3D Cardiac Tissue Co-Culture
Transcription
3D Cardiac Tissue Co-Culture
Protocol and Handling Guide 3D Cardiac Tissue Generation Using Cor.4U® Cardiomyocytes and CorF.4UTM Cardiac Fibroblasts 1 of 12 Notes Content 1. GENERAL INFORMATION ............................................................. 2 2. SAFETY INFORMATION AND GUIDANCE FOR USE ........................ 2 3. MATERIAL ................................................................................... 3 3.1 CELL FORMATS (CRYOPRESERVED COR.4U® CARDIOMYOCYTES AND CORF.4UTM CARDIAC FIBROBLASTS).....................................................................................3 3.2 REAGENTS DELIVERED WITH COR.4U® CARDIOMYOCYTES ...............................3 3.3 TRANSPORT OF CRYOPRESERVED COR.4U® CARDIOMYOCYTES AND CORF.4UTM CARDIAC FIBROBLASTS .......................................................................................3 3.4 STORAGE CONDITIONS ........................................................................................................4 3.5 REQUIREMENTS .......................................................................................................................4 4. PREPARATIONS ........................................................................... 5 4.1 COATING WITH 0.1% GELATIN SOLUTION FOR SEEDING OF CORF.4UTM CARDIAC FIBROBLASTS..................................................................................................................5 4.2 COATING WITH FIBRONECTIN FOR SEEDING OF COR.4U® CARDIOMYOCYTES ............................................................................................................................5 5. THAWING AND SEEDING OF CRYOPRESERVED COR.4U® CARDIOMYOCYTES AND CORF.4UTM CARDIAC FIBROBLASTS ........... 6 5.1 THAWING OF 5 X 106 CRYOPRESERVED COR.4U® CARDIOMYOCYTES TM AND/OR CORF.4U CARDIAC FIBROBLASTS .....................................................................6 5.2 COUNTING OF COR.4U® CARDIOMYOCYTES OR CORF.4UTM FIBROBLASTS WITH A NEUBAUER HAEMOCYTOMETER ..................................................................................6 5.3 MEDIUM CHANGE....................................................................................................................8 6. DISSOCIATION OF COR.4U® CARDIOMYOCYTES AND CORF.4UTM CARDIAC FIBROBLASTS .................................................................. 8 6.1 DISSOCIATION OF CORF.4UTM CELLS SEEDED ON GELATIN AND COR.4U® CELLS SEEDED ON FIBRONECTIN..............................................................................................8 7. FORMATION OF 3D CARDIAC TISSUE ........................................ 10 8. APPENDIX............................................................................... 11 3D Cardiac Tissue.v.2 Axiogenesis AG - Nattermannallee 1 - 50829 - Cologne Scientific Support: [email protected] 2 of 12 Notes 1. General Information This protocol provides fundamental information on how to culture and maintain Cor.4U® cardiomyocytes in a 3D model in combination with cardiac fibroblasts. The protocol covers coating with different substrates, thawing, plating, maintenance, and dissociation of Cor.4U® cardiomyocytes and CorF.4UTM cardiac fibroblasts. Please read the entire protocol first before you start your experiment. 2. Safety Information and Guidance for Use 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 protocol from human skin fibroblasts. These pure cardiomyocytes (100%) 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 analysis and multi-electrode array (MEA) recordings, demonstrate expected electrophysiological properties of these cells. Cor.4U® cardiomyocytes are particularly useful for cell-based in vitro assays in pharmacology, safety, and toxicology. The cells are ideal for electrophysiological applications as well as for high content and high throughput screening applications. CorF.4UTM cardiac fibroblasts were separated from Cor.4U® cardiomyocytes (both have a common cardiac precursor) at earliest stage of puromycin selection and verified by positive immunostaining for FSA (Fibroblast Surface Antigen), Col-1a (collagen-1a) and DDR2 (Discoidin Domain Receptor) • Cor.4U® cardiomyocytes and CorF.4UTM cardiac fibroblasts are intended for in vitro research use only. The Kit is not intended for diagnostics, therapeutic or clinical use and is not approved for human in vivo applications. • Both cell types are genetically modified human cells and should be handled according to local directives (Biosafety level 1). The cells can be inactivated by autoclaving at 121°C for 20 minutes. • Both cell types should be cultured in a sterile environment according to good cell culture and good laboratory practices. It is highly recommended that gloves, eye protection 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. 