Assessment of anti-inflammatory bioactivity of extracellular vesicles is susceptible to error via media component contamination.

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.

Viscosity of Plasma as a Key Factor in Assessment of Extracellular Vesicles by Light Scattering.

Extracellular vesicles (EVs) isolated from biological samples are a promising material for use in medicine and technology. However, the assessment methods that would yield repeatable concentrations, sizes and compositions of the harvested material are missing. A plausible model for the description of EV isolates has not been developed. Furthermore, the identity and genesis of EVs are still obscure and the relevant parameters have not yet been identified. The purpose of this work is to better understand the mechanisms taking place during harvesting of EVs, in particular the role of viscosity of EV suspension. The EVs were harvested from blood plasma by repeated centrifugation and washing of samples. Their size and shape were assessed by using a combination of static and dynamic light scattering. The average shape parameter of the assessed particles was found to be ρ ~ 1 (0.94-1.1 in exosome standards and 0.7-1.2 in blood plasma and EV isolates), pertaining to spherical shells (spherical vesicles). This study has estimated the value of the viscosity coefficient of the medium in blood plasma to be 1.2 mPa/s. It can be concluded that light scattering could be a plausible method for the assessment of EVs upon considering that EVs are a dynamic material with a transient identity.

Molecular assessment of circulating exosomes toward liquid biopsy diagnosis of Ewing sarcoma family of tumors.

Ewing sarcoma was first described in 1921 in the Proceedings of the New York Pathological Society by an eminent American pathologist from Cornell named James R. Ewing as a "diffuse endothelioma of bone." Since this initial description, more has been discovered regarding Ewing sarcoma and in the 1980's both Ewing sarcoma and peripheral primitive neuroectodermal tumors due to their similar features and shared identical genetic abnormality were grouped into a class of cancers entitled Ewing sarcoma family of tumors (ESFTs). Ewing sarcoma is the second most common pediatric osseous malignancy followed by osteosarcoma, with highest incidence among 10-20 years old. Ewing sarcoma is consistently associated with chromosomal translocation and functional fusion of the EWSR1 gene to any of several structurally related transcription factor genes of the E26 transformation-specific family. These tumor-specific molecular rearrangements are useful for primary diagnosis, may provide prognostic information, and present potential therapeutic targets. Therefore, ways to rapidly and efficiently detect these defining genomic alterations are of clinical relevance. Within the past decade, liquid biopsies including extracellular vesicles (EVs), have emerged as a promising alternative and/or complimentary approach to standard tumor biopsies. It was recently reported that fusion mRNAs from tumor-specific chromosome translocations can be detected in Ewing sarcoma cell-derived exosomes. Within this review, we overview the current advances in Ewing sarcoma and the opportunities and challenges in exploiting circulating exosomes, primarily small bioactive EVs (30-180 nm), as developing sources of biomarkers for diagnosis and therapeutic response monitoring in children and young adult patients with ESFT.

Extracellular vesicles: a promising tool for assessment of embryonic competence.

PURPOSE OF REVIEW: Extracellular vesicles have recently emerged as a promising field of research due to their pivotal roles in intercellular communication and potential to serve as biomarkers. This review focuses on extracellular vesicles secreted by the human preimplantation embryo. The most recent findings on embryo-derived extracellular vesicles are described and discussed, as well as current technical challenges to study them. RECENT FINDINGS: So far, only a few studies have addressed extracellular vesicles of embryonic origin and explored their potential as biomarkers for embryo selection. Two main hypotheses have driven interest in studying extracellular vesicles in IVF embryo-conditioned culture media. On the one hand, the potential roles of extracellular vesicles in mediating the embryo-endometrial crosstalk for proper implantation. On the other hand, the profile of secreted extracellular vesicles as an indicator of embryonic fitness irrespective of any involvement or communication with the endometrium. Embryo-derived extracellular vesicles have already been investigated to design diagnostic tests for embryo viability, however with small sample sizes or without extensive technology validation. SUMMARY: Extracellular vesicles offer indeed a novel means to assess embryonic fitness. Further validation studies, technology development and more complex study designs are certainly required to implement the profiling of embryonic extracellular vesicles as a diagnostic test for embryo selection.