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^2,3.

The membrane of sEVs is enriched with multiple functional proteins. Among them, tetraspanins such as CD63 are associated with vesicle membrane organization and fusion⁴. Furthermore, cytosolic proteins associated with the endosomal sorting complex required for transport (ESCRT), such as ALIX and TSG101, are integral to sEV biogenesis and secretion. Consequently, they serve as reliable markers for confirming the endosomal origin of the isolated vesicles^5,6.

The infrapatellar fat pad (IPFP) has been recognized as an active joint-associated tissue closely linked to the initiation and progression of osteoarthritis (OA)^7,8. Under inflammatory and degenerative conditions, sEVs derived from IPFP are hypothesized to carry inflammatory mediators and pathogenic signals, thereby contributing to synovial inflammation and cartilage degradation⁹. Characterizing sEVs released by IPFP tissues from OA patients may therefore provide critical insights into disease-associated intercellular communication networks.

Given the increasing interest in sEVs as molecular mediators and potential therapeutic agents, the establishment of a high-yield, high-purity, and reproducible isolation approach is essential. Although differential ultracentrifugation remains one of the most widely used purification strategies¹⁰, protocols optimized for cell culture supernatants or biofluids may not be directly applicable to solid adipose-derived tissues such as the IPFP. The high lipid content, dense extracellular matrix, and variable enzymatic digestion conditions can influence vesicle recovery and purity.

Tissue explant culture provides a physiologically relevant approach that preserves native cellular architecture and cell–cell interactions while minimizing excessive enzymatic disruption, thereby enabling the collection of sEVs released under near-native conditions. However, a standardized workflow specifically tailored for isolating sEVs from IPFP explant cultures is currently lacking.

Here, we describe a step-by-step protocol for isolating and characterizing small extracellular vesicles from infrapatellar fat pad explant cultures of osteoarthritis patients using differential ultracentrifugation, providing a reproducible methodological framework for downstream functional and translational studies.

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/mL, respectively). Filter-sterilize through a 0.22 µm filter.
2. Prepare western blot running buffer by mixing 950 mL of distilled water (dH₂O) with 50 mL of 20x running buffer (Tris/MOPS/SDS).
3. Prepare western blot transfer buffer by mixing 800 mL of dH₂O with 100 mL of 10x transfer buffer and 100 mL of absolute ethanol.
   NOTE: This rapid transfer buffer has been specifically optimized for high-current wet transfer (400 mA) at room temperature. Due to its low heat generation, an ice bath is not required for transfer durations under 45 min, which has been shown to maintain high transfer efficiency for proteins within the 10–250 kDa range.
   CAUTION: Absolute ethanol is flammable. Handle in a well-ventilated area and keep away from heat sources.
4. Prepare washing buffer by mixing 50 mL of 20 x TBS (200 mM Tris and 3 M NaCl at pH 7.5–7.6) and 2 mL Tween-20 with 950 mL of dH₂O.
5. Prepare chemiluminescent working solution by mixing equal volumes of luminol/enhancer solution and peroxide buffer (approximately 0.1 mL of working solution is needed per square centimeter of membrane).
   ​CAUTION: Chemiluminescent substrates can be irritating. Wear gloves and eye protection. Dispose of chemical waste according to local institutional regulations.

2. Preparation of IPFP tissue and collection of conditioned medium

1. Wash freshly obtained IPFP tissue (approx. 10 g) three times with 50 mL of pre-cooled PBS per wash by gentle manual agitation in the centrifuge tube to thoroughly remove residual blood.
2. Mince the IPFP tissue into pieces around 2mm x 2mm x 2mm using sterile scissors or a scalpel.
3. Transfer IPFP tissue pieces into T175 flasks. Add 50 mL of exosome-depleted DMEM/F12 complete medium. Incubate at 37 °C with 5% CO₂ for 48 h with gentle agitation.
4. Collect the DMEM/F12 complete medium (i.e., conditioned medium) in 50 mL tubes on ice. Filter through a 70 µm strainer to remove tissue debris.

