Impact of donor pool size on the variability of platelet lysate-derived extracellular vesicles for regenerative medicine

Abstract

Aim: 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).

Methods: 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.

Results: 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).

Conclusion: 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.

Keywords

Variability / coefficient of variability / wound healing / platelet lysate-derived extracellular vesicles / platelet concentrate (PC) / multiple platelet concentrate (MPC)

References

[1] Théry C,Aikawa E.Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the international society for extracellular vesicles and update of the MISEV2014 guidelines.J Extracell Vesicles2018;7:1535750 PMCID:PMC6322352

[2] Wiklander OPB,Lötvall J,El Andaloussi S.Advances in therapeutic applications of extracellular vesicles.Sci Transl Med2019;11 PMCID:PMC7104415

[3] Antich-Rosselló M,Monjo M.Platelet-derived extracellular vesicles for regenerative medicine.Int J Mol Sci2021;22:8580 PMCID:PMC8395287

[4] Torreggiani E,Roncuzzi L,Baglìo SR.Exosomes: novel effectors of human platelet lysate activity.Eur Cell Mater2014;28:137-51; discussion 151

[5] Antich-Rosselló M,Calvo J,Monjo M.Platelet-derived extracellular vesicles promote osteoinduction of mesenchymal stromal cells.Bone Joint Res2020;9:667-74 PMCID:PMC7563034

[6] Antich-Rosselló M,Forteza-Genestra MA.Evaluation of platelet-derived extracellular vesicles in gingival fibroblasts and keratinocytes for periodontal applications.Int J Mol Sci2022;23:7668 PMCID:PMC9321144

[7] Lovisolo F,Gino S,Renò F.Platelet rich plasma-derived microvesicles increased in vitro wound healing.Eur Rev Med Pharmacol Sci2020;24:9658-64

[8] Miyazawa B,Togarrati PP.Regulation of endothelial cell permeability by platelet-derived extracellular vesicles.J Trauma Acute Care Surg2019;86:931-42 PMCID:PMC7381393

[9] Lee JH,Song J,You G.Activated platelet-derived vesicles for efficient hemostatic activity.Macromol Biosci2020;20:e1900338

[10] Forteza-Genestra MA,Ramis-Munar G.Comparative effect of platelet- and mesenchymal stromal cell-derived extracellular vesicles on human cartilage explants using an ex vivo inflammatory osteoarthritis model.Bone Joint Res2023;12:667-76 PMCID:PMC10584413

[11] Xu N,Guan J.Wound healing effects of a Curcuma zedoaria polysaccharide with platelet-rich plasma exosomes assembled on chitosan/silk hydrogel sponge in a diabetic rat model.Int J Biol Macromol2018;117:102-7

[12] Sadallah S,Martin PJ.Microparticles (ectosomes) shed by stored human platelets downregulate macrophages and modify the development of dendritic cells.J Immunol2011;186:6543-52

[13] Xu Y,He L.Platelet-rich plasma-derived exosomal USP15 promotes cutaneous wound healing via deubiquitinating EIF4A1.Oxid Med Cell Longev2021;2021:9674809 PMCID:PMC8371654

[14] Guo SC,Yin WJ,Yuan T.Exosomes derived from platelet-rich plasma promote the re-epithelization of chronic cutaneous wounds via activation of YAP in a diabetic rat model.Theranostics2017;7:81-96 PMCID:PMC5196887

[15] Amengual-Tugores AM,Forteza-Genestra MA,Ramis JM.Extracellular vesicle-based hydrogels for wound healing applications.Int J Mol Sci2023;24:4104 PMCID:PMC9967521

[16] Lucien F,Lenassi M.MIBlood-EV: minimal information to enhance the quality and reproducibility of blood extracellular vesicle research.J Extracell Vesicles2023;12:e12385 PMCID:PMC10704543

[17] Collins T,Barkatali B.Platelet-rich plasma: a narrative review.EFORT Open Rev2021;6:225-35 PMCID:PMC8142058

[18] Noulsri E.Effects of donor age, donor sex, blood-component processing, and storage on cell-derived microparticle concentrations in routine blood-component preparation.Transfus Apher Sci2018;57:587-92

