Functional Characterization of Human Stem Cell–Derived Cardiomyocytes

Glenn E. Kirsch1, Carlos A. Obejero‐Paz1, Andrew Bruening‐Wright1

1 ChanTest Corporation, Cleveland, Ohio
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 11.12
DOI:  10.1002/0471141755.ph1112s64
Online Posting Date:  March, 2014
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Abstract

Cardiac toxicity is a leading contributor to late stage attrition in the drug discovery process and to withdrawal of approved drugs from the market. In vitro assays that enable earlier and more accurate testing for cardiac risk provide early stage predictive indicators that aid in mitigating risk. Human cardiomyocytes, the most relevant subjects for early stage testing, are severely limited in supply, but human stem cell–derived cardiomyocytes (SC‐hCM) are readily available from commercial sources and are increasingly used in academic research, drug discovery, and safety pharmacology. As a result, SC‐hCM electrophysiology has become a valuable tool for assessing cardiac risk associated with drug administration. Described in this unit are techniques for recording individual sodium, calcium, and potassium currents, as well as single‐cell action potentials and impedance recordings from contracting syncytia of thousands of interconnected cells. Curr. Protoc. Pharmacol. 64:11.12.1‐11.12.26. © 2014 by John Wiley & Sons, Inc.

Keywords: stem cell–derived cardiomyocytes; electrophysiology; impedance

     
 
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Table of Contents

  • Basic Protocol 1: Measurement of Action Potentials in Stem Cell–Derived Human Cardiomyocytes
  • Support Protocol 1: Preparation of Stem Cell‐Derived Human Cardiomyocytes for Recording Action Potentials or Ion Channel Currents
  • Basic Protocol 2: Voltage‐Clamp Studies of Ionic Currents in Stem Cell–Derived Human Cardiomyocytes
  • Support Protocol 2: Voltage Protocol to Record Calcium Currents
  • Support Protocol 3: Voltage Protocol to Record Sodium Currents
  • Support Protocol 4: Voltage Protocols to Record Transient Outward Currents
  • Basic Protocol 3: Measurement of Contractile Activity of iCELL Cardiomyocyte Monolayers in the xCELLigence RTCA Cardio System
  • Support Protocol 5: Preparation of iCELL Cardiomyocyte Monolayers for Impedance Recordings Using the xCELLigence RTCA Cardio System
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Measurement of Action Potentials in Stem Cell–Derived Human Cardiomyocytes

  Materials
  • Cardiomyocyte cultures in 35‐mm dishes ( protocol 2)
  • Action potential extracellular solution (see recipe)
  • Action potential intracellular (micropipet) solution (see recipe)
  • Positive control compounds (e.g., quinidine; Sigma‐Aldrich)
  • Electrophysiology workstation
    • Computer running pCLAMP software (Molecular Devices)
    • Patch‐clamp amplifier (Axon Instruments 200B)
    • Analog to digital converter
    • Antivibration table (e.g., Air table, Newport, cat. no. LW3036B‐OPT)
    • Inverted microscope (Zeiss Axiovert 25 CFL)
    • Faraday cage
    • Data acquisition system (Digidata; Axon Instruments, cat. no. 1320A)
    • Laboratory DC power supply (Tenma, cat. no. 72‐2005)
    • Headstage (Axon Instruments)
    • Cable assembly (Warner Instruments, cat. no. CC‐28)
    • Manipulator for perfusion (Fine Science Tools)
    • Magnetic stand (Fine Science Tools)
    • Manipulator stand (Sutter Instruments, cat. no. MT‐75)
    • Manipulator for in‐line solution preheater (World Precision Instruments KITE‐L)
  • Perfusion and heating systems
    • Dual channel heater controller (Warner Instruments, cat. no. TC‐344B)
    • Culture dish heater (Warner Instruments, cat. no. DH‐35)
    • 10‐ml syringe
    • Stopcock (Cole‐Parmer, cat. no. 30600‐06)
    • Female luer (Cole‐Parmer, cat. no. 45508‐00)
    • Silicone tubing, size 24 (Cole‐Parmer Instrument)
    • T‐fittings (Cole‐Parmer Instrument, cat. no. 06365‐77)
    • Tygon tubing (Cole‐Parmer Instrument, cat. nos. AAQ‐0127 and AAC02529‐CP)
    • In‐line solution preheater (Warner Instruments, cat. no. SH‐27B)
    • Suction device (24‐gauge needle connected to vacuum source)
    • Thermistor probe (Warner Instruments, cat. no. CC‐28)
  • Borosilicate glass tubing (World Precision Instruments, cat. no. TW150F‐3)
  • P‐97 pipet puller (Sutter Instruments)
  • Electrode holder (Molecular Devices, cat. no. 1‐HL‐N)
  • Micromanipulator assembly (Burleigh, PCS‐6000 EXPO series)
    • Micromanipulator axis control unit
    • Micromanipulator power supply
    • Micromanipulator assembly base

