Biotechnol. Appl. Biochem. (2010) 56, 151–160 (Printed in Great Britain) doi:10.1042/BA20100151 151 A novel virally inactivated human platelet lysate preparation rich in TGF-β , EGF and IGF, and depleted of PDGF and VEGF Pierre-Alain Burnouf*† 1 , Po-Kai Juan‡ 1 , Chen-Yao Su§, Ya-Po Kuo‖, Ming-Li Chou†, Ching-Hua Su†, Yu-Hung Tseng¶, Che-Tong Lin** and Thierry Burnouf* , ** 2 *Human Protein Process Sciences (HPPS), 59 800 Lille, France, †Department of Microbiology and Immunology, Taipei Medical University, Taipei, Taiwan, ‡Department of Dentistry, Taipei Medical University, Taipei, Taiwan, §Department of Dentistry, National Yang-Ming University, 11221 Taipei, Taiwan, ‖Institute of Oral Biology, National Yang-Ming University, 11221 Taipei, Taiwan, ¶Gwo-Wei Dental Implant Center, 11221 Taipei, Taiwan, and **College of Oral Medicine, Taipei Medical University, Taipei, Taiwan There is emerging interest in the use of standardized virally inactivated human platelet lysate preparations rich in GFs (growth factors) for cell cultures, cell therapy and clinical applications. In the present paper, we report a simple process to prepare a virally inactivated platelet lysate preparation rich in TGF-β 1 (transforming growth factor-β 1), EGF (epidermal growth factor) and IGF (insulin-like growth factor) and depleted of PDGF (platelet-derived growth factor) and VEGF (vascular endothelial growth factor). Apheresis platelet concentrates were treated by the S/D (solvent/detergent) viral inactivation procedure, then subjected to an oil extraction followed by adsorption with activated charcoal and finally sterile- filtered. The resulting preparation contained a mean of 368.4, 2.4 and 54.7 ng/ml of TGF-β 1, EGF and IGF respectively. PDGF-AB and VEGF were essentially completely removed by the charcoal treatment. The mean albumin, IgG, IgM and IgA and fibrinogen contents were approx. 40.0, 8.5, 0.87, 1.66 and 2.65 mg/ml respectively, cholesterol and triglycerides were at 15 and 20.7 mg/ml respectively and TnBP (tri- n-butyl phosphate) and Triton X-45 were at 8.7 and 8.8 p.p.m. respectively. Supplementing MEM (minimum essential medium) with 1–10 % of this S/D-treated platelet lysate promoted the proliferation of MG63 and SIRC cell lines as well as, or better than, 10 % (v/v) FBS (fetal bovine serum), as based on the MTS [3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)- 2-(4-sulfophenyl)-2H-tetrazolium] assay. The process used to prepare such S/D-treated platelet lysates is easily scalable for industrial production. Our results open up the possibility to evaluate the potential of this new preparation for stem cell expansion and/or bone tissue engineering and regeneration. Introduction As is evident by proteomics and functional analyses, human platelets contain a myriad of molecules exhibiting important physiological functions [1]. These include the GFs (growth factors) that are stored in the α-granules [2]. PGFs (platelet growth factors) include three PDGF (platelet- derived growth factor) isoforms (PDGF-AA, -AB and -BB), VEGF (vascular endothelial growth factor), TGF-β (transforming growth factor-β ; TGF-β 1 and TGF-β 2), EGF (epidermal growth factor), FGF (fibroblast growth factor) and some IGF (insulin-like growth factor). There is increasing interest in the use of human PGFs both as therapeutic biological products in the field of regenerative medicine as well as for various applications in cell cultures and cell therapy as a replacement of FBS (fetal bovine serum). Such preparations need, however, to be standardized [3]. Currently, the major therapeutic applications of platelet lysates rich in GF are to stimulate bone regeneration in oral, maxillofacial, plastic and orthopaedic surgery, or to accelerate wound healing of soft tissues, in particular in the treatment of recalcitrant hard-to-cure leg ulcers [4–6]. For such clinical applications, a single-donor PC (platelet concentrate), or a platelet-rich-plasma donation, of autologous or allogeneic origin, is used as a topical product, as such or after activation by exogenous thrombin to induce the release and temporary entrapment of the GF into a fibrin-rich biomaterial, called platelet gel [7]. The GF-rich fraction can be applied on tissues, either alone or in combination with a carrier, such as collagen or Key words: fetal bovine serum (FBS), growth factor, lysate, platelet, solvent/detergent, stem cell. Abbreviations used: BMP , bone morphogenetic protein; EGF, epidermal growth factor; FBS, fetal bovine serum; GF, growth factor; HAV, hepatitis A virus; HIC, hydrophobic interaction chromatography; IGF, insulin-like growth factor; MEM, minimum essential medium; MSC, mesenchymal stem cell; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium; NAT, nucleic acid testing; PC, platelet concentrate; PDGF, platelet-derived growth factor; PGF, platelet growth factor; PMS, phenazine methosulfate; RBC, red blood cell concentrate; rh, recombinant human; S/D, solvent/detergent; S/D-PC, S/D-treated PC; TGF-β, transforming growth factor-β; TnBP , tri-n-butyl phosphate; VEGF, vascular endothelial growth factor. 1 These two authors contributed equally to the experiments. 2 To whom correspondence should be addressed (email tburnou@attglobal.net). C  2010 Portland Press Limited