Immunomagnetic Separation Method Integrated with the Strep-Tag II System for Rapid Enrichment and Mild Release of Exosomes

Abstract

Exosomes are important participants in numerous pathophysiological processes and hold promising application value in cancer diagnosis, monitoring, and prognosis. However, the small size (40−160 nm) and high heterogeneity of exosomes make it still challenging to enrich exosomes efficiently from the complex biological fluid microenvironment, which has largely restricted their downstream analysis and clinical application. In this work, we introduced a novel method for rapid isolation and mild release of exosomes from the cell culture supernatant. A Strep-tag II-based immunomagnetic isolation (SIMI) system was constructed by modifying the capture antibodies onto magnetic nanoparticles through specific and reversible recognition between Strep-Tactin and Strep-tag II. Due to their high affinity and binding selectivity, exosomes could be isolated within 38 min with an isolation efficiency of 82.5% and a release efficiency of 62%. Compared with the gold-standard ultracentrifugation, the SIMI system could harvest nearly 59% more exosomes from the 293 T cell culture medium with shorter isolation time and higher purity. In addition, cellular uptake assay indicated that exosomes released from magnetic nanoparticles could maintain their high biological activity. These superior characteristics show that this novel method is a fast, efficient, and nondestructive exosome isolation tool and thus could potentially be further utilized in various exosome-related applications, e.g., disease diagnosis and drug delivery.

Magnetic Nanoparticle-Based Microfluidic Platform for Automated Enrichment of High-Purity Extracellular Vesicles

“…SIMI magnetic beads were prepared using a previously reported method Figure c,d displays the SEM image and hysteresis curve (saturation magnetization value 40.5 emu/g) of SIMI.…”

“…The platform is easy to fabricate and automation, with no requirement for precise fluidic control, providing a simple tool for EV-based analysis. Our previously developed Strep-tag II-based immunomagnetic isolation (SIMI) system 27 offers good performance for rapid capture and nondestructive EV release. The ExoCPR platform had a 75.8% isolation efficiency and a 62.7% release efficiency, and the entire process could be accomplished within 29 min.…”

“…SIMI magnetic beads were prepared using a previously reported method. 27 μL) and SIMI magnetic beads were added to the capture chamber using a pipet. Then, silicone oil (50 μL) and wash buffer (50 μL) were introduced into the corresponding immiscible phase chambers.…”

Isolation and usage of exosomes in central nervous system diseases

“…45 tion method, the SIMI system extracted approximately 59% more exosomes from the 293T cell culture medium, while also offering a shorter isolation time, higher purity, and improved biological activity. 82 A novel method was developed to isolate exosomes from serum in a precise and time-dependent manner. This approach used magnetic beads for capturing exosomes and employed lightactivated elution.…”

Application and advances of biomimetic membrane materials in central nervous system disorders

“…Then centrifuge at 2000 g and 10,000 g for 30 min and 60 min respectively at 4℃ to remove cell debris; Finally, EVs was obtained after 120,000 g centrifugation at 4℃ for 1 h. In order to purify EVs, EVs can be re-suspended with sterile PBS, filtered with 0.22µM sterile filter, re-centrifuged at 120,000 g at 4℃ for 1 h, and then re-suspended in sterile PBS or RIPA buffer and stored at -80 °C until further use. In addition to ultracentrifugation, other methods for extracting EVs include ultrafiltration [ 75 , 76 ], size exclusion [ 77 ], polymer-based precipitation [ 78 ], immunoaffinity capture [ 79 ] and microfluidics [ 80 ]. Ultrafiltration is a technique that utilizes the difference in pressure across the ultrafiltration membrane and separates EVs based on their characteristic size; size exclusion is a technique that allows the sample to flow through the column, where substances larger than the pore size of the gel particles are not allowed to enter the pores, and they elute through the space between the porous gel and the mobile phase; polymer co-precipitation technique is based on the principle that hydrophilic polymers interact with the hydrophilic bonds of the sample’s EVs to form a hydrophobic microenvironment around the EVs, which results in the formation of precipitates and extraction of EVs; the immunoaffinity capture technique is based on the principle of isolating and enriching EVs by identifying specific proteins of EVs; and microfluidics is a signal-detection-based technique for EVs extraction.…”