Cytiva™ Plus Cardiomyocytes - GE Healthcare Life Sciences
Contents
1. 8 Determine the total Volume of Cell Suspension Note We use a 1000 uL pipette to establish this volume 9 Calculate the number of viable cells in the cell suspension Viable cells ml 10 x 5 x Total Number of Cells ml Non Viable Cells mll Total number of viable cells Viable cells ml x Volume of Cell Suspension ml Worked example for a typical 3 5 x 105 vial of Cytiva Plus Cardiomyocytes Total Number of Cells ml determined in step 6 1 83 x 105 Non Viable Cells ml determined in step 5 1 64 x 10 Viable cells ml 10 x 3 x 1 83 x 10 1 64 x 10 3 85 x 105 cells ml Total number of viable cells 3 85 x 106 cells ml x 1 ml 3 85 x 10 cells Note There are a total of 5 5 x 106 cells in a 3 5 x 106 vial of Cytiva Plus Cardiomyocytes and we routinely record a value of gt 70 post thaw cell viability i e 3 85 x 105 viable cells 27 6 5 Seeding Cytiva Plus Cardiomyocytes into cell culture plates for HCA Time 30 minutes Cells should be seeded at a density of 3 6 x 104 viable cells in 200 UL RPMI 1640 B27 per well of a 96 well cell culture plate or 9 x 10 viable cells in 50 uL RPMI 1640 B27 per well of a 384 well cell culture plate i e 1 8 x 10 viable cells ml Note Assuming the volume of the cell suspension is 1 ml and the number of viable cells is 3 85 x 109 the current concentration is 3 85 x 108 viable cells ml Consequently the next step involves diluting the cell suspension2. counter is outlined below However this method could be adapted for other commercial cell counters 15 Consumables e RPMI 1640 B27 medium filter sterilized e 4x 1 5 ml tubes e 4 x NucleoCassettes ChemoMetec e Reagent A100 ChemoMetec e Reagent B ChemoMetec Equipment e 100 uL pipette amp sterile 1 200 uL tips e 1000 uL pipette amp sterile 1 1000 uL tips e NucleoCounter NC 100 ChemoMetec Protocol Perform the following steps aseptically inside the biosafety cabinet BSC 1 Determine viable cell number using the NucleoCounter NC 100 ChemoMetec To do this transfer 40 uL of the cell suspension into a 1 5 ml tube Add to the tube 360 uL of RPMI 1640 B27 medium i e now a 1 10 dilution Gently agitate to achieve an even cell suspension 2 The remaining steps can be performed outside of the BSC 3 Transfer 100 uL of 1 10 diluted cell suspension into each of three more 1 5 ml tubes 4 Use a 100 uL of 1 10 diluted cell suspension sample to calculate he number of Non Viable Cells mi i e directly load the sample into a NucleoCassette 5 Repeat step 4 for a second time and calculate the average result 6 Use another 1 x 100 uL of 1 10 diluted cell suspension sample o calculate the Total Number of Cells ml To do this add 100 uL of Reagent A100 and then 100 uL of Reagent B to the 100 uL of 1 10 diluted cell suspension sample Mix by pipetting then load into a NucleoCassette 16 7 Repeat ste
3. 9 After seeding all wells put the lid on the tissue culture dish and incubate the MEA plate in a standard cell culture incubator at 37 C for 2 3 hours The sterile distilled water in the gaps between the wells will prevent the evaporation of the bead of cell suspension 19 10 After 2 3 hours gently add 500 uL of warm RPMI 1640 B27 medium to the corner of each well of the 12 well MEA plate or 150 uL of warm RPMI 1640 B27 medium to the corner of each well of the 48 well MEA plate Pipette against the side of the well so as not to disturb the plated cells 11 Gently add another 500 pL of warm RPMI 1640 B27 medium to the corner of each well of the 12 well MEA plate or 150 uL of warm RPMI 1640 B27 medium to the corner of each well of the 48 well MEA plate 12 Gently add another 1000 uL of warm RPMI 1640 B27 medium to the corner of each well of the 12 well MEA plate i e total well volume is now 2 ml or another 150 uL of warm RPMI 1640 B27 medium to the corner of each well of the 48 well MEA plate i e total well volume is now 450 ul 13 Aspirate the 500 uL of sterile distilled H2O from the gaps between the wells in the 12 well MEA plate or 200 uL of sterile distilled H O from the gaps between the wells in the 48 well MEA plate 14 Confirm cell attachment at this point by observing the MEA wells under the microscope if using a transparent plate see Fig 3A and 3B 15 Incubate in a standard cell c
4. Cardiomyocytes onto coverslips Time 30 minutes Cells should be seeded at a density of 4 6 x 104 viable cells in 650 uL RPMI 1640 B27 per well of a 24 well cell culture plate uL i e 7 1 x 10 viable cells ml Note Assuming the volume of the cell suspension is 1 ml and the number of viable cells is 1 12 x 105 the current concentration is 1 12 x 105 viable cells ml Consequently the next step involves diluting the cell suspension to achieve the appropriate cell concentration 39 Consumables e RPMI 1640 B27 medium filter sterilized e 60 ml bottle Equipment e 24 well cell culture plate containing Matrigel coated coverslips e 200 uL pipette amp sterile 1 200 uL tips e 37 C water bath Protocol Perform the following steps aseptically inside the biosafety cabinet BSC 1 Warm 25 ml RPMI 1640 B27 medium in a sterile 60 ml bottle sealed in a 37 C water bath 2 Dilute the cell suspension to 7 1 x 104 viable cells ml using warm RPMI 1640 B27 medium Required volume ml Viable cells 7 1 x 104 viable cells ml Example A total viable count of 1 12 x 105 cells would be divided by 7 1 x 104 viable cells ml to obtain a final required volume of 15 8 ml 3 Aspirate the Matrigel from each well of the 24 well cell culture plate and immediately replace with 650 uL of Cytiva Plus Cardiomyocyte suspension i e 7 1 x 104 viable cells ml 4 After seeding all the required wells put the lid on the cell culture plate and incub
