Standardized Cryopreservation of Pluripotent Stem Cells

Rick I. Cohen1, Maria L. Thompson2, Brian Schryver2, Rolf O. Ehrhardt2

1 Rutgers University, Piscataway, New Jersey, 2 BioCision LLC, San Rafael, California
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1C.14
DOI:  10.1002/9780470151808.sc01c14s35
Online Posting Date:  November, 2015
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The successful exploitation of human cells for research, translational, therapeutic, and commercial purposes requires that effective and simple cryopreservation methods be applied for storage in local and master cell banks. Of all the cell types utilized in modern research, human embryonic stem cells and their more recent relatives, induced pluripotent stem cells, are two of the most sensitive to cryopreservation. It is frequently observed that the lack of quality control and proper processing techniques yield poor recovery of pluripotent stem cells. The procedures in this unit have been optimized for handling some of the most recalcitrant stem cell lines, and provide a method for controlled‐rate freezing, using minimal equipment that affords levels of cell viability comparable to expensive controlled‐rate freezers, and standardized cell thawing. The protocol provides a clinically relevant, inexpensive, reliable, and user‐friendly method that successfully prepares cells for long‐term cold storage and ensures maximum levels of cell viability post thaw. © 2015 by John Wiley & Sons, Inc.

Keywords: stem cell culture; stem cell biology; stem cell isolation and storage; cryopreservation; cell thawing

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Table of Contents

  • Introduction
  • Basic Protocol 1: Freezing Stem Cell Lines
  • Basic Protocol 2: Thawing Pluripotent Stem Cell Lines
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Freezing Stem Cell Lines

  • 70% ethanol
  • Cell culture: actively growing pluripotent stem cells plated on 6‐well dish (each well near confluency can accommodate up to 5 million cells per well)
  • 0.5 mM PBS‐EDTA, pH 7.4
  • Phosphate‐buffered saline, pH 7.4, containing 0.5 mM EDTA (PBS‐EDTA)
  • Standard stem cell medium: mTeSR1 (StemCell Technologies) is the most widely used medium; however, there are several well functioning alternatives that are defined and/or xenobiotic‐free (newer‐generation low‐protein media may not function as well in this protocol):
    • PeproGrow‐hESC (BSA‐containing defined medium; PeproTech)
    • PsGro hESC/IPSC Growth Medium (human serum albumin–containing xenobiotic‐free medium; System Bioscience,
  • 1× or 2× freezing medium (see recipe) in 15‐ml tubes
  • Liquid N 2
  • Spray bottle for 70% ethanol
  • Cryogenic vials, leak‐free (liquid N 2 tested), sterile (TruCool brand recommended;
  • Indelible marker, resistant to cold, water, and ethanol
  • CoolBox 2XT workstation with CoolRack CFT30 thermo‐conductive cryogenic tube module and CoolRack 15‐ml conical tube module (BioCision,
  • CoolCell cell freezing container (BioCision,
  • Serological pipet assortment, including 5‐ml glass serological pipets
  • Cell scraper (Sarstedt brand recommended)
  • 15‐ml conical‐bottom centrifuge tubes, sterile (e.g., BD Falcon)
  • Beckman refrigerated centrifuge with TH‐4 rotor (or equivalent)
  • ThawSTAR CFT Transporter (
  • Liquid N 2 tank, preferably vapor tank
NOTE: The use of sterile technique throughout these procedures is assumed.

Basic Protocol 2: Thawing Pluripotent Stem Cell Lines

  • Stem cell medium: this medium should match the medium the cells have been cultured in; once the cells are thawed, they can be transitioned into a new medium (transitioning frozen cells into a new medium may be risky, as thawed cells will normally spontaneously differentiate to some degree due to the cryopreservation process, and adding the stress of adapting to a new medium at the thawing step should be avoided, if possible)
  • Frozen 2‐ml cryogenic vial containing stem cell line ( protocol 1)
  • Dry ice
  • 70% ethanol
  • Appropriate cell culture vessel (6‐well dish coated with Matrigel or other appropriate matrix)
  • 15‐ml sterile conical tube
  • CoolBox 2XT workstation with CoolRack CFT30 thermo‐conductive cryogenic tube module and CoolRack 15‐ml conical tube module (BioCision,
  • Ice pan for dry ice (recommended)
  • Foam floater to accommodate cryogenic vials in water bath
  • ThawSTAR automated cell thawing system (BioCision,
  • ThawSTAR CFT Transporter (BioCision, (optional)
  • CryoPod Carrier (BioCision, (recommended)
  • Additional CoolRack CFT30 cryogenic vial module (BioCision, (optional)
  • ThermalTray HP platform (optional)
  • Inverted microscope
  • Laboratory timer with alarm
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Literature Cited

Literature Cited
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