Isolation and Therapeutic Application of Extracellular Vesicles Derived from Self-Assembled Mesenchymal Stem Cell Aggregates

Summary

This protocol details the efficient isolation of large extracellular vesicles (LEVs) from self-assembled stem cell aggregates and their therapeutic application for mandibular bone regeneration.

Abstract

Extracellular vesicles (EVs) are vital mediators of intercellular communication and promising cell-free agents for regenerative medicine. Among EV subpopulations, large extracellular vesicles (LEVs) are particularly suited for tissue repair due to their enriched pro-regenerative cargo. However, standardized and reproducible methods to generate functional dental stem cell-derived LEVs for downstream characterization and therapeutic evaluation remain limited. Utilizing the exceptional osteogenic potential of stem cells from the apical papilla (SCAPs), we developed a 3D culture model under low-adhesion conditions to induce self-assembled SCAP aggregates. This model mimics physiological mesenchymal condensation, significantly enhancing cell-cell interactions and boosting LEV secretion. Key culture parameters and handling notes are provided to improve aggregate consistency and maximize LEV output while maintaining cell viability. Here, we present a standardized protocol for the efficient production of functional SCAP-derived LEVs, encompassing 3D aggregate generation, isolation via differential centrifugation, nanoparticle tracking analysis (NTA)-based particle size distribution and concentration analysis, and therapeutic delivery using a thermally crosslinked hydrogel for mandibular bone defect repair. This approach streamlines LEV production for downstream mechanistic studies and preclinical evaluation in craniofacial regeneration. This optimized workflow ensures the reproducible preparation of bioactive LEVs with consistent yields and enriched pro-osteogenic cargo, facilitating fundamental research and accelerating the clinical translation of EV-mediated craniofacial reconstruction. In representative applications, hydrogel-loaded LEVs provide a localized depot at defect sites and facilitate convenient handling during implantation. Collectively, this method provides a scalable platform to prepare SCAP-derived LEVs and highlights key considerations for standardized reporting and translational study design. This approach supports broader adoption across craniofacial EV laboratories.

Introduction

Extracellular vesicles (EVs) are membrane-bound nanoparticles released by cells that mediate intercellular communication through the transfer of bioactive cargoes such as proteins, miRNAs, and lipids^1,2,3. Among EV subpopulations, large extracellular vesicles (LEVs) are distinctive for their direct release from the cell membrane and inherent enrichment of pro-regenerative molecules, making them well-suited for tissue repair and regeneration demands^4,5. In regenerative medicine, EVs derived from mesenchymal stem cells (MSCs) are increasingly recognized as a viable cell-free alternative to whole-cell therapy^6,7,8. They recapitulate the paracrine regenerative effects of parental MSCs while avoiding limitations of direct cell transplantation, such as low engraftment efficiency, immunogenicity, and batch variability^7,8,9,10. Among MSC sources, stem cells from the apical papilla (SCAPs) exhibit superior osteogenic and odontogenic differentiation potential^11,12,13, rendering SCAP-derived EVs particularly attractive for orofacial bone and dental tissue repair⁸.

Notably, the biogenesis and functionality of EVs are profoundly influenced by the cellular microenvironment^14,15. Conventional two-dimensional (2D) monolayer cultures significantly deviate from the native tissue context^16,17, frequently yielding EVs with compromised regenerative capacity¹⁸. In contrast, three-dimensional (3D) self-assembled MSC aggregates replicate key aspects of physiological mesenchymal condensation, a developmental stage characterized by high cell density, intensive cell–cell contact, and enriched extracellular matrix (ECM), which provides structural support and delivers biochemical and mechanical cues that regulate cell adhesion, migration, and fate decisions^19,20,21. This biomimetic 3D microenvironment not only enhances intercellular communication and paracrine signaling^22,23 but also promotes the secretion of EVs with higher yield and improved bioactive cargo profiles^24,25,26. Utilizing 3D self-assembled MSC aggregates thus emerges as a highly promising strategy for obtaining functionally potent EVs for regenerative applications¹⁸.