5. Hemocytometer or automated cell counter 37 C water bath Centrifuge Application dependent Patch clamp system Sub cellular imaging system for High Content Analysis Multi electrode array system Impedance system Calcium transient system 10 5 Protocols for MEA 5 1 Preparation of MEA coated plates Time 4 hours Consumables FBS at 4 C e Sterile distilled H O e Fibronectin 1 mg human BD Biosciences 354008 at 4 C e 50 ml tube e D PBS Equipment e MEA plate e g Axion BioSystems 12 well plate e 20 uL pipette amp sterile 1 20 uL tips e 1000 uL pipette amp sterile 1 1000 uL tips Protocol Perform the following steps aseptically inside the biosafety cabinet BSC The first step in preparing an MEA plate for use is to ensure that the surface is hydrophilic The surface of a new MEA plate is hydrophobic and even hydrophilic MEAs tend to become hydrophobic again during storage A hydrophobic surface will prevent attachment and growth of the hydrophilic cells FBS treatment renders the surface hydrophilic 1 Place a 4 uL bead of FBS over the recording electrode area of each well of the MEA plate see Fig 1 Note If a bead fails to form ignore that recording site 2 Fora 12 well MEA plate add 500 uL of sterile distilled HO to the gaps between the wells to prevent the evaporation of the bead of FBS see Fig 1 For a 48 well MEA plate add 200 uL of sterile dis
6. a 10 ml stripette remove 8 mls of the supernatant Remove a further 1 ml with a 1000 uL pipette taking care not to disturb the cell pellet Resuspend the cells in the residual liquid approx 1 ml with gentle agitation 7 7 Determination of post thaw viability of Cytiva Plus Cardiomyocytes for manual patch clamp Time 30 minutes Determine the viable cell number and viable cell density using preferred method of choice We use a NucleoCounter NC 100 ChemoMetec cell counter the method for which is outlined below However this method could be adapted for other commercial cell counters Consumables e RPMI 1640 B27 medium filter sterilized e 4x 1 5 ml tubes e 4 x NucleoCassettes ChemoMetec e Reagent A100 ChemoMetec e Reagent B ChemoMetec 37 Equipment e 100 uL pipette amp sterile 1 200 uL tips e 1000 uL pipette amp sterile 1 1000 uL tips e NucleoCounter NC 100 ChemoMetec Protocol Perform the following steps aseptically inside the biosafety cabinet BSC 1 Determine viable cell number using the NucleoCounter NC 100 ChemoMetec To do this transfer 40 uL of the cell suspension into a 1 5 ml tube Add to the tube 360 uL of RPMI 1640 B27 medium i e now a 1 10 dilution Gently agitate to achieve an even cell suspension 2 The remaining steps can be performed outside of the BSC 3 Transfer 100 uL of 1 10 diluted cell suspension into each of three more 1 5 ml tubes 4 Use a 100 pL of 1 10 diluted ce
7. for internal research and development but not for any commercial purposes Commercial purposes shall include sale lease license or other transfer of the material or any material derived or produced from it sale lease license or other grant of rights to use this material or any material derived or produced from it use of this material to perform services for a fee for third parties A license to use the Cytiva Plus Cardiomyocytes for commercial purposes is subject to a separate license agreement with GE Healthcare IF YOU REQUIRE A COMMERCIAL LICENSE TO USE THIS MATERIAL AND DO NOT HAVE ONE PLEASE CONTACT YOUR LOCAL GE HEALTHCARE REPRESENTATIVE AND RETURN THIS MATERIAL UNOPENED TO GE HEALTHCARE UK LTD THE MAYNARD CENTRE FOREST FARM WHITCHURCH CARDIFF CF14 7YT UK AND ANY MONEY PAID FOR THE MATERIAL WILL BE REFUNDED GE Healthcare Cytiva Plus Cardiomyocytes the Products are sold under licence from Asterias Biotherapeutics Inc and Wisconsin Alumni Research Foundation WARF under US patent and publication numbers US 5 843 780 US 6 200 806 US 6 602 711 US 6 800 480 US 7 005 252 US 7 029 913 US 7 297 539 US 7 413 902 US 7 425 448 US 7 452 718 US 7 582 479 US 7 732 199 US 7 763 464 US 7 781 216 US 7 851 167 US 7 897 389 and US 2007 0010012 and equivalent patent and patent applications in other countries Important note For in vitro research use only Not to be used for any therapeutic or diagnostic applications
8. to achieve the appropriate cell concentration Consumables e RPMI 1640 B27 medium filter sterilized e 60 ml bottle Equipment e Fibronectin coated cell culture plate e 200 uL pipette amp sterile 1 200 uL tip Protocol Perform the following steps aseptically inside the biosafety cabinet BSC 1 Warm 25 ml RPMI 1640 B27 medium in a sterile 60 ml bottle sealed in a 37 C water bath 2 Dilute the cell suspension to 1 8 x 105 viable cells ml using warm RPMI 1640 B27 medium Required volume ml Viable cells 1 8 x 105 viable cells ml 28 Example A total viable count of 3 85 x 105 cells would be divided by 1 8 x 10 viable cells ml to obtain a final required volume of 21 4 ml 3 Remove fibronectin coated cell culture plate from the incubator 4 Aspirate the fibronectin from each well of the cell culture and immediately replace with 200 LL of Cytiva Plus Cardiomyocyte suspension i e 1 8 x 10 viable cells ml per well of a 96 well cell culture plate or 50 uL per well of a 384 well cell culture plate 5 After seeding all the required wells put the lid on the cell culture plate and incubate in a standard cell culture incubator at 37 C 5 CO 6 6 Media change on day 4 post thaw Time 20 minutes 1 Warm 30 ml RPMI 1640 B27 medium in a 60 ml bottle sealed in a 37 C water bath 2 Perform the following steps aseptically inside the biosafety cabinet BSC 3 After 96 hours flick the media out of t
9. Cytiva Plus Cardiomyocytes Product booklet Codes 29 0918 80 gt 1 x 109 1 000 000 viable cells 29 0918 81 gt 5 5 x 106 5 500 000 viable cells Page finder 1 Legal 2 Handling 2 1 Safety warnings and precautions 2 2 Storage 2 3 Expiry 2 4 Packaging 3 Introduction 4 Components and other materials required 4 1 Components 4 2 Materials to be supplied by user 4 3 Equipment needed 5 Protocols for MEA 5 1 Preparation of MEA coated plates Time 4 hours 5 2 Preparation of RPMI 1640 B27 medium for MEA Time 30 minutes 5 3 Thawing of Cytiva Plus Cardiomyocytes for MEA Time 30 minutes 5 4 Determining Post Thaw Cell Viability of Cytiva Plus Cardiomyocytes for MEA Time 30 minutes 5 5 Seeding Cytiva Plus Cardiomyocytes onto MEA plates Time 3 hours 5 6 Media change on day 4 post thaw Time 20 minutes 6 Protocols for HCA 6 1 Preparation of cell culture plates for HCA Time 2 hours 6 2 Preparation of RPMI 1640 B27 medium for HCA Time 20 minutes 6 3 Thawing of Cytiva Plus Cardiomyocytes for HCA Time 30 minutes 3 Wehe O O O WO DA NN CB GD m a 20 22 22 23 24 6 4 Determining Post thaw Cell Viability of Cytiva Plus Cardiomyocytes for HCA Time 30 minutes 6 5 Seeding Cytiva Plus Cardiomyocytes into cell culture plates for HCA Time 30 minutes 6 6 Media change on day 4 post thaw Time 20 minutes 7 Protocols for manual patch clamp 7 1 St
