats. 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 (see table 2). Table 1: Overview of available Cor.4U products Material Container Content Storage Shelf life Cryopreserved Cor.4U Cryovial 0.25, 1 or 4 Million cells Liquid nitrogen See label (max. 1.5 years) Pre-cultured Cor.4U Cell culture flasks or specific culture plates various Incubator 37 C N/A Cor.4U Culture Medium Bottle 250 ml Frozen -20 C See label BMCC Medium (serum-free) Bottle 250 ml Frozen -20 C See label Puromycin Cryovial 10 µl Frozen -20 C See label

5 4 of 13 Table 2: Overview of Cor.4U product formats for various assays Assay Frozen cells Assay-related plates Cells per well Plate reader assays (e.g. calcium transients) 4 Mio/ vial 384-well plate 96-well plate CardioExcyte96, Nanion Technologies 4 Mio/ vial NSP well xcelligence, Acea Biosciences 4 Mio/ vial E-Plate 96-well MEA, Maestro System 4 Mio/ vial 96-well MEA Manual Patch Clamp 3 vials of 0.25 Mio /vial N/A N/A 3.2 Storage Conditions Cryopreserved: Upon receipt of cryopreserved Cor.4U, transfer the vials directly to the vapor phase of liquid nitrogen. Do not expose the vials to room temperature as recrystallization will harm the cells. Pre-cultured: Upon receipt, transfer flasks with pre-cultured Cor.4U 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 shipped with the package and transfer the plates immediately to a humidified incubator (37 C, 5% CO2). Upon receipt of fresh Cor.4U seeded on multi-well plates, transfer the plate into a sterile hood, carefully remove the sealing mat, aspirate the medium and add 200 µl per well (96 well plate) or 50 µl per well (384 well plate) of fresh, prewarmed Cor.4U Culture Medium. Transfer the plates immediately to a humidified incubator (37 C, 5% CO2). Store frozen Cor.4U Culture Medium and BMCC medium at _ 20 C. Thaw medium overnight at 4 C and avoid excessive exposure to light. Once thawed, medium can be kept at 4 C up to 4 weeks. The media delivered with fresh Cor.4U is not frozen and can be stored at 4 C for up to 4 weeks.

6 5 of Requirements Table 3: Overview of required equipment (please read assay-specific protocols for required media and devices) Item Inverse microscope Sterile laminar flow hood (Bio safety level 1) Freezer (-20 C) refrigerator (+4 C) Liquid nitrogen container Pipettor for serological pipettes Cell culture incubator (37 C, 95% humidity, 5% CO2) Water bath Neubauer Hemocytometer 4. Preparation 4.1 Surfaces Cor.4U can be cultivated on various surfaces. Plastic Cor.4U 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 Cor.4U on glass surfaces (e.g. standard MEA probes, glass slides for Patch Clamp) is not as tight as on plastic ware. Please contact Axiogenesis technical support for additional information to improve attachment rates.

7 6 of Coating of Culture Surfaces Different coating reagents are suitable for culturing Cor.4U. Due to the specific needs of various assays, a proper choice of coating reagent is recommended. Table 4: Overview of suggested coating agents Coating agent Vendor Assay Comment Fibronectin Sigma F1141 Standard cultivation Calcium transients CardioExcyte96 xcelligence RTCA - Not suitable for hypertrophy assays since it induces hypertrophy - Susceptible to shear stress - Do not vortex and avoid harsh pipetting - Do not refreeze Geltrex ready-touse Life Technologies A MEA - Results in a very tight cell contact - Work with ice-cold solution to avoid polymerization - Detachment is difficult Gelatin Sigma G1393 Manual Patch Clamp, hypertrophy assay - Provides only loose cell contact - Maintains a more round cell shape that is beneficial for Manual Patch Clamp