Despite the clear advantages of aggregate-derived EVs, standardized protocols for their isolation and characterization from 3D systems remain underdeveloped. Current EV isolation methodologies, such as differential centrifugation, are well-established and offer a scalable framework for EV production²⁷. Similarly, methods for generating MSC aggregates have been reported, often relying on vitamin C-induced cellular curling and aggregation from 2D-cultured cells²⁸. However, the ECM architecture within these vitamin C-induced aggregates may still differ from the spatially organized, condensation-like microenvironment of native mesenchymal tissues. In contrast, scaffold-free self-assembly under low-adhesion conditions facilitates the formation of compact, spherical aggregates that approximate key features of mesenchymal condensation^19,29. Presently, a systematic and reproducible workflow for isolating and applying EVs from such low-adhesion-induced 3D SCAP aggregates is lacking. From a practical standpoint, LEV production from conventional 2D SCAP cultures typically falls in the ~10¹¹ particles/mL range by nanoparticle tracking analysis (NTA), and 3D low-adhesion aggregation is expected to increase LEV output by several-fold while maintaining reproducibility for downstream applications. To address this gap, we herein detail a comprehensive protocol that leverages SCAPs’ inherent osteogenic/odontogenic functionality and optimizes the MSC microenvironment through 3D low-adhesion aggregation. This protocol enables the reliable production of high-quality LEVs, providing a standardized tool to advance cell-free regenerative therapies in craniofacial medicine.

Protocol

All animal experiments were approved by the Animal Care and Use Committee of the Fourth Military Medical University and were conducted in compliance with the ARRIVE guidelines. The collection and use of human samples were approved by the Ethics Committee of the Fourth Military Medical University (approval no. IRB-REV-2022187). Written informed consent was obtained from all donors prior to sample collection; for donors who were minors or otherwise unable to provide consent, written informed consent was obtained from their legal guardians. All reagents were sterile-filtered, and cell culture procedures were conducted in a biosafety cabinet to maintain sterility (see Table of Materials). Treat all human-derived materials (teeth, tissues, primary cells, conditioned media, and EV preparations) as potentially infectious. Perform all procedures involving human samples in a certified biosafety cabinet using appropriate personal protective equipment (PPE), including a lab coat, gloves, and eye protection. For biosafety and waste disposal, follow institutional BSL-2 guidelines: Handle all human-derived materials and contaminated disposables as BSL-2 biohazard waste; decontaminate surfaces/equipment with an institutionally approved disinfectant after use. Collect liquid biohazard waste in labeled containers and inactivate it using approved methods before disposal. Dispose of sharps immediately in approved sharps containers (no recapping). Treat animal tissues/carcasses and anesthetic/chemical residues as regulated biohazard/chemical waste and dispose of them via institutional services per applicable regulations.

1. Isolation and culture of human SCAPs

NOTE: P/S is a bioactive antibiotic mixture and may cause skin/eye irritation or allergic reactions. Handle in a BSL-2 biosafety cabinet when opening; wear gloves and eye protection; avoid aerosol generation; and dispose of waste in accordance with institutional chemical waste guidelines. Collagenase type I (solution) is a bioactive enzyme and may cause skin/eye irritation or allergic reactions. Handle in a BSL-2 biosafety cabinet; wear gloves and eye protection; avoid splashes and aerosol generation during pipetting; and decontaminate spills promptly according to institutional procedures. Protect collagenase type I from light during storage and use (see the Table of Materials).