3D Cardiac Tissue.v.2 Axiogenesis AG - Nattermannallee 1 - 50829 - Cologne Scientific Support: [email protected] 3 of 12 Notes 3. Material 3.1 Cell Formats (Cryopreserved Cor.4U® cardiomyocytes and CorF.4UTM cardiac fibroblasts) • 1 or more vials of 5 x 106 Cor.4U® cardiomyocytes: cat.-no. Ax-BHC02-5M • Alternatively: 1 or more vials of 1 x 106 Cor.4U® cardiomyocytes: cat.no. Ax-B-HC02-1M • 1 or more vials of 2 x 106 CorF.4UTM cardiac fibroblasts: cat.-no. Ax-CHF02-2M 3.2 Reagents delivered with Cor.4U® cardiomyocytes • Thawing Medium • Cor.4U® Culture Medium • Puromycin (Cardiomyocyte selection agent) NOTE: Do not use Puromycin with the CorF.4U® cardiac fibroblasts - only with the Cor.4U cardiomyocytes! Puromycin treatment will select for cardiomyocytes; however, cardiac fibroblasts will not survive. Remove puromycin from the cellular media (by performing a full media change) within 24 hours to prevent chronic exposure/death of Cor.4U cardiomyocytes. 3.3 Transport of cryopreserved Cor.4U® cardiomyocytes and CorF.4UTM cardiac fibroblasts • Both cell types are delivered in a liquid nitrogen container. It is not recommended to ship the cells on dry ice or to store them on dry ice, because CO2 can diffuse through the vial and damage the cells. Additionally at -80°C (dry ice) the planar ice can turn into so-called dendritic ice which harms the cells. This risk does not exist if delivered in a liquid nitrogen container. 3D Cardiac Tissue.v.2 Axiogenesis AG - Nattermannallee 1 - 50829 - Cologne Scientific Support: [email protected] 4 of 12 Notes 3.4 Storage Conditions • Cryopreserved Cor.4U® cardiomyocytes and CorF.4UTM cardiac fibroblasts: Upon receipt, transfer the vials immediately to the vapor phase of liquid nitrogen. Do not store the cells at -80°C, recrystallisation can occur which may damage the cells. • Thawing Medium, Cor.4U® Culture: You will receive each medium frozen. Store the media at -20°C. Prior to use thaw medium overnight and avoid exposure to light. Once thawed, keep at 4°C for up to 4 weeks. 3.5 Requirements Item Vendor Cat. No. Inverse microscope with phase contrast equipment various Sterile laminar flow hood various Freezer (-20°C), refrigerator (+4°C) various Liquid Nitrogen storage container various 100µl, 1000µl pipette various Pipettor for serological pipettes various Water bath various Sterile 50 mL Polypropylene Tubes various PBS with and w/o Ca2+ and Mg2+ various Neubauer Haemocytometer various Trypan Blue Solution Sigma T8154 Invitrogen 15575-038 Sigma G1393 Geltrex Life Tech. A1413202 NutriStem® hESC XF Culture Media Biological Industry 05-100-1 EDTA 0.5 M UltraPure TM Gelatin Solution 3D Cardiac Tissue.v.2 Axiogenesis AG - Nattermannallee 1 - 50829 - Cologne Scientific Support: [email protected] 5 of 12 Notes Day 0 4. Preparations 4.1 Coating with 0.1% gelatin solution for seeding of CorF.4UTM cardiac fibroblasts 1. Dilute gelatin solution to 0.1% in PBS containing Ca2+ and Mg2+. Prepare 20 mL gelatin coating solution for 2 x T75 flasks. 2. Pipette 10 mL of the gelatin coating solution into each T75 flask and incubate for at least 30 min at 37°C, 5% CO2, and 95% humidity. Alternatively, coating can be done over night at 4°C. 4.2 Coating with fibronectin for seeding of Cor.4U ® cardiomyocytes 1. Dilute fibronectin to 10 µg/mL (1:100 dilution) in sterile PBS containing Mg2+ and Ca2+. Prepare 20 mL of the fibronectin solution for coating of 2 x T75 flasks, e.g 200 µl fibronectin stock solution into 20 mL of PBS with Mg2+ and Ca2+. Mix the solution very gently. 2. Pipette the fibronectin solution into the flasks, 10mL each and incubate for 3 h at 37°C, 5% CO2, and 95% humidity. Alternatively, coating can be done overnight at 4°C. INFO: Fibronectin is susceptible to shear stress, avoid harsh pipetting and do not vortex or spin the solution. 