Isolation and Characterization of Small Extracellular Vesicles from Infrapatellar Fat Pads of Osteoarthritis Patients

Summary

SUMMARY This protocol presents a standardized workflow for isolating and characterizing small extracellular vesicles (sEVs) from infrapatellar fat pad tissues of osteoarthritis patients.

Abstract

Extracellular vesicles (EVs), particularly small extracellular vesicles (sEVs) defined as vesicles smaller than 200 nm, serve as essential mediators of intercellular communication and released by nearly all cell types into various biological fluids. The infrapatellar fat pad (IPFP) is closely associated with the development and progression of knee osteoarthritis (OA). However, standardized methods for isolating sEVs directly from IPFP explants remain limited. We present a reproducible protocol for the isolation and characterization of sEVs derived from IPFP tissues obtained from OA patients. Conditioned medium collected from IPFP tissue cultures is sequentially cleared of cells and debris by low-speed centrifugation, followed by ultracentrifugation to enrich vesicles within the sEV size range. The resulting vesicles are characterized according to minimal information for studies of extracellular vesicles (MISEV2023) recommendations using a set of representative protein markers across distinct functional categories: CD63 as a membrane-associated tetraspanin, ALIX and TSG101 as cytosolic proteins involved in biogenesis mediated by the Endosomal Sorting Complex Required for Transport (ESCRT) machinery, and Calnexin as an endoplasmic reticulum-resident protein to monitor potential non-vesicular contamination. This workflow yields well-defined sEV preparations suitable for downstream applications such as functional assays or proteomic profiling in osteoarthritis research.

Introduction

Small extracellular vesicles (sEVs) are a heterogeneous subtype of extracellular vesicles that, according to the operational classification proposed by the International Society for Extracellular Vesicles (ISEV) in the minimal information for studies of extracellular vesicles (MISEV2023) guidelines, are defined as vesicles typically smaller than 200 nm in diameter¹. sEVs are released by virtually all cell types into biological fluids and have attracted increasing attention due to their essential role as mediators of intercellular communication in both physiological and pathological contexts^2,....

Protocol

The use of IPFP tissues from osteoarthritis patients in this study was approved by the Ethics Committee of the First Hospital of Hebei Medical University (Approval No. [2024] 018). Sample collection was performed within the approved period, and written informed consent was obtained from all participants prior to tissue acquisition.

1. Preparation of medium and solutions

1. Prepare 500 mL of exosome-depleted complete DMEM/F12 medium by mixing 445 mL of basal DMEM/F12 medium, 50 mL of exosome-free fetal bovine serum (10%), and 5 mL of Penicillin/Streptomycin/Amphotericin B (100 U/mL, 100 µg/mL, and 0.25 µg/....

Results

Successful isolation of small extracellular vesicles (sEVs) from human infrapatellar fat pad (IPFP) tissues was confirmed through multimodal characterization.

Transmission electron microscopy (TEM) revealed that the isolated vesicles exhibited the characteristic cup-shaped morphology with clearly defined lipid bilayer membranes, consistent with the structural features of sEVs (Figure 1)¹¹.

Discussion

This protocol provides a reproducible and practical workflow for isolating small extracellular vesicles (sEVs) from human infrapatellar fat pad (IPFP) tissues using explant culture followed by differential ultracentrifugation. The isolated vesicles were validated through complementary characterization methods, including transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot detection of established EV markers¹. TEM confirmed the presence of membrane-bound ve.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
40 mL PET Centrifuge Tubes	himac (Eppendorf Himac Technologies)	5720411148	Polyethylene terephthalate, 26 x 90 mm
70 µm Cell Strainer	Beijing Lanjieke Technology	560049	For tissue debris removal
Anti Alix antibody	Shanghai Abways Biotechnology	CY7215	sEV positive marker
Anti Calnexin antibody	Shanghai Abways Biotechnology	CY5839	Negative marker (ER contamination)
Anti CD63 antibody	Affinity Biosciences	DF2305	Tetraspanin marker
Anti TSG101 antibody	Shanghai Abways Biotechnology	CY5985	sEV positive marker
BCA Protein Assay Kit	Shanghai YaMei Biotechnology	ZJ102	For protein quantification
Cell culture flask (T175)	NEST Biotechnology	560229	For IPFP explant culture
Centrifuge tube (50 mL)	BIOFIL	560041	For initial low-speed steps
Exosome-depleted Fetal Bovine Serum	Cyagen Biosciences	FBSNE-01061	For tissue culture
HRP Goat anti-Rabbit IgG	Shanghai Abways Biotechnology	AB0101	Secondary antibody for WB
Instant Protein Loading Buffer (Denaturing, Reducing, 5×)	Shanghai YaMei Biotechnology	LT101	For protein denaturation
Micro BCA Protein Assay Kit	Thermo Scientific	23235	For protein quantification
Millex-GP Filter Unit (0.22 µm)	Beijing Lanjieke Technology	560360	Sterile, for vesicle filtration
Nanoparticle Tracking Analyzer	Malvern Panalytical	NanoSight NS300	For size and concentration analysis
Omni-Flash Ice-Free Rapid Transfer Buffer	Shanghai YaMei Biotechnology	PS201S	For Western blot protein transfer
Phosphotungstic acid (2%)	Sigma-Aldrich	P4006	For TEM negative staining
Protein Loading Buffer (Denaturing, Non-Reducing, 5×)	Shanghai YaMei Biotechnology	LT103	For protein denaturation
Protein-Free Rapid Blocking Buffer (5×)	Shanghai YaMei Biotechnology	PS108	For block the membranes
RIPA lysis buffer	Beijing Solarbio Science & Technology Co.,Ltd	R0010	For sEV protein extraction
TBS(20×)	Beijing Solarbio Science & Technology Co.,Ltd	T1080	For prepare washing buffer
Transmission Electron Microscope	Hitachi	HT7800	For morphological characterization
Tris/MOPS/SDS Electrophoresis Buffer	Shanghai YaMei Biotechnology	PS120	For protein separation
Tween-20	Beijing Solarbio Science & Technology Co.,Ltd	T8220	For prepare washing buffer
Ultracentrifuge	himac (Eppendorf Himac Technologies)	CP100NX	High-speed isolation system
Ultra-sensitive chemiluminescent detection kit	Shanghai YaMei Biotechnology	SQ201	For protein band visualization
Universal Antibody Dilution Buffer	Shanghai YaMei Biotechnology	PS119L	For primary/secondary antibodies