8 7 of 13 Coating with Fibronectin 1. Prepare fibronectin coating solution by diluting fibronectin stock solution (1 mg/ml) in PBS with Ca 2+ and Mg 2+ to a final concentration of 10 µg/ml fibronectin (Dilution 1:100). Mix the solution carefully. Please find the specific volumes in the corresponding assay-specific handling guides. 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, you can incubate the coating solution at 4 C overnight. Coating a MEA with Geltrex 1. Place sterile MEAs under laminar flow hood. 2. Transfer an appropriate amount of cold Geltrex ready-to-use solution in a sterile 50 ml tube on ice. 3. Place a 5 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. 5. Incubate the coated MEA for at least 30 min at 37 C in a humidified chamber. Coating with Gelatin 1. Dilute sterile gelatin solution to 0.1% in PBS 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. 4. Incubate the coverslips in an incubator at 37 C for at least 1h. 5. Thawing and Seeding of Cor.4U 5.1 Thawing of cryopreserved Cor.4U (4 x 10 6 cells) Use this procedure when performing the following experiments: Impedance recordings using the xcelligence RTCA or CardioExcyte96 device, automated patch clamp recordings and plate reader assays like calcium transient measurements. 1. Transfer 3 ml Cor.4U Culture Medium into a 50 ml tube (tube A) and warm to 37 C. 2. Pre-warm 1 ml Cor.4U Culture Medium in another 50 ml tube (tube B). 3. Quickly transfer the cells from liquid nitrogen on dry ice 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). 4. Disinfect and transfer the vial to the laminar flow hood and pipette the cell suspension carefully into tube A. 5. Rinse the cryo vial with 1 ml Cor.4U Culture Medium from tube B and add it to the cell suspension in tube A. The total volume is now 5 ml. 6. Withdraw 20 µl for the determination of cell numbers and viability (see chapter 5.4)

9 8 of 13 Attention: Do not spin down the cells! Centrifugation directly after thawing will damage the cells and will lead to cell loss. 7. Count the number of viable cells (e.g. in a hemocytometer; see chapter 5.4). 5.2 Thawing of Cryopreserved Cor.4U (1 x 10 6 cells) Use this procedure when performing the following experiments: MEA recordings or automated patch clamp. 1. Transfer 3 ml Cor.4U Culture Medium into a 50 ml tube (tube A) and warm to 37 C. 2. Pre-warm 1 ml Cor.4U Culture Medium in another 50 ml tube (tube B). 3. Quickly transfer the cells from liquid nitrogen on dry ice 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. 40 sec). 4. Disinfect and transfer the vial to the laminar flow hood and withdraw 20 µl for the determination of cell numbers and viability (see chapter 5.4). 5. Then transfer the residual cell suspension into tube A. 6. Rinse the cryo vial with 1 ml Cor.4U Culture Medium from tube B and transfer it to the cell suspension in tube A. The total volume in tube A is now 4.5 ml. 7. In a volume of 4.5 ml, the DMSO, present in the freezing medium, is diluted to a non-harmful concentration. Attention: Do not spin down the cells! Centrifugation directly after thawing will damage the cells and will lead to cell loss. 8. Count the number of viable cells (e.g. in a hemocytometer; see chapter 5.4). 5.3 Thawing of cryopreserved Cor.4U (0.25 x 10 6 cells) Use this procedure when performing manual patch clamp recordings. 1. Transfer 3 ml Cor.4U Culture Medium into a 50 ml tube (tube A) and warm to 37 C. 2. Pre-warm 1 ml Cor.4U Culture Medium in another 50 ml tube (tube B). 3. Quickly transfer the cells from liquid nitrogen on dry ice 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. 40 sec). 4. Disinfect and transfer the vial to the laminar flow hood and withdraw 20 µl for the determination of cell numbers and viability (see chapter 5.4). 5. Then transfer the residual cell suspension into tube A. 6. Rinse the cryo vial with 1 ml Cor.4U Culture Medium from tube B and transfer it to the cell suspension in tube A. The total volume in tube A is now 4.5 ml. 7. In a volume of 4.5 ml, the DMSO, present in the freezing medium, is diluted to a non-harmful concentration.

10 9 of 13 Attention: Do not spin down the cells! Centrifugation directly after thawing will damage the cells and will lead to cell loss. 8. Count the number of viable cells (e.g. in a hemocytometer; see chapter 5.4). 5.4 Counting of Cor.4U in a Neubauer Hemocytometer 1. Add 20 µl trypan blue solution to 20 µl of the cell suspension in a tube. 2. Apply 10 µl of the 1:1 mixture into a Neubauer hemocytometer and count viable (clear), dead (blue) and total cells. 3. Count the number of cells in each of the four outer boxes highlighted in yellow of Figure 2. Calculate the mean number of cells per yellow box. Fig 2. Neubauer hemocytometer 4. Calculate the number of cells corrected by chamber factor (1 x 10 4 ), dilution factor (2), and total volume (e.g. 5 ml). E.g.: Mean number of cells per box = x x 2 x 5 = 5,000,000 (5 million cells in the cell suspension) 5. Adjust the cell suspension (viable cells/ml) adequately with Cor.4U Culture Medium depending on the suggested assay conditions and mix the suspension carefully. 6. Proceed with seeding of Cor.4U according to the assay-specific protocol.