Support Protocol 1: Preparation of Stem Cell‐Derived Human Cardiomyocytes for Recording Action Potentials or Ion Channel Currents

  Materials
  • Matrigel (BD Biosciences, cat. no. 354235)
  • 50× B27 medium (Invitrogen, cat. no. 17504‐044)
  • RPMI 1640 medium (Invitrogen, cat. no. 22400‐089)
  • Cytiva cardiomyocytes (1e5m, GE Healthcare, cat. no. 28‐9774‐35), stored in liquid nitrogen
  • 50‐ml conical vials
  • 35‐mm cell culture plates (CELLSTAR, cat. no. 627 170)
  • Biosafety cabinet
  • 37°C water bath
  • Additional reagents and equipment for determining cell number and viability (e.g., Phelan, )

Basic Protocol 2: Voltage‐Clamp Studies of Ionic Currents in Stem Cell–Derived Human Cardiomyocytes

  Materials
  • Extracellular I Ca recording solution (see recipe)
  • Intracellular I Ca recording solution (see recipe)
  • Extracellular I Na recording solution (see recipe)
  • Intracellular I Na recording solution (see recipe)
  • Extracellular I to recording solution (see recipe)
  • Intracellular I to recording solution (see recipe)
  • Electrophysiology work station with perfusion system (e.g., see Fig. and protocol 1)
  • pClamp computer program for analyzing results (Molecular Devices)

Support Protocol 2: Voltage Protocol to Record Calcium Currents

  Materials
  • Maintenance medium (Cellular Dynamics International)
  • Fibronectin‐coated E‐Plate (E‐Plate Cardio 96, ACEA Biosciences; protocol 8)
  • Cultures of iCell cardiomyocytes ( protocol 8)
  • 2×solutions of test compounds
  • xCELLigence RTCA Cardio System (ACEA Biosciences)
  • 20‐ and 200‐µl 12‐channel or 8‐channel multichannel pipettors
  • Humidified, 37°C, 5% CO 2 cell culture incubator

Support Protocol 3: Voltage Protocol to Record Sodium Currents

  Materials
  • Dulbecco's phosphate buffered saline (DPBS) without Ca2+ and Mg2+ (Invitrogen)
  • 1 mg/ml fibronectin (Sigma‐Aldrich) reconstituted in sterile water, according to the manufacturer's instructions; store 1‐ml aliquots up to 6 months at −20°C
  • iCell cardiomyocytes kit (Cellular Dynamics International)
  • 0.5% (w/v) Trypsin‐EDTA (5×), no phenol red (Invitrogen): store in 3‐ml aliquots for single use up to 6 months at −20°C
  • 20‐ and 200‐µl 12‐channel or 8‐channel multichannel pipettors
  • 6‐well cell culture plates (Greiner Bio‐One, cat. no. 657160)
  • Humidified, 37°C, 5% CO 2 cell culture incubator
  • Biological safety cabinet with UV lamp
  • 15‐ and 50‐ml conical tubes, sterile
  • and 5‐ml serological pipets
  • Sterile reagent reservoirs (VWR, cat. no. 89094‐662)
  • E‐Plate Cardio 96 (ACEA Biosciences)
  • Additional reagents and equipment for determining cell number (Phelan, )
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Figures

Videos

Literature Cited

Literature Cited
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