3. Isolation of sEVs using differential ultracentrifugation

NOTE: Perform all the following steps at 4 °C or on ice.

1. Initial low-speed centrifugation to remove tissue debris
   1. Centrifuge the conditioned medium at 300 x g for 10 min at 4 °C. Carefully collect the supernatant and transfer it into new 50 mL centrifuge tubes.
   2. Centrifuge the supernatant at 3,000 x g for 10 min at 4 °C. Collect the supernatant and transfer it into new tubes, ensuring the pellet is not disturbed.
2. Intermediate centrifugation and filtration.
   1. Centrifuge the supernatant at 10,000 x g for 30 min at 4 °C.
   2. Collect the supernatant and filter it through a 0.22 µm vacuum filter to remove larger vesicles and contaminants.
3. First ultracentrifugation: sEV pelleting.
   1. Transfer the filtered supernatant into 40 mL polycarbonate centrifuge tubes.
   2. Ultracentrifuge the samples at 110,000 x g for 70 min at 4 °C using a fixed angle rotor (k-factor = 70). Carefully remove the supernatant using a pipette.
   3. Resuspend the pellet in 10 mL of pre-cooled, sterile PBS. Filter the resuspended solution through a 0.22 µm syringe filter to ensure the removal of any remaining aggregates.
4. Second ultracentrifugation: sEV purification
   1. Ultracentrifuge the suspension again at 110,000 x g for 70 min at 4 °C using the fixed angle rotor (k-factor = 70).
   2. Carefully discard the supernatant, leaving the sEV pellet at the bottom of the tube. Resuspend the final sEV pellet in 200 µL of sterile PBS.
   3. Divide the sEV suspension into 20 µL aliquots to prevent damage from repeated freeze-thaw cycles. Store the aliquots at -80 °C for long-term use.

4. Characterization of sEVs by western blot analysis

1. Prepare the sEV protein samples.
   1. Mix the sEV pellet (from step 3.5.2) with 80–120 µL of RIPA lysis buffer supplemented with protease inhibitors.
      NOTE: Aim for a ratio of approximately 1 µL of RIPA buffer per 0.5–1.0 µg of estimated protein. A final protein concentration of at least 1.0 µg/µL is recommended to ensure efficient electrophoretic separation and high-quality signal detection during western blot analysis.
      CAUTION: RIPA buffer contains detergents and irritants. Use personal protective equipment.
   2. Vortex the mixture briefly and incubate the samples on ice for 15–30 min with gentle rocking.
      NOTE: Thorough mixing at this stage is critical for complete membrane disruption and optimal protein release. Insufficient vortexing may result in incomplete lysis and inconsistent protein concentrations across replicates.
   3. Centrifuge the lysate at 12,000–14,000 x g for 10–15 min. Carefully collect the supernatant.
   4. Determine the total protein concentration of the sEV lysate using a high-sensitivity bicinchoninic acid (BCA) protein assay.
      NOTE: A high-sensitivity BCA assay is recommended over the standard BCA assay for extracellular vesicle samples due to its lower detection range (0.5–20 µg/mL), which enables accurate quantification of the typically low protein concentrations obtained from purified sEV preparations. Perform the assay according to the kit protocol.
2. Western Blot Analysis.
   1. Mix the protein samples with 5 x SDS-PAGE sample loading buffer.
      NOTE: For CD63 detection, prepare samples using a non-reducing 5x loading buffer (without β-mercaptoethanol) and incubate at 70 °C for 10 min. These conditions preserve conformational epitopes and prevent heat-induced aggregation of this tetraspanin protein. For other markers, including ALIX (ALG-2-interacting protein X), TSG101 (tumor susceptibility gene 101), and Calnexin, prepare samples using reducing 5x loading buffer containing 10% β-mercaptoethanol and heat at 95 °C for 5 min to ensure complete protein denaturation.
   2. Load the prepared samples (typically 20–40 µg of total protein) into a precast gradient gel (4%–12%).
   3. Load the protein molecular weight marker into the assigned well according to the manufacturer's recommendations.
   4. Run the gel in running buffer at a constant voltage of 100 V.
   5. Continue the run for 1–1.5 h, or until the loading dye reaches the bottom of the gel.
      NOTE: The running time may differ depending on the electrophoresis system and the percentage of the gel.
   6. Transfer the proteins from the gel to PVDF membranes using a wet transfer system at 400 mA for 30 min.
      NOTE: The transfer duration may vary depending on the brand of transfer buffer used.
   7. Block the membranes in a protein-free blocking buffer prepared in tris-buffered saline containing 0.2% Tween-20 (TBS-T) for 30 min at room temperature with gentle agitation. The blocking buffer is formulated without serum proteins, biotin, or phosphorylated proteins to minimize nonspecific binding and background interference.
   8. Incubate the membrane with specific primary antibodies diluted in a serum-free antibody dilution buffer containing protein-based stabilizers and antibody-preserving additives at 4 °C overnight (12–16 h) with gentle agitation.
   9. Wash the membranes in washing buffer (TBST containing 0.2% Tween-20) under gentle agitation on a shaker.
   10. Incubate the membranes with the appropriate secondary antibodies at room temperature for 1 h with gentle agitation.
   11. Wash the membranes again in washing buffer under gentle agitation on a shaker.
   12. Visualize the protein bands using chemiluminescent substrate.
       ​CAUTION: Chemiluminescent substrates can be irritating to the skin and eyes. Wear appropriate gloves and safety glasses. Dispose of the substrate according to institutional hazardous waste guidelines.