1. Collect third molars with immature apices from healthy donors (16–21 years old) and rinse thoroughly with phosphate-buffered saline (PBS) containing 1% penicillin/streptomycin (P/S) (see Table of Materials).
2. Dissect the apical papilla tissue from the root apex using sterile ophthalmic tweezers and scissors, then mince it into small fragments (<1 mm³).
3. Transfer all tissue fragments into a 15 mL centrifuge tube. Digest the fragments with 2 mg/mL collagenase type I at 37 °C for 1 h, with gentle shaking every 15 min.
4. Stop digestion by adding an equal volume of complete α-minimum essential medium (α-MEM) (see Table of Materials).
   NOTE: The digestion time of collagenase type I is not fixed and depends on tissue digestion status. Monitor closely, terminate digestion when tissues become semi-eroded by adding medium. Over-digestion may impair cell viability.
5. Centrifuge the cell suspension at 100 × g for 5 min at 4 °C, discard the supernatant, and resuspend the pellet in 2 mL of complete α-MEM.
6. Seed the digested tissue suspension into a 10 cm Petri dish and add 8 mL of complete α-MEM to a final volume of 10 mL per dish.
7. Culture SCAPs at 37 °C in a humidified incubator with 5% CO₂.
   NOTE: Monitor cultures continuously, cells typically migrate out from tissue fragments and attach to the dish in a radial pattern within 3–7 days. If tissue viability is poor or no cell migration is observed within 5 days, switch to complete α-MEM supplemented with 20% serum.
8. For cell passaging, cells were washed with 5 mL of PBS to remove residual medium once they reached 80–90% confluence. Subsequently, they were incubated with 2 mL of 0.25% trypsin-1 mM EDTA at 37 °C for 2 min (see Table of Materials).
   NOTE: Trypsin–EDTA is an irritant and can damage skin/eyes. Handle with gloves and eye protection, avoid splashes and aerosol formation, and rinse immediately with plenty of water if contact occurs. Dispose of trypsin-containing liquid waste according to institutional guidelines.
9. When cells become rounded with disrupted intercellular junctions under a microscope, gently detach cells from the dish without excessive structural damage. Neutralize trypsin by adding 4 mL of complete α-MEM.
10. Collect the cells by gentle, repeated pipetting into a fresh 15 mL centrifuge tube. After centrifugation at 100 × g for 5 min, discard the supernatant and resuspend the cell pellet in complete α-MEM. Seed the resuspended cells into fresh culture dishes or well plates. Replace the medium every two days.
    NOTE: Ensure that SCAPs used for aggregate culture are within passage 5. Stemness and aggregation capacity decline with increased passage numbers^30,31, which may reduce aggregate formation efficiency and functional performance.

2. Self-assembly of SCAP aggregates

1. Harvest passage 3–5 SCAPs by trypsinization, centrifuge at 100 × g for 5 min, and resuspend in α-MEM supplemented with 10% EV-depleted fetal bovine serum (FBS) and 1% P/S (see Table of Materials).
   NOTE: Prepare EV-depleted FBS by centrifuging FBS at 150,000 × g at 4 °C overnight to remove EVs and other impurities³².
2. Count cells using a hemocytometer and seed 1 × 10⁵ cells per well in ultra-low attachment 24-well plates. Adjust the medium volume to 500 μL per well to maintain a suspension state.
3. Incubate the plates at 37 °C with 5% CO₂ for 72 h, and replace the culture medium every 48 h.
4. Monitor aggregate formation daily under an inverted optical microscope. Collect EVs when aggregates reach 200–400 μm in diameter and exhibit a compact, smooth-edged structure.
   NOTE: Define one production “batch” as LEVs isolated from the pooled conditioned medium collected from SCAP aggregates seeded on the same day from the same cell passage and maintained under identical culture conditions within a single collection window (72 h). Process each batch independently through centrifugation. Replace the culture medium at 48 h after seeding and replenish with the same volume of fresh medium. For LEV production, collect conditioned medium at 48 h and again at 72 h after seeding, and pool the harvested media (0–48 h and 48–72 h fractions) as a single batch. Keep the pooled conditioned medium at 4 °C and process it for LEVs isolation.

