Isolation and Characterization of Dental Pulp Stem Cell-Derived Exosomes Across Cell Passages

Summary

This protocol outlines a standardized workflow for isolating and characterizing dental pulp stem cells (DPSC)-derived exosomes across passages, enabling reproducible assessment of exosome quality and functional activity for regenerative applications.

Abstract

Dental pulp stem cells (DPSCs) represent an accessible and clinically relevant source of mesenchymal stem cells, and their derived exosomes have emerged as promising bioactive vesicles for cell-free applications in tissue engineering and regenerative medicine. However, reproducible and standardized methods for isolating and functionally validating DPSC-derived exosomes remain limited, particularly with respect to variability introduced during cell expansion across passages. Here, we describe a comprehensive, standardized, kit-based workflow for the isolation and purification of exosomes from DPSC-conditioned medium that is compatible with routine laboratory practice. This method enables consistent recovery of intact vesicles suitable for morphological, molecular, and functional characterization using commonly available techniques. Exosome identity is assessed based on morphology, size distribution, and marker expression, while biological activity is evaluated using a defined in vitro anti-inflammatory assay. To examine passage-related effects, exosomes derived from different DPSC passages are compared using this functional readout, providing a practical framework for assessing consistency during cell expansion. By integrating isolation, purification, characterization, and functional validation into a single workflow, this protocol offers a reproducible and scalable approach for generating functionally validated DPSC-derived exosomes for biomaterials research and regenerative applications.

Introduction

Exosomes derived from mesenchymal stem cells serve as crucial mediators of intercellular communication and demonstrate significant therapeutic potential in the fields of tissue engineering and regenerative medicine. Exosomes are vesicles measuring 30–150 nm in diameter, capable of delivering bioactive molecules and regulating various functions in recipient cells, such as proliferation, differentiation, and immune response^1,2. Among the various mesenchymal stem cell (MSC) sources, dental pulp stem cells (DPSCs) are of particular interest because of their ready accessibility, robust proliferative capa....

Protocol

This study was conducted in accordance with the principles of the Declaration of Helsinki and approved by the institutional ethics committees. Human dental pulp tissues were obtained from discarded third molars or orthodontic premolars collected at Ningbo Stomatology Hospital in accordance with institutional ethical guidelines (Protocol No. NBKQYY2025LS-04; approval date: May 30 2025). Human skin tissues were obtained from discarded foreskin samples collected during routine circumcision procedures in the Department of Urology at the Affiliated People's Hospital of Ningbo University, in accordance with institutional ethical guidelines (Protocol No. 2024104; approva....

Results

Identification and Characterization of Dental Pulp Stem Cells
Primary dental pulp stem cells (DPSCs) isolated using the described protocol exhibited stable growth and typical mesenchymal morphology during in vitro expansion. As shown in Figure 1, DPSCs at passages 2, 4, and 6 displayed a homogeneous population of spindle-shaped, fibroblast-like cells, with no apparent morphological differences or signs of spontaneous differentiation across pa.......

Discussion

The standardized protocol described in this study provides a reproducible framework for the isolation of dental pulp stem cells (DPSCs), the production of DPSC-derived exosomes (DPSC-Exos), and the functional validation of these vesicles across multiple cell passages. The discussion below highlights critical procedural considerations, optimization strategies, methodological limitations, and the broader relevance of this workflow for regenerative medicine research.

