Study Setting

All experiments were conducted at the Central Research Laboratory of a tertiary-care hospital affiliated with Nitte (Deemed to be University) in Mangalore, Karnataka, India. The facility is fully accredited in accordance with National Accreditation Board for Testing and Calibration Laboratories (NABL) and International Organization for Standardization (ISO) guidelines.

Ethical Considerations

The study was conducted in compliance with the Declaration of Helsinki (1964, as revised in 2013) and received approval from the Institutional Central Ethics Committee (Ref: NU/CEC/2021/230, dated January 13, 2022). The committee reviewed and approved the use of 150 mL of hHAT, ensuring the strict protection of donor rights and the appropriate handling of all biological specimens.

Type of Sampling

A purposive sampling strategy was employed to ensure the consistent quality and biological relevance of the source material for sEV isolation. A cohort of six donors was selected, based on established EV literature indicating that five or more biological replicates are sufficient to achieve reproducible vesicle yields and ensure biological consistency.

Eligibility Criteria

Adipose tissue samples were procured from healthy, non-obese adults (body mass index [BMI] < 30 kg/m) aged 18–45 years who were undergoing elective liposuction procedures. Individuals falling outside this age range, presenting with significant medical comorbidities, or possessing a BMI ≥ 30 kg/m were excluded from the study. All donors underwent standard screening to confirm the absence of infectious diseases, including HIV, hepatitis B, and hepatitis C. SVF from healthy donors was specifically utilized to minimize variability arising from metabolic or infectious conditions and to yield physiologically normal, immunomodulatory vesicles.

Research Participant Consent

Written informed consent was obtained from all participants prior to enrollment. Confidentiality was strictly maintained by anonymizing samples utilizing coded identifiers, and participants retained the right to withdraw their consent at any time without penalty. Tissue collection was performed by trained surgical personnel during routine clinical procedures to mitigate any associated risks and minimize patient discomfort.

Study Design and Reporting

This original, in vitro, observational study was designed to investigate the innate antibacterial activity of SVF-sEVs against MRSA.

hHAT-SVF-sEV Preparation Protocol and Antibacterial Property Data Collection

All samples were processed within 2 hours of collection to preserve vesicle integrity and prevent the degradation of bioactive molecules. All experiments were outcome-driven and performed under controlled in vitro laboratory conditions, without randomization or the involvement of live human or animal subjects. Isolated SVF-sEVs were characterized for vesicle morphology, protein profiles, and antibacterial properties utilizing nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and optical density measurements at 600 nm (OD). This purposive design facilitated the evaluation of 100% neat (undiluted and intact) SVF-sEVs against MRSA under standardized conditions, as delineated in Figure 1.

The study adhered to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines to ensure methodological transparency, comprehensive reporting, and reproducibility. Detailed information regarding sample procurement, vesicle isolation, quality control procedures, data validation, and the complete STROBE checklist is provided in the Supplementary File.

hHAT Sample Collection

A total volume of 150 mL of residual hHAT—also referred to as subcutaneous adipose tissue or lipoaspirate, and typically considered biological waste—was collected from the 360° collection sites of six consenting research participants. The cohort comprised healthy, non-obese Indian males and females aged 18 to 45 years with a normal body mass index (BMI). The date of sample collection, total volume of lipoaspirate, number of freeze-thaw cycles, and specific storage conditions for downstream analyses were systematically documented.

SVF Isolation

To isolate the SVF, the aliquoted adipose tissue samples were washed with an equal volume of Dulbecco’s phosphate-buffered saline (DPBS) and subsequently centrifuged at 500 × g for 5 minutes at 37 °C. This washing procedure was repeated a minimum of three times until macroscopic blood and debris were removed. Next, an equal volume of 0.1% type II collagenase tissue digestion enzyme (TDE) was added to the washed tissue (up to 20 mL of wet tissue per conical tube), and the suspension was incubated at 37 °C with moderate agitation for 4.5 hours. Following incubation, enzymatic digestion was terminated by the addition of cold PBS, and the mixture was centrifuged at 1500 × g for 5 minutes. Finally, the resulting SVF pellet was resuspended in PBS, filtered through a 100 µm cell strainer, and subjected to a final centrifugation step.

