Urine is commonly used for clinical diagnosis and biomedical research. The discovery of extracellular vesicles (EV) in urine opened a new fast‐growing scientific field. In the last decade urinary extracellular vesicles (uEVs) were shown to mirror molecular processes as well as physiological and pathological conditions in kidney, urothelial and prostate tissue. Therefore, several methods to isolate and characterize uEVs have been developed. However, methodological aspects of EV separation and analysis, including normalization of results, need further optimization and standardization to foster scientific advances in uEV research and a subsequent successful translation into clinical practice. This position paper is written by the Urine Task Force of the Rigor and Standardization Subcommittee of ISEV consisting of nephrologists, urologists, cardiologists and biologists with active experience in uEV research. Our aim is to present the state of the art and identify challenges and gaps in current uEV‐based analyses for clinical applications. Finally, recommendations for improved rigor, reproducibility and interoperability in uEV research are provided in order to facilitate advances in the field.

Extracellular vesicles (EVs) in human blood are a potential source of biomarkers. To which extent anticoagulation affects their concentration, cellular origin and protein composition is largely unexplored. To study this, blood from 23 healthy subjects was collected in acid citrate dextrose (ACD), citrate or EDTA, or without anticoagulation to obtain serum. EVs were isolated by ultracentrifugation or by size-exclusion chromatography (SEC) for fluorescence-SEC. EVs were analyzed by micro flow cytometry, NTA, TEM, Western blot, and protein mass spectrometry. The plasma EV concentration was unaffected by anticoagulants, but serum contained more platelet EVs. The protein composition of plasma EVs differed between anticoagulants, and between plasma and serum. Comparison to other studies further revealed that the shared EV protein composition resembles the “protein corona” of synthetic nanoparticles incubated in plasma or serum. In conclusion, we have validated a higher concentration of platelet EVs in serum than plasma by contemporary EV methods. Anticoagulation should be carefully described (i) to enable study comparison, (ii) to utilize available sample cohorts, and (iii) when preparing/selecting biobank samples. Further, the similarity of the EV protein corona and that of nanoparticles implicates that EVs carry both intravesicular and extravesicular cargo, which will expand their applicability for biomarker discovery.

The endoplasmic reticulum (ER) is a multifaceted cellular organelle both structurally and functionally, and its cell cycle–dependent morphological changes are poorly understood. Our quantitative confocal and EM analyses show that the ER undergoes dramatic reorganization during cell division in cultured mammalian cells as mitotic ER profiles become shorter and more branched. 3D modeling by electron tomography reveals that the abundant interphase structures, sheets, are lost and subsequently transform into a branched tubular network that remains continuous. This is confirmed by observing the most prominent ER subdomain, the nuclear envelope (NE). A NE marker protein spreads to the mitotic ER tubules, although it does not show a homogenous distribution within the network. We mimicked the mitotic ER reorganization using puromycin to strip the membrane-bound ribosomes from the interphase ER corresponding to the observed loss of ribosomes normally occurring during mitosis. We propose that the structural changes in mitotic ER are linked to ribosomal action on the ER membranes.

Body fluids are a rich source of extracellular vesicles (EVs), which carry cargo derived from the secreting cells. So far, biomarkers for pathological conditions have been mainly searched from their protein, (mi)RNA, DNA and lipid cargo. Here, we explored the small molecule metabolites from urinary and platelet EVs relative to their matched source samples. As a proof-of-concept study of intra-EV metabolites, we compared alternative normalization methods to profile urinary EVs from prostate cancer patients before and after prostatectomy and from healthy controls.

Fetal bovine serum (FBS) is the most commonly used supplement in studies involving cell‐culture experiments. However, FBS contains large numbers of bovine extracellular vesicles (EVs), which hamper the analyses of secreted EVs from the cell type of preference and, thus, also the downstream analyses. Therefore, a prior elimination of EVs from FBS is crucial. However, the current methods of EV depletion by ultracentrifugation are cumbersome and the commercial alternatives expensive. In this study, our aim was to develop a protocol to completely deplete EVs from FBS, which may have wide applicability in cell‐culture applications. We investigated different EV‐depleted FBS prepared by our novel ultrafiltration‐based protocol, by conventionally used overnight ultracentrifugation, or commercially available depleted FBS, and compared them with regular FBS. All sera were characterized by nanoparticle tracking analysis, electron microscopy, Western blotting and RNA quantification. Next, adipose‐tissue mesenchymal stem cells (AT‐MSCs) and cancer cells were grown in the media supplemented with the three different EV‐depleted FBS and compared with cells grown in regular FBS media to assess the effects on cell proliferation, stress, differentiation and EV production. The novel ultrafiltration‐based protocol depleted EVs from FBS clearly more efficiently than ultracentrifugation and commercial methods. Cell proliferation, stress, differentiation and EV production of AT‐MSCs and cancer cell lines were similarly maintained in all three EV‐depleted FBS media up to 96 h. In summary, our ultrafiltration protocol efficiently depletes EVs, is easy to use and maintains cell growth and metabolism. Since the method is also cost‐effective and easy to standardize, it could be used in a wide range of cell‐culture applications helping to increase comparability of EV research results between laboratories.

The endoplasmic reticulum (ER) is both structurally and functionally complex, consisting of a dynamic network of interconnected sheets and tubules. To achieve a more comprehensive view of ER organization in interphase and mitotic cells and to address a discrepancy in the field (i.e., whether ER sheets persist, or are transformed to tubules, during mitosis), we analyzed the ER in four different mammalian cell lines using live-cell imaging, high-resolution electron microscopy, and three dimensional electron microscopy. In interphase cells, we found great variation in network organization and sheet structures among different cell lines. In mitotic cells, we show that the ER undergoes both spatial reorganization and structural transformation of sheets toward more fenestrated and tubular forms. However, the extent of spatial reorganization and sheet-to-tubule transformation varies among cell lines. Fenestration and tubulation of the ER correlates with a reduced number of membrane-bound ribosomes.