3D Cardiac Tissue.v.2 Axiogenesis AG - Nattermannallee 1 - 50829 - Cologne Scientific Support: [email protected] 6 of 12 Notes Day 1 5. Thawing and seeding of cryopreserved Cor.4U® cardiomyocytes and CorF.4UTM cardiac fibroblasts INFO: The thawing procedure for Cor.4U® and CorF.4UTM vials is the same. Cor.4U® thawing and culture medium can be used for CorF.4UTM cells as well. 5.1 Thawing of 5 x 106 cryopreserved Cor.4U® cardiomyocytes and/or CorF.4UTM cardiac fibroblasts 1. Warm 30 ml of Cor.4U® Culture Medium and Cor.4U® Thawing Medium to 37°C in a water bath. 2. Pipette 6 mL of Cor.4U® Thawing Medium into two 50 mL tubes (3 ml into each tube) and warm to 37°C. 3. Transfer the cells from the liquid nitrogen on dry ice directly to the laboratory. Ensure the cells are transferred as quickly as possible and then immediately place the vial into a water bath at 37°C until the frozen cell suspension detaches from the bottom of the vial, approximately 2 minutes. Dry and disinfect the vial and transfer it into the laminar hood. 4. Carefully transfer the cell suspension from the vial into one 50 ml tube containing 3 mL warm Cor.4U® Thawing Medium using a P1000 pipette. 5. Rinse the vial with 1 mL Cor.4U® Thawing Medium using the Thawing medium of the remaining 50 ml tube and add this rinsing solution to the cell suspension; the tube with the cell solution has now a total volume of 5 mL. 5.2 Counting of Cor.4U® cardiomyocytes or CorF.4UTM fibroblasts with a Neubauer haemocytometer 1. Pipette 10 µl trypan blue solution and 10 µL of the cell suspension into a tube. Incubate the mixture for 3 min at 37°C. Apply 10 µl of the 1:1 mixture into a Neubauer Haemocytometer and count live (clear) cells, 3D Cardiac Tissue.v.2 Axiogenesis AG - Nattermannallee 1 - 50829 - Cologne Scientific Support: [email protected] 7 of 12 Notes dead (blue) and total cells. 2. Count the number of viable cells in each of the four outer boxes highlighted in yellow of Figure 1. Divide the counted number by 4 to receive the mean value. E.g. Number counted for all 4 boxes: 200 200 / 4 = 50 Fig 1. Neubauer Haemocytometer 3. Calculate the number of viable cells correct for chamber factor (1 x 104), dilution factor (2), and total volume (5 mL). E.g.: Mean value of viable cells: 50 50 x 10000 x 2 x 5 = 5 000 000 cells (5 million viable cells in the cell suspension) 4. Seed 2 – 2.5 x 106 viable Cor.4U® cells in a total volume of 15 mL with warm Cor.4U® Culture Medium per T75 flask to receive a semiconfluent density of cells. 5. Seed 2 – 2.5 x 106 viable CorF.4UTM cells in a total volume of 15 mL with warm Cor.4U® Culture Medium per T75 flask to receive a semiconfluent density of cells. 6. Shake the flasks gently in a ‘figure eight’ motion before transferring them into the incubator. 7. Culture the flasks for 4-5 days before dissociating them for 3D culture. 3D Cardiac Tissue.v.2 Axiogenesis AG - Nattermannallee 1 - 50829 - Cologne Scientific Support: [email protected] 8 of 12 Notes Day 2 - 5 5.3 Medium Change 1. Change the medium as necessary (2 -3 times a week). The Cor.4U® Culture medium can be used to cultivate both cell types: Cor.4U® cardiomyocytes and CorF.4UTM cardiac fibroblasts. 2. Warm 15 mL of Cor.4U® Culture Medium for each flask to 37°C, e.g. 60 mL for 4 x T75 flasks. 3. Place the T75 flask into the laminar hood. Aspirate the medium carefully and add 15 mL of warm, fresh Cor.4U® Culture Medium per flask. NOTE: Aspirate the medium at slowest speed to avoid disruption of the cell layer. Do not add the medium directly onto the cells to avoid damage and removal of the cells. 6. Dissociation of Cor.4U® cardiomyocytes and CorF.4UTM cardiac fibroblasts INFO: The dissociation procedure for Cor.4U® and CorF.4UTM cells is the same. 6.1 Dissociation of CorF.4UTM cells seeded on gelatin and Cor.4U® cells seeded on fibronectin. 1. Prepare sterile PBS without Ca2+ Mg2+ with 2mM EDTA as described in the appendix. You will need 20 mL per T75 flask. 2. Warm 5 mL 1x Trypsin/EDTA solution for each T75 flask to 37°C. 3. Warm Cor.4U® Culture Medium to 37°C. You will need 10 mL for each T75 flask. 4. Aspirate the medium, and carefully wash each T75 flask of cells twice with 10 mL PBS without Ca2+ Mg2+. 