Co-continuous structural effect of size-controlled macro-porous glass membrane on extracellular vesicle collection for the analysis of miRNA

Abstract

Recent studies have shown that extracellular vesicles (EVs) can be utilized as appropriate and highly specific biomarkers in liquid biopsy for the diagnosis and prognosis of serious illness. However, there are few methods that can collect and isolate miRNA in EVs simply, quickly and efficiently using general equipment such as a normal centrifuge. In this paper, we developed an advanced glass membrane column (AGC) device incorporating a size-controlled macro-porous glass (MPG) membrane with a co-continuous structure to overcome the limitations of conventional EV collection and miRNA extraction from the EVs. The size of macro-pores in the MPG membrane could be accurately controlled by changing the heating temperature and time on the basis of spinodal decomposition of B2O3, Na2O, and SiO2 in phase separation. The AGC device with an MPG membrane could collect the EVs simply and quickly (< 10 min) from cell culture supernatant, serum and urine. This AGC device could extract miRNA from the EVs captured in the MPG membrane with high efficiency when combined with a miRNA extraction solution. We suggest that the AGC device with an MPG membrane can be useful for the diagnosis and prognosis of serious illness using of EVs in various kinds of body fluids.

Clinical impact of bile‐derived exosomal microRNAs as novel diagnostic and prognostic biomarkers for biliary tract cancers

“…In addition, standard exosome markers, CD9 and CD63, were detected by a CD9/CD63 ELISA kit to quantify the exosomes, as previously reported. 9 Subsequently, the amount of RNA in exosomes was quantified. As shown in Figure 2A , three independent samples processed from the bile of three BTC patients using the AGC device were confirmed to contain clearly significant amounts of exosomes.…”

“…To confirm the captured exosomes and exosome RNAs by the AGC device, we first quantified the exosomes by western blotting. In addition, standard exosome markers, CD9 and CD63, were detected by a CD9/CD63 ELISA kit to quantify the exosomes, as previously reported 9 . Subsequently, the amount of RNA in exosomes was quantified.…”

“…Furthermore, we previously showed that the AGC column with an MPG membrane could collect exosomes simply and quickly (<10 min) from various liquid samples. 9 In addition, the AGC device could extract miRNAs from the exosomes captured in the MPG membrane with high efficiency using the miRNA extraction solution. We have also shown that the number of miRNAs detected by miRNA array in the AGC device was greater than those collected by three major methods, UC, magnetic beads, and precipitation reagent.…”

“…We have also shown that the number of miRNAs detected by miRNA array in the AGC device was greater than those collected by three major methods, UC, magnetic beads, and precipitation reagent. 9 This isolation protocol could be expected to help standardize the sample quality for the following analysis targeting exosomal miRNAs.…”

“…To collect exosomes efficiently from a limited volume liquid sample, we utilized an AGC device in this study, as has been published previously. 9 One milliliter of the liquid samples, that is, bile juice, serum, and cell culture supernatants, was filtered through a 0.22 μm filter; 200 μl of the filtered samples was transferred to the upper part of the AGC device and centrifuged at 6000 g for 5 min. Total RNA was then isolated from the exosomes captured in the AGC device using a miRNeasy Serum/Plasma kit (Qiagen).…”