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Open Access 01-12-2015 | Research

Mechanical fibrinogen-depletion supports heparin-free mesenchymal stem cell propagation in human platelet lysate

Authors: Sandra Laner-Plamberger, Thomas Lener, Doris Schmid, Doris A. Streif, Tina Salzer, Michaela Öller, Cornelia Hauser-Kronberger, Thorsten Fischer, Volker R. Jacobs, Katharina Schallmoser, Mario Gimona, Eva Rohde

Published in: Journal of Translational Medicine | Issue 1/2015

Abstract

Background

Pooled human platelet lysate (pHPL) is an efficient alternative to xenogenic supplements for ex vivo expansion of mesenchymal stem cells (MSCs) in clinical studies. Currently, porcine heparin is used in pHPL-supplemented medium to prevent clotting due to plasmatic coagulation factors. We therefore searched for an efficient and reproducible medium preparation method that avoids clot formation while omitting animal-derived heparin.

Methods

We established a protocol to deplete fibrinogen by clotting of pHPL in medium, subsequent mechanical hydrogel disruption and removal of the fibrin pellet. After primary culture, bone-marrow and umbilical cord derived MSCs were tested for surface markers by flow cytometry and for trilineage differentiation capacity. Proliferation and clonogenicity were analyzed for three passages.

Results

The proposed clotting procedure reduced fibrinogen more than 1000-fold, while a volume recovery of 99.5 % was obtained. All MSC types were propagated in standard and fibrinogen-depleted medium. Flow cytometric phenotype profiles and adipogenic, osteogenic and chondrogenic differentiation potential in vitro were independent of MSC-source or medium type. Enhanced proliferation of MSCs was observed in the absence of fibrinogen but presence of heparin compared to standard medium. Interestingly, this proliferative response to heparin was not detected after an initial contact with fibrinogen during the isolation procedure.

Conclusions

Here, we present an efficient, reproducible and economical method in compliance to good manufacturing practice for the preparation of MSC media avoiding xenogenic components and suitable for clinical studies.

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Title: Mechanical fibrinogen-depletion supports heparin-free mesenchymal stem cell propagation in human platelet lysate
Authors: Sandra Laner-Plamberger
Thomas Lener
Doris Schmid
Doris A. Streif
Tina Salzer
Michaela Öller
Cornelia Hauser-Kronberger
Thorsten Fischer
Volker R. Jacobs
Katharina Schallmoser
Mario Gimona
Eva Rohde
Publication date: 01-12-2015
Publisher: BioMed Central
Published in: Journal of Translational Medicine / Issue 1/2015
Electronic ISSN: 1479-5876
DOI: https://doi.org/10.1186/s12967-015-0717-4

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