3. Isolation and characterization of EVs from SCAP aggregates

1. Differential centrifugation for isolation of large LEVs
   1. Collect the culture medium containing SCAP aggregates into 15 mL centrifuge tubes.
   2. Centrifuge at 800 × g for 10 min at 4 °C to remove intact aggregates and large cell debris.
   3. Transfer the supernatant to new 15 mL tubes and centrifuge at 2,500 × g for 15 min at 4 °C to eliminate residual platelets and small cell fragments.
   4. Carefully transfer the supernatant to sterile 1.5 mL conical tubes and centrifuge at 16,800 × g for 30 min at 4 °C to pellet LEVs.
      NOTE: Use 1.5 mL conical tubes to enhance LEVs yield.
   5. Discard the supernatant, resuspend the pellet in 1 mL of sterile PBS, and centrifuge again at 16,800 × g for 30 min at 4 °C using a fixed-angle rotor (compatible with a standard benchtop centrifuge) to pellet the LEVs.
   6. Set the deceleration/brake to a "slow" or "low" setting (typically brake Level 1 or 2) to prevent the resuspension of the LEV pellet during the final deceleration phase.
   7. Discard the supernatant and resuspend the final LEV pellet in 50 μL of sterile PBS. Use the suspension immediately (within 24 h) or store it at 4 °C. Avoid freezing to minimize loss of bioactivity.
      NOTE: Prepare approximately 80 wells of aggregates to obtain sufficient LEVs for typical downstream experiments. LEVs samples are recommended for immediate characterization or therapeutic application rather than long-term storage. If longer storage is required, aliquot the LEV suspension (e.g., 5–10 μL per tube) to avoid repeated temperature fluctuations and freeze–thaw cycles. Snap-freeze aliquots on dry ice and store at −80 °C for short-term extended storage (e.g., up to 1–3 months). Thaw once on ice and use immediately. Note that freezing may reduce bioactivity and can alter particle recovery/size distribution; therefore, whenever possible, perform functional studies using freshly prepared LEVs. For quality control after storage, remeasure particle concentration and size distribution using NTA, and proceed only if the values remain within the predefined acceptance range. Additionally, in this protocol, NTA particle concentration is used as the primary quantification method to avoid the loss of vesicles associated with protein-based assays like BCA, thereby maximizing yield for downstream applications.
2. NTA for particle size and concentration
   NOTE: NTA is performed using a commercially available nanoparticle tracking analyzer (see Table of Materials) to evaluate size distribution and particle concentration.
   1. Dilute 1 μL of the LEVs suspension (from Section 3.1, Step 6) in 1,499 μL of sterile PBS in a sterile 1.5 mL conical tube and mix thoroughly.
   2. Start the compatible software and fill the sample cell with 10 mL of ultrapure water. Confirm that the background particle count is < 10 per frame to ensure the system is clean (see Table of Materials).
   3. Calibrate the instrument with a prepared standard solution. Set the software parameters to: Sensitivity 70, Shutter 70, Min Area 5, and Min Brightness 20. Perform "AutoAlignment" to ensure the laser is focused. Adjust the standard dilution until the particle count shown in the software is 50–400 (preferably ~200) and then run the calibration program.
      NOTE: Prepare the standard solution by reconstituting 1 μL of calibration beads in 1 mL of distilled water to make a primary solution. Dilute 100 μL of the primary solution in 25 mL of distilled water (1:250,000 dilution) and store at 4 °C for up to 1 week.
   4. Rinse the sample cell with 5 mL of distilled water before analysis.
   5. Flush the channel with 1 mL of distilled water and confirm the particle count on the detection interface is <10.
   6. Inject 1 mL of the diluted LEVs sample at a constant speed of 0.5 mL/s using a sterile 1 mL syringe.
      CAUTION: Manually control the syringe to maintain a steady injection speed.
   7. Perform particle analysis and generate reports. In the software, create a new measurement and select Size/Concentration mode. Set acquisition to 3 recordings, 60 s per recording, at 25 °C using the same detection parameters as in Step 3 (Sensitivity 70, Shutter 70, Min Area 5, Min Brightness 20).
   8. Start acquisition and verify that the on-screen particle count remains within 50–400 particles/frame (preferably ~200); if outside this range, stop and adjust the sample dilution, then repeat Step 6. After acquisition, run the analysis to obtain mean size, mode size, D10/D50/D90, and particle concentration (particles/mL). Then export the results and size distribution plots as .csv and .pdf (or equivalent report formats) and save the measurement file for record keeping.

