Human platelets represent a promising source of bioactive substances as growth factors not just for in vivo wound healing and tissue repair, but also for the expansion of human stem and progenitor cells in vitro. The replacement of fetal bovine serum (FBS) as a standard culture supplement by human platelet-derived growth factors now allows for the GMP-compliant implementation of various cell therapeutics in the growing field of regenerative medicine.
For this purpose a protocol for the preparation of human platelet lysate (HPL) by several freeze–thaw cycles has been developed, resulting in platelet fragmentation and the release of stored growth factors. By pooling up to 15 U of HPL derived from individual blood donors, a virtually standardized product is achieved. The depletion of platelet particles and fragments in a final centrifugation step reduces the risk of alloimmunization against platelet antigens and the formation of aggregates in cell culture.
The successful application of pooled human platelet lysate (pHPL) as a culture medium supplement for the ex vivo propagation of human mesenchymal stem/progenitor cells (MSPCs) and endothelial colony forming progenitor cells (ECFCs) indicates the feasibility of this animal serum-free source of growth factors. Further studies will evaluate efficacy and safety of pHPL.
Platelet-rich plasma Pooled human platelet lysate Apheresis Buffy coat Platelet concentrate Platelet-derived growth factors Mesenchymal stem/progenitor cells (MSPCs) Endothelial colony-forming progenitor cells (ECFCs)
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This work was supported by the Austrian Research Foundation (FWF, grant N211-NAN; DS) and the Adult Stem Cell Research Foundation (KS). The authors thank Eva Rohde and Konrad Rosskopf for critical review, Claudia Url and Marianne Keller for excellent technical assistance, Tina Schreiner for graphics editing, and Monica Farrell for editorial assistance.
Reed GL (2007) Platelets. Elsevier Science, San Diego, CaliforniaGoogle Scholar
Nurden AT, Nurden P, Sanchez M, Andia I, Anitua E (2008) Platelets and wound healing. Front Biosci 13:3532–3548PubMedGoogle Scholar
Barrientos S, Stojadinovic O, Golinko MS, Brem H, Tomic-Canic M (2008) Growth factors and cytokines in wound healing. Wound Repair Regen 16:585–601PubMedCrossRefGoogle Scholar
Borzini P, Mazzucco L (2005) Tissue regeneration and in loco administration of platelet derivatives: clinical outcome, heterogeneous products, and heterogeneity of the effector mechanisms. Transfusion 45:1759–1767PubMedCrossRefGoogle Scholar
Anitua E, Sanchez M, Orive G, Andia I (2008) Delivering growth factors for therapeutics. Trends Pharmacol Sci 29:37–41PubMedCrossRefGoogle Scholar
Martinez-Zapata MJ, Marti-Carvajal A, Sola I, Bolibar I, Angel Exposito J, Rodriguez L et al (2009) Efficacy and safety of the use of autologous plasma rich in platelets for tissue regeneration: a systematic review. Transfusion 49:44–56PubMedCrossRefGoogle Scholar
Foster TE, Puskas BL, Mandelbaum BR, Gerhardt MB, Rodeo SA (2009) Platelet-rich plasma: from basic science to clinical applications. Am J Sports Med 37:2259–2272PubMedCrossRefGoogle Scholar
Doucet C, Ernou I, Zhang YZ, Llense JR, Begot L, Holy X et al (2005) Platelet lysates promote mesenchymal stem cell expansion: a safety substitute for animal serum in cell-based therapy applications. J Cell Physiol 205:228–236PubMedCrossRefGoogle Scholar
Schallmoser K, Bartmann C, Rohde E, Reinisch A, Kashofer K, Stadelmeyer E et al (2007) Human platelet lysate can replace fetal bovine serum for clinical-scale expansion of functional mesenchymal stromal cells. Transfusion 47:1436–1446PubMedCrossRefGoogle Scholar
Schallmoser K, Rohde E, Reinisch A, Bartmann C, Thaler D, Drexler C et al (2008) Rapid large-scale expansion of functional mesenchymal stem cells from unmanipulated bone marrow without animal serum. Tissue Eng Part C Methods 14:185–196PubMedCrossRefGoogle Scholar
Reinisch A, Bartmann C, Rohde E, Schallmoser K, Bjelic-Radisic V, Lanzer G et al (2007) Humanized system to propagate cord blood-derived multipotent mesenchymal stromal cells for clinical application. Regen Med 2:371–382PubMedCrossRefGoogle Scholar
Bieback K, Hecker A, Kocaomer A, Lannert H, Schallmoser K, Strunk D et al (2009) Human alternatives to fetal bovine serum for the expansion of mesenchymal stromal cells from bone marrow. Stem Cells 27:2331–2341PubMedCrossRefGoogle Scholar
Rohde E, Schallmoser K, Bartmann C, Reinisch A, Strunk D (2008) GMP-compliant propagation of human multipotent mesenchymal stromal cells. Pharmaceutical manufacturing handbook: regulations and quality. Wiley, Hoboken, New JerseyGoogle Scholar
Weibrich G, Kleis WK, Hafner G, Hitzler WE (2002) Growth factor levels in platelet-rich plasma and correlations with donor age, sex, and platelet count. J Craniomaxillofac Surg 30:97–102PubMedCrossRefGoogle Scholar
Frechette JP, Martineau I, Gagnon G (2005) Platelet-rich plasmas: growth factor content and roles in wound healing. J Dent Res 84:434–439PubMedCrossRefGoogle Scholar
Schallmoser K, Strunk D (2009) Preparation of pooled human platelet lysate (pHPL) as an efficient supplement for animal serum-free human stem cell cultures. J Vis Exp 32:1523PubMedGoogle Scholar
Strunk D, Schallmoser K, Rohde E (2008) Plasma-free platelet lysate for use as a supplement in cell cultures and for the preparation of cell therapeutics. Patent (WO/2008/034803)Google Scholar
Reinisch A, Hofmann NA, Obenauf AC, Kashofer K, Rohde E, Schallmoser K et al (2009) Humanized large-scale expanded endothelial colony-forming cells function in vitro and in vivo. Blood 113:6716–6725PubMedCrossRefGoogle Scholar
Reinisch A, Strunk D (2009) Isolation and animal serum free expansion of human umbilical cord derived mesenchymal stromal cells (MSCs) and endothelial colony forming progenitor cells (ECFCs). J Vis Exp 32:1525PubMedGoogle Scholar
Hofmann NA, Reinisch A, Strunk D (2009) Isolation and large scale expansion of adult human endothelial colony forming progenitor cells. J Vis Exp 32:1524PubMedGoogle Scholar
Guide to the preparation, use and quality assurance of blood components (2008). European Directorate for the Quality of Medicine & Healthcare of the Council of Europe, Strasbourg, FranceGoogle Scholar
Bartmann C, Rohde E, Schallmoser K, Purstner P, Lanzer G, Linkesch W et al (2007) Two steps to functional mesenchymal stromal cells for clinical application. Transfusion 47:1426–1435PubMedCrossRefGoogle Scholar