10. Incubate the cell culture plate overnight at 2 8 C before use 3 Matrigel coated vessels are stable for 10 days when stored at 2 8 C 34 7 5 Preparation of RPMI 1640 B27 medium for manual patch clamp Time 20 minutes Consumables e RPMI 1640 Glutamine medium 500 ml at 4 C e B27 supplement 10 ml at 4 C e 10 ml sterile serological pipette Equipment e 500 ml 0 22 um cellulose acetate filter unit Corning 430769 e Vacuum line e Pipette gun Protocol Perform the following steps aseptically inside the biosafety cabinet BSC 1 Using a 10 ml sterile serological pipette add 10 ml of B27 supplement to 500 ml bottle of RPMI 1640 Glutamine medium Swirl bottle several times to mix 2 Place the filtration unit into the BSC Ww Carefully pour 510 ml of the RPMI B27 medium into the reservoir of the filtration unit Place the lid on the filter unit Connect the filter unit to a vacuum source When filtration is complete disconnect the vacuum source Detach the upper reservoir of the filtration unit oN DWH Place sterile cap on the bottle portion of the filter unit Note Store the medium at 2 8 C Use within one week of preparation Avoid repeated warming of the RPMI 1640 B27 medium Warm only the required volume of medium to complete the task 35 7 6 Thawing Cytiva Plus Cardiomyocytes for manual patch clamp Time 30 minutes Consumables e 24 well cell culture plate containing Matri
11. Not recommended or intended for diagnosis of disease in humans or animals Do not use internally or externally in humans or animals 2014 General Electric Company All rights reserved First published April 2014 All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them A copy of these terms and conditions is available on request Contact your local GE Healthcare representative for the most current information http www gelifesciences com GE Healthcare UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK 2 Handling A Material Safety Data Sheet MSDS for the dimethyl sulphoxide DMSO in which Cytiva Plus Cardiomyocytes are frozen is included with this shipment and available at http www gelifesciences com msds 2 1 Safety warnings and precautions Warning For research use only Not recommended or intended for diagnosis of disease in humans or animals Do not use internally or externally in humans or animals All chemicals should be considered as potentially hazardous We therefore recommend that this product is handled only by those persons who have been trained in laboratory techniques and that it is used in accordance with the principles of good laboratory practice Wear suitable protective clothing such as laboratory overalls safety glasses and gloves Care should be taken to avoid conta
12. a is a trademark of General Electric Company or one of its subsidiaries B27 and KnockOut are trademarks of Gibco Matrigel is a trademark of Becton Dickinson and Company NucleoCounter NucleoCassette and NC 100 are trademarks of Chemometec A S Clear is a registered trademark of Greiner Bio One GmbH All other third party trademarks are the property of their respective owner License statements Notice to purchaser Important license information The purchase of Cytiva Plus Cardiomyocytes includes a limited license to use the Cytiva Plus Cardiomyocytes for internal research and development but not for any commercial purposes Commercia purposes shall include sale lease license or other transfer of the material or any materia derived or produced from it sale lease license or other grant of rights to use this Materia or any material derived or produced from it use of this material to perform services for a fee for third parties A license to use the Cytiva Plus Cardiomyocytes for commercia purposes is subject to a separate license agreement with GE Healthcare IF YOU REQUIRE A COMMERCIAL LICENSE TO USE THIS MATERIAL AND DO NOT HAVE ONE PLEASE CONTAC YOUR LOCAL GE HEALTHCARE REPRESENTATIVE AND RETURN THIS MATERIAL UNOPENED TO GE HEALTHCARE UK LTD THE MAYNARD CENTRE FOREST FARM WHITCHURCH CARDIFF CF14 7YT UK AND ANY MONEY PAID FOR THE MATERIAL WILL BE REFUNDED GE Healthcare Cytiva Plus Cardiomyocytes the Products are sold under licen
13. ate in a standard cell culture incubator at 37 C 5 CO 40 7 9 Media change on day 4 post thaw Time 20 minutes 1 Warm 30 ml RPMI 1640 B27 medium in a 60 ml bottle sealed in a 37 C water bath 2 Perform the following steps aseptically inside the biosafety cabinet BSC 3 After 96 hours carefully aspirate off 300 uL of the medium from each well leaving 350 uL residual medium and replace with 300 uL of warmed RPMI 1640 B27 medium so as not to disturb the seeded cells Incubate plate at 37 C 5 COs 4 Repeat a media change at day 7 replacing half the seeded medium with fresh pre warmed RPMI 1640 B27 medium 5 Perform manual patch clamp 4 7 days after seeding in cell culture plates It is the responsibility of the user to determine the optimal culture time for this application 41 Figure 5 Examples of cell morphologies patched manually 42 8 Troubleshooting Problems Solutions Poor cardiomyocyte viability Poor cell attachment 43 Check storage shipping conditions Follow pack leaflet to revive cells from cryostore Follow recommended procedure for thawing and dilution of cardiomyocytes as improper handling of Cytiva Plus Cardiomyocytes could cause low viability Ensure pre warmed medium is added drop wise to the cardiomyocytes Make sure plate being used is tissue culture treated and sterile Follow recommended procedure for preparation and storage of fibronectin plates Aspira