5. Characterization of sEVs by Nanoparticle Tracking Analysis (NTA)

1. Turn on the nanoparticle tracking analyzer and the syringe pump module according to the manufacturer's instructions, and allow the system to equilibrate for at least 30 min before measurement.
2. Gently thaw the EV samples on ice and dilute the samples in sterile, particle-free phosphate-buffered saline (PBS) to achieve an optimal particle concentration within the recommended measurement range (approximately 1 x 10⁸ to 1 x 10⁹ particles/mL).
3. Load 1 mL of the diluted sample into a sterile syringe and slowly inject the sample into the measurement chamber, ensuring no air bubbles are introduced.
4. Adjust the camera level and focus to clearly visualize individual particles exhibiting Brownian motion.
5. Record three videos of 60 s duration for each sample under constant flow conditions using the syringe pump to ensure consistent particle distribution.
6. Analyze the recorded videos using NTA software with a detection threshold set to 12, blur size set to Auto, and maximum jump distance set to Auto.
7. Generate particle size distribution and concentration profiles using the software’s automated analysis function.
8. Export the raw data, particle size distribution graphs, and concentration measurements for further statistical analysis.
9. Clean the measurement chamber thoroughly with sterile particle-free water followed by 70% ethanol, and flush again with sterile particle-free water to prevent cross-contamination between samples.

6. Characterization of sEVs by Transmission Electron Microscopy (TEM)

1. Use carbon-coated 200-mesh copper grids. Perform glow discharge on the grids for 30 s using a glow discharge system, if available.
2. Apply 10 µL of the sEV suspension (resuspended in sterile, filtered PBS) onto the grid and incubate for 2–5 min at room temperature.
3. Remove excess liquid by gently touching the edge of the grid with filter paper.
4. Wash the grid by sequentially placing the sample-loaded surface onto three separate drops of ultrapure water.
5. Apply 10 µL of 2% phosphotungstic acid solution (pH 7.0) onto the grid and incubate for 1–2 min at room temperature.
   CAUTION: Phosphotungstic acid is a corrosive acidic reagent that may cause irritation to the skin, eyes, and respiratory tract. Handle in accordance with institutional safety guidelines while wearing appropriate personal protective equipment, including gloves and eye protection. Dispose of staining waste and contaminated materials following approved hazardous chemical waste procedures.
6. Remove excess stain using filter paper and allow the grid to air-dry completely at room temperature for at least 10 min.
7. Examine the grids using a transmission electron microscope operated at an accelerating voltage of 80 kV.
8. Acquire representative images at appropriate magnifications to assess vesicle morphology.

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