[19] Taniguchi Y,Sugaya H.Growth factor levels in leukocyte-poor platelet-rich plasma and correlations with donor age, gender, and platelets in the Japanese population.J Exp Orthop2019;6:4 PMCID:PMC6359998

[20] Agostini F,Battiston M.Standardization of platelet releasate products for clinical applications in cell therapy: a mathematical approach.J Transl Med2017;15:107 PMCID:PMC5437585

[21] Visnovitz T,Sódar BW.An improved 96 well plate format lipid quantification assay for standardisation of experiments with extracellular vesicles.J Extracell Vesicles2019;8:1565263 PMCID:PMC6352952

[22] Suarez-Arnedo A,Clavijo C,Cruz JC.An image J plugin for the high throughput image analysis of in vitro scratch wound healing assays.PLoS One2020;15:e0232565 PMCID:PMC7386569

[23] Małys MS,Papp K.Small extracellular vesicles are released ex vivo from platelets into serum and from residual blood cells into stored plasma.J Extracellular Bio2023;2:e88

[24] Marino LS,Julius A.Lyophilized human platelet lysate as a supplementation in the culture of umbilical cord derived mesenchymal stem cells.Tissue Cell2023;82:102092

[25] Forteza-Genestra MA,Calvo J,Monjo M.Purity determines the effect of extracellular vesicles derived from mesenchymal stromal cells.Cells2020;9:422 PMCID:PMC7072280

[26] Osteikoetxea X,Szabó-Taylor K.Improved characterization of EV preparations based on protein to lipid ratio and lipid properties.PLoS One2015;10:e0121184 PMCID:PMC4370721

[27] Koliha N,Heider U.A novel multiplex bead-based platform highlights the diversity of extracellular vesicles.J Extracell Vesicles2016;5:29975 PMCID:PMC4762227

[28] Loron G,Olivier P,Baud O.Inhaled nitric oxide promotes angiogenesis in the rodent developing brain.Int J Mol Sci2023;24:5871 PMCID:PMC10054632

[29] Kenny FN.Integrin-mediated adhesion and mechano-sensing in cutaneous wound healing.Cell Tissue Res2015;360:571-82

[30] Karimi N,Dias T,Lässer C.Tetraspanins distinguish separate extracellular vesicle subpopulations in human serum and plasma - contributions of platelet extracellular vesicles in plasma samples.J Extracell Vesicles2022;11:e12213 PMCID:PMC9077141

[31] Görgens A,Hagey DW.Identification of storage conditions stabilizing extracellular vesicles preparations.J Extracell Vesicles2022;11:e12238 PMCID:PMC9206228

[32] Sonnaert M,Luyten FP.Quantitative validation of the presto blue metabolic assay for online monitoring of cell proliferation in a 3D perfusion bioreactor system.Tissue Eng Part C Methods2015;21:519-29 PMCID:PMC4442584

[33] Ye Y,Ma YY.Exosomal miR-141-3p regulates osteoblast activity to promote the osteoblastic metastasis of prostate cancer.Oncotarget2017;8:94834-49 PMCID:PMC5706916

[34] Hu Y,Wang ZX.Exosomes from human umbilical cord blood accelerate cutaneous wound healing through miR-21-3p-mediated promotion of angiogenesis and fibroblast function.Theranostics2018;8:169-84 PMCID:PMC5743467

[35] Berger DR,Steinmetz NJ.Platelet lysates from aged donors promote human tenocyte proliferation and migration in a concentration-dependent manner.Bone Joint Res2019;8:32-40 PMCID:PMC6359887

[36] Hofmann E,Pignet AL.Human in vitro skin models for wound healing and wound healing disorders.Biomedicines2023;11:1056 PMCID:PMC10135654

[37] Burnouf T,Lundy DJ,Tseng CL.Expanding applications of allogeneic platelets, platelet lysates, and platelet extracellular vesicles in cell therapy, regenerative medicine, and targeted drug delivery.J Biomed Sci2023;30:79 PMCID:PMC10500824