3D Cardiac Tissue.v.2 Axiogenesis AG - Nattermannallee 1 - 50829 - Cologne Scientific Support: [email protected] 9 of 12 Notes 5. Discard the PBS, apply 10 ml of the pre-warmed PBS/EDTA solution for a T75 flask. Incubate the cells for 5 min before proceeding to step 6. 6. Discard the PBS/EDTA solution and apply 5 mL of the warm trypsin/EDTA solution to a T75 flask. Incubate the cells for a maximum of 2 minutes at 37°C in the incubator. 7. In the meantime, pipette 10 ml of warm Cor.4U® Culture Medium for each T75 flask into a 50 mL tube. 8. After the 2 minute incubation, check if the cells have detached. In case the cells have not completely detached, tap the flask 3 - 4 times. If larger amounts of cells are still attached, incubate the cells for another minute at 37°C, tap the flask, and check again for detachment of the cells. NOTE: Do not incubate with 1x Trypsin/EDTA for longer than 5 min! Longer treatments will cause loss of viable cells. 2 min or less is recommended. 9. Stop the trypsin dissociation by adding 5 ml of warm Cor.4U® Culture medium to the T75 flask. 10. Carefully detach remaining cells from the culture surface by rinsing the cell suspension 2 -3 times over the culture surface with a serological pipette. 11. Transfer the cell suspension into a 50 mL tube. 12. Rinse the flask with an additional 5 mL of Cor.4U® Culture Medium for a T75 flask and transfer it into the 50 mL tube. The total volume of the tube containing the cell suspension is now 15 mL. 13. Centrifuge the cell suspension for 4-5 min at 100 x g, and discard the supernatant. 14. Re-suspend the cell pellet in 1 mL Cor.4U® Culture Medium and count the cells as described in step 5.2. 3D Cardiac Tissue.v.2 Axiogenesis AG - Nattermannallee 1 - 50829 - Cologne Scientific Support: [email protected] 10 of 12 Notes 7. Formation of 3D Cardiac tissue INFO: Use the following recommendations to receive functional cardiac tissue: To produce a solid gel, the geltrex volume needs to be higher than the volume of cell suspension! Keep geltrex on ice during the whole procedure to avoid polymerization. ® The optimal ratio of Cor.4U and CorF.4UTM cells should be 2:1 to 3:1 (or 25-50% fibroblasts) 1. Depending on the 3D tissue size desired, mix Cor.4U® and CorF.4UTM cells according to Table 2, mix gently, then centrifuge again for 4-5 min at 100 x g. 2. Discard the supernatant and re-suspend the Cor.4U®/CorF.4UTM cell pellet in the indicated amount of Nutristem according to the size of cardiac tissue you want to generate (see Table 2). 3. Place the cell suspension on ice and mix it with a certain volume of the ice cold Geltrex. Use preliminary cooled pipette tips (at -20oC) ® CorF.4UTM Geltrex Nutristem Small 3D Tissue 70 µL 30 µL 2x106 1x106 Medium 3D Tissue 140 µL 60 µL 4x106 2x106 Large 3D Tissue 210 µL 90 µL 6x106 3x106 Cor.4U Table 2: Recommended volumes (Geltrex and Nutristem) and seeding ® densities of Cor.4U and CorF.4UTM cells. 3. Transfer the cell-geltrex solution with a pipette as a drop into a well of a 6-well multi plate. Place only one drop/well. 4. Incubate the cell-geltrex drops for 15 – 20 min at 37°C in the incubator until they become solid. 3D Cardiac Tissue.v.2 Axiogenesis AG - Nattermannallee 1 - 50829 - Cologne Scientific Support: [email protected] 11 of 12 Notes 5. After formation of the solid gel, cover the drops with 5 mL pre-warmed Nutristem Medium and incubate at 37°C, 5% CO2 and 95% humidity for 48 hours. 6. After 48 hours, warm the necessary amount of Cor.4U medium to 37°C. ® Culture 7. Transfer the 6-well multi plate into the laminar hood and carefully aspirate the medium. ® 8. Replace the Nutristem medium with warm, fresh Cor.4U Culture medium and place the 6-well multi plate back into the incubator. 9. Replace medium every 3 - 4 days. NOTE: the first asynchronous contractions of single cardiomyocytes will appear 24 – 36 h after change to Cor.4U® Culture medium. Regular synchronous beating can be detected after 7 – 10 days. Within these days, the volume of the cardiac tissue constructs will decrease 3 – 4 fold and thereby lead to an increased cell density of 100 x 106 per cm3, a density that is consistent with normal tissue. 8. Appendix Preparation of PBS/EDTA solution: Dilute 2 ml cell-culture tested 0.5 M EDTA solution (pH 8.0) in 500 ml PBS or DPBS without Ca2+/Mg2+. 3D Cardiac Tissue.v.2 Axiogenesis AG - Nattermannallee 1 - 50829 - Cologne Scientific Support: [email protected]