Activation of Inflammation by MCF-7 Cells-Derived Small Extracellular Vesicles (sEV): Comparison of Three Different Isolation Methods of SEV

Overview

Journal Pharm Res

Specialties Pharmacology
Pharmacy

Date 2022 Aug 24

PMID 36002612

Authors

Kohei Suzuki

Masataka Nakano

Shimon Nakashima

Tatsuki Fukami

Miki Nakajima

Affiliations

Soon will be listed here.

Abstract

Purpose: Small extracellular vesicles (sEV) containing proteins and RNAs play important roles as intercellular signal mediators. A critical issue is that there are multiple methods to prepare sEV fractions. The purpose of this study was to examine whether cancer cell-derived sEV fractions prepared by different isolation methods show similar responses for the induction of inflammatory cytokines in macrophages.

Methods: sEV fractions from the conditioned medium of MCF-7 cells were prepared by ultracentrifugation (UC), the MagCapture Exosome Isolation Kit PS (PS), or the ExoQuick-TC kit (EQ). The mRNA levels of inflammatory cytokines in differentiated THP-1 cells treated with the sEV fractions were evaluated.

Results: The yields of sEV fractions obtained from 1 mL conditioned medium by UC, PS, or EQ were 3.2×10 particles (0.27 μg protein), 12.8×10 particles (0.87 μg protein) and 23.5 ×10 particles (4.50 μg protein), respectively. The average particle sizes in the UC, PS, and EQ fractions were 184.8 ± 1.8 nm, 157.8 ± 1.3 nm and 165.8 ± 1.1 nm, respectively. CD9 and CD81, markers of sEV, were most highly detected in the PS fraction, followed by the EQ and UC fractions. These results suggest that PS gave sEV with relatively high purity, and many protein contaminants appear to be included in the EQ fraction. The mRNA levels of inflammatory cytokines in THP-1 macrophages were most prominently increased by treatment with the UC fraction, followed by the EQ and PS fractions, suggesting that contaminants rather than sEV may largely induce an inflammatory response.

Conclusion: The isolation method affects the evaluation of sEV function.

Citing Articles

The role of exosomes in liver cancer: comprehensive insights from biological function to therapeutic applications.

Zhang Y, Zhang C, Wu N, Feng Y, Wang J, Ma L Front Immunol. 2024; 15:1473030.

PMID: 39497820 PMC: 11532175. DOI: 10.3389/fimmu.2024.1473030.

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