Improved isolation strategies to increase the yield and purity of human urinary exosomes for biomarker discovery

Abstract

Circulating miRNAs are detected in extracellular space and body fluids such as urine. Circulating RNAs can be packaged in secreted urinary extracellular vesicles (uEVs) and thus protected from degradation. Urinary exosome preparations might contain specific miRNAs, relevant as biomarkers in renal and bladder diseases. Major difficulties in application of uEVs into the clinical environment are the high variability and low reproducibility of uEV isolation methods. Here we used five different methods to isolate uEVs and compared the size distribution, morphology, yield, presence of exosomal protein markers and RNA content of uEVs. We present an optimized ultracentrifugation and size exclusion chromatography approach for highly reproducible isolation for 50–150 nm uEVs, corresponding to the exosomes, from 50 ml urine. We profiled the miRNA content of uEVs and total urine from the same samples with the NanoString platform and validated the data using qPCR. Our results indicate that 18 miRNAs, robustly detected in uEVs were always present in the total urine. However, 15 miRNAs could be detected only in the total urine preparations and might represent naked circulating miRNA species. This is a novel unbiased and reproducible strategy for uEVs isolation, content normalization and miRNA cargo analysis, suitable for biomarker discovery studies.

Isolation and quantification of blood apoptotic bodies in neurological patients

“…Research into EVs has been rising during the last decade, being microvesicles and exosomes the best characterized vesicle populations [25]. Indeed, there is available and updated information elucidating the most efficient methods for obtaining high yields of these highly-purified vesicles from both cell culture and complex biological fluids, such as plasma [31][32][33]. However, it is striking that apoptotic bodies have often been overlooked in studies of circulating vesicles.…”

Exosome Biochemistry and Advanced Nanotechnology for Next‐Generation Theranostic Platforms

“…Although all isolations contained 40–100 nm nanovesicles, and were positive for exosome markers (Alix, TSG101, HSP70) based on electron microscopy and western blotting, immunoaffinity capture method is the most effective method to isolate exosomes with desirable structural/compositional integrity. Gheinani et al also optimized ultracentrifugation and size exclusion chromatography approach for highly reproducible isolation of exosomes of 50–150 nm in diameter, which is beneficial to subsequent diagnostic applications.…”

Roles for Exosome in Various Kidney Diseases and Disorders

“…However, isolation or purification of exosomes from animal model or raw biological fluids is still challenging because some components, such as high-density lipoproteins, chylomicrons, and microvesicles, have their size ranges close to that of exosomes (Yuana et al, 2014). Recently, several efforts have been made to optimize the isolation/purification of exosomes (Gheinani et al, 2018;Ayala-Mar et al, 2019;Doyle and Wang, 2019;Hou et al, 2019;Huang et al, 2019;Wang et al, 2019a;Yang et al, 2019). Nevertheless, the outcome is still unsatisfactory.…”