4. Therapeutic application of LEVs in mandibular bone regeneration

1. Preparation of LEV-loaded hydrogel
   1. Prepare Pluronic F-127 stock (20% w/v). Add Pluronic F-127 powder to sterile PBS to a final concentration of 20% (w/v) and stir at 4 °C overnight until fully dissolved and homogeneous (see Table of Materials). Keep the solution at 4 °C throughout preparation to avoid premature gelation.
      NOTE: 20% (w/v) Pluronic F-127 is liquid at 4 °C and undergoes thermogelation at ~20–25 °C (tube/room dependent); it becomes a stable gel at 37 °C.
   2. Load LEVs under cold conditions. Pre-cool tubes, pipette tips, and the hydrogel to 4 °C. Mix 50 µg LEVs (equivalent to LEVs from two 24-well plates of aggregates) with 1 mL of 20% Pluronic F 127 by gentle pipetting and brief low-speed vortexing to ensure uniform distribution. Maintain the mixture on ice or at 4 °C until implantation.
   3. Handling/use guidance (for reproducibility). Perform loading and syringe/tip loading quickly on ice. If the mixture warms above ~20–25 °C, viscosity increases and gelation may begin; in that case, return to 4 °C until the material becomes fully flowable again before use. After placement in the defect, allow gelation to occur in situ at body temperature (37 °C).
2. Establishment of murine mandibular bone defect model
   1. Anesthetize 8-week-old C57Bl/6 mice (male or female) via intraperitoneal injection of pentobarbital sodium (50 mg/kg) (see Table of Materials).
      CAUTION: Pentobarbital sodium is a potent sedative/anesthetic and is toxic to humans if accidentally injected, ingested, or inhaled. Prepare and administer using safe sharps practices (do not recap needles; discard immediately into a sharps container), wear gloves and eye protection, avoid skin contact, and store/record use according to institutional controlled-drug policies. Confirm proper anesthesia in the mice by checking for an absent corneal reflex and no limb withdrawal upon footpad pinch. Apply a protective ophthalmic ointment to prevent corneal drying.
   2. Shave the fur over the left mandibular region and disinfect alternately with 10% povidone-iodine and 75% ethanol.
      CAUTION: 75% ethanol is highly flammable; use it away from ignition sources and ensure good ventilation. Povidone-iodine may cause skin/eye irritation or allergic reactions; wear gloves, avoid contact with eyes/mucosa, and clean spills promptly.
   3. Make a 1 cm longitudinal incision along the mandibular edge and bluntly dissect soft tissues to expose the mandibular angle.
   4. Create a circular bone defect (2 mm in diameter) using a dental drill with continuous saline irrigation to prevent thermal damage (see Table of Materials).
      CAUTION: Monitor intraoperative bleeding closely, control hemorrhage with small cotton balls to maintain a clear surgical field, and reduce infection risk. Avoid contacting surrounding soft tissues with the drill to prevent entanglement.
3. Implantation of LEV-loaded hydrogel
   1. Load the LEV-hydrogel mixture into a sterile syringe and inject it into the bone defect, ensuring complete filling.
      NOTE: Administer ~10 μg LEVs per mouse. Two 24-well plates of SCAP aggregates are required to yield sufficient LEVs for an experiment with n = 5 mice.
   2. For the control group, inject an equal volume of Pluronic F-127 hydrogel without LEVs.
   3. Suture the incision with 4-0 absorbable sutures and administer subcutaneous analgesia (Carprofen, 5 mg/kg) to alleviate postoperative pain (see Table of Materials).
      CAUTION: Carprofen is a pharmacologically active NSAID and may be harmful if accidentally injected or if it contacts skin/eyes. Wear gloves, avoid sharp injury, discard needles/syringes in a sharps container, and dispose of unused drugs and contaminated materials per institutional pharmaceutical waste procedures.
   4. Place mice on a rewarming blanket until recovery from anesthesia, then transfer to individual cages with free access to food and water.
      NOTE: Do not leave mice unattended until they regain sufficient consciousness to maintain sternal recumbency. Return mice to group housing only after full recovery.

