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Isolation, Culture, and Characterization of Human Umbilical Cord Blood-Derived Mesenchymal Stromal Cells

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Mesenchymal Stem Cells

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1416))

Abstract

Umbilical cord blood (CB) is considered one of the youngest available sources of adult stem cells. Besides hematopoietic stem cells, CB has been shown to contain endothelial progenitor cells as well as mesenchymal stromal/stem cells (MSC). To isolate MSC from cord blood, CB is collected into a sterile bag containing the anticoagulant citrate-phosphate-dextrose (CPD). The CB is then processed by density-gradient centrifugation to obtain mononuclear cells (MNC). These are cultured until the outgrowth of fibroblastoid cell colonies appears. After reaching a subconfluent stage, cells are harvested, expanded, and characterized as cord blood mesenchymal stromal cells (CB-MSC) according to standard criteria: plastic adherence, fibroblast morphology, CFU-f assay, proliferation potential, immune phenotype, and differentiation potential.

Apparently, the frequency of MSC in CB is extremely low. Thus, not every CB unit will provide adequate MSC isolation yields. Different strategies have been proposed aiming to optimize the isolation success by selecting CB units of optimal quality. It is commonly agreed on that a high CB volume, a high cellular content, and a short time frame between birth and MSC isolation are criteria that will enhance the MSC isolation success.

The procedures in this chapter are standardized protocols that were established and optimized in the authors’ research laboratory; however, various modifications of the protocols are possible.

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Acknowledgment

This work was supported by the German Jose-Carreras Leukemia-Foundation e.V., DJCLS-R03/18, the German Ministry of Education and Research (BMBF) 01GN0531 and 01GN0939, and by the European Community, LSHB-CT-2005-018999.

We greatly acknowledge the work of Susanne Pilopp née Kern, Andrea Hecker, Cora Ecker, Susanne Elvers-Hornung, and Stephanie Uhlig. P.N. was supported by the “Stiftung Transfusionsmedizin und Immunhämatologie.”

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Authors and Affiliations

  1. Medical Faculty Mannheim, Institute of Transfusion Medicine and Immunology, Heidelberg University, Heidelberg, Germany

    Karen Bieback & Philipp Netsch

  2. German Red Cross Blood Service Baden-Württemberg – Hessen, Friedrich-Ebert Str. 107, Mannheim, D-68167, Germany

    Karen Bieback & Philipp Netsch

Authors
  1. Karen Bieback
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  2. Philipp Netsch
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Correspondence to Karen Bieback .

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Editors and Affiliations

  1. Dept of Molecular Med,Unit of Cardiology, University of Pavia, PAVIA, Italy

    Massimiliano Gnecchi

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Bieback, K., Netsch, P. (2016). Isolation, Culture, and Characterization of Human Umbilical Cord Blood-Derived Mesenchymal Stromal Cells. In: Gnecchi, M. (eds) Mesenchymal Stem Cells. Methods in Molecular Biology, vol 1416. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3584-0_14

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  • DOI: https://doi.org/10.1007/978-1-4939-3584-0_14

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3582-6

  • Online ISBN: 978-1-4939-3584-0

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