SVF Cell Counting, Viability Assessment, and Conditioning

The isolated SVF pellet was resuspended in 8 mL of plain Dulbecco’s Modified Eagle Medium (DMEM), and initial cell quantification and viability assessments were performed utilizing a trypan blue exclusion assay. For standard cell culture propagation, a routine 1% antibiotic-antimycotic mixture (comprising penicillin and streptomycin) was supplemented. However, to ensure unbiased outcomes and prevent experimental interference, an alternate conditioning protocol entirely devoid of antibiotics and antifungals was strictly utilized for all samples designated for downstream microbiological evaluations.

SVF-sEV Isolation, Cryopreservation, and Thawing

The resulting cell suspension was aliquoted into T-75 tissue culture flasks at a seeding density of approximately 6 × 10 SVF cells per flask. The cultures were incubated at 37 °C in a humidified atmosphere containing 5% CO₂. Once the heterogeneous SVF cell populations reached >60% sub-confluence, the conditioned medium was harvested at 16 and 20 hours. This conditioned medium was then processed via sequential differential centrifugation: 300 × g for 10 minutes to remove cells and large debris; 2,000 × g for 20 minutes to eliminate apoptotic bodies; and 10,000 × g for 30 minutes to pellet larger extracellular vesicles. The resulting supernatant was subjected to ultracentrifugation at 200,000 × g for 70 minutes at 4 °C utilizing a Beckman Coulter Optima ultracentrifuge equipped with a Type 70 Ti rotor to precipitate the SVF-sEVs. The obtained pellets were washed once with PBS and re-ultracentrifuged at 100,000 × g for 70 minutes. To enhance vesicle purity and remove non-EV-associated proteins and lipids, the final SVF-sEV pellets were resuspended in PBS and subjected to ultrafiltration using a 10 kDa Amicon Ultra-15 centrifugal filter unit (Millipore, Merck KGaA, Ireland) at 4,000 × g for 30 minutes at 4 °C. The purified SVF-sEVs were resuspended in an equal volume of PBS and aliquoted into 1.5 mL click-lock microcentrifuge tubes, which were either stored at −80 °C for long-term cryopreservation or maintained at 4 °C for immediate downstream analyses.

SVF-sEV Physicochemical Assessment to Confirm Vesicle Identity

To confirm vesicle identity in strict accordance with the MISEV2023 guidelines, the isolated SVF-sEVs were characterized for size, morphology, and molecular markers utilizing complementary orthogonal techniques. The specific methodologies employed were as follows:

• Nanoparticle Tracking Analysis (NTA): Particle size distribution and vesicle concentration were assessed utilizing a NanoSight LM10 instrument (Malvern Instruments, Malvern, UK) equipped with a 405 nm laser. The SVF-sEV samples were diluted 1:10,000 in DPBS prior to analysis. Instrument calibration was performed using National Institute of Standards and Technology (NIST)-traceable 200 nm polystyrene beads. The detection threshold and camera level were standardized at 7 and 14, respectively.

• Transmission Electron Microscopy (TEM): Vesicle morphology and membrane integrity were evaluated using an FEI Tecnai T20 transmission electron microscope operating at 200 kV. The acquired images were subsequently processed utilizing Gatan Microscopy Suite software.

• Fluorescence Microscopy (FM): Fluorescence microscopy was employed to visualize vesicle ultrastructure and to further assess the integrity of the lipid membrane and the characteristic cup-shaped morphology.

• Western Blotting (WB): Biochemical validation of the SVF-sEVs was conducted via Western blotting, in accordance with MISEV2023 recommendations. This involved probing for the EV-enriched positive markers CD63 and TSG101, alongside the negative marker GRP94. The absence of GRP94 was utilized to confirm minimal contamination by intracellular components. Samples were concentrated, lysed, sonicated, and centrifuged, after which the total protein content was quantified utilizing a bicinchoninic acid (BCA) assay (Thermo Fisher Scientific, Waltham, MA, USA). Equivalent amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently detected utilizing horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10,000). β-actin (1:1,000) served as an internal loading control. Primary antibodies directed against CD63, TSG101, and GRP94 were sourced from Invitrogen (Cat# SAB4301607, PA531260, and SAB2101094, respectively), while secondary antibodies were procured from Thermo Fisher Scientific (Cat# G21234 and G21040).16

