RESUMO
BACKGROUND: Human platelet lysate (HPL) is emerging as the preferred xeno-free supplement for the expansion of mesenchymal stromal cells (MSCs) for bone tissue engineering (BTE) applications. Due to a growing demand, the need for standardization and scaling-up of HPL has been highlighted. However, the optimal storage time of the source material, i.e., outdated platelet concentrates (PCs), remains to be determined. The present study aimed to determine the optimal storage time of PCs in terms of the cytokine content and biological efficacy of HPL. METHODS: Donor-matched bone marrow (BMSCs) and adipose-derived MSCs (ASCs) expanded in HPL or fetal bovine serum (FBS) were characterized based on in vitro proliferation, immunophenotype, and multi-lineage differentiation. Osteogenic differentiation was assessed at early (gene expression), intermediate [alkaline phosphatase (ALP) activity], and terminal stages (mineralization). Using a multiplex immunoassay, the cytokine contents of HPLs produced from PCs stored for 1-9 months were screened and a preliminary threshold of 4 months was identified. Next, HPLs were produced from PCs stored for controlled durations of 0, 1, 2, 3, and 4 months, and their efficacy was compared in terms of cytokine content and BMSCs' proliferation and osteogenic differentiation. RESULTS: BMSCs and ASCs in both HPL and FBS demonstrated a characteristic immunophenotype and multi-lineage differentiation; osteogenic differentiation of BMSCs and ASCs was significantly enhanced in HPL vs. FBS. Multiplex network analysis of HPL revealed several interacting growth factors, chemokines, and inflammatory cytokines. Notably, stem cell growth factor (SCGF) was detected in high concentrations. A majority of cytokines were elevated in HPLs produced from PCs stored for ≤ 4 months vs. > 4 months. However, no further differences in PC storage times between 0 and 4 months were identified in terms of HPLs' cytokine content or their effects on the proliferation, ALP activity, and mineralization of BMSCs from multiple donors. CONCLUSIONS: MSCs expanded in HPL demonstrate enhanced osteogenic differentiation, albeit with considerable donor variation. HPLs produced from outdated PCs stored for up to 4 months efficiently supported the proliferation and osteogenic differentiation of MSCs. These findings may facilitate the standardization and scaling-up of HPL from outdated PCs for BTE applications.