14. aterials to be supplied by user Product Supplier Product code 15 ml Centrifuge tubes Corning 430052 50 ml Centrifuge tubes Corning 430290 Serological pipettes 5 ml Corning 4487 Serological pipettes 10 ml Corning 4488 Serological pipettes 25 ml Corning 4489 500 ml Cellulose acetate filter units Corning 430769 12 or 48 well MEA plate for MEA applications e g Axion BioSystems M768 GLx or M768 KAP 48 Multi well cell culture plates Various e g 96 well culture plate for HCA and Ca2 transient Greiner uClear 781091 applications Multi well plates for impedance e g ACEA E plate Cardio 96 applications Coverslips electrophysiological applications VWR 631 0149 1 5 ml tubes Corning 430290 Sterile bottle e g 30 or 60 ml not available in North America Standard glass coverslips of NALG2019 0030 0060 approximately 13 mm diameter and 0 13 mm thickness are suitable 9 Product Supplier Product code RPMI 1640 Glutamine Gibco 21875034 D PBS Sigma D8537 B27 50x Gibco 17504 004 Fibronectin BD Biosciences 354008 FBS Gibco 26140 079 Matrigel Becton Dickinson 356231 KnockOut D MEM KO DMEM Gibco 10829 018 Sterile distilled water Fresenius Kabi 22 96 985 4 3 Equipment needed Adjustable pipettes and tips Liquid nitrogen vapour store Biosafety cabinet Ice bucket with dry ice Cryovial rack Vacuum pump and line
15. ce from Asterias Biotherapeutics Inc and Wisconsin Alumni Research Foundation WARF under US patent and publication numbers US 5 843 780 US 6 200 806 US 6 602 711 US 6 800 480 US 7 005 252 US 7 029 913 US 7 297 539 US 7 413 902 US 7 425 448 US 7 452 718 US 7 582 479 US 7 732 199 US 7 763 464 US 7 781 216 US 7 851 167 US 7 897 389 and US 2007 0010012 and equivalent patent and patent applications in other countries 2014 General Electric Company All rights reserved All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them A copy of these terms and conditions is available on request Contact your local GE Healthcare representative for the most current information http www gelifesciences com contact http www gelifesciences com GE Healthcare UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK imagination at work rr ey
16. ct with skin or eyes In the case of contact with skin or eyes wash immediately with water See material safety data sheet s and or safety statement s for specific advice 2 2 Storage Cytiva Plus Cardiomyocyte product codes 29 0918 80 and 29 0918 81 are supplied in 1 ml cryovials of gt 1 x 106 1 000 000 viable cells 1 6 x 106 total cells and 23 5 x 105 3 500 000 viable cells 5 x 105 total cells respectively cryopreserved in 10 DMSO and 90 foetal bovine serum Upon receipt the frozen vials of cells should immediately be removed from the outer packaging and transferred to the vapor phase of a liquid nitrogen storage unit at 140 C 2 3 Expiry Please refer to the Certificate of Analysis for further details 2 4 Packaging Cytiva Plus Cardiomyocytes are provided as cryopreserved single cell suspensions in 1 ml cryovials 3 Introduction GE Cytiva Plus Cardiomyocytes are human cardiomyocytes derived from the NIH approved stem cell line NIH hESC 10 0061 Cytiva Plus Cardiomyocytes have been extensively characterized and functionally verified by flow cytometry sub cellular imaging and electrophysiology Cytiva Plus Cardiomyocytes are supplied cryopreserved in a ready to use format 4 Components and other materials required 4 1 Components e GE Cytiva Plus Cardiomyocytes e Certificate of Analysis including product specifications e Material Safety Data Sheet e User manual 4 2 M
17. erilization of glass coverslips Time 4 hours 7 2 Preparation of 1 2 diluted Matrigel aliquots Time 10 minutes with O N thaw 7 3 Preparation of 1 30 diluted Matrigel Time 10 minutes 7 4 Preparation of Matrigel coated coverslips Time 10 minutes 7 5 Preparation of RPMI 1640 B27 medium for manual patch clamp Time 20 minutes 7 6 Thawing Cytiva Plus Cardiomyocytes for manual patch clamp Time 30 minutes 7 7 Determination of post thaw viability of Cytiva Plus Cardiomyocytes for manual patch clamp Time 30 minutes 7 8 Seeding Cytiva Plus Cardiomyocytes onto coverslips Time 30 minutes 7 9 Media change on day 4 post thaw Time 20 minutes 8 Troubleshooting 9 Related products 25 28 29 31 31 32 33 34 35 36 37 39 41 43 46 1 Legdl GE GE monogram and imagination at work are trademarks of General Electric Company Cytiva is a trademark of General Electric Company or one of its subsidiaries B27 and KnockOut are trademarks of Gibco Matrigel is a trademark of Becton Dickinson and Company NucleoCounter NucleoCassette and NC 100 are trademarks of Chemometec A S uCleor is a registered trademark of Greiner Bio One GmbH All other third party trademarks are the property of their respective owner License statements Notice to purchaser Important license information The purchase of Cytiva Plus Cardiomyocytes includes a limited license to use the Cytiva Plus Cardiomyocytes
18. gel coated coverslips 2 8 C e 1x 106 vial of Cytiva Plus Cardiomyocytes e RPMI 1640 B27 medium filter sterilized e 50 ml sterile tube e 10 ml sterile serological pipette Equipment e Ice bucket with dry ice e Cryovial rack e 37 C water bath e Centrifuge e 1000 uL pipette amp sterile 1 1000 uL tips e Pipette gun Protocol Perform the following steps aseptically inside the biosafety cabinet BSC 1 Before thawing the cryovial of cells take the 24 well cell culture plate containing the Matrigel coated coverslips out of 2 8 C storage Leave the 24 well cell culture plate at room temperature for 1 hour before seeding the cells 2 Remove the cryovial from the cryostore and place onto dry ice until ready to thaw 3 Thaw cell suspension in a 37 C water bath with gentle agitation until ice crystals just disappear Note Take care not to immerse the whole cryovial into the water bath Avoid extended incubation at 37 C 36 4 Wipe the outside of the cryovial with 70 isopropanol and transfer to BSC 5 Carefully transfer the cell suspension into a sterile 50 ml centrifuge tube using a 1000 uL pipette 6 Rinse the inside of the cryovial with 1 ml of room temperature RPMI 1640 B27 and combine with the cell suspension drop wise with gentle mixing 7 Slowly over the course of 2 minutes add 8 ml of RPMI 1640 B27 to the 50 ml centrifuge tube 8 Centrifuge at 300 g for 5 minutes at 20 C 9 Carefully using
