We previously described and characterized a highly standardized and clinical grade human platelet lysate (hPL). We also demonstrated that it constituted a powerful substitute for fetal bovine serum (FBS) for human mesenchymal stem cell (hMSC) expansion required in cell therapy procedures, avoiding xenogenic risks and ethical issues. Even if hPL is manufactured from controlled platelet concentrates released by blood transfusion centres, the recently published general chapter 5.2.12 of the European Pharmacopeia requires the addition of a step of viral inactivation during the production process of such raw material of biological origin used for cell-based and gene therapy medicinal products.

Human platelet lysate (hPL) is rich in growth factors (GF) and nutritive elements and represents a powerful xeno-free alternative to fetal bovine serum (FBS) notably for mesenchymal stem cell (hMSC) proliferation. However, there is a large variability in hPL sources and production protocols, resulting in discrepancies in product quality, low management of product safety and poor batch-to-batch standardization. We describe here the development and the characterization of a standardized hPL prepared from outdated transfusional grade screened normal human donor platelet concentrates (PCs), manufactured on an industrial scale (batch sizes of 10 L; 250 donors) and following a highly qualified process (clean room, trained operators, validated aseptic filtration). PCs were frozen at −80°C and thawed at +4°C to lyse platelets. Cell debris were removed by centrifugation and the supernatant (hPL) was recovered. Clinical grade 10L batches of aseptic filtered hPL were characterized. First, we showed that hPL prepared from a limited number of donors displayed a variability in terms of GF contents. On the contrary, we observed a robust standardization between 10L-industrial batches of hPL in terms of GF contents (bFGF, EGF, VEGF, PDGF-AB, TGF-beta1 and IGF-1), biochemical analyses (total proteins, albumin, fibrinogen and iron) and efficacy on bone marrow (BM)-hMSC proliferation. Secondly, we compared expansion and functional characteristics of BM-hMSCs grown in clinical grade hPL versus MSC-screened FBS batch. We showed a reproducible increase in cell growth kinetics using hPL, a maintenance of BM-hMSC clonogenic potential and membrane marker expression (with however a strong overexpression of CD90). We observed a similar adipogenic and osteogenic differentiation potential and finally that immunosuppressive properties of BM-hMSCs (inhibition of T-cell proliferation) cultivated in parallel in both conditions remained also identical. Finally, we documented the stability over time of hPL stored at −80°C and −20°C. In conclusion, we demonstrated the feasibility to use a standardized, efficient and clinical grade hPL for research and cell therapy applications.

BACKGROUND:We recently developed and characterized a standardized and clinical grade human Platelet Lysate (hPL) that constitutes an advantageous substitute for fetal bovine serum (FBS) for human mesenchymal stem cell (hMSC) expansion required in cell therapy procedures, avoiding xenogenic risks (virological and immunological) and ethical issues. Because of the progressive use of pathogen-reduced (PR) labile blood components, and the requirement of ensuring the viral safety of raw materials for cell therapy products, we evaluated the impact of the novel procedure known as THERAFLEX UV-Platelets for pathogen reduction on hPL quality (growth factors content) and efficacy (as a medium supplement for hMSC expansion). This technology is based on short-wave ultraviolet light (UV-C) that induces non-reversible damages in DNA and RNA of pathogens while preserving protein structures and functions, and has the main advantage of not needing the addition of any photosensitizing additives (that might secondarily interfere with hMSCs).METHODOLOGY / PRINCIPAL FINDINGS:We applied the THERAFLEX UV-Platelets procedure on fresh platelet concentrates (PCs) suspended in platelet additive solution and prepared hPL from these treated PCs. We compared the quality and efficacy of PR-hPL with the corresponding non-PR ones. We found no impact on the content of five cytokines tested (EGF, bFGF, PDGF-AB, VEGF and IGF-1) but a significant decrease in TGF-ß1 (-21%, n = 11, p<0.01). We performed large-scale culture of hMSCs from bone marrow (BM) during three passages and showed that hPL or PR-hPL at 8% triggered comparable BM-hMSC proliferation as FBS at 10% plus bFGF. Moreover, after proliferation of hMSCs in an hPL- or PR-hPL-containing medium, their profile of membrane marker expression, their clonogenic potential and immunosuppressive properties were maintained, in comparison with BM-hMSCs cultured under FBS conditions. The potential to differentiate towards the adipogenic and osteogenic lineages of hMSCs cultured in parallel in the three conditions also remained identical.CONCLUSION / SIGNIFICANCE:We demonstrated the feasibility of using UV-C-treated platelets to subsequently obtain pathogen-reduced hPL, while preserving its optimal quality and efficacy for hMSC expansion in cell therapy applications.