Cryopreservation of Human Pluripotent Stem Cells in Defined Medium

Weiwei Liu1, Guokai Chen2

1 Faculty of Health Sciences, University of Macau, Taipa, Macau, 2 Center for Molecular Medicine, National Heart, Lung and Blood Institute, Bethesda, Maryland
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1C.17
DOI:  10.1002/9780470151808.sc01c17s31
Online Posting Date:  November, 2014
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Abstract

This unit describes a cryopreservation procedure using an enzyme‐free dissociation method to harvest cells and preserve cells in albumin‐free chemically defined E8 medium for human pluripotent stem cells (hPSCs). The dissociation by EDTA/PBS produces small cell aggregates that allow high survival efficiency in passaging and cryopreservation. Cryopreservation in E8 medium eliminates serum and other animal products, and is suitable for dealing with the increasing demand for high‐quality hPSCs in translational research. In combination with the special feature of EDTA/PBS dissociation, the protocols in this unit allow for efficient cryopreservation in a more time‐saving manner. © 2014 by John Wiley & Sons, Inc.

Keywords: pluripotent stem cells; induced pluripotent stem cells; embryonic stem cells; dissociation; cell culture; cryopreservation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Passaging of Human ESCs/iPSCs With EDTA Dissociation—Preparing Cells 2 to 3 Days Before Cryopreservation
  • Alternate Protocol 1: Passaging of Human ESCs/iPSCs Prior to Cryopreservation When Large Numbers of Cell Lines are Involved
  • Basic Protocol 2: Cryopreservation of Human ESCs/iPSCs With EDTA Dissociation
  • Alternate Protocol 2: Cryopreservation of Human ESCs/iPSCs When Large Numbers of Colonies are Involved
  • Basic Protocol 3: Recovery of Human ESC/iPSC Colonies After Cryopreservation
  • Alternate Protocol 3: Human ESC/iPSc Recovery After Cryopreservation When Large Numbers of Vials are Involved
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Passaging of Human ESCs/iPSCs With EDTA Dissociation—Preparing Cells 2 to 3 Days Before Cryopreservation

  Materials
  • Matrigel, growth factor–reduced (BD Biosciences, cat. no. 354230); always store in a non‐frost‐free freezer, preferably at −80°C
  • DMEM/F12 medium (e.g., Life Technologies)
  • EDTA dissociation solution (see recipe)
  • Complete E8 medium: E8 medium can be prepared as previously described (Beers et al., ), and is also available as Essential 8 Medium from Life Technologies (cat. no. A14666SA) or from Stem Cell Technologies as TeSR‐E8 (cat. no. 05840)
  • Rho‐associated protein kinase (ROCK) inhibitor Y‐27632 (Tocris, cat. no. 1254; optional): dissolve the Y‐27632 in sterile H 2O or sterile DMSO at a final concentration of 10 mM (1000×), then aliquot and store it at −80°C (stable for at least 1 year; Y‐27632 is the ROCK inhibitor that we commonly use; other ROCK inhibitors may also be used)
  • Human ESCs (hESCs): H1 cells, WA01 [U.S. National Institutes of Health (NIH), human ESC registry no. 0043] or human iPSCs from fibroblasts: ND2 (NIH control iPSC line), growing in 6‐well plates6‐well Nunclon Delta Surface tissue culture dishes (Thermo Scientific, cat. no. 140675 or equivalent)

Alternate Protocol 1: Passaging of Human ESCs/iPSCs Prior to Cryopreservation When Large Numbers of Cell Lines are Involved