Results

According to the experimental workflow, key steps including SCAP isolation, aggregate formation, LEVs extraction, and therapeutic implantation were successfully executed (Figure 1). Clinical impacted third molars from donors exhibited immature apices with intact apical papilla tissue attached around the open root canal orifices, confirming the suitable source of SCAP (Figure 2A). When cultured in vitro, spindle-shaped SCAPs with abundant cellular proces...

Discussion

The growing interest in cell-free regenerative therapies has positioned mesenchymal stem cell-derived LEVs as promising alternatives to whole-cell transplantation, owing to their ability to deliver bioactive molecules without the risks associated with cell-based approaches. However, the clinical translation of LEV-based therapies has been hampered by the lack of physiologically relevant culture systems that can fully preserve and enhance LEVs functionality. Conventional 2D monolayer cultures significantly diverge from th...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 mL Syringe with Needle	BD	300841	Anesthetization
10 cm Cell Culture Dish	Corning	353003	Cell culture
15 mL Polystyrene Centrifuge Tube	Corning	352095	Cell culture
75% Ethanol Disinfectant	Hai Shi Hai Nuo	HS-750	Operation
Absorbable Suture 4-0	Jinhuan Medical	R413	Operation
Benchtop centrifuge	WIGGENS	CE187	EV isolation
CO2 Incubator, 150 L, Universal Type	ThermoFisher	51032874	Cell culture
Collagenase Type I	ThermoFisher	17100017	Cell culture
Cotton Balls	Deroyal	30-033	Operation
Curved Operating Scissor	JZ Surgical Instrument	J21040	Operation
Dental Mobile Turbine Unit	KaVo	S609C	Operation
Electronic Balance	Zhi Ke	ZK-DST	Cell culture
Eppendorf Tube	Eppendorf	3810X	EV isolation
Forcep,fine	JZ Surgical Instrument	JD1020	Operation
Hair Removal Cream	Veet	1.00097E+11	Operation
High-speed Ball Bur	Dentsply Sirona	E0123	Operation
High-speed Handpiece	KaVo	KV-1-008-1644	Operation
Inverted Microscope	Mshot	MF53-N	Cell culture
MEM α (Minimum Essential Medium α)	ThermoFisher	12561056	Cell culture
Nanoparticle Tracking Video Microscope PMX-120	Particle Metrix	ZetaView PMX120	Nanoparticle tracking analysis
Penicillin-Streptomycin (P/S)	ThermoFisher	15140122	Cell culture
Pentobarbital sodium	Sigma-Aldrich	57-33-0	Anesthetization
Phosphate-Buffered Saline (PBS)	ThermoFisher	10010023	Cell culture
Pluronic F-127	Sigma-Aldrich	P2443	EV implantation
Povidone Iodine Solution	Baxter	PROSL500	Operation
Specialty Fetal Bovine Serum (FBS)	ThermoFisher	12664025	Cell culture
Sterile Gauze Block	Winning	10-000-681	Operation
Tabletop High-Speed Micro Centrifuge	Hitachi	CT15E	EV isolation
Trephine Drill	Biomet Microfixation	01-9182	Operation
Trypsin-EDTA	ThermoFisher	25200072	Cell culture
Ultra-Low Attachment 24-Well Plate	Corning	3473	Cell culture
Vortex Mixer Genie	Scientific Industries	SI0425	EV implantation