Cellular iCell User manual

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iCell®Cardiomyocytes
User’s Guide
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Trademarks
iCell is a registered trademark, and Cellular Dynamics and the logo are trademarks of Cellular Dynamics International, Inc. and may not be used
without the express written permission of Cellular Dynamics International, Inc. (CDI).
All other brands, product names, company names, trademarks, and service marks are the properties of their respective owners.
RestrictionsandLiabilities
This document is provided “as is.” CDI assumes no responsibility for any typographical, technical, or other inaccuracies in this document. CDI reserves the
right to periodically change information that is contained in this document; however, CDI makes no commitment to provide any such changes, updates,
enhancements, or other additions to this document to you in a timely manner or at all.
OTHER THAN THE EXPRESS LIMITED WARRANTY SET FORTH IN THIS USER’S GUIDE, CDI MAKES NO REPRESENTATIONS, WARRANTIES,
CONDITIONS OR COVENANTS, EITHER EXPRESS OR IMPLIED (INCLUDING WITHOUT LIMITATION, ANY EXPRESS OR IMPLIED WARRANTIES
OR CONDITIONS OF FITNESS FOR A PARTICULAR PURPOSE, NON-INFRINGEMENT, MERCHANTABILITY, DURABILITY, TITLE, OR RELATED
TO THE PERFORMANCE OR NON-PERFORMANCE OF ANY PRODUCT REFERENCED HEREIN OR PERFORMANCE OF ANY SERVICES
REFERENCED HEREIN).
This document might contain references to third-party sources of information, hardware or software, products or services and/or third-party websites
(collectively the “Third-Party Information”). CDI does not control, and is not responsible for, any Third-Party Information, including, without limitation the
content, accuracy, copyright compliance, compatibility, performance, trustworthiness, legality, decency, links, or any other aspect of Third-Party
Information. The inclusion of Third-Party Information in this document does not imply endorsement by CDI of the Third-Party Information or the third party
in any way.
CDI does not in any way guarantee or represent that you will obtain satisfactory results from using iCell Cardiomyocytes as described herein. The only
warranties provided to you are included in the Limited Warranty enclosed with this guide. You assume all risk in connection with your use of iCell
Cardiomyocytes.
ConditionsofUse
iCell Cardiomyocytes are for life science research use only and subject to the use restrictions as contained in Appendix A. You are responsible for
understanding and performing the protocols described within. CDI does not guarantee any results you may achieve. These protocols are provided as
CDI’s recommendations based on its use and experience with iCell Cardiomyocytes.
Origin
iCell Cardiomyocytes are manufactured in the United States of America.
CopyrightNotice
© 2015 Cellular Dynamics International, Inc. All rights reserved. This document may not be reproduced, distributed, modified or publicly displayed without
the express written permission of Cellular Dynamics International, Inc.
RevisionHistory
Version 1.8: July 2015
Version 1.7: August 2014
Version 1.6.1: November 2013
Version 1.6: October 2013
Version 1.5.1: November 2012
Version 1.5: September 2011
Version 1.0: December 2009

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iCell Cardiomyocytes User’s Guide i
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Table of Contents
Before You Begin.................................................................................................... ii
Chapter 1. Introduction .......................................................................................... 1
Components Supplied by Cellular Dynamics ............................................................2
Required Equipment and Consumables....................................................................2
Technical Support and Training.................................................................................3
Workflow Diagram .....................................................................................................4
Chapter 2. Handling and Storage.......................................................................... 5
Handling iCell Cardiomyocytes..................................................................................5
Handling iCell Cardiomyocytes Media.......................................................................5
Chapter 3. Preparing Cell Culture Surfaces......................................................... 6
Preparing 0.1% (w/v) Gelatin Solution.......................................................................6
Preparing the Gelatin Cell Culture Vessel .................................................................6
Chapter 4. Thawing Medium and iCell Cardiomyocytes..................................... 7
Thawing iCell Cardiomyocytes Plating Medium ........................................................7
Thawing iCell Cardiomyocytes ..................................................................................7
Chapter 5. Plating iCell Cardiomyocytes ............................................................. 9
Determining Viable Cell Density ..............................................................................10
Plating iCell Cardiomyocytes ...................................................................................10
Chapter 6. Maintaining iCell Cardiomyocytes ................................................... 13
Appendices ........................................................................................................... 14
Appendix A. Intellectual Property Rights, Use Restrictions, and Limited License...14
Appendix B. Limited Warranty .................................................................................14
Appendix C. Limited Liability....................................................................................16
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ii Cellular Dynamics International, Inc. 
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Notes
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Before You Begin
Immediately transfer the frozen vials to liquid nitrogen storage.
Read this entire iCell®Cardiomyocytes User’s Guide before handling or using
iCell Cardiomyocytes.
iCell Cardiomyocytes are for life science research use only. See Appendix A
for more information and other restrictions.
A Safety Data Sheet (SDS) for dimethyl sulfoxide (DMSO), in which iCell
Cardiomyocytes are frozen, is available online at www.cellulardynamics.com/lit/
or on request from Cellular Dynamics International. Only technically qualified
individuals experienced in handling DMSO and human biological materials
should access, use, or handle iCell Cardiomyocytes.