  Materials
  • Matrigel‐coated 6‐well and 12‐well plates (see protocol 1, step 1, for coating technique)
  • 70% ethanol
  • Complete E8 medium: E8 medium can be prepared as previously described (Beers et al., ), and is also available as Essential 8 Medium from Life Technologies (cat. no. A14666SA) or from Stem Cell Technologies as TeSR‐E8 (cat. no. 05840)
  • Rho‐associated protein kinase (ROCK) inhibitor Y‐27632 (Tocris, cat. no. 1254; optional): dissolve the Y‐27632 in sterile H 2O or sterile DMSO at a final concentration of 10 mM (1000×), then aliquot and store it at −80°C (stable for at least 1 year; Y‐27632 is the ROCK inhibitor that we commonly use; other ROCK inhibitors may also be used)
  • Human ESC (hESC) or iPSC cell lines of interest, growing in 6‐well plates
  • EDTA dissociation solution (see recipe)
  • Inverted phase‐contrast microscope (×4 and ×10 objectives, Zeiss or equivalent)

Basic Protocol 2: Cryopreservation of Human ESCs/iPSCs With EDTA Dissociation

  Materials
  • 2× cryopreservation medium: 20% (v/v) DMSO in complete E8 medium (optionally with 20 μM ROCK inhibitor
  • Complete E8 medium: E8 medium can be prepared as previously described (Beers et al., ), and is also available as Essential 8 Medium from Life Technologies (cat. no. A14666SA) or from Stem Cell Technologies as TeSR‐E8 (cat. no. 05840)
  • Rho‐associated protein kinase (ROCK) inhibitor Y‐27632 (Tocris, cat. no. 1254; optional): dissolve the Y‐27632 in sterile H 2O or sterile DMSO at a final concentration of 10 mM (1000×), then aliquot and store it at −80°C (stable for at least 1 year; Y‐27632 is the ROCK inhibitor that we commonly use; other ROCK inhibitors may also be used)
  • hESC or iPSC growing in 6‐well plates ( protocol 1, step 10)
  • EDTA dissociation solution (see recipe)
  • 1.2‐ml cryovials (USA Scientific, cat. no. 1412‐9100, or equivalent) and cryovial labels
  • Isopropanol cell‐freezing container (e.g., Mr. Frosty from Nalgene)
  • Liquid nitrogen tank
    • Additional reagents and solutions for dissociating cells ( protocol 1, steps 3 to 5)

Alternate Protocol 2: Cryopreservation of Human ESCs/iPSCs When Large Numbers of Colonies are Involved

Basic Protocol 3: Recovery of Human ESC/iPSC Colonies After Cryopreservation

  Materials
  • hESCs or iPSCs cryopreserved in liquid nitrogen ( protocol 3 or protocol 4)
  • 70% ethanol
  • Complete E8 medium: E8 medium can be prepared as previously described (Beers et al., ), and is also available as Essential 8 Medium from Life Technologies (cat. no. A14666SA) or from Stem Cell Technologies as TeSR‐E8 (cat. no. 05840)
  • Rho‐associated protein kinase (ROCK) inhibitor Y‐27632 (Tocris, cat. no. 1254; optional): dissolve the Y‐27632 in sterile H 2O or sterile DMSO at a final concentration of 10 mM (1000×), then aliquot and store it at −80°C (stable for at least 1 year; Y‐27632 is the ROCK inhibitor that we commonly use; other ROCK inhibitors may also be used)
  • Matrigel‐coated 6‐well plates (see protocol 1, step 1, for coating technique)
  • 15‐ml conical tubes (Corning Falcon, cat. no. 352097, or equivalent)
  • Centrifuge (Beckman Coulter Allegra X15‐R with SX4750 swinging‐bucket rotor or equivalent)

Alternate Protocol 3: Human ESC/iPSc Recovery After Cryopreservation When Large Numbers of Vials are Involved

  Additional Materials
  • Four‐way flipper rack (Thermo Scientific)
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Figures

Videos

Literature Cited

Literature Cited
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  Beers, J., Gulbranson, D.R., George, N., Siniscalchi, LI., Jones, J., Thomson, J.A., and Chen, G.K. 2012. Passaging and colony expansion of human pluripotent stem cells by enzyme‐free dissociation in chemically defined culture conditions. Nat. Protoc. 7:2029‐2040.
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