title: Mechanical fibrinogen-depletion supports heparin-free mesenchymal stem cell propagation in human platelet lysate.
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.
authors.affiliation: Spinal Cord Injury and Tissue Regeneration Center Salzburg, Paracelsus Medical University, Salzburg, Austria. s.laner-plamberger@salk.at.|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. s.laner-plamberger@salk.at.
authors.forename: Sandra
authors.initials: S
authors.lastname: Laner-Plamberger
authors.affiliation: Spinal Cord Injury and Tissue Regeneration Center Salzburg, Paracelsus Medical University, Salzburg, Austria. t.lener@salk.at.|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. t.lener@salk.at.
authors.forename: Thomas
authors.initials: T
authors.lastname: Lener
authors.affiliation: Spinal Cord Injury and Tissue Regeneration Center Salzburg, Paracelsus Medical University, Salzburg, Austria. doris.schmid@pmu.ac.at.|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. doris.schmid@pmu.ac.at.
authors.forename: Doris
authors.initials: D
authors.lastname: Schmid
authors.affiliation: Spinal Cord Injury and Tissue Regeneration Center Salzburg, Paracelsus Medical University, Salzburg, Austria. doris.streif@pmu.ac.at.|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. doris.streif@pmu.ac.at.
authors.forename: Doris A
authors.initials: DA
authors.lastname: Streif
authors.affiliation: 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. t.salzer@salk.at.
authors.forename: Tina
authors.initials: T
authors.lastname: Salzer
authors.affiliation: Spinal Cord Injury and Tissue Regeneration Center Salzburg, Paracelsus Medical University, Salzburg, Austria. m.oeller@salk.at.|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. m.oeller@salk.at.
authors.forename: Michaela
authors.initials: M
authors.lastname: Öller
authors.affiliation: Department of Pathology, Paracelsus Medical University, Salzburg, Austria. c.kronberger@salk.at.
authors.forename: Cornelia
authors.initials: C
authors.lastname: Hauser-Kronberger
authors.affiliation: Department of Gynecology and Obstetrics, Paracelsus Medical University, Salzburg, Austria. th.fischer@salk.at.
authors.forename: Thorsten
authors.initials: T
authors.lastname: Fischer
authors.affiliation: Department of Gynecology and Obstetrics, Paracelsus Medical University, Salzburg, Austria. v.jacobs@salk.at.
authors.forename: Volker R
authors.initials: VR
authors.lastname: Jacobs
authors.affiliation: Spinal Cord Injury and Tissue Regeneration Center Salzburg, Paracelsus Medical University, Salzburg, Austria. k.schallmoser@salk.at.|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. k.schallmoser@salk.at.
authors.forename: Katharina
authors.initials: K
authors.lastname: Schallmoser
authors.affiliation: Spinal Cord Injury and Tissue Regeneration Center Salzburg, Paracelsus Medical University, Salzburg, Austria. mario.gimona@pmu.ac.at.|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. mario.gimona@pmu.ac.at.
authors.forename: Mario
authors.initials: M
authors.lastname: Gimona
authors.affiliation: Spinal Cord Injury and Tissue Regeneration Center Salzburg, Paracelsus Medical University, Salzburg, Austria. e.rohde@salk.at.|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. e.rohde@salk.at.
authors.forename: Eva
authors.initials: E
authors.lastname: Rohde
mesh_terms: D001792:Blood Platelets / Q000166:cytology* / Q000378:metabolism; D002454:Cell Differentiation; D005340:Fibrinogen / Q000378:metabolism*; D005434:Flow Cytometry; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism
chemical_list: D005340:Fibrinogen; D006493:Heparin