Optimization of Isolation Method for Extracellular Vesicles from Pancreatic Juice and Impact of Protease Activity

Clinical application of pancreatic juice‐derived small extracellular vesicles of pancreatic ductal adenocarcinoma

“…Pure PJ samples can usually be collected during ERP; however, contamination with contrast agent, which is a little viscous and hyperosmotic, should be avoided. Endoscopic placement of a naso‐pancreatic drainage (ENPD) tube is often useful for increasing the total amount of PJ that is collected, even in cases in which an insufficient amount of PJ can be collected during ERP and/or when a PJ sample is highly viscous, as we described in a previous study 29 . Alternatively, the collection of duodenal fluid close to the ampullary orifice during diagnostic endoscopic ultrasonography (EUS) after intravenous secretin stimulation was also reported 28 .…”

“…Endoscopic placement of a naso-pancreatic drainage (ENPD) tube is often useful for increasing the total amount of PJ that is collected, even in cases in which an insufficient amount of PJ can be collected during ERP and/or when a PJ sample is highly viscous, as we described in a previous study. 29 Alternatively, the collection of duodenal fluid close to the ampullary orifice during diagnostic endoscopic ultrasonography (EUS) after intravenous secretin stimulation was also reported. 28 This method is ideal for screening of PDAC detection because there is no risk of post-ERP pancreatitis; however, the contamination of duodenal contents is unavoidable.…”

“…Nakamura et al reported that the expression of microRNA (miRNA)-21 in PJ-derived sEVs was stable in such alkaline conditions at 37 • C for 48 h in vitro, suggesting miRNAs in sEVs would be stable in the pancreatic duct. 27 We investigated the stability of PDAC cell lines, PANC-1-derived sEVs under alkaline conditions and under protease activation, as well in vitro, and found that the morphology-as observed by TEM-of PANC-1-derived sEVs was unchanged under alkaline conditions for 12 h at 37 • C, while the sEVs collapsed at 12 h after exposure to pancreatin (an activated pancreatic enzyme) at 37 • C. 29 In addition, according to an in vitro evaluation using PJ samples with protease activation, the expression of TSG101 (one of the markers present within sEVs) obviously decreased in comparison to the control (no exposure) after more than 6 h of exposure to room temperature and after 3 h of exposure, thus suggesting that protease activity would certainly affect the degradation of sEVs in PJ. Considering the time required for degradation, collected PJ samples that have just flowed out into the ENPD tube would be suitable as PJ samples.…”

“…To our knowledge, there have been five reports on human PJ‐derived sEVs, and these have mainly been related to the identification of biomarkers of PDAC (Table 1). 25–29 …”

“… 31 According to our analysis using transmission electron microscopy (TEM) and Western blotting of sEV markers, SEC could be an optimal isolation method due to its higher collection rate and the purity of PJ‐derived sEVs. 29 SEC is a simple and reproducible sEVs collection method and readily recover morphologically intact functionally competent EVs from small volumes. 31 On the other hand, dUC has disadvantages such as low collection and high time consumption, and is unsuitable for viscous samples, 27 and the precipitation can yield higher sEVs amounts but achieves lower purity, because of co‐precipitated proteins,and is time‐consuming.…”