Comparison of the Variability of Small Extracellular Vesicles Derived from Human Liver Cancer Tissues and Cultured from the Tissue Explants Based on a Simple Enrichment Method

Jie Chen,Tong Yang,Minghong Zhao,Xiaomei Yi,Yu Jiang,Die Hu,Fangfang Xie,Zhigang Jiao,Defa Huang,Wenjuan Zhang,Xiaoxing Wang,Tianyu Zhong,Zhengzhe Li,Jianwen Mo

doi:10.1007/s12015-021-10264-1

Abstract

A potential use of small extracellular vesicles (sEVs) for diagnostic and therapeutic purposes has recently generated a great interest. sEVs, when purified directly from various tissues with proper procedures, can reflect the physiological and pathological state of the organism. However, the quality of sEV is affected by many factors during isolation, including separation of sEV from cell and tissues debris, the use of enzymes for tissue digestion, and the storage state of tissues. In the present study, we established an assay for the isolation and purification of liver cancer tissues-derived sEVs (tdsEVs) and cultured explants-derived sEVs (cedsEVs) by comparing the quality of sEVs derived from different concentration of digestion enzyme and incubation time. The nano-flow cytometry (NanoFCM) showed that the isolated tdsEVs by our method are purer than those obtained from differential ultracentrifugation. Our study thus establishes a simple and effective approach for isolation of high-quality sEVs that can be used for analysis of their constituents.Graphical abstract

Highlights

Small extracellular vesicles are a kind of bilayer phospholipid membrane vesicles (30–200 nm in diameter) [1, 2] that are derived from various types of cells and released into the various body fluids and the interstitial space of tissuesJie Chen, Zhigang Jiao and Jianwen Mo contributed to this work.[3,4,5,6]
The samples were divided into three parts, which were used for the preparation of fresh tissues-derived sEVs (tdsEVs), frozen tdsEVs and cultured explants-derived sEVs (cedsEVs)
NanoFCM and BCA methods showed that were the numbers of particle and concentration of protein isolated by method 1 significantly higher compared with the other two methods (Fig. 2a-b), but were the CV% values of method 1 derived from the ratio of particles larger (Fig. 2f)

Summary

Introduction

Small extracellular vesicles (sEVs) are a kind of bilayer phospholipid membrane vesicles (30–200 nm in diameter) [1, 2] that are derived from various types of cells and released into the various body fluids and the interstitial space of tissuesJie Chen, Zhigang Jiao and Jianwen Mo contributed to this work.[3,4,5,6]. The released sEV play an important role in intercellular communication [7, 8]. These vesicles are packaged with proteins, mRNA, miRNA, rRNA and DNA specific for the cells, which partially reflect the physiological and pathological state of the body [9,10,11]. Due to the physical characteristic of sEV such as smaller size and low buoyant density, the isolation of sEVs from mixed components in the body fluids is challenging and currently requires tremendous effort. Current sample preparation kits and procedures for sEV isolation can be divided into physical and affinity separation approaches.

Methods

Results

Conclusion