TRANSPLANTATION AND CELLULAR ENGINEERING Human platelet lysate can replace fetal bovine serum for clinical-scale expansion of functional mesenchymal stromal cells Katharina Schallmoser,* Christina Bartmann,* Eva Rohde, Andreas Reinisch, Karl Kashofer, Elke Stadelmeyer, Camilla Drexler, Gerhard Lanzer, Werner Linkesch, and Dirk Strunk BACKGROUND: Human multipotent mesenchymal stromal cells (MSCs) are promising candidates for a growing spectrum of regenerative and immunomodula- tory cellular therapies. Translation of auspicious experi- mental results into clinical applications has been limited by the dependence of MSC propagation from fetal bovine serum (FBS). STUDY DESIGN AND METHODS: The capacity of human platelet lysate (HPL) to replace FBS for clinical- scale MSC propagation was analyzed. RESULTS: HPL could be efficiently produced from buffy coats. Multiplex analyses allowed a distinct HPL growth factor profile to be delineated. With a previously estab- lished two-step clinical-scale procedure, HPL was reproducibly more efficient than FBS in supporting MSC outgrowth. With only 3 ¥ 10 5 primary culture-derived MSCs, a mean of 4.36 ¥ 10 8 HPL-MSCs (range, 3.01 ¥ 10 8 -5.40 ¥ 10 8 ) was obtained within a single sec- ondary 11- to 13-day culture step. Although morphologi- cally distinct, HPL-MSCs and FBS-MSCs did not differ significantly in terms of immunophenotype, differentia- tion potential in vitro, and lack of tumorigenicity in nude mice in vivo. CONCLUSIONS: Replacing FBS with HPL prevents bovine prion, viral, and zoonose contamination of the stem cell product. This new efficient FBS-free two-step procedure for clinical-scale MSC propagation may rep- resent a major step toward challenging new stem cell therapies. M ultipotent mesenchymal stromal cells (MSCs) are currently undergoing clinical trials to explore their potential regulation of hematopoiesis, immune response, and tissue regeneration (http://www.clinicaltrials.gov/). There is already evidence for the safety and measurable effi- ciency of systemic MSC infusions in various disease conditions. 1-10 MSCs represent less than 0.1% of nucleated cells in adult bone marrow (BM) aspirates. Ex vivo expan- sion to obtain sufficient amounts of MSCs for a suggested clinical dose of at least 2 ¥ 10 6 MSCs per kg body weight of the adult patient is a challenge. 7 With a two-step preclini- cal protocol we were able to reproducibly generate a minimum of 1.5 ¥ 10 8 MSCs from 10-mL BM aspirates within 4 weeks in fetal bovine serum (FBS)-supplemented ABBREVIATIONS: a-MEM = alpha-modified minimum essen- tial medium; bFGF = basic fibroblast growth factor; CFU- F = fibroblast colony-forming unit; EGF = epidermal growth factor; HPL = human platelet lysate; MSC(s) = mesenchymal stromal cell(s); PDGF(s) = platelet-derived growth factor(s); PRP = platelet-rich plasma; VEGF = vascular endothelial cell growth factor. From the Department of Blood Group Serology and Transfusion Medicine, the Department of Internal Medicine, Division of Hematology and Stem Cell Transplantation, the Institute of Pathology, the Department of Orthopedic Surgery, and StemCell Cluster, Medical University, Graz, Austria. Address reprint requests to: Dirk Strunk, MD, Department of Internal Medicine, Division of Hematology and Stem Cell Transplantation, Medical University, Auenbrugger Pl. 38, A-8036 Graz, Austria; e-mail: dirk.strunk@klinikum-graz.at. *Both authors contributed equally. This work has been supported by the Austrian Genome Program II Project GATiB and by The Adult Stem Cell Research Foundation. Received for publication November 24, 2006; revision received January 25, 2007, and accepted January 31, 2007. doi: 10.1111/j.1537-2995.2007.01220.x TRANSFUSION 2007;47:1436-1446. 1436 TRANSFUSION Volume 47, August 2007