Extracellular vesicles selective capture by peptide-functionalized hollow fiber membranes.

Recently, membrane devices and processes have been applied for the separation and concentration of subcellular components such as extracellular vesicles (EVs), which play a diagnostic and therapeutic role in many pathological conditions. However, the separation and isolation of specific EV populations from other components found in biological fluids is still challenging. Here, we developed a peptide-functionalized hollow fiber (HF) membrane module to achieve the separation and enrichment of highly pure EVs derived from the culture media of human cardiac progenitor cells. The strategy is based on the functionalization of PSf HF membrane module with BPt, a peptide sequence able to bind nanovesicles characterized by highly curved membranes. HF membranes were modified by a nanometric coating with a copoly azide polymer to limit non-specific interactions and to enable the conjugation with peptide ligand by click chemistry reaction. The BPt-functionalized module was integrated into a TFF process to facilitate the design, rationalization, and optimization of EV isolation. This integration combined size-based transport of species with specific membrane sensing ligands. The TFF integrated BPt-functionalized membrane module demonstrated the ability to selectively capture EVs with diameter < 200 nm into the lumen of fibers while effectively removing contaminants such as albumin. The captured and released EVs contain the common markers including CD63, CD81, CD9 and syntenin-1. Moreover, they maintained a round shape morphology and structural integrity highlighting that this approach enables EVs concentration and purification with low shear stress. Additionally, it achieved the removal of contaminants such as albumin with high reliability and reproducibility, reaching a removal of 93%.

Salerno S ，Piscioneri A ，Morelli S ，Gori A ，Provasi E ，Gagni P ，Barile L ，Cretich M ，Chiari M ，De Bartolo L ... -  《-》

Highly-purified exosomes and shed microvesicles isolated from the human colon cancer cell line LIM1863 by sequential centrifugal ultrafiltration are biochemically and functionally distinct.

Secretion and exchange of extracellular vesicles (EVs) by most cell types is emerging as a fundamental biological process. Although much is known about EVs, there is still a lack of definition as to how many naturally occurring EV subtypes there are and how their properties and functionalities might differ. This vexing issue is critical if EVs are to be fully harnessed for therapeutic applications. To address this question we have developed and describe here a sequential centrifugal ultrafiltration (SCUF) method to examine, in an unbiased manner, what EV subtypes are released in vitro into cell culture medium using the human colon carcinoma cell line LIM1863 as a model system. Using the culture medium from ∼7.2×10(9) LIM1863 cells, SCUF was performed using hydrophilic PVDF membranes with low protein binding properties (Millipore Durapore™ Ultrafree-CL filters with 0.1, 0.22, 0.45 and 0.65 μm pore size). EV particle sizing was measured using both dynamic light scattering and cryo-electron microscopy. Comparative proteome profiling was performed by GeLC-MS/MS and qualitative protein differences between EV subtypes determined by label-free spectral counting. The results showed essentially two EV subtypes; one subtype (fraction Fn1) comprised heterogeneous EVs with particle diameters of 30-1300 nm, the other (fraction Fn5) being homogeneous EVs of 30-100 nm diameter; based on cryo-EM both EV subtypes were round shaped. Western blot analysis showed Fn5 (SCUF-Exos) contained traditional exosome marker proteins (Alix(+), TSG101(+), CD81(+), CD63(+)), while Fn1 (SCUF-sMVs) lacked these protein markers. These findings were consistent with sMVs isolated by differential centrifugation (10,000 g, DC-sMVs) and exosomes (100,000 g EVs depleted of 10,000 g material). The buoyant density of sMVs determined by OptiPrep™ density gradient centrifugation was 1.18-1.19 g/mL and exosomes 1.10-1.11 g/mL. Comparative protein profiling of SCUF-Exos/-sMVs revealed 354 and 606 unambiguous protein identifications, respectively, with 256 proteins in common. A salient finding was the first report of 350 proteins uniquely identified in sMVs may of which have the potential to enable discrimination of this EV subtype from exosomes (notably, members of the septin family, kinesin-like protein (KIF23), exportin-2/chromosome segregation like-1 protein (CSE1L), and Rac GTPase-activating protein 1 (RACGAP1)). We report for the first time that both SCUF-Exos and SCUF-sMVs isolated from LIM1863 colon cancer cells induce invasion of recipient NIH3T3 cells. Interestingly, the SCUF-sMVs promote invasion to a significantly greater extent (3-fold) than SCUF-Exos. This analytical SCUF method for fractionating EVs is potentially scalable using tangential flow filtration, thereby providing a solid foundation for future in-depth functional studies of EV subtypes using diverse cell types and functional assays.

Xu R ，Greening DW ，Rai A ，Ji H ，Simpson RJ ... -  《-》

Novel Electrochemically Switchable, Flexible, Microporous Cloth that Selectively Captures, Releases, and Concentrates Intact Extracellular Vesicles.

Akbarinejad A ，Hisey CL ，Brewster D ，Ashraf J ，Chang V ，Sabet S ，Nursalim Y ，Lucarelli V ，Blenkiron C ，Chamley L ，Barker D ，Williams DE ，Evans CW ，Travas-Sejdic J ... -  《-》

A comprehensive proteomics profiling identifies NRP1 as a novel identity marker of human bone marrow mesenchymal stromal cell-derived small extracellular vesicles.

Munshi A ，Mehic J ，Creskey M ，Gobin J ，Gao J ，Rigg E ，Muradia G ，Luebbert CC ，Westwood C ，Stalker A ，Allan DS ，Johnston MJW ，Cyr T ，Rosu-Myles M ，Lavoie JR ... -  《Stem Cell Research & Therapy》

Standardized Method to Functionalize Plasma-Extracellular Vesicles via Copper-Free Click Chemistry for Targeted Drug Delivery Strategies.

Ciferri MC ，Bruno S ，Rosenwasser N ，Gorgun C ，Reverberi D ，Gagliani MC ，Cortese K ，Grange C ，Bussolati B ，Quarto R ，Tasso R ... -  《-》