Large-scale heparin-based bind-and-elute chromatography identifies two biologically distinct populations of extracellular vesicles.

Purifying extracellular vesicles (EVs) has been challenging because EVs are heterogeneous in cargo yet share similar sizes and densities. Most surface marker-based affinity separation methods are limited to research or diagnostic scales. We report that heparin chromatography can separate purified EVs into two distinct subpopulations as ascertained by MS/MS: a non-heparin-binding (NHB) fraction that contains classical EV markers such as tetraspanins and a heparin-binding (HB) fraction enriched in fibronectins and histones. Both fractions were similarly fusogenic but induced different transcriptional responses in endothelial cells. While EVs that were purified by conventional, non-affinity methods alone induced ERK1/2 phosphorylation and Ki67, the NHB fraction did not. This result suggests heparin chromatography as an additional novel fractionation step that is inherently scalable, does not lead to loss of material, and separates inflammatory and pyrogenic EVs from unreactive EVs, which will improve clinical applications.

Extracellular Vesicle Isolation by a Tangential-Flow Filtration-Based Large-Scale Purification Method.

Extracellular vesicle (EV) isolation from conditioned cell culture medium has been a challenging topic. It is particularly difficult to obtain pure and intact EVs at a large scale. The commonly used methods such as differential centrifugation, ultracentrifugation, size exclusion chromatography, polyethylene glycol (PEG) precipitation, filtration, and affinity-based purification each have their advantages and limitations. Here, we present a tangential-flow filtration (TFF) based, multi-step purification protocol that combines filtration, PEG precipitation, and Capto Core 700 multimodal chromatography (MMC) to isolate EVs at high purity from large volumes of cell culture conditioned medium. Inserting the TFF step before PEG precipitation removes proteins, which may aggregate in subsequent steps and co-purify with EVs.

Research on the Synergistic Effect of Total Ionization and Displacement Dose in GaN HEMT Using Neutron and Gamma-Ray Irradiation.

This paper studies the synergistic effect of total ionizing dose (TID) and displacement damage dose (DDD) in enhancement-mode GaN high electron mobility transistor (HEMT) based on the p-GaN gate and cascode structure using neutron and 60Co gamma-ray irradiation. The results show that when the accumulated gamma-ray doses are up to 800k rad(Si), the leakage-current degradations of the two types of GaN HEMTs with 14 MeV neutron irradiation of 1.3 × 1012 n/cm2 and 3 × 1012 n/cm2 exhibit a lower degradation than the sum of the two separated effects. However, the threshold voltage shifts of the cascode structure GaN HEMT show a higher degradation when exposed to both TID and DDD effects. Moreover, the failure mechanisms of the synergistic effect in GaN HEMT are investigated using the scanning electron microscopy technique. It is shown that for the p-GaNHEMT, the increase in channel resistance and the degradation of two-dimensional electron gas mobility caused by neutron irradiation suppresses the increase in the TID leakage current. For the cascode structure HEMT, the neutron radiation-generated defects in the oxide layer of the metal-oxide-semiconductor field-effect transistor might capture holes induced by gamma-ray irradiation, resulting in a further increase in the number of trapped charges in the oxide layer.