19. he plate onto clean tissues and replace with 40 HL of warmed RPMI 1640 B27 medium into each well so as not to disturb the seeded cells Incubate plate at 37 C 5 CO3 4 Repeat a media change at day 7 replacing the seeded medium with fresh pre warmed RPMI 1640 B27 medium 5 Perform HCA 7 8 days after seeding in cell culture plates It is the responsibility of the user to determine the optimal culture time for this application 29 Figure 4 Brightfield images of Cytiva Plus Cardiomyocytes seeded in microplates for HCA after 8 days post thaw 30 7 Protocols for Manual Patch Clamp 7 1 Sterilization of glass coverslips Time 4 hours Consumables e Borosilicate glass coverslips 13 mm e Isopropanol e 24 well cell culture plate e 50 ml sterile tube Equipment e Sterile forceps Protocol Perform the following steps aseptically inside the biosafety cabinet BSC 1 Sterilize the coverslips by soaking in isopropanol for a minimum of 2 hours in a sealed sterile 50 ml tube Ensure complete wetting of both sides of each coverslip 2 Using sterile forceps remove a coverslip from the isopropanol and shake the excess isopropanol from the coverslip Place the coverslip into the well of a sterile 24 well plastic cell culture plate 3 Repeat steps 1 3 for the number of wells required 4 Let the coverslips air dry in a BSC for a minimum of 2 hours 31 7 2 Preparation of 1 2 diluted Matrigel aliquots Time 10 minutes
20. hod could be adapted for other commercial cell counters 25 Consumables e RPMI 1640 B27 medium filter sterilized e 4x 1 5 ml tubes 4 x NucleoCassettes ChemoMetec e Reagent A100 ChemoMetec e Reagent B ChemoMetec Equipment e 100 uL pipette amp sterile 1 200 uL tips e 1000 uL pipette amp sterile 1 1000 uL tips e NucleoCounter NC 100 ChemoMetec Protocol Perform the following steps aseptically inside the biosafety cabinet BSC 1 Determine viable cell number using the NucleoCounter NC 100 ChemoMetec To do this transfer 40 uL of the cell suspension into a 1 5 ml tube Add to the tube 360 uL of RPMI 1640 B27 medium i e now a 1 10 dilution Gently agitate to achieve an even cell suspension 2 The remaining steps can be performed outside of the BSC 3 Transfer 100 uL of 1 10 diluted cell suspension into 4 x 1 5 ml tubes 4 Use a 100 uL of 1 10 diluted cell suspension sample to calculate the number of Non Viable Cells rni i e directly load the sample into a NucleoCassette 5 Repeat step 4 for a second time and calculate the average result 6 Use another 1 x 100 uL of 1 10 diluted cell suspension sample to calculate the Total Number of Cells ml To do this add 100 uL of Reagent A100 and then 100 uL of Reagent B to the 100 uL of 1 10 diluted cell suspension sample Mix by pipetting then load into a ucleoCassette 26 7 Repeat step 6 for a second time and calculate the average result
21. ion 5 5 9 to dry out The cell droplet should still be visible post 2 3 hour incubation prior to adding medium to the well 5 5 10 Take care when adding the medium to the well so as not to disturb the seeded cells 5 5 10 If using re usable MEA plates ensure that the MEA plates are A Sterile and the plating surface is dry and free of cellular debris B Maintained and stored as directed by the manufacturer 9 Related Products Product name Code Cytiva Cell Health Assay for 29 0244 68 high content analysis Cytiva Cell Integrity Assay for 29 0244 69 high content analysis Hardware Code Cell Analyzer 2200 Imaging System 29 0278 86 Cell Analyzer 6000 Imaging System 28 9939 14 Options Code Cell Analyzer Transmitted Light Module 28 9534 87 Cell Analyzer Temperature Control Module 28 9534 73 Cell Analyzer Environmental Control Module 28 9534 85 Cell Analyzer Liquid Handling Module 28 9798 62 Software Code Cell Investigator single seat license 28 4089 71 Cell Investigator Zebrafish Analysis 28 9826 95 software plug in Cell Miner HCM single academic use 28 9624 55 Cell Miner HCM single commercial use 28 9624 56 46 Page intentionally left blank 47 GE Healthcare offices GE Healthcare Bio Sciences AB Bj rkgatan 30 751 84 Uppsala Sweden GE Healthcare Europe GmbH Munzinger Strasse 5 D 79111 Freiburg Germany GE Healthcare Bio Sciences Corp 800 Centennial Avenue PO Bo
22. ll suspension sample to calculate the number of Non Viable Cells ml i e directly load the sample into a NucleoCassette 5 Repeat step 4 for a second time and calculate the average result 6 Use another 1 x 100 uL of 1 10 diluted cell suspension samples to calculate the Total Number of Cells ml To do this add 100 uL of Reagent A100 and then 100 uL of Reagent B to the 100 uL of 1 10 diluted cell suspension sample Mix by pipetting then load into a NucleoCassette 7 Repeat step 6 for a second time and calculate the average result 8 Determine the total Volume of Cel Suspension Note We use a 1000 uL pipette to establish this volume 38 9 Calculate the number of viable cells in the cell suspension Viable cells ml 10 x 3 x Total Number of Cells ml Non Viable Cells ml Total number of viable cells Viable cells ml x Volume of Cell Suspension ml Worked example for a typical 1 0 x 106 vial of Cytiva Plus Cardiomyocytes Total Number of Cells ml determined in step 6 5 33 x 104 Non Viable Cells ml determined in step 5 4 80 x 104 Viable cells ml 10 x 3 x 5 33 x 104 4 80 x 104 1 12 x 105 cells ml Total number of viable cells 1 12 x 105 cells ml x 1 ml 1 12 x 108 cells Note There are a total of 1 6 x 106 cells in a 1 0 x 106 vial of Cytiva Plus Cardiomyocytes and we routinely record a value of gt 70 post thaw cell viability i e 1 12 x 105 viable cells 7 8 Seeding Cytiva Plus
