Platelet Lysate as source of growth factors for Bone Marrow derived Mesenchymal Stromal cells clinical expansion: a study of starting platelet concentration dose-dependent effects

Abstract

Background & Aim Background In the last decades the replacement of fetal bovine serum (FBS)with human Platelet Lysate (hPL) for ATMPs clinical expansion has been for a long time investigated to overcome FBS related issues.Despite several studies confirm hPL safety and efficiency in Mesenchymal Stromal Cell(MSC)expansion, there are still some gaps in the knowledge of hPL as cell culture supplement, starting from composition and quality release criteria. Aim of the study As growth factors are released after thrombocytes lysis during hPL production, starting platelet concentration may affect hPL quality.The aim of this study was to investigate hPL starting platelet concentration effects on bone marrow derived MSC(BM-MSC). Methods, Results & Conclusion Material and methods MSC were isolated from the bone marrow of 7 donors and cultured from p1 to p7 in 4 different conditions: DMEM 10% FBS and DMEM 5% hPL varying starting platelet concentration.Particularly hPL was produced according to standardized protocol by in-hospital Transfusion Service, in three different starting platelet concentrations (sPLTC): high sPLTC(4 × 109 PLTS/ml), medium(2 × 109 PLTS/ml) and low(1 × 109 PLTS/ml).The study focused on analysis of parameters that are mostly affected by hPL such as proliferation, immunophenotype, telomeric length, differentiation and cell senescence. Results and Conclusions Evaluation of proliferation indexes(PDT and PD) underlined dose dependent effects of sPLTC (figure 1), also confirmed by flow cytometry cell cycle analysis of BrdU incorporation. Immunophenotype seems not to be affected by sPLTC, as classical MSC markers meet ISCT criteria (table 1). Differences were instead detected by analyzing mean fluorescence intensity (MFI) in a dose dependent manner (figure 2), both for intrinsic and extrinsic parameters. Differentiation potential seems to be unaffected by different sPLTC as all cells batches differentiated into osteoblasts, adipocytes and chondrocytes. On the contrary senescence and relative telomeric length RTL (detected by SA-β-GAL activity and PNA-FITC flow cytometry) are strongly affected by sPLTC, in a dose dependent manner. Particularly high sPLTC results in increased senescent cells associated with decreased RTL (figure 3). In conclusion our data showed that sPLTC affects some BM MSCs properties, underlying its importance during hPL preparation. According to this study we suggest a medium sPLTC for hPL preparation, as best compromise between increase in proliferation index and effects on senescence.