Amengual-Tugores Andreu Miquel, Ráez-Meseguer Carmen, Forteza-Genestra Maria Antònia, Calvo Javier, Gayà Antoni, Monjo Marta, Ramis Joana Maria
Group of Cell Therapy and Tissue Engineering (TERCIT), Research Institute on Health Sciences (IUNICS), University of the Balearic Islands (UIB), Palma 07122, Spain.
Health Research Institute of the Balearic Islands (IdISBa), Palma 07122, Spain.
Extracell Vesicles Circ Nucl Acids. 2024 May 29;5(2):259-270. doi: 10.20517/evcna.2024.05. eCollection 2024.
The objective of the present study was to determine the variability of platelet lysate-derived extracellular vesicles (pEV), in terms of characteristics and functionality through wound healing assays, when isolated either from platelet concentrates (PC, obtained from 5 donors) or from multiple PC (MPC, that is 50 donors). pEV were isolated under GMP-like conditions in a clean room using Size Exclusion Chromatography (SEC). The differential characteristics between pEV obtained from PC (PC-EV) or MPC (MPC-EV) were evaluated by means of protein concentration, Nanoparticle Tracking Analysis (NTA), Transmission Electron Microscopy (TEM), and flow cytometry using the MACSPlex™ arrays for surface analysis profiling of EV. The functionality of the isolated pEV was determined in cell culture by metabolic activity and LDH activity determination and through a wound healing assay after 24 h treatment. No significant differences were observed in the pEV characteristics evaluated, whether isolated from PC or MPC. As regards functionality, a higher wound closure percentage was obtained in those pEV pools isolated from PC (5 donors). No differences in the coefficient of variation (CV) were found when comparing all the evaluated variables of pEV derived either from PC (5 donors) or from MPC (50 donors). Our findings challenge the necessity of a larger donor pool for pEV isolation, revealing no significant variations in the analyzed variables of MPC-EV and PC-EV. Notably, our results suggest that, unlike platelet concentrates, a high number of donors is not required to reduce the variability of pEV, showing that the pool of only 5 donors can provide a consistent and reliable therapeutic product.
本研究的目的是通过伤口愈合试验，确定从血小板浓缩物（PC，来自5名供体）或多个PC（MPC，即50名供体）中分离出的血小板裂解物衍生的细胞外囊泡（pEV）在特性和功能方面的变异性。pEV在洁净室中使用尺寸排阻色谱法（SEC）在类似GMP的条件下分离。通过蛋白质浓度、纳米颗粒跟踪分析（NTA）、透射电子显微镜（TEM）以及使用MACSPlex™阵列进行流式细胞术以分析EV的表面特征，来评估从PC（PC-EV）或MPC（MPC-EV）获得的pEV之间的差异特性。通过代谢活性和LDH活性测定以及在24小时处理后的伤口愈合试验，在细胞培养中确定分离出的pEV的功能。无论从PC还是MPC中分离，在所评估的pEV特性方面均未观察到显著差异。关于功能，从PC（5名供体）中分离出的那些pEV池获得了更高的伤口闭合百分比。比较从PC（5名供体）或MPC（50名供体）衍生的pEV的所有评估变量时，未发现变异系数（CV）有差异。我们的研究结果对从更大供体库中分离pEV的必要性提出了质疑，表明MPC-EV和PC-EV的分析变量没有显著差异。值得注意的是，我们的结果表明，与血小板浓缩物不同，不需要大量供体来降低pEV的变异性，这表明仅5名供体的库就能提供一致且可靠的治疗产品。