Yield, Purity, Endotoxin, and Sterility Considerations

SVF-sEV yield was quantified via NTA as particles per frame and normalized to the initial mass of the adipose tissue processed. This normalization facilitated relative comparisons across preparations and demonstrated consistent vesicle recovery, thereby supporting workflow reproducibility and scalability. An Amicon ultrafiltration step was utilized to minimize co-isolated impurities, such as lipoproteins and protein aggregates. The purity of the resulting SVF-sEV preparations was subsequently verified via Western blot analysis. Formal endotoxin quantification utilizing a Limulus amebocyte lysate (LAL) assay was not performed; thus, potential endotoxin contributions cannot be entirely excluded and remain a key limitation of this study. However, all SVF-sEV isolations were performed aseptically utilizing sterile reagents and consumables, and no microbial contamination was observed during processing or functional assays. Sterility was assessed qualitatively by the absence of turbidity, visible microbial growth, or unexpected cytotoxicity in downstream experiments. Future studies incorporating formal endotoxin quantification and rigorous sterility testing will be requisite to support regulatory compliance and clinical translation. The prepared SVF-sEVs were subsequently utilized in triplicate microbiological experiments to assess their capacity to inhibit MRSA and MSSA. All experiments were conducted in compliance with Good Clinical and Laboratory Practice (GCLP) standards utilizing the bacterial strains detailed below.

Bacterial Strains

The antimicrobial activity of the SVF-sEVs was evaluated against Staphylococcus aureus. Reference strains of MRSA (ATCC 43300) and MSSA (ATCC 29213), alongside clinical MRSA and MSSA isolates, were procured from the Central Research Laboratory. Methicillin resistance was genetically confirmed via polymerase chain reaction (PCR) detection of the mecA gene.

SVF-sEV In Vitro Antibacterial Activity Assays

• Agar Diffusion Assay:Inoculum Preparation: Three to five isolated colonies were selected from the ATCC reference and clinical strain plates, inoculated into Mueller-Hinton (MH) broth, and incubated for 4 hours at 37 °C. The turbidity of the bacterial suspension was subsequently adjusted to 0.5 McFarland units (approximately 1.5 × 10⁸ CFU/mL). Inoculation of Agar Plates: A sterile cotton swab was immersed in the standardized inoculum and utilized to uniformly inoculate the entire surface of a Mueller-Hinton agar (MHA) plate by swabbing in three intersecting directions. Following inoculation, a sterile, heated 5 mm cork borer was forcefully pressed into the agar to create wells; each plate contained three to six wells. Varying concentrations of SVF-sEVs (0.5%, 1%, 5%, 10%, 25%, 50%, 75%, and 100%) were then dispensed into the respective wells to ensure effective diffusion into the S. aureus lawns. A commercially available single-dose ciprofloxacin disk (5 µg) served as a positive control. The plates were incubated at 37 °C for 24 hours, after which the zones of inhibition were measured in strict accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines.17

• Time-Kill Assay: Following the confirmation that 100% neat SVF-sEVs (EVN) demonstrated the highest efficacy among the tested concentrations, their bactericidal impact against the S. aureus strains was evaluated longitudinally from 4 to 24 hours, with the results compared against a blank control.

• Colony-Forming Unit (CFU) Enumeration via Spread Plate Method: This assay was conducted to quantitatively assess the inherent antibacterial efficacy of the purified SVF-sEVs. Assays were performed in 48-well microtiter plates under two experimental conditions: with and without the addition of SVF-sEVs. In the untreated control wells ("without SVF-sEVs"), a mixture of 90 µL of fresh Luria-Bertani (LB) broth and 10 µL of the respective bacterial inoculum was utilized. Conversely, in the treatment wells ("with SVF-sEVs"), 10 µL of EVN was added to 100 µL of the respective inoculated culture broth. The microtiter plates were incubated, and samples were drawn at 0, 6, and 24 hours. At each time point, a 10 µL (0.01 mL) aliquot from each well was spread onto fresh MHA plates using a sterile glass spreader and incubated overnight. The plates were subsequently examined for visible colony inhibition, and viable microbial counts were determined both before and after EVN treatment. The concentration of viable bacteria was calculated using the following formula: CFU/mL = (Number of colonies × Dilution factor) / Volume plated (mL). Given that direct plating was performed without prior dilution, the dilution factor was 1, simplifying the calculation to: Number of colonies × 100.

