Rs for instance cell passage and cell seeding density influence EV biogenesis and bioactivity has the possible to enhance therapeutic EV production. Here, we investigate the influence of these parameters on MSC-derived EV production and vascularisation bioactivity. Methods: Conditioned media was collected right after 24 h from MSCs seeded at distinct densities (1E2, 5E2, 1E3, 1E4 cells/cm2) or passages (P2-P5). EVs were isolated from the conditioned media by means of differential centrifugation and quantified by nanoparticle tracking evaluation (NTA) utilizing aScientific Plan ISEVNanosight LM10 and CD63 ExoELISA. Vascularisation bioactivity of isolated EVs was assessed within a wound healing assay. Results: NTA and ExoELISA final results indicated increased EV production prices per cell when MSCs were seeded at reduced initial densities, regardless of the cell passage. The average fold decrease in EVs production per cell among cells seeded at 1E2 cells/cm2 and 1E4 cells/cm2 for P2, P3, P4, and P5 was 100, 85, 110, and 50, respectively (n = five, p 0.01). In addition, several EV collection time points (12 and 24 h) in the identical cells improved total EV production more than 3 fold in comparison with a single collection more than precisely the same time period (24 h) (n = 3, p 0.05). Seeding density had no affect around the vascularisation bioactivity of MSC EVs made as assessed by the wound-healing assay (n = 3). In contrast, growing cell passage was correlated with diminished EV bioactivity (n = three). Conclusion: These benefits recommend that high EV production rates may be achieved by seeding cells at reduce initial seeding densities. Low cell passage number is critical to retaining MSC EV vascularisation bioactivity. The implications of these findings are that greater amounts of bioactive EVs can be achieved making use of a lower number of producer cells with increased frequency of collection. This may possibly allow for important reduction in expense of EV production and start to inform the rational design of a large-scale biomanufacturing method for therapeutic EV production.eGFP-positive EVs. Cell proliferation kinetics within bioreactors are monitored by glucose uptake, and the production of EVs both below serum-supplemented and defined serum-free conditions is presently evaluated. The concentration and size distribution are measured by nanoparticle tracking analysis (NTA) and surface marker expression profiles and VEGFR Synonyms uptake kinetics in recipient cells of harvested EVs are analysed via flow cytometry. All parameters are in comparison with classical 2D culture. Also, PRMT4 Compound unique schedules for EV harvesting are compared in order to optimise and standardise the production. Preliminary final results and experiences applying hollow fibre bioreactors for the large-scale production of EVs from diverse cell forms will likely be presented right here.PT02.Purifying and molecular profiling extracellular vesicles (EVs) from different biological specimens Abiodun Ogunjimi1 and Liang ZhangLunenfeld-Tanenbaum Study Institute; 2City University of Hong Kong, ChinaPT02.Evaluation and optimisation of a hollow fibre bioreactor system for standardisation of massive scale production of extracellular vesicles Ulrika Felldin1, Giulia Corso1, Bernd Giebel1,2, Helmut Hanenberg3, Joel Z. Nordin1, Samir El-Andaloussi1,four and AndrG gens1,1 Division of Laboratory Medicine, Karolinska Instiutet, Stockholm, Sweden; 2Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg-Essen, Essen, Germany; 3Department of Pediatrics III, University C.