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Heparin concentration is critical for cell culture with human platelet lysate

  • Author Footnotes
    ∗ These authors contributed equally to this work.
    Hatim Hemeda
    Footnotes
    ∗ These authors contributed equally to this work.
    Affiliations
    Helmholtz-Institute for Biomedical Engineering, RWTH Aachen University Medical School, Aachen, Germany
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  • Author Footnotes
    ∗ These authors contributed equally to this work.
    Jana Kalz
    Footnotes
    ∗ These authors contributed equally to this work.
    Affiliations
    Helmholtz-Institute for Biomedical Engineering, RWTH Aachen University Medical School, Aachen, Germany
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  • Author Footnotes
    ∗ These authors contributed equally to this work.
    Gudrun Walenda
    Footnotes
    ∗ These authors contributed equally to this work.
    Affiliations
    Helmholtz-Institute for Biomedical Engineering, RWTH Aachen University Medical School, Aachen, Germany
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  • Michael Lohmann
    Affiliations
    Helmholtz-Institute for Biomedical Engineering, RWTH Aachen University Medical School, Aachen, Germany
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  • Wolfgang Wagner
    Correspondence
    Correspondence: Wolfgang Wagner, MD, PhD, Helmholtz-Institute for Biomedical Engineering, Stem Cell Biology and Cellular Engineering, RWTH Aachen University Medical School, Pauwelsstrasse 20, 52074 Aachen, Germany.
    Affiliations
    Helmholtz-Institute for Biomedical Engineering, RWTH Aachen University Medical School, Aachen, Germany
    Search for articles by this author
  • Author Footnotes
    ∗ These authors contributed equally to this work.

      Abstract

      Background aims

      Culture media for mesenchymal stromal cells (MSCs) are generally supplemented with fetal bovine serum. Human platelet lysate (hPL) has been proven to be a very effective alternative without the risk of xenogeneic infections or immune reactions. In contrast to fetal bovine serum, hPL comprises plasma, and anticoagulants—usually unfractionated heparin (UFH)—need to be added to prevent gel formation.

      Methods

      Cultures of MSCs in hPL media with various concentrations of UFH and enoxaparin, a low-molecular-weight heparin (LMWH), were systematically compared with regard to proliferation, fibroblastoid colony-forming unit frequency, immunophenotype and in vitro differentiation.

      Results

      At least 0.61 IU/mL UFH or 0.024 mg/mL LMWH was necessary for reliable prevention of coagulation of hPL pools used in this study. Higher concentrations impaired cellular proliferation in a dose-dependent manner even without benzyl alcohol, which is commonly added to heparins as a bacteriostatic agent. Colony-forming unit frequency was also reduced at higher heparin concentrations, particularly with LMWH, whereas no significant effect was observed on cellular morphology or immunophenotype. High concentrations of heparins reduced the in vitro differentiation toward adipogenic and osteogenic lineages.

      Conclusions

      Heparin concentration is critical for culture of MSCs in hPL media; this is of particular relevance for cellular therapy where cell culture procedures need to be optimized and standardized.

      Key Words

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