• Antibiofilm Activity Assay: The biofilm formation capacity of both ATCC and clinical MRSA and MSSA strains was evaluated utilizing a standard microtiter plate method.18 The S. aureus strains were inoculated into brain heart infusion (BHI) broth and incubated for 4 hours to achieve a turbidity equivalent to 0.5 McFarland units (1.5 × 10⁸ CFU/mL). For the baseline biofilm formation assay, 180 µL of fresh BHI broth was dispensed into the first four wells of a 96-well microtiter plate, followed by the addition of 20 µL of the standardized inoculum for each strain. A fifth well, containing only plain BHI broth, served as the negative control. Each treatment group was evaluated in triplicate, and the plate was incubated at 37 °C for 24 hours. Post-incubation, the wells were gently rinsed three times with 1× PBS, inverted, and tapped to remove planktonic cells and residual media, thereby minimizing background staining. Adherent biofilms were fixed by adding 200 µL of 95% ethanol, followed by staining with 200 µL of a 1% crystal violet (CV) solution per well. The plate was incubated at room temperature for 15 minutes, washed three times with 1× PBS, and blotted with paper towels to remove excess dye. Finally, 200 µL of 30% glacial acetic acid was added to solubilize the bound CV. After a 15-minute incubation at room temperature, the plate was inverted to dry. Uninoculated broth was utilized as a control to verify sterility and account for the non-specific binding of media components.

Statistical Analysis

Data derived from the agar diffusion assays were analyzed using GraphPad Prism version 9.0.2 (GraphPad Software, San Diego, CA, USA). Data normality was assessed using the Shapiro–Wilk test, and statistical significance was established at p < 0.05, corresponding to a 95% confidence interval. Comparisons between MRSA and MSSA strains treated with SVF-sEVs were conducted utilizing unpaired, two-tailed Student’s t-tests or a one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test, as appropriate. Descriptive statistics were generated utilizing SPSS version 26.0 (IBM Corp., Armonk, NY, USA). Data from the time-kill kinetic assays are presented as the mean ± standard deviation (SD) derived from a minimum of three independent experiments. Statistical differences across time points and between treatment conditions were evaluated utilizing a two-way repeated-measures ANOVA. For the CFU enumeration assays, CFU/mL values were log₁₀-transformed to stabilize variance and subsequently assessed for normality using the Shapiro–Wilk test. In instances where normality assumptions were satisfied, intergroup comparisons were performed using a one-way ANOVA with Tukey’s post hoc test; conversely, non-parametric data were analyzed utilizing the Kruskal–Wallis test followed by Dunn’s post hoc analysis. A post hoc power assessment indicated sufficient statistical power to detect large antimicrobial effect sizes; however, given the exploratory nature of this investigation, the analyses were interpreted as hypothesis-generating. Although multiple SVF-sEV concentrations were evaluated experimentally, only data corresponding to the 100% concentration are presented. No correction for multiple comparisons was applied, as these specific analyses were restricted to MRSA and MSSA strains evaluated at a single concentration.

Baseline biofilm formation and subsequent antibiofilm activity were assessed utilizing a semi-quantitative ordinal scale (categorized as no biofilm, weak, or moderate). For image-based analyses, qualitative scores were converted into ordinal numerical values (0–2) and evaluated using the Kruskal–Wallis test to ascertain overall intergroup differences. Because the data were categorical and the sample sizes per group were relatively small, post hoc pairwise comparisons were omitted, and the results are presented descriptively. These analyses are intended to illustrate comparative biofilm behavior and should be strictly interpreted as exploratory in nature.

Identification of Isolated SVF Cells

Assessing the viability of SVF cells based on membrane integrity is essential for the production of therapeutic-grade SVF-sEVs. Utilizing a trypan blue exclusion assay, we quantified the cell count, viability, and overall cellular composition of the heterogeneous SVF population, confirming it was comparable to its parent tissue source. This analysis demonstrated 80% to 90% cell viability and revealed the presence of a diverse immune cell population, which accounted for approximately 80% of the hHAT cellular composition (Figure 2i, ii).

