Isolation and Characterization of Exosomes Derived from Mouse Spleen Tissues

Summary

Here, we present a protocol to isolate mouse spleen-derived exosomes using a combination of digestion with collagenase type I and ultracentrifugation.

Abstract

Exosomes (Exo) are lipid-bilayer structures secreted by various cells, including those of animals, plants, and prokaryotes. Previous studies have revealed that Exo derived from humoral or cell-supernatant are promising targets for novel diagnostic or prognostic biomarkers, underscoring their significant role in disease pathogenesis. Tissue-derived Exo (Ti-Exo) have attracted increasing attention due to its ability to accurately reflect tissue specificity and the microenvironment. Ti-Exo, present in interstitial space, play crucial roles in intercellular communication and cross-organ signaling. Despite their recognized value in elucidating disease mechanisms, isolating Ti-Exo remains challenging due to the complexity of tissue matrices and variability in extraction methods. In this study, we developed a practical protocol for isolating exosomes from mice spleen tissue, providing a reproducible technique for subsequent identification analysis and functional studies. We used Type I collagenase digestion combined with differential ultracentrifugation to isolate spleen-derived Exo. The characteristics of isolated Exo were determined through electron microscopy, the nano-flow cytometer, and the western blot. The isolated spleen-derived Exo displayed the typical morphology of lipid bilayer vesicles, with particle sizes ranging from 30 nm to 150 nm. In addition, the expression profile of exosome markers confirmed the presence and purity of exosomes. Taken together, we successfully established a practical protocol for isolating spleen-derived Exo in mice.

Introduction

Exosomes (Exo) are a subgroup of extracellular vesicles (EVs), ranging from 30 to 150 nm in size, encapsulating a diverse array of biomolecules, including proteins, nucleic acids, and lipids¹. These biomolecules are derived from the parental cells and are released into the extracellular space. Exo facilitate biomolecular information exchange between cells and their surrounding microenvironment, playing roles in both prokaryotic and eukaryotic systems². The characteristics of exosomes, such as content, size, membrane components, and cellular origin, are highly variable and influenced by the originating cell type, cellular....

Protocol

The samples were obtained from mice, with ethical approval granted by the Ethics Committee of Guangdong Medical University. Detailed descriptions of materials, equipment, and software used in this protocol are provided in the Table of Materials. The details of the preparation prior to extraction are illustrated in Figure 1, while the process of Spleen-Exo extraction and enrichment is described in Figure 2.

1. Pr.......

Discussion

Recent research has reported that spleen-derived exosomes (Spleen-Exo) play a critical role in the treatment of gastric cancer¹². To further elucidate the functions of Spleen-Exo, it is necessary to establish a reproducible and optimal method for their extraction from spleen tissue. While protocols exist for isolating exosomes from brain and liver cancer^13,14, methods for other tissues remain underdeveloped and require further validation. .......

Materials

Name	Company	Catalog Number	Comments
70 µm Cell Strainer	Biologix Group Co., Ltd.USA	15-1070
Anti CD9 antibody	Cell Signaling Technology, Inc (CST), USA	983275
Anti GM130 antibody	Proteintech Group, Inc.China	66662-1-lg
Anti TSG101 antibody	Abcam Plc, UK	125011
Cell Culture Dish	Wuxi NEST Biotechnology Co.,Ltd, China	704001
Centrifuge tube	Wuxi NEST Biotechnology Co.,Ltd, China	788211
Collagenase Type I	Sigma-Aldrich Corp., USA	C2674
Desktop Thermostatic Shaker	Shanghai bluepard instruments Co., Ltd., China	THZ-100
Electrophoresis buffer	Wuhan Servicebio Technology Co., Ltd., China	G2081-1L
Enhanced BCA Protein Assay Kit	Shanghai Beyotime Biotechnology Co., Ltd., China	P0010S
Flow NanoAnalyzer	NanoFCM Inc., China	N30E
Fluorescence/Chemiluminescence imaging system	Guangzhou Biolight Biotechnology Co., Ltd., China	GelView 6000Plus
HRP Goat anti-Mouse IgG	Proteintech Group, Inc.China	15014
HRP Goat anti-Rabbit IgG	Proteintech Group, Inc.China	15015
microplate reader	Molecular Devices, USA	CMax Plus
Microscissors	Shanghai Medical Instruments (group) Co., Ltd.,China	WA1010
Microscopic tweezers	Shanghai Medical Instruments (group) Co., Ltd.,China	WA3010
Multifuge X1R Pro centrifuge	Thermo Fisher Scientific, USA	75009750
Ophthalmic scissors	Shanghai Medical Instruments (group) Co., Ltd.,China	Y00030
Ophthalmic tweezers	Shanghai Medical Instruments (Group) Co., Ltd., China	JD1060
Optima XPN-100 Ultrafiltration centrifuge	Beckman Coulter, USA	A94469
phosphate buffered saline (PBS)	Beijing Solarbio Science & Technology Co.,Ltd., China	P1003
RIPA lysis buffer (strong, without inhibitors)	Shanghai Beyotime Biotechnology Co., Ltd., China	P0013K
Ruler	Deli Manufacturing Company, China
SDS-PAGE Sample Loading Buffer, 5x	Shanghai Beyotime Biotechnology Co., Ltd., China	P0015
Transfer Pipet	Biologix Group Co., Ltd.USA	30-0238A1
Transmission Electron Microscope	Hitachi, Japan	H-7650
Ultracentrifugation tube	Beckman Coulter, USA	355618
Western Transfer Buffer	Wuhan Servicebio Technology Co., Ltd., China	G2028-1L