23. mables e RPMI 1640 B27 medium filter sterilized e 50 ml tube e 60 ml bottle Equipment e Fibronectin coated well MEA plate e 20 uL pipette amp sterile 1 20 uL tips e 200 uL pipette amp sterile 1 200 uL tips e 1000 uL pipette amp sterile 1 1000 uL tips Protocol Perform the following steps aseptically inside the biosafety cabinet BSC 1 Warm 25 ml RPMI 1640 B27 medium in a 60 ml bottle sealed in a 37 C water bath 2 Centrifuge the cell suspension in a 50 ml tube at 300 g for 5 minutes at 20 C 3 Carefully using a 1000 uL pipette remove the supernatant 4 Resuspend the cells in the residual liquid with gentle agitation 5 Determine the total volume of the cell suspension Note We use a 100 UL pipette to establish this volume 18 6 Dilute the cell suspension to 1 5 x 10 viable cells ml using warm RPMI 1640 B27 medium Required volume ml Viable cells 1 5 x 107 viable cells ml Note Assuming number of viable cells is 3 85 x 106 the volume should be made up to 260 pL Remove fibronectin coated well MEA plate from incubator 8 Aspirate the fibronectin bead from each well of the MEA plate use a pipette set to dispense 8 uL and immediately replace with a 4 LL bead of Cytiva Plus Cardiomyocyte suspension 1 5 x 107 viable cells ml over the recording electrode area see Fig 2 Cytiva Plus bead of cells Figure 2 Fibronectin bead replaced with Cytiva Plus cardiomyocyte bead
24. on step perform the following steps aseptically inside the biosafety cabinet BSC 1 Remove the cryovial from the cryostore and place onto dry ice until ready to thaw 24 2 Thaw cell suspension in a 37 C water bath with gentle agitation until ice crystals just disappear Note Take care not to immerse the whole cryovial into the water bath Avoid extended incubation at 37 C 3 Wipe the outside of the cryovial with 70 isopropanol and transfer to BSC 4 Carefully transfer the cell suspension into a sterile 50 ml centrifuge tube using a 1000 uL pipette 5 Rinse the inside of the cryovial with 1 ml of room temperature RPMI 1640 B27 and combine with the cell suspension drop wise with gentle mixing 6 Slowly over the course of 2 minutes add 8 ml of RPMI 1640 B27 to the 50 ml centrifuge tube 7 Centrifuge at 300 g for 5 minutes at 20 C 8 Carefully using a 10 ml stripette remove 8 mls of the supernatant Remove a further 1 ml with a 1000 uL pipette taking care not to disturb the cell pellet Resuspend the cells in the residual liquid approx 1 ml with gentle agitation 6 4 Determining Post thaw Cell Viability of Cytiva Plus Cardiomyocytes for HCA Time 30 minutes During the 2 hour fibronectin incubation step determine the viable cell number and viable cell density using preferred method of choice We use a NucleoCounter NC 100 ChemoMetec cell counter the method for which is outlined below However this met
25. ose to light for extended periods of time Perform the following steps aseptically inside the biosafety cabinet BSC 2 Wipe the required number of RPMI 1640 Glutamine medium bottle s and B27 Supplement vialls with 70 isopropanol and transfer to a BSC 3 Place the filtration unit into the BSC 4 Using a 10 ml sterile serological pipette add 10 ml of B27 supplement to 500 ml bottle of RPMI 1640 Glutamine medium Swirl bottle several times to mix 5 Carefully pour 510 ml of the RPMI 1640 B27 medium into the reservoir of the filtration unit 23 6 Place the lid on the filter unit 7 Connect the filter unit to a vacuum source 8 When filtration is complete disconnect the vacuum source 9 Detach the upper reservoir of the filtration unit 10 Place sterile cap on the bottle portion of the filter unit Note Store the medium at 2 8 C Use within one week of preparation Avoid repeated warming of the RPMI 1640 B27 medium Warm only the required volume of medium to complete the task 6 3 Thawing Cytiva Plus Cardiomyocytes for HCA Time 30 minutes Consumables e 3 5 x 105 vial of Cytiva Plus Cardiomyocytes e RPMI 1640 B27 medium filter sterilized e 50 ml sterile tube e 10 ml sterile serological pipette Equipment e Ice bucket with dry ice e Cryovial rack e 37 C water bath e Centrifuge e 1000 uL pipette amp sterile 1 1000 uL tips e Pipette gun Protocol During the 2 hour fibronectin incubati
26. p 6 for a second time and calculate the average result 8 Determine the total Volume of Cell Suspension Note We use a 1000 uL pipette to establish this volume 9 Calculate the number of viable cells in the cell suspension Viable cells ml 10 x 5 x Total Number of Cells ml Non Viable Cells mi Total number of viable cells Viable cells ml x Volume of Cell Suspension ml Worked example for a typical 3 5 x 105 vial of Cytiva Plus Cardiomyocytes Total Number of Cells ml determined in step 6 1 83 x 105 Non Viable Cells ml determined in step 5 1 64 x 10 Viable cells ml 10 x 3 x 1 83 x 105 1 64 x 10 3 85 x 105 cells ml Total number of viable cells 3 85 x 106 cells ml x 1 ml 3 85 x 108 cells Note There are a total of 5 5 x 10 cells in a 3 5 x 10 vial of Cytiva Plus Cardiomyocytes and we routinely record a value of gt 70 post thaw cell viability i e 3 85 x 106 viable cells 17 5 5 Seeding Cytiva Plus Cardiomyocytes onto MEA plates Time 3 hours Cells should be seeded at a density of 6 x 10 viable cells in 4 uL i e 1 5 x 107 viable cells ml over the recording electrode area Note Assuming the volume of the cell suspension is 1 ml and the number of viable cells is 3 85 x 109 the current concentration is 3 85 x 108 viable cells ml Consequently the next step involves centrifuging and resuspending the cells from section 5 4 to achieve the appropriate cell concentration Consu
