Cryopreservation of Dissociated Human Embryonic Stem Cells in the Presence of ROCK Inhibitor

Raquel Martín‐Ibáñez1, Anne Marie Strömberg1, Outi Hovatta1, Josep M. Canals2

1 Department of Clinical Science, Intervention and Technology, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden, 2 Departament de Biologia Cellular, Immunologia i Neurosciències, Facultad de Medicina, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Universitat de Barcelona and Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Barcelona, Spain
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1C.8
DOI:  10.1002/9780470151808.sc01c08s10
Online Posting Date:  July, 2009
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Abstract

Two different methods have been adopted for the cryopreservation of human embryonic stem cells (hESCs): vitrification and conventional slow freezing/rapid thawing. However, these methods present poor viability and high differentiation rates. Therefore, the development of an efficient cryopreservation protocol for hESCs is one of the major challenges for the application of these cells in clinical therapy and regenerative medicine. A novel method for the cryopreservation of dissociated hESCs in the presence of a selective Rho‐associated kinase (ROCK) inhibitor that increases cell survival and the efficiency of colony formation of cryopreserved hESCs has been developed. Moreover, this protocol improves the existing methods presenting short recovery times and hardly any differentiation rates. Thus, an easy handling protocol that allows the cryopreservation of large amounts of hESCs is described. Curr. Protoc. Stem Cell Biol. 10:1C.8.1‐1C.8.15. © 2009 by John Wiley & Sons, Inc.

Keywords: freezing; thawing; Y‐27632; survival; differentiation; hESCs

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Cryopreservation of Dissociated Human Embryonic Stem Cells in the Presence of ROCK Inhibitor
  • Support Protocol 1: hESC Maintenance Culture
  • Support Protocol 2: Preparing Human Foreskin Fibroblast Feeder Layers
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Cryopreservation of Dissociated Human Embryonic Stem Cells in the Presence of ROCK Inhibitor

  Materials
  • hESCs
  • 5 mM ROCK inhibitor Y‐27632 (Calbiochem, cat. no. 688001)
  • Dulbecco's phosphate buffered saline without calcium and magnesium (CMF‐DPBS; Invitrogen, cat. no. 14190)
  • TrypLE Express (Invitrogen, cat. no. 12604021)
  • hESC culture medium (see recipe)
  • Basic fibroblast growth factor (bFGF; R&D Systems, cat. no. 234‐FSE/CF)
  • Trypan blue
  • Mitotically inactivated human foreskin fibroblast feeders (see protocol 3)
  • DMSO
  • 70% ethanol
  • Stereomicroscope
  • Sterile 15‐ml conical tubes (e.g., BD Falcon)
  • 35‐mm cell culture dishes (BD Falcon, cat. no. 353001)
  • 200‐µl micropipets
  • Centrifuge
  • Neubauer counting chamber or hemacytometer
  • 1.8‐ml cryotubes (Nunc, cat. no. 377267)
  • Cryo 1°C freezing container (e.g., Mr. Frosty; Nalgene, cat. no. 5100)
  • −80°C freezer
  • −150°C freezer or liquid nitrogen tank
  • 37°C water bath
NOTE: ROCK inhibitor stock solution should be stored in small aliquots at −20°C to avoid repeated freeze/thaw cycles and also should be protected from light.NOTE: To work in sterile conditions, all steps involving cell manipulation must be done in a laminar flow hood.

Support Protocol 1: hESC Maintenance Culture

  Materials
  • hESCs
  • Mitotically inactivated human foreskin fibroblast feeders (see protocol 3)
  • hESC culture medium (see recipe)
  • Scalpels
  • Additional reagents and equipment for hESC culture (see protocol 1)

Support Protocol 2: Preparing Human Foreskin Fibroblast Feeder Layers

  Materials
  • Human foreskin fibroblasts (ATCC # CRL‐2429)
  • Culture medium for feeders (see recipe)
  • Additional reagents and equipment for washing and detaching cells (see protocol 1)
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Figures

Videos

Literature Cited

Literature Cited
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