Dataset Information

Comprehensive evaluation of methods for small extracellular vesicles separation from human plasma, urine and cell culture medium.

ABSTRACT: One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the lack of a consensus method for EV separation. This may also explain the diversity of the experimental results, as co-separated soluble proteins and lipoproteins may impede the interpretation of experimental findings. In this study, we comprehensively evaluated the EV yields and sample purities of three most popular EV separation methods, ultracentrifugation, precipitation and size exclusion chromatography combined with ultrafiltration, along with a microfluidic tangential flow filtration device, Exodisc, in three commonly used biological samples, cell culture medium, human urine and plasma. Single EV phenotyping and density-gradient ultracentrifugation were used to understand the proportion of true EVs in particle separations. Our findings suggest Exodisc has the best EV yield though it may co-separate contaminants when the non-EV particle levels are high in input materials. We found no 100% pure EV preparations due to the overlap of their size and density with many non-EV particles in biofluids. Precipitation has the lowest sample purity, regardless of sample type. The purities of the other techniques may vary in different sample types and are largely dependent on their working principles and the intrinsic composition of the input sample. Researchers should choose the proper separation method according to the sample type, downstream analysis and their working scenarios.

SUBMITTER: Dong L

PROVIDER: S-EPMC7810129 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Comprehensive evaluation of methods for small extracellular vesicles separation from human plasma, urine and cell culture medium.

Dong Liang L Zieren Richard C RC Horie Kengo K Kim Chi-Ju CJ Mallick Emily E Jing Yuezhou Y Feng Mingxiao M Kuczler Morgan D MD Green Jordan J Amend Sarah R SR Witwer Kenneth W KW de Reijke Theo M TM Cho Yoon-Kyoung YK Pienta Kenneth J KJ Xue Wei W

Journal of extracellular vesicles 20201201 2

One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the lack of a consensus method for EV separation. This may also explain the diversity of the experimental results, as co-separated soluble proteins and lipoproteins may impede the interpretation of experimental findings. In this study, we comprehensively evaluated the EV yields and sample purities of three most popular EV separation methods, ultracentrifugation, precipitation and size exclusion chromat ...[more]

PMID: 33489012

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Project description:Extracellular vesicles that are derived from stem cells play an important role in the treatment of disease. To obtain high-quality small extracellular vesicles (sEVs), we optimized the culture conditions of human induced pluripotent stem cells (hiPSCs), the supernatant collection time, and sEVs extraction methods. Firstly, hiPSCs were cultured in extracellular vesicles-production medium (EVs-PM) containing different concentrations (0%, 0.25%, 0.5%, 2%, 5%, and 20%) of extracellular vesicle-depleted knockout serum replacement (ED-KSR), and the culture supernatants were collected continuously for 5 days. Then, the sEVs were isolated, followed by an evaluation of their characteristics. The survival rates of the hiPSCs lines that were cultured in EVs-PM containing 0.5% to 20% ED-KSR were not significantly different (P > 0.05). The survival rates of the hiPSCs in 0.5% ED-KSR after the culture supernatants were continuously collected for day 1, day 3, and day 5 were not statistically significant (P > 0.05). After 5 days of continuous collection of the supernatant, the hiPSCs expressed some pluripotent markers, while SSEA4 and TRA-1-60 expression changed gradually. The sEVs that were extracted by the two methods were all 50-200 nm, double-layered and oval or round cellular vesicles and expressed the marker proteins CD63, TSG101, and HSP70. The characteristics of sEVs extracted on day 1, day 3, and day 5 were almost identical on morphology, size and the relative quantity of annexin V-positive subpopulations. The PKH67 staining showed that the sEVs could be endocytosed by HepG2 cells and aggregated in the cytoplasm. The proliferation experiments showed that the sEVs can promote cell proliferation. In Conclusion, the 0.5% ED-KSR is the optimal concentration, and that the hiPSCs culture supernatant can be continuously collected for 5 days while maintaining high cell viability and some pluripotent characteristics. Both of the methods extraction can be used to obtain biologically active sEVs.

Project description:Cell therapy is witnessing a notable shift toward cell-free treatments based on paracrine factors, in particular, towards small extracellular vesicles (sEV), that mimic the functional effect of the parental cells. While numerous sEV-based applications are currently in advanced preclinical stages, their promised translation depends on overcoming the manufacturing hurdles posed by the large-scale production of purified sEV. Unquestionably, the culture medium used with the parental cells plays a key role in the sEV's secretion rate and content. An essential requisite is the use of a serum-, xeno-, and blood-free medium to meet the regulatory entity requirements of clinical-grade sEV's production. Here, we evaluated OxiumTMEXO, a regulatory complying medium, with respect to production capacity and conservation of the EV's characteristics and functionality and the parental cell's phenotype and viability. A comparative study was established with standard DMEM and a commercially available culture medium developed specifically for sEV production. Under similar conditions, OxiumTMEXO displayed a three-fold increase of sEV secretion, with an enrichment of particles ranging between 51 and 200 nm. These results were obtained through direct quantification from the conditioned medium to avoid the isolation method's interference and variability and were compared to the two culture media under evaluation. The higher yield obtained was consistent with several harvest time points (2, 4, and 6 days) and different cell sources, incluiding umbilical cord-, menstrual blood-derived mesenchymal stromal cells and fibroblasts. Additionally, the stem cell phenotype and viability of the parental cell remained unchanged. Furthermore, OxiumTMEXO-sEV showed a similar expression pattern of the vesicular markers CD63, CD9, and CD81, with respect to sEV derived from the other conditions. The in vitro internalization assays in different target cell types and the pharmacokinetic profile of intraperitoneally administered sEV in vivo indicated that the higher EV production rate did not affect the uptake kinetics or the systemic biodistribution in healthy mice. In conclusion, the OxiumTMEXO medium sustains an efficient and robust production of large quantities of sEV, conserving the classic functional properties of internalization into acceptor target cells and biodistribution in vivo, supplying the amount and quality of EVs for the development of cell-free therapies.

Dataset Information

Comprehensive evaluation of methods for small extracellular vesicles separation from human plasma, urine and cell culture medium.

Publications

Comprehensive evaluation of methods for small extracellular vesicles separation from human plasma, urine and cell culture medium.

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