ORIGINAL ARTICLE Human platelet lysate allows expansion and clinical grade production of mesenchymal stromal cells from small samples of bone marrow aspirates or marrow filter washouts C Capelli 1,3 , M Domenghini 1,3 , G Borleri 1 , P Bellavita 2 , R Poma 2 , A Carobbio 1 , C Mico` 1 , A Rambaldi 1 , J Golay 1 and M Introna 1 1 Laboratorio di Terapia Cellulare e Genica ‘G. Lanzani’, Unita` di Ematologia, Ospedali Riuniti di Bergamo, Bergamo, Italy and 2 Servizio di Immunoematologia e Medicina Trasfusionale Ospedali Riuniti di Bergamo, Bergamo, Italy We compared two protocols for the expansion of human mesenchymal stromal cells (hMSCs) starting from diagnostic samples of BM aspirates (2–5ml) or using the remnants in the bag and filter at the end of the BM infusions. The protocols differed in the presence of either 10% fetal bovine serum (FBS) or 5% platelet lysate (PL). We obtained a significantly (P ¼ 0.02) better expansion with PL, obtaining a median 1010-fold compared to 198- fold with a selected batch of FBS and in fewer days (29.8 in PL versus 41.4 in FBS). Overall, we recovered a variable number from 54.8  10 6 to 365  10 6 hMSCs in PL versus a variable number from 2.7  10 6 to 31  10 6 in FBS. No difference could be found in terms of gross morphology, differentiation potential, surface markers and immunological properties (inhibition of allogeneic PHA response and mixed lymphocyte reaction) of cells expanded with PL or FBS. The preparations were found within the range of acceptability for all the quality control criteria. Due to the clinical grade nature of the PL and the reproducibility of separate preparations, we propose this method to obtain hMSCs even from minute amounts of BM cells. Bone Marrow Transplantation (2007) 40, 785–791; doi:10.1038/sj.bmt.1705798; published online 6 August 2007 Keywords: MSC; platelet lysate; bone marrow stromal cells Introduction Since their first description by Friedenstein et al., 1 human mesenchymal stem cells (hMSCs) hold promises for the treatment of several human diseases from acute graft versus host reactions in allogeneic BMTs and support of marrow failure to heart ischemia. 2–4 Nonetheless, the most utilized methods to produce mesenchymal stem cells require the addition of fetal bovine serum (FBS), which represents a concern for the safe administration in patients and is often criticized by the regulatory authorities responsible for the approval of somatic cell therapy experimental protocols. Furthermore, different batches of FBS give variable efficiencies of hMSCs expansion in vitro. Finally, relatively large amounts of BM harvest are generally required (50– 100 ml) to reach sufficient numbers of hMSCs (from 0.5 to 5  10 6 /kg). 2,5–9 In order to try and improve the method of expansion of hMSCs in clinical grade conditions, we have added the platelet lysate (PL) commonly produced in the Blood Collection Facility for dermatologic or orthopedic proce- dures to substitute for the FBS and have performed a close comparison between a selected FBS batch and PL to reach clinically relevant numbers of hMSCs starting from minute amounts of BM cells. We will use throughout the text the acronym hMSCs as referred to human mesenchymal stem cells as well as human mesenchymal stromal cells, as very recently debated in an International Commission. 10,11 Materials and methods Platelet lysate preparation Each platelet lysate (PL) was obtained from a single allogenic platelet unit stored at 401C prepared by the Hospital Transfusion center. The platelet unit had been prepared from a whole blood (WB) donation following Standard Blood Bank Procedures. Briefly, the WB dona- tion (450 ml) was collected into a quadruple blood-bag system (Baxter Health Care Corporation, Deerfield, IL, USA). Platelet-rich plasma was separated from WB by ‘light-spin’ centrifugation and the platelets were concen- trated by a second ‘heavy-spin’ centrifugation, with subsequent removal of supernatant plasma (AABB Tech- nical Manual, 14th edition, American Association of Blood Banks Ed, 8101 Glenbrook Road, Bethesda, MD 20814, Received 6 December 2006; revised 25 June 2007; accepted 27 June 2007; published online 6 August 2007 Correspondence: Dr M Introna, Laboratory of Cellular and Gene Therapy ‘G. Lanzani’, Unit of Hematology, Presidio Matteo Rota, via Garibaldi 11/13, Ospedali Riuniti di Bergamo, Bergamo 24124, Italy. E-mail: mintrona@ospedaliriuniti.bergamo.it 3 These authors contributed equally to this work. Bone Marrow Transplantation (2007) 40, 785–791 & 2007 Nature Publishing Group All rights reserved 0268-3369/07 $30.00 www.nature.com/bmt