The reliability of this proto.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.05% Trypsin-EDTA Solution	Gibco	25300054	Due to its digestive strength, trypsin is widely used for cell dissociation, routine cell culture passaging, and primary tissue dissociation.
100 mm Cell and Tissue Culture Dish	Biofil	TCD010100	Surface Treated/Non-Treated Sterile&Non-Pyrogenic DNase/RNase-Free
100 nm PS beads	Thermo Fisher	3100A	Prior to conducting nanoparticle tracking analysis (NTA) experiments, the instrument must be calibrated using a standard with a precisely known particle size (100 nm).
15-mL Bulk Conical Centrifuge Tube	KIRGEN	KG2611	Sterile, RNase/Dnase and pyrogenic free
200-mesh Formvar-carbon-coated copper grid	Zhongjing Scientific Instruments	AZH200	Supports exosome samples for TEM imaging. The Formvar-carbon coating enhances sample adhesion and conductivity, while the 200-mesh grid provides adequate open area for even particle distribution and stable imaging.
37 °C Incubator	Thermo Scientific	51032877	The 37-degree CO2 incubator provides an ideal in vitro environment.
50-mL Bulk Conical Centrifuge Tube	KIRGEN	KG2811	Sterile, RNase/Dnase and pyrogenic free
Anti-CD63 antibody [MX-49.129.5]	Abcam	ab193349	Anti-CD63 antibody [MX-49.129.5] (ab193349) is a mouse monoclonal antibody detecting CD63 in Western Blot, Flow Cytometry (Intra), Flow Cytometry, IHC-P, ICC/IF. Suitable for Human.
Anti-TSG101 antibody [EPR7130(B)]	Abcam	ab125011	Anti-TSG101 antibody [EPR7130(B)] (ab125011) is a rabbit monoclonal antibody detecting TSG101 in Western Blot, Flow Cytometry (Intra), Flow Cytometry, IHC-P, ICC/IF. Suitable for Human, Mouse, Rat.
Automated cell counter	Countstar	IC1000	Automated cell counter for fast, precise cell counting
BCA Protein Assay Kit	Beyotime	P0012	Have good linear relationship within the concentration range of 50 - 2000 μg/ml.
Benchtop Centrifuge	BIORIDGE	TD5	Benchtop centrifuge for quick and efficient sample separation in laboratory settings
Biological safety cabinet	Thermo Scientific	1379	Biological Safety Cabinet is a sterile containment device that protects operators, samples, and environments from biohazards by filtering airborne pathogens and preventing cross-contamination during microbiological work.
Cell Culture Plate,12-Well	Eppendorf	0030721110	Tissue culture treated,with lid,flat bottom Sterile,free of detectable pyrogens,RNase & DNase,human & bact.DNA Non-cytotoxic
Cell Culture Plate,96-Well	NEST	701001 0714B	Tissue Culture Treated,Polystyrene,Non-Pyrogenic,Sterile
Cellsaving	NCM	C40100	Serum-free,animal protein-free Cell Freezing Medium
Collagenase, Type I, powder	Gibco	17100017	Collagenase is a protease that cleaves the bond between a neutral amino acid (X) and glycine in the sequence Pro-X-Gly-Pro, which is found with high frequency in collagen.
Dispase II, powder	Gibco	17105041	Dispase II (neutral protease) is an amino-endo peptidase that hydrolyzes the N-terminal peptide bonds of non-polar amino acid residues.
Dulbecco's Modified Eagle Medium	Gibco	C11995500BT	[+]4.5g/L D-Glucose [+]L-Glutamine [+]110mg/L Sodium Pyruvate
Evo M-MLV Reverse Transcription Kit	AG	AG11707	A dedicated reverse transcription reagent for Real Time RT-PCR. It utilizes the Evo M-MLV reverse transcriptase with strong extension ability, enabling efficient synthesis of cDNA within a short period of time.
Exosome Concentration Solution	Umibio	UR52111	Extract exosomes from the cell supernatant
Exosome Depleted Fetal Bovine Serum	Umibio	UR50202	The exosome-free fetal bovine serum was obtained by filtering the sterilely collected healthy fetal bovine serum, and ≥99% of the endogenous exosomes in the fetal bovine serum were removed. It had the same cell growth rate and morphology as the fetal bovine serum before processing.
Fetal Bovine Serum, qualified, Australia	Gibco	10099141	Hemoglobin level: ≤30 mg/dL (levels routinely ≤25 mg/dL)
Human-H36B4	Sangon Biotech	SB002	Forward: 5'-GCAATGTTGCCAGTGTCTGT-3'; Reverse: 5'-GCCTTGACCTTTTCAGCAAG-3'
Human-IL-23A	Sangon Biotech	SB002	Forward: 5'-GAAGAGGGAGATGAAGAGAC-3'; Reverse: 5'-TATCCGATCCTAGCAGCTTC-3'
Inverted Biological Microscope	Leica	DMI1	Directly observe the living cells or tissues that are adhering to the walls of containers such as culture flasks and petri dishes.
Minimum Essential Medium	Gibco	C12571500BT	[+]L-Glutamine [+]Ribonucleosides [+]Deoxyribonucleosides
NcmSpin Rapid RNA Extraction Kit	NCM	M5106	This kit can rapidly extract total RNA from up to 3,000,000 cells and up to 10mg of tissue.
Penicillin-Streptomycin	NCM	C100C5	Contains 10,000 units/ml of penicillin and 10,000 micrograms/ml of streptomycin. It has been filtered to remove bacteria and can be directly used for cell culture. It effectively prevents bacterial contamination of the cells.
PerfectStart Green qPCR SuperMix	TransGen	AQ601-02-V2	A fluorescent dye that binds to the minor groove of all double-stranded DNA and emits green light upon excitation. During PCR amplification, it incorporates into newly amplified DNA, resulting in increased fluorescence. During the denaturation step, the DNA strands separate, releasing the dye and causing a significant drop in the fluorescence signal.
Phosphate Buffered Saline (PBS)	Viva Cell Biosciences	C3582-0500	Its function is to keep the pH of the medium within physiological range and maintain the osmotic pressure balance inside and outside the cell.
Phosphotungstic acid	Solarbio	G1870	Used as a negative stain for TEM sample preparation.
SW Shaking Water Baths	Julabo	(DIN 12876-1)I	Working temperature range from +20 to +99.9 °C
Syringe Filter,0.22 μm/33 mm,PES	Beyotime	FF362	DNase/RNase Free Low Protein Binding Non-Pyrogenic Individually Wrapped
Transmission Electron Microscope (TEM)	HITACHI	HT7700	Used for high-resolution imaging of nanoparticle morphology, including exosomes. Operated at an accelerating voltage of 80–100 kV to visualize negatively stained samples and confirm particle size, shape, and structural integrity.
ZetaView_Particle Metrix	Particle Metrix	PMX-120	Used for measuring the particle size, concentration, Zeta potential, and fluorescence properties of nanoparticles.