27. plate e g Greiner uClear 781091 e 96 well cell culture plate e g Greiner uClear 655090 e 20 uL pipette amp sterile 1 40 uL tips e 1000 uL pipette amp sterile 1 1000 uL tips Protocol Perform the following steps aseptically inside the biosafety cabinet BSC 1 Prepare a 1 mg ml solution of fibronectin by adding 1 ml sterile distilled water to the 1 mg fibronectin 2 Take 125 uL of this 1 mg ml fibronectin solution and add it to 9875 uL D PBS in a 50 ml tube for a final concentration of 12 5 ug ml fibronectin Add 100 uL of 12 5 ug ml fibronectin solution to each well of the 96 well cell culture plate or 30 uL to each well of a 384 well cell culture plate 3 Put the lid on the cell culture plate and incubate for 2 hours in a standard cell culture incubator at 37 C 5 CO 22 6 2 Preparation of RPMI 1640 B27 medium for HCA Time 20 minutes Consumables e RPMI 1640 Glutamine medium 500 ml at 4 C e B27 supplement 10 ml at 20 C e 10 ml sterile serological pipette Equipment e 500 ml 0 22 um cellulose acetate filter unit Corning 430769 e Vacuum line e Pipette gun Protocol During the 2 hour fibronectin incubation step prepare medium Note Medium may be prepared immediately before thawing Cytiva Plus Cardiomyocytes or prepared and stored at 2 8 C and used within one week of preparation 1 Thaw frozen 10 ml B27 supplement vidl s for 10 minutes in a 37 C water bath Do not incubate at 37 C or exp
28. rature Keep the solution and pipettes cold at all stages of handling Perform the following steps aseptically inside the biosafety cabinet BSC 1 Slowly thaw a 2 ml 1 2 diluted Matrigel aliquot prepared in step 7 2 at 4 C for at least 2 hours to avoid the formation of a gel Once thawed transfer to the BSC 2 Cool a sterile 5 ml serological pipette by drawing 5 ml of cold KO DMEM into the pipette 3 Dilute the 2 ml 1 2 diluted Matrigel aliquot with 5 ml cold KO DMEM Carefully mix Matrigel solution avoiding the formation of bubbles 4 Cool a sterile 25 ml pipette by drawing 25 ml of cold KO DMEM into the pipette 5 Add a further 23 ml cold KO DMEM for a final dilution of 1 30 Carefully mix Matrigel solution avoiding the formation of bubbles 33 7 4 Preparation of Matrigel coated coverslips Time 10 minutes Consumables e 30 ml 1 30 diluted Matrigel 4 C e 24 well cell culture plate containing glass coverslips e KO DMEM 2 8 C e 10 ml sterile serological pipette 2 8 C e 10 x 50 ml tubes Equipment e Pipette gun Protocol Do not allow the Matrigel solution to reach room temperature Keep the solution pipettes and KO DMEM cold at all stages of handling Avoid repeated freeze thawing of diluted Matrigel aliquots Perform the following steps aseptically inside the biosafety cabinet BSC 1 Add 400 uL of 1 30 diluted Matrigel to each well of the 24 well cell culture plate containing a glass coverslip 2
29. te fibronection just before seeding Be careful not to touch the bottom of plate Make sure the surface of the well has not been marked during the removal of fibronectin Do not let the plate dry out Place into incubator on a flat surface for 2 hours Make sure viable count number is used to seed Problems Solutions Problems Solutions Poor cell attachment Uneven coating of Matrigel Aggregation of Matrigel 44 Follow feeding steps in pack leaflet Ensure RPMI 1640 B27 is warmed immediately before use and avoid prolonged exposure to light Use RPMI 1640 B27 within 7 days of preparation and avoid repeated warming Poor ion channel activity Poor performance on MEA Ensure that Matrigel coverage is uniform by microscopic inspection Matrigel coated plates should be warmed to room temperature for at least 30 minutes before use Ensure culture vessels are stored on a level surface Use only cell culture treated plastic ware Follow recommended procedure for preparation of Matrigel coating of plates Avoid warming of Matrigel keep on ice during processing if necessary Avoid room temperature plastic coming in contact with Matrigel 45 Allow at least 72 hour post thaw before use depending on the application lon channel activity will increase as cells recover from thaw Ensure correct seeding density of Cytiva Plus for the application Do not allow the droplets of cell suspens
30. te gun Protocol During the 2 hour fibronectin incubation step perform the following steps aseptically inside the biosafety cabinet BSC 1 Remove the cryovial from the cryostore and place onto dry ice until ready to thaw 14 2 Thaw cell suspension in a 37 C water bath with gentle agitation until ice crystals just disappear Note Take care not to immerse the whole cryovial into the water bath Avoid extended incubation at 37 C 3 Wipe the outside of the cryovial with 70 isopropanol and transfer to BSC 4 Carefully transfer the cell suspension into a sterile 50 ml centrifuge tube using a 1000 LL pipette 5 Rinse the inside of the cryovial with 1 ml of room temperature RPMI 1640 B27 and combine with the cell suspension drop wise with gentle mixing 6 Slowly over the course of 2 minutes add 8 ml of RPMI 1640 B27 to the 50 ml centrifuge tube 7 Centrifuge at 300 g for 5 minutes at 20 C 8 Carefully using a 10 ml stripette remove 8 mls of the supernatant Remove a further 1 ml with a 1000 UL pipette taking care not to disturb the cell pellet Resuspend the cells in the residual liquid approx 1 ml with gentle agitation 5 4 Determining Post Thaw Cell Viability of Cytiva Plus Cardiomyocytes for MEA Time 30 minutes During the 2 hour fibronectin incubation step determine the viable cell number and viable cell density using preferred method of choice A method for the NucleoCounter NC 100 ChemoMetec cell
31. tilled H20 to the gaps between the wells 11 3 Put the lid on the MEA plate and incubate the MEA plate for 1 5 hours at room temperature 4 Prepare a 1 mg ml solution of fibronectin by adding 1 ml sterile distilled H20 to the 1 mg fibronectin 5 Take 12 5 uL of this 1 mg ml fibronectin solution and add it to 987 5 uL D PBS in a 50 ml tube for a final concentration of 12 5 pg ml fibronectin 6 Aspirate the FBS bead from each well of the MEA plate use a pipette set to dispense 8 uL and immediately replace with a 4 uL bead of 12 5 ug ml fibronectin solution over the recording electrode area 7 Put the lid on the tissue culture dish and incubate the MEA plate for 2 hours in a standard cell culture incubator at 37 C Figure 1 12 well MEA plate showing location of sterile water addition to the inter well space to prevent evaporation of the FBS and fibronectin drop 12 5 2 Preparation of RPMI 1640 B27 medium for MEA Time 30 minutes Consumables e RPMI 1640 Glutamine medium 500 ml at 4 C e B27 supplement 10 ml at 4 C e 10 ml sterile serological pipette Equipment e 500 ml 0 22 um cellulose acetate filter unit Corning 430769 e Vacuum line Pipette gun Protocol During the 2 hour fibronectin incubation step prepare medium Note Medium may be prepared immediately before thawing Cytiva Plus Cardiomyocytes or prepared and stored at 2 8 C and used within one week of preparation 1 Thaw frozen 10 ml B27 s