Notes
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iCell Cardiomyocytes User’s Guide 1
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Chapter 1. Introduction
Cellular Dynamics International’s (CDI) iCell Cardiomyocytes are highly purified,
human cardiomyocytes derived from induced pluripotent stem (iPS) cells using
CDI’s proprietary differentiation and purification protocols. iCell Cardiomyocytes are
a mixture of spontaneously electrically active atrial-, nodal-, and ventricular-like
myocytes with typical biochemical, electrophysiological, and mechanical
characteristics and expected responses upon exposure to exogenous agents.
Thus, these cells provide a reliable source of human cardiomyocytes suitable for
use in targeted drug discovery, toxicity testing, and other life science research.
When thawed and plated with iCell Cardiomyocytes Plating Medium and
maintained in iCell Cardiomyocytes Maintenance Medium as instructed in this
User’s Guide, iCell Cardiomyocytes will begin to beat spontaneously within 24 - 48
hours. When plated at appropriate densities, iCell Cardiomyocytes also will form
electrically connected syncytial layers that beat in synchrony. A wash step at 48
hours post-plating using the Maintenance Medium will remove non-adhered cells,
leaving a population of plated, electrically and mechanically active cardiomyocytes
that are ready for use.
iCell Cardiomyocytes Maintenance Medium is antibiotic-free and has been
specially formulated to maintain the health and function of the cardiomyocytes
while limiting the proliferation of the small percentage of non-cardiomyocyte cells.
iCell Cardiomyocytes therefore can be maintained in culture for at least 2 weeks in
the Maintenance Medium without appreciable loss of purity, enabling longer term
studies. Thus, the combination of CDI’s purification process and adherence to the
procedures described in this User’s Guide makes additional use of antibiotics
unnecessary.

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2Cellular Dynamics International, Inc. 
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Notes
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Components Supplied by Cellular Dynamics
Item Catalog Number
iCell Cardiomyocytes1CMC-100-110-001 (1 unit)+
CMC-100-110-005 (5 unit)+
CMC-100-010-000.5 (0.5 unit)#
CMC-100-010-001 (1 unit)#
CMC-100-010-005 (5 unit)#
iCell Cardiomyocytes Plating Medium1CMM-100-110-001 (1 unit)
CMM-100-110-005 (5 unit)
iCell Cardiomyocytes Maintenance Medium1CMM-100-120-001 (1 unit)
CMM-100-120-005 (5 unit)
iCell Cardiomyocytes User’s Guide1
Certificate of Analysis2
Certificate of Origin
If required for shipping purposes
+ Contains monomeric red fluorescent protein (RFP) expressed under control of the endogenous Myh6 promoter
# No RFP expression
1 Safety Data Sheets and User’s Guide available online at www.cellulardynamics.com/lit/
2 Available online at www.cellulardynamics.com/coa/
Required Equipment and Consumables
Item Vendor Catalog Number
Equipment
37°C Water Bath Multiple Vendors
Biological Safety Cabinet with UV Lamp Multiple Vendors
Cell Culture Incubator Multiple Vendors
Hemocytometer or Automated Cell Counter Multiple Vendors
Liquid Nitrogen Storage Unit Multiple Vendors
Phase Contrast Microscope Multiple Vendors
Pipettors Multiple Vendors
Tabletop Centrifuge Multiple Vendors
Consumables
50 ml Centrifuge Tubes Multiple Vendors
Cell Culture Vessels, Sterile, TC Grade Multiple Vendors
Sterile Distilled Water Multiple Vendors
Gelatin, Type A, Cell Culture Tested Sigma-Aldrich G1890
Pipettes Multiple Vendors
Trypan Blue Gibco 15250