SVF-sEV Phenotypic Characterization

Within the PBS suspension of the prepared SVF-sEVs, dispersed nanoparticles were visualized in the raw images acquired via nanoparticle tracking analysis (NTA) (Figure 3). Transmission electron microscopy (TEM) identified nanoscale vesicles morphologically consistent with SVF-sEVs; intact, spherical structures encapsulating protein cargo were clearly evident (indicated by red arrows, Figure 4i–iii). Furthermore, fluorescence microscopy confirmed the presence of the intact, cup-shaped ultrastructure characteristic of sEVs (Figure 5).

Confirmation of MRSA Strains

A 533-base pair (bp) mecA gene amplicon was detected in both the ATCC MRSA reference strain and the clinical MRSA isolates. In contrast, the MSSA strains exhibited no amplification, thereby confirming their methicillin susceptibility (Figure 6).

SVF-sEV In Vitro Antibacterial Activity

• Agar Diffusion Assay—MRSA Inhibition by SVF-sEVs: All tested S. aureus strains exhibited dose-dependent susceptibility to SVF-sEVs, with maximal inhibition observed at a 100% concentration. Neat SVF-sEVs generated zones of inhibition ranging from 12 mm to 30 mm across the tested strains (Figure 7i, ii). The ATCC MSSA and clinical MRSA isolates demonstrated the most pronounced inhibition. In contrast, diluted SVF-sEVs (≤25%) produced substantially smaller or negligible zones, indicating diminished efficacy. Ciprofloxacin disks (positive control) consistently yielded zones of inhibition comparable to those of the 100% SVF-sEVs, while PBS (negative control) exhibited no inhibition.

• Time-Kill Assay—Bactericidal Activity Over Time: The bactericidal effects of the SVF-sEVs were monitored longitudinally. Treatment with 100% neat SVF-sEVs induced a rapid reduction in viable S. aureus counts within 4 hours, followed by a substantial decrease in the overall bacterial load at 24 hours (Figure 8). This time-dependent killing kinetic demonstrates the potent and sustained antibacterial activity of the SVF-sEVs.

• Colony-Forming Unit (CFU) Assay: Exposure to 100% SVF-sEVs resulted in substantial reductions in CFU counts in both MRSA and MSSA cultures at 6 and 24 hours compared with untreated controls. The ATCC MRSA and clinical MRSA/MSSA isolates exhibited enhanced susceptibility, demonstrating pronounced CFU declines by 24 hours. Consistent bacteriostatic and bactericidal effects were observed across all experimental replicates (Figure 9i, ii). Log₁₀-transformed CFU/mL analyses revealed statistically significant decreases following SVF-sEV treatment compared with the baseline (evaluated via a one-way ANOVA with Tukey’s post hoc test or a Kruskal–Wallis test with Dunn’s post hoc test, as applicable), thereby supporting a robust antimicrobial effect. Given the exploratory design of this study, these results should be regarded as preliminary yet biologically relevant.

• Antibiofilm Activity of SVF-sEVs: SVF-sEV treatment resulted in a notable suppression of biofilm formation in both the ATCC reference and clinical S. aureus strains. Untreated controls maintained moderate biofilm formation, whereas SVF-sEV-treated wells exhibited a reduction in biofilm intensity to weak or undetectable levels, with complete inhibition observed in the MRSA cultures. These antibiofilm effects were consistent across the tested strains; residual weak biofilm was detected solely in the CS-MSSA isolate at 24 hours (Figure 10). Based on OD₅₇₀ measurements and predefined classification criteria, the treated wells shifted from the strong or moderate categories to the weak or non-biofilm categories. The negative controls remained unstained, whereas the positive controls retained strong staining, indicative of mature biofilms. Table 1 summarizes the baseline biofilm formation (BF) and subsequent antibiofilm activity (ABF) derived from these analyses.

For quantitative classification, biofilm producers were categorized using an ordinal scale (corresponding to codes 0 through 3) based on the cut-off optical density (ODc). The negative control (NC) consisted solely of plain media, while the positive control (PC) comprised media inoculated with ATCC MRSA. The classification criteria were formally defined as follows:

• 0 (Non-biofilm producer): OD ≤ ODc

• 1 (Weak producer): ODc < OD ≤ 2 × ODc

• 2 (Moderate producer): 2 × ODc < OD ≤ 4 × ODc

• 3 (Strong producer): OD > 4 × ODc