Comparison of ultracentrifugation and a commercial kit for isolation of exosomes derived from glioblastoma and breast cancer cells

Abstract

Exosomes are small extracellular vesicles around 30-100 nm in diameter that are secreted from cells and can be found in most body fluids. Exosomes can be a vital source of biomarkers as they contain various substances (e.g. lipids, RNAs, metabolites and proteins) that can reflect the cell of origin (e.g. cancer cells). For isolation of exosomes present in biological matrices, ultracentrifugation (UC)-based procedures are most common. Other approaches exist, including commercial kits developed for easy and low sample volume isolation. In this study, differential UC and an isolation kit from a major vendor (Total Exosome Isolation Reagent from Thermo Fisher Scientific) were compared. Exosomes were isolated from cell culture media of two different cell sources (patient derived cells from glioblastoma multiforme and the breast cancer cell line MDA-MB-231). For both isolation methods, transmission electron microscopy, dynamic light scattering and western blotting indicated the presence of exosomes. The kit- and UC isolates contained similar amounts of protein measured by the bicinchoninic acid (BCA) assay with absorbance at 562 nm. Using western blot, positive exosome markers were identified in all isolates, and additional exosome markers were identified using MS-based proteomics. For the glioblastoma exosome isolates, the number of proteins identified with liquid chromatography tandem MS (LC-MS/MS) was higher for the UC isolates than the kit isolates when injecting equal protein amounts, contrary to that for the breast cancer exosome isolates. However, negative exosome markers were also found in glioblastoma isolates using LC-MS/MS. Thus, we would not use the term “exosome isolation” as impurities may be present with both isolation methods. Notably, potential biomarkers for both diseases were identified in the isolates using LS-MS/MS. In our opinion, the two isolation methods had rather similar performance, although with some minor differences based on cell of origin.

Distinct mechanisms of microRNA sorting into cancer cell-derived extracellular vesicle subtypes

“…We decided to focus on the top three candidates, nucleolin (NCL), Lupus La (La) and nucleophosmin (NPM1). These three RBPs have been reported to be present in crude high-speed pellet preparations from conditioned media from different carcinoma cell lines (Liang et al, 2013; Demory Beckler et al, 2013), including from our model cell line MDA-MD-231 (Skottvoll et al, 2018).…”

Parallelized multidimensional analytic framework applied to mammary epithelial cells uncovers regulatory principles in EMT

“…Serum-free conditioned media were centrifuged at 800 × g for 10 min at 4 °C and sequentially passed first using 0.22 µm filter (Fisher) and then through a 100 kDa cut-off filter (Thermo Scientific). The retentate was resuspended in 1 mL PBS and used for EXOS extraction using the Total Exosome Isolation Reagent (from cell culture media) as per manufacturer's instructions (Thermo Scientific) 50 . Exos pellets were stored at -80 °C.…”

Synergistic effects of exosomal crocin or curcumin compounds and HPV L1-E7 polypeptide vaccine construct on tumor eradication in C57BL/6 mouse model

“…Based on the results presented by Skottvoll et al ., the quality of ultracentrifuge/ commercial kit isolation methods were approximately equal. Moreover, kit isolation needs less starting material than conventional ultracentrifuge equipment [ 43 ]. In this study, ExoQuick-TC ULTRA kit was used to isolate exosomes from human embryonic kidney (HEK)-293T cells [ 44 ].…”