Assessment of anti-inflammatory bioactivity of extracellular vesicles is susceptible to error via media component contamination |
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| Institution: | 1. Fischell Department of Bioengineering, University of Maryland, College Park, Maryland, USA;2. Program in Molecular and Cell Biology, University of Maryland, College Park, Maryland, USA;1. Division of Hematology, Department of Internal Medicine, Faculty of Medicine, Kagawa University, Takamatsu, Japan;2. Department of Hematology, Takamatsu Red Cross Hospital, Takamatsu, Japan;3. Department of Hematology, Kagawa Prefectural Central Hospital, Takamatsu, Japan;1. Developmental Biology and Cancer Department, UCL GOS Institute of Child Health, London, UK;2. Centre for Cell, Gene & Tissue Therapeutics, Royal Free London NHS Foundation Trust, London, United Kingdom;3. Department of Zoology, Faculty of Science, South Valley University, Qena, Egypt;4. Cancer Institute, UCL, London, United Kingdom;5. Department of Plastic Surgery, Great Ormond Street Hospital for Children NHS Foundation Trust, London, United Kingdom;1. Centro de Oncologia, Hospital Sirio Libanes, São Paulo, São Paulo, Brazil;2. Disciplina de Hematologia, Universidade Federal de São Paulo/Escola Paulista de Medicina, São Paulo, Brazil;3. Grupo Fleury, São Paulo, Brazil;1. Department of Biomedical Engineering, University of Texas at Austin, Austin, Texas, USA;2. Department of Kinesiology and Health Education, University of Texas at Austin, Austin, Texas, USA |
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| Abstract: | Extracellular vesicles (EVs) are widely implicated as novel diagnostic and therapeutic modalities for a wide range of diseases. Thus, optimization of EV biomanufacturing is of high interest. In the course of developing parameters for a human embryonic kidney cells (HEK293T) EV production platform, we examined the combinatorial effects of cell culture conditions (i.e., static versus dynamic) and isolation techniques (i.e., ultracentrifugation versus tangential flow filtration versus size-exclusion chromatography) on functional characteristics of HEK293T EVs, including anti-inflammatory bioactivity using a well-established lipopolysaccharide-stimulated mouse macrophage model. We unexpectedly found that, depending on culture condition and isolation strategy, HEK293T EVs appeared to significantly suppress the secretion of pro-inflammatory cytokines (i.e., interleukin-6, RANTES regulated upon activation, normal T cell expressed and secreted]) in the stimulated mouse macrophages. Further examination revealed that these results were most likely due to non-EV fetal bovine serum components in HEK293T EV preparations. Thus, future research assessing the anti-inflammatory effects of EVs should be designed to account for this phenomenon. |
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