Isolation of Mononuclear Cells from Human Cord Blood by Ficoll‐Paque Density Gradient

Taina Jaatinen1, Jarmo Laine1

1 Finnish Red Cross Blood Service, Helsinki
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2A.1
DOI:  10.1002/9780470151808.sc02a01s1
Online Posting Date:  June, 2007
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Abstract

When preparing stem cell specimens from cord blood, pre‐enrichment of mononuclear cells is highly recommended to improve the recovery of rare stem cells. Mononuclear cells are easily isolated by density gradient centrifugation. In Ficoll‐Paque density gradient centrifugation, anticoagulant‐treated and diluted cord blood is layered on the Ficoll‐Paque solution and centrifuged. During centrifugation, erythrocytes and granulocytes sediment to the bottom layer. Lower density lymphocytes, together with other slowly sedimenting cells such as platelets and monocytes, are retained at the interface between the plasma and Ficoll‐Paque, where they can be collected and subjected to subsequent isolation of hematopoietic stem cells or to the culture of mesenchymal stem cells. Curr. Protoc. Stem Cell Biol. 1:2A.1.1‐2A.1.4. © 2007 by John Wiley & Sons, Inc.

Keywords: cord blood; fresh; cryopreserved; mononuclear cell; density gradient

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1:

  Materials
  • Anticoagulated cord blood (citrate dextrose, citrate phosphate dextrose, citrate, heparin, or EDTA)
  • PBS/EDTA: Phosphate‐buffered saline (PBS; see recipe)/2 mM disodium EDTA
  • Ficoll‐Paque Plus (Amersham Biosciences) or equivalent
  • PBS/0.5% (w/v) bovine serum albumin/2 mM EDTA
  • 500‐ml Erlenmeyer flask or similar container
  • 50‐ml centrifuge tubes
  • Centrifuge (preferably swinging‐bucket rotor)
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Figures

Videos

Literature Cited

Literature Cited
   Bøyum, A. 1964. Separation of white blood cells. Nature 204:793‐794.
   Bøyum, A. 1968. Isolation of leucocytes from human blood. Further observations. Methylcellulose, dextran, and ficoll as erythrocyteaggregating agents. Scand. J. Clin. Lab. Invest. Suppl. 97:31‐50.
   Kekarainen, T., Mannelin, S., Laine, J., and Jaatinen, T. 2006. Optimization of immunomagnetic separation for cord blood‐derived hematopoietic stem cells. B.M.C. Cell. Biol. 7:30.
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