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

Abstract

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0717-4) contains supplementary material, which is available to authorized users.

Venous Blood Derivatives as FBS-Substitutes for Mesenchymal Stem Cells: A Systematic Scoping Review

“…VBD concentrations added to culture medium ranged from 0.5 to 30% (54)(55)(56). Overall, VBD allowed for either, maintenance of MCS's fibroblast-like morphology (17,43,57), high proliferation (58-60), high colony-formation ability (52,61,62) and maintenance of multipotency (63)(64)(65)(66)(67). A linear dose-dependent response regarding medium concentration was not observed for evaluated VBD (54,56,68).…”

Integrated culture platform based on a human platelet lysate supplement for the isolation and scalable manufacturing of umbilical cord matrix-derived mesenchymal stem/stromal cells

“…Nevertheless, the use of HPL‐supplemented medium in this downstream step of the process would have greater potential to comply with GMP standards when envisaging a cell manufacturing scenario in a clinical setting. In addition, although heparin of xenogeneic (porcine) origin was used in this work to prevent coagulation of HPL in culture, this can be easily overcome by using fibrinogen‐depleted HPL products (Copland et al ., ; Laner‐Plamberger et al ., ), which are already commercially available.…”

A functional corona around extracellular vesicles enhances angiogenesis during skin regeneration and signals in immune cells

“…For ECFC culture EGM-2 was supplemented with hydrocortisone, hFGF, VEGF, IGF, EGF and ascorbic acid from the supplied bullet Kit (all Lonza) and FBS was replaced by an equivalent volume of pHPL [26], [41]. For particle depletion media were prepared by clotting supplemented -MEM as described without heparin to avoid possible inhibition of EV function [15], [18], [19], [27], [42]. The collapsed fibrin clot was removed by centrifugation for 10 min, 3000 x g at room temperature.…”