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

Journal of Translational Medicine

Research

Tina Salzer¹  Thorsten Fischer²  Volker R. Jacobs²  Cornelia Hauser-Kronberger³  Doris A. Streif⁴  Doris Schmid⁴  Katharina Schallmoser⁴  Sandra Laner-Plamberger⁴  Michaela Öller⁴  Eva Rohde⁴  Thomas Lener⁴  Mario Gimona⁴ 

[1] Department of Blood Group Serology and Transfusion Medicine, Spinal Cord Injury and Tissue Regeneration Center Salzburg, Paracelsus Medical University, Lindhofstrasse 20-22, 5020, Salzburg, Austria;Department of Gynecology and Obstetrics, Paracelsus Medical University, Salzburg, Austria;Department of Pathology, Paracelsus Medical University, Salzburg, Austria;Spinal Cord Injury and Tissue Regeneration Center Salzburg, Paracelsus Medical University, Salzburg, Austria;Department of Blood Group Serology and Transfusion Medicine, Spinal Cord Injury and Tissue Regeneration Center Salzburg, Paracelsus Medical University, Lindhofstrasse 20-22, 5020, Salzburg, Austria;

关键词: Fibrinogen;    Heparin;    Mesenchymal stem cells;    Pooled human platelet lysate;   

DOI  :  10.1186/s12967-015-0717-4

 received in 2015-08-27, accepted in 2015-10-29,  发布年份 2015

来源: Springer

【 摘 要 】

BackgroundPooled 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.MethodsWe 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.ResultsThe 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.ConclusionsHere, 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.

【 授权许可】

CC BY   
© Laner-Plamberger et al. 2015