11 10 of 13 Table 5: Overview of Cor.4U seeding densities according to assay type Assay and Format No. cells per well Volume of cell suspension per well Density in cell suspension (including dead volume) Calcium transient 384-well 96-well µl 200 µl 4 Mio cells/10ml 3.3 Mio cells/22ml CardioExcyte9 6 NSP-96 xcelligence RTCA E-plate (96 well) µl 3.3 Mio cells/22ml µl 3.3 Mio cells/19.8ml MEA Axion (96-well) 48-well 12-well droplet 5 µl 2 Mio/ ml MEA MCS 96-well 48-well droplet 5 µl 2 Mio/ ml 6. Puromycin treatment Cor.4U are puromycin resistant and survive exposure of 2 µg/ml puromycin for up to 24h (longer exposure can be toxic). The puromycin selection step hinders the outgrowth of fibroblasts which can occur during long-term experiments. For shortterm experiments (up to a week) fibroblasts do not interfere with the experiments. When planning long-term experiments extending one week, an inhibition of the fibroblast proliferation using puromycin can be performed if necessary. The puromycin treatment should be started earliest after cells have been settled and started regular beating (after 24-48h). 1. Warm the necessary amount of Cor.4U Culture Medium to 37 C and add puromycin to a final concentration of 2 µg/ml. 2. Aspirate the medium very carefully and add the medium with puromycin without pipetting directly onto the cells. 3. Transfer the dish back into the incubator and incubate the cells over night, maximally for 24h. 4. Next day, pre-warm the required amount of Cor.4U Culture Medium to 37 C. 5. Carefully aspirate the medium from the dishes and replace with fresh, warm medium without puromycin. Incubate the cells again in the incubator until you want to perform the experiment. Inspect the cells frequently and change the medium when necessary.

12 11 of 13 NOTE: Do not incubate Cor.4U with puromycin for longer than 24h to avoid damage of the cells. Do not thaw and seed the cells in Cor.4U Culture Medium containing puromycin. 7. 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 Cor.4U are seeded in culture flasks, the medium should be changed every 2-3 days to maintain a good relation of medium to cells. When Cor.4U are seeded in culture plates or assay-related plates, the medium should be changed on daily basis due to a much lower relation of medium to cells compared to flasks. Please also follow the instruction in the corresponding assay-specific protocols (available at Table 5: Overview of medium change dependent on the assay type Assay and Format Medium change Change to BMCC medium Calcium transient CardioExcyte96 xcelligence RTCA MEA Axion 100% daily 4 x 50% twice a day 4 x 50% twice a day 100% every 2nd day 100%, at least 2h before starting experiments 4 x 50%, 24h before starting experiments and 2 hours before addition of compounds 4 x 50%, 24h before starting experiments 100%, 2 h before starting experiments and 2 hours before addition of compounds Manual patch clamp 1-2 x per week, 100% N/A Automated patch clamp 3 x per week, 100% N/A

13 12 of Detachment To detach Cor.4U, we recommend using Accumax (Sigma-Aldrich cat. A7089). 1. Please equilibrate Accumax to room temperature before use, do not warm to 37 C. 2. Wash the cells with PBS w/o Ca 2+ and Mg 2+ for better detachment rates. 3. Incubate the cells for 5-10 min in Accumax at 37 C (use 2 ml per T25 flask and 5 ml per T75 flask). 4. Check the detaching process of the cells: After 5 min the cells normally start to detach, but tent to cluster. It is beneficial to incubate them a little longer at 37 C to get single isolated cells. 5. Once the cells detach very easily, add 2 ml (T25 flask) or 5 ml (T75 flask) of Cor.4U Culture medium and transfer the cell suspension to a 50 ml tube. 6. Carefully detach remaining cells from the flask surface by rinsing the surface of the flask with additional 2 ml or 5 ml and transfer the solution to the 50 ml tube containing the cell suspension. 7. Centrifuge the cell suspension for 3 min at 200 x g, and discard the supernatant. Resuspend the cell pellet in Cor.4U Culture Medium according to your assay protocol. NOTE: Do not force the cells to detach by pipetting. This harsh procedure will harm and stress the cells.

14 13 of 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 info@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 and Cor.4U are registered trademarks of AXIOGENESIS AG, Cologne, Germany. Mel.Cor is a trademark of AXIOGENESIS AG, Cologne, Germany. TurboGFP and TurboRFP are trademarks of Evrogen, Moscow, Russia. Peri.4U, Dopa.4U, CNS.4U and Astro.4U neural cells are trademarks of AXIOGENESIS AG, Cologne, Germany. For information on the patents, patent applications, and licenses associated with the product contact the AXIOGENESIS Business Development Department, AXIOGENESIS AG at: patent@axiogenesis.com.