Cryopreservation and Thawing of Cells

Wayne M. Yokoyama1, Maria L. Thompson2, Rolf O. Ehrhardt2

1 University of California School of Medicine, San Francisco, California, 2 BioCision LLC, Larkspur, California
Publication Name:  Current Protocols in Immunology
Unit Number:  Appendix 3G
DOI:  10.1002/0471142735.ima03gs99
Online Posting Date:  November, 2012
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Abstract

Successful cryopreservation of cells requires not only that the cells be handled in a proper fashion for harvesting with equipment in place to ensure consistency, reproducibility, and sterility, but also that a correct choice and amount of cryoprotective agent is added. In general, a controlled freezing rate of 1°C/min is necessary to retain optimal viability of the recovered cells. There are many variations of cell freezing methods in use, including costly electronically regulated control rate freezers, unstandardized, passive isopropyl alcohol freezing containers, and crude rudimentary devices constructed from Styrofoam boxes or paper insulation. However, for the freezing and recovery of cell lines, primary cells, and stem cell cultures, the protocol described in this unit is simple, reproducible, and successful. Not only does it eliminate the need for isopropanol, as well as the costs and hazards associated with its use and disposal, but it provides a uniform method with improved cell viability and recovery. Curr. Protoc. Immunol. 99:A.3G.1‐A.3G.5. © 2012 by John Wiley & Sons, Inc.

Keywords: cell culture mammalian; cell biology; cell isolation and culture

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Freezing Cell Lines or Hybridomas
  • Basic Protocol 2: Thawing Cell Lines or Hybridomas
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Freezing Cell Lines or Hybridomas

  Materials
  • Cell line to be cryopreserved
  • Ice
  • Freezing medium (see recipe)
  • 75‐cm2 tissue culture flask, sterile
  • Cryogenic vials, leak‐free (LN 2‐tested), sterile (e.g., TruCool or equivalent)
  • Indelible marker, resistant to cold, water and ethanol
  • CoolRack CFT30 thermo‐conductive cryogenic tube module (or equivalent)
  • 50‐ml conical centrifuge tube, sterile
  • Beckman TH‐4 rotor (or equivalent)
  • Pipets
  • CoolCell cell freezing container
  • −80°C freezer
  • Liquid nitrogen freezer
NOTE: The use of sterile technique throughout these procedures is critical.

Basic Protocol 2: Thawing Cell Lines or Hybridomas

  Materials
  • Frozen cryogenic vial containing cell line (see protocol 1)
  • 70% ethanol
  • Medium ( appendix 2A; used for growth of cell line)
  • Dry ice
  • CoolRack CFT30 thermo‐conductive cryogenic tube module (or equivalent)
  • 37°C water bath
  • Thermal Tray HP platform (optional)
  • Sterilized tissue culture hood
  • 1‐ml pipet, sterile
  • 12‐ml tube with cap, sterile
  • Beckman TH‐4 rotor (or equivalent)
  • Tissue culture flasks
  • Inverted microscope
  • Additional reagents and equipment for trypan blue exclusion ( appendix 3B)
NOTE: The use of sterile technique throughout these procedures is critical.
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Figures

Videos

Literature Cited

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