32. ulture incubator at 37 C 5 CO gt 5 6 Media change on day 4 post thaw Time 20 minutes 1 Warm 30 ml RPMI 1640 B27 medium in a 60 ml bottle sealed in a 37 C water bath 2 Perform the following steps aseptically inside the biosafety cabinet BSC 3 For the 12 well MEA plate using a 1000 uL pipette carefully aspirate 1000 uL of media from each well of the 12 well MEA plate then gently add 1000 pL of warm RPMI 1640 B27 medium 20 to the corner of each well of the 12 well MEA plate to give a total volume of 2 ml 4 For the 48 well plate using a 1000 uL pipette carefully aspirate 250 LL of media from each well of the 48 well MEA plate then using a 200 uL pipette gently add 200 uL of warm RPMI 1640 B27 medium to the corner of each well of the 48 well MEA plate to give a total well volume of 400 pL 5 Perform MEA recordings 5 7 days after plating onto the MEA plate It is the responsibility of the user to determine the optimal culture time for this application 3A 3B Figure 3A Wells containing Cytiva Plus Cardiomyocytes and media Figure 3B Cytiva Plus Cardiomyocytes as viewed under a microscope 5 days after seeding 21 6 Protocols for HCA 6 1 Preparation of cell culture plates for HCA Time 2 hours Consumables e Sterile distilled H O Fibronectin 1 mg human BD Biosciences 354008 at 4 C e 50 ml sterile tube e D PBS sigma D8537 Equipment e 384 well cell culture
33. upplement vialls in a 37 C water bath for 10 minutes Do not incubate at 37 C or expose to light for extended periods of time Perform the following steps aseptically inside the biosafety cabinet BSC 2 Wipe the required number of RPMI 1640 Glutamine medium bottle s and B27 Supplement vidl s with 70 isopropanol and transfer to a BSC 3 Place the filtration unit into the BSC 4 Using a 10 ml sterile serological pipette add 10 ml of B27 supplement to a 500 ml bottle of RPMI1640 Glutamine medium Swirl bottle several times to mix 5 Carefully transfer the medium and supplement into the reservoir of the filter unit 13 6 Place the lid on the filter unit 7 Connect the filter unit to a vacuum source 8 When filtration is complete disconnect the vacuum source 9 Detach the upper reservoir of the filtration unit 10 Place sterile cap on the bottle portion of the filter unit Note Store the medium at 2 8 C Use within one week of preparation Avoid repeated warming of the RPMI 1640 B27 medium Warm only the required volume of medium to complete the task 5 3 Thawing Cytiva Plus Cardiomyocytes for MEA Time 30 minutes Consumables e 3 5 x 105 vial of Cytiva Plus Cardiomyocytes e RPMI 1640 B27 medium filter sterilized e 50 ml tube e 10 ml sterile serological pipette Equipment e Ice bucket with dry ice e Cryovial rack e 37 C water bath e Centrifuge e 1000 uL pipette amp sterile 1 1000 uL tips e Pipet
34. with O N thaw Consumables e Matrigel BD Biosciences 356231 20 C e KO DMEM 2 8 C e 10 ml sterile serological pipette 2 8 C e 10 x 50 ml sterile tubes 2 8 C Equipment e Pipette gun Protocol Do not allow the Matrigel solution to reach room temperature Keep the solution pipettes and KO DMEM cold at all stages of handling Avoid repeated freeze thawing of diluted Matrigel aliquots Perform the following steps aseptically inside the biosafety cabinet BSC 1 Slowly thaw Matrigel at 2 8 C to avoid the formation of a gel Note This process is usually performed overnight 2 Cool a sterile 10 ml serological pipette by drawing and releasing 10 ml of cold KO DMEM into the pipette repeatedly without removing the pipette from the bottle of KO DMEM 3 Add 10 ml of cold KO DMEM to the vial containing 10 ml Matrigel 4 Working quickly mix the Matrigel and KO DMEM with a 10 ml pipette avoiding the formation of bubbles 5 Aliquot 2 ml of diluted Matrigel into each pre chilled sterile 50 ml tube store at 20 C until required Diluted Matrigel solution is stable for 3 months when stored at 20 C 32 7 3 Preparation of 1 30 diluted Matrigel Time 10 minutes Consumables e 2 ml 1 2 diluted Matrigel 20 C e KO DMEM 2 8 C e 5 ml sterile serological pipette 2 8 C e 25 ml sterile serological pipette 2 8 C Equipment e Pipette gun Protocol Do not allow the Matrigel solution to reach room tempe
35. x 1327 Piscataway NJ 08855 1327 USA GE Healthcare Japan Coporation Sanken Bldg 3 25 1 Hyakunincho Shinjuku ku Tokyo 169 0073 Japan For your local office contact information visit www gelifesciences com contact GE Healthcare UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK http www gelifesciences com imagination at work 28 0918 80 AA 04 2014 Cytiva Plus Cardiomyocytes User Guide Product protocol card Codes 29 0918 80 gt 1 x 105 1 000 000 viable cells 29 0918 81 23 5 x 105 3 500 000 viable cells Thaw vial of cardiomyocytes at 37 C Remove vial contents to a 50 ml tube with a 1 ml pipette M Rinse vial with 1 ml RPMI 1640 B27 and slowly add to the cardiomyocytes y Add a further 8 mls of RPMI 1640 B27 to the cell suspension M Centrifuge cell suspension at 300 g for 5 minutes M Remove supernatant and resuspend cell pellet in RPMI 1640 B27 Perform a cell count Dilute cell suspension to required cell density M Dispense into appropriate cell culture device M ncubate at 37 C for 4 7 days depending on application Feed 4 days post thaw and every 3 days thereafter with RPMI 1640 B27 Warning For research use only Not recommended or intended for diagnosis of disease in humans or animals Do not use internally or externally in humans or animals GE GE monogram and imagination at work are trademarks of General Electric Company Cytiv
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