Notes
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iCell Cardiomyocytes User’s Guide 3
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Technical Support and Training
CDI’s Technical Support Scientists have the necessary laboratory and analytical
experience to respond to your inquiries. In addition, hands-on training is available
(www.cellulardynamics.com/training/) or watch our Handling iCell Cardiomyocytes
Training Video (www.cellulardynamics.com/cm_handling/). In-lab training may be
available upon request.
Telephone [phone] (US toll-free) / [phone] x5
Monday - Friday, 8:30 am - 5:00 pm US Central Time
Fax [phone]
Email [e-mail]
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4Cellular Dynamics International, Inc. 
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Notes
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Workflow Diagram
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Upon receipt, immediately
transfer to LN2storage!
Culture Preparation
Culture Maintenance
Handling and Storage
p. 5
Thawing
p. 7
Plating
p. 9
Replacing the Medium
p. 13
Experimental Utilization
(native biology, drug discovery,
disease modeling, functionality, etc.)
Applications
Preparing Cell Culture Surfaces
p. 6

Notes
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iCell Cardiomyocytes User’s Guide 5
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Chapter 2. Handling and Storage
Handling iCell Cardiomyocytes
iCell Cardiomyocytes are provided as cryopreserved single-cell suspensions in
1.5 ml cryovials. Upon receipt, directly transfer the cryobox containing iCell
Cardiomyocytes to the vapor phase of a liquid nitrogen storage dewar. CDI strongly
recommends transferring the entire cryobox into the storage rack to avoid
transferring individual vials.
Handling iCell Cardiomyocytes Media
iCell Cardiomyocytes Plating Medium and iCell Cardiomyocytes Maintenance
Medium are shipped frozen on dry ice. Upon receipt, store iCell Cardiomyocytes
media at -20°C until ready for use and at 4°C for up to 2 weeks after thaw. If media
will be used for longer than 2 weeks, aliquot and freeze again after the initial thaw.
Do not subject media to more than a single refreeze and thaw cycle.
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It is critical to maintain cryopreserved iCell Cardiomyocytes at a stable
temperature. Minimize exposure of cryopreserved iCell Cardiomyocytes to
ambient temperature when transferring vials to liquid nitrogen storage.

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6Cellular Dynamics International, Inc. 
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Notes
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Chapter 3. Preparing Cell Culture Surfaces
iCell Cardiomyocytes will plate and function on a variety of substrates including
freshly prepared gelatin and fibronectin, which have been shown to support
attachment, viability, and function of iCell Cardiomyocytes with similar efficiencies.
Coating plates with a 0.1% gelatin solution is an economical, simple, and
recommended method for preparing cell culture plates for culturing iCell
Cardiomyocytes.
CDI provides application protocols and notes that recommend assay-specific
substrates. See www.cellulardynamics.com/lit/ for a list of available application
protocols and notes for iCell Cardiomyocytes. Regardless of the substrate of
choice, prepare plating surfaces before thawing iCell Cardiomyocytes.
Preparing 0.1% (w/v) Gelatin Solution
1. Prepare bottles for mixing the 0.1% gelatin solution.
Do not use bottles that have previously contained or been exposed to
detergent.
Acid-wash new bottles before use.
Use only high-purity, 18 Msterile water for washing bottles.
2. Prepare 0.1% gelatin solution by dissolving 500 mg of gelatin (Type A, powder,
cell culture tested) in 500 ml of sterile water in each bottle.
3. Sterilize the gelatin solution by autoclaving using liquid cycle. The sterile 0.1%
gelatin solution is stable for 3 months when prepared as described above and
stored at room temperature.
Preparing the Gelatin Cell Culture Vessel
1. Select the cell culture vessel appropriate for your experimental use. Add the
volume of 0.1% gelatin solution specified in the table below. Scale volumes
appropriately for other vessel formats.
Culture
Vessel
Surface Area
(cm2)
Volume of 0.1% Gelatin
Solution (ml)
6-well Cell Culture Plate 9.5 2
12-well Cell Culture Plate 3.8 1
24-well Cell Culture Plate 1.9 0.6
96-well Cell Culture Plate 0.32 0.1
384-well Cell Culture Plate 0.06 0.025
T25 Flask 25 8
Table 1: Summary of Useful Volumes and Measures
All volumes and measures are per well, if applicable.
Note: For glass coverslips for immunocytochemistry or electrophysiological
applications, see the iCell Cardiomyocytes Application Protocols available
online at www.cellulardynamics.com/lit/.
2. Incubate the vessel(s) in a 37°C cell culture incubator for at least 1 hour.
3. Aspirate gelatin solution immediately before addition of the cell suspension.