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Mesenchymal Stromal Cell Phenotype is not Influenced by Confluence during Culture Expansion

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Abstract

Background

Accumulating preclinical and clinical evidence indicates that human mesenchymal stromal cells (MSCs) are good candidates for cell therapy. For clinical applications of MSCs extensive in vitro expansion is required to obtain an adequate number of cells. It is evident that the pursuit for cell quantity must not affect quality, but it is also a fact that in vitro culture conditions affect MSC phenotype. One possible variable is the degree of cell confluence during expansion.

Methods

We investigate the influence of cell density on homogeneity and differentiation during culture expansion of un-stimulated MSCs isolated from the bone marrow in DMEM and fetal bovine serum (FBS).

MSC morphology, phenotype and differentiation were investigated weekly during 5 weeks culture expansion using electron microscopy, flow cytometry, immunocytochemistry, qualitative RT-PCR and quantitative Q-PCR.

Results

The morphological observation and the phenotypic analyses showed that MSCs after 3 weeks cultivation constituted a phenotypically homogenous MSC cell population, which at low levels expressed genes for different cell lineages, confirming their multilineage plasticity, without actual differentiation. This phenotype persisted independent of increasing cell densities.

Discussion

These data demonstrate that MSC characteristics and plasticity can be maintained during culture expansion from bone marrow mononuclear cells to MSCs and that a homogeneous phenotype of undifferentiated MSCs which persists independent of cell density can be used for clinical therapies.

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Acknowledgements

We thank Irene Lynfort, Louise Degn Neilsen, Robert Burdorf and Hanne Holm for their technical support. The study was supported by research grants from the Aase and Ejnar Danielsen Foundation, the Research Foundation at Rigshospitalet, FP7 Health-2007-2.4.2.-5, grand no. 222995and the Danish Medical Research Council.

Competing interest statement

The authors declare no competing financial interests.

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Authors and Affiliations

  1. Cardiology Stem Cell Laboratory, The Heart Centre, Copenhagen University Hospital Rigshospitalet, Copenhagen, Denmark

    Mandana Haack-Sørensen, Susanne Kofoed Hansen, Louise Hansen, Annette Ekblond & Jens Kastrup

  2. Department of Pathology and Department of Endocrinology (KMEB), Odense University Hospital, Odense C, Denmark

    Michael Gaster

  3. Department of Basic Animal and Veterinary Science, Faculty of Life Science, University of Copenhagen, Frederiksberg C, Denmark

    Poul Hyttel

  4. Cardiac Catheterization Laboratory 2014, The Heart Centre, Copenhagen University Hospital Rigshospitalet, 2100, Copenhagen Ø, Denmark

    Jens Kastrup

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  1. Mandana Haack-Sørensen
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Correspondence to Jens Kastrup.

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Haack-Sørensen, M., Hansen, S.K., Hansen, L. et al. Mesenchymal Stromal Cell Phenotype is not Influenced by Confluence during Culture Expansion. Stem Cell Rev and Rep 9, 44–58 (2013). https://doi.org/10.1007/s12015-012-9386-3

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