Extracellular vesicles (EVs) play a key role in many physiological and pathophysiological processes and hold great potential for therapeutic and diagnostic use. Despite significant advances within the last decade, the key issue of EV storage stability remains unresolved and under investigated. Here, we aimed to identify storage conditions stabilizing EVs and comprehensively compared the impact of various storage buffer formulations at different temperatures on EVs derived from different cellular sources for up to 2 years. EV features including concentration, diameter, surface protein profile and nucleic acid contents were assessed by complementary methods, and engineered EVs containing fluorophores or functionalized surface proteins were utilized to compare cellular uptake and ligand binding. We show that storing EVs in PBS over time leads to drastically reduced recovery particularly for pure EV samples at all temperatures tested, starting already within days. We further report that using PBS as diluent was found to result in severely reduced EV recovery rates already within minutes. Several of the tested new buffer conditions largely prevented the observed effects, the lead candidate being PBS supplemented with human albumin and trehalose (PBS‐HAT). We report that PBS‐HAT buffer facilitates clearly improved short‐term and long‐term EV preservation for samples stored at ‐80°C, stability throughout several freeze‐thaw cycles, and drastically improved EV recovery when using a diluent for EV samples for downstream applications.
Extracellular vesicles (EVs) have recently emerged as a highly promising cell-free biotherapeutics. While a range of engineering strategies have been developed to functionalize the EV surface, current approaches fail to address the limitations associated with endogenous surface display, pertaining to the heterogeneous display of commonly used EV-loading moieties among different EV subpopulations. Here we present a novel engineering platform to display multiple protein therapeutics simultaneously on the EV surface. As proof-of-concept, we screened multiple endogenous display strategies for decorating the EV surface with cytokine binding domains derived from tumor necrosis factor receptor 1 (TNFR1) and interleukin 6 signal transducer (IL6ST), which can act as decoys for the pro-inflammatory cytokines TNFα and IL6, respectively. Combining synthetic biology and systematic screening of loading moieties, resulted in a three-component system which increased the display and decoy activity of TNFR1 and IL6ST, respectively. Further, this system allowed for .
Extracellular vesicles (EVs) are released from basically all cells. Over the last decade, small EVs (sEVs; 50–150 nm) have gained enormous attention in diagnostics and therapy. However, methodological limitations coupled to the lack of EV standards leave many questions in this quickly evolving field unresolved. Recently, by using enhanced green fluorescent protein (eGFP)‐labeled sEVs as biological reference material, we systematically optimized imaging flow cytometry for single sEV analysis. Furthermore, we showed that sEVs stained with different fluorescent antibodies can be analyzed in a multiparametric manner. However, many parameters potentially affecting the sEV staining procedure still require further evaluation and optimization. Here, we present a concise, systematic evaluation of the impact of the incubation temperature (4°C, room temperature and 37°C) during sEV antibody staining on the outcome of experiments involving the staining of EVs with fluorescence‐conjugated antibodies. We provide evidence that both the staining intensity and the sample recovery can vary depending on the incubation temperature applied, and that observed differences are less pronounced following prolonged incubation times. In addition, this study can serve as an application‐specific example of parameter evaluation in EV flow cytometry. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.
Thioredoxins (Trx) and glutaredoxins (Grx) are thiol oxidoreductases that are ubiquitously expressed, and are involved in several biological processes. The expression of thioredoxins and glutaredoxins is induced in many neoplasms, and correlates with prognosis in gallbladder and colorectal carcinoma. The aim of the present study was to examine the expression pattern of these proteins (redoxins) in hepatocellular carcinoma (HCC) and to correlate their levels with clinical features. Paraffin-embedded tissues from 25 patients resected for HCC and 15 patients resected for colorectal carcinoma (CRC) liver metastases were analyzed with immunohistochemistry. Our results showed that Trx1, Trx2 and Grx5 were upregulated in HCCs as compared to the respective surrounding liver. In comparison, almost all redoxins were upregulated in CRC liver metastases, with Trx1 and Grx3 being significantly more increased in the CRC liver metastases than in the primary HCC tumors. In HCC, Trx1 correlated significantly with cell proliferation, and with a trend towards increased levels with micro-vascular invasion, while expression of Trx2 decreased with tumor size. Trx1 levels were lower in tumors of males, smokers, and patients with high alcohol consumption. Grx2 levels were significantly higher in patients with metabolic syndrome. In conclusion, this study illustrates specific correlations of individual redoxins to clinical features of HCC, and implicates the redoxins in the pathogenesis of HCC.
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