Ethical declaration

This study was conducted in accordance with the ethical principles contained in the Declaration of Helsinki, the Good Clinical Practice Guidelines (ICH GCP E6R2), the Good Clinical Practice guidelines of the Vietnamese Ministry of Health, and current regulatory requirements and policies of DNA International General Hospital (Ho Chi Minh City Vietnam) on ethics in biomedical research. The study on participants was approved by the Ethics Committee of DNA International General Hospital (Ho Chi Minh City 700,000, Vietnam) for biomedical research (decision number was No.21/CN-HĐĐĐ), the National Ethics Committee (decision no. 13/CN-HĐĐĐ), and the Vietnam Ministry of Health (1690/QĐ-BYT). Before participation, all patients provided written informed consent after receiving adequate information. This research has been registered with ClinicalTrials.gov (NCT05827757).

Patient recruitment

The current trial is a phase I single-group, open-label, controlled before-after clinical trial to evaluate the safety and efficacy of autologous AD-MSC transplantation on proinflammatory cytokines and anti-inflammatory cytokines in aging-related low-grade inflammation patients. The study was conducted at DNA International General Hospital (Ho Chi Minh City, Vietnam). The participant enrollment took place from December 2020 to October 2022. Study subjects who meet the inclusion and do not meet the exclusion criteria of the study will be recruited with a minimum sample size of 12 patients (the minimum number of patients required by the Ministry of Health of Vietnam for the phase I trial).

The following are the inclusion criteria: (1) males or females aged 40–64 years; (2) TNF-α index > 11 pg/ml and IL6 index > 1.23 pg/ml (blood samples were tested on the multiplex system at Cho Ray Hospital, Ho Chi Minh City, Vietnam); (3) possesion of at least two of the following three comorbidities: diabetes, dyslipidemia, and obesity (the diagnosis of the comorbidities was made according to the Ministry of Health’s general guidelines); (4) stable use of medications for the previous 3 months to treat the previously mentioned comorbidities (diabetes, dyslipidemia, and obesity); and (5) agreement to participate in the study and to comply with the research examination and evaluation process.

The following are the exclusion criteria: (1) patients with coagulopathy, (2) history of or current severe heart failure, (3) acute respiratory disease at the time of screening, (4) patients with cancer or other acute illness requiring treatment, (5) history of allergy to anesthetics and antibiotics, (6) currently/planning to participate in another clinical trial during the study period, and (7) possessing additional conditions or circumstances that make it difficult to provide treatment, according to the researcher. Pathology of disease in the exclusion criteria was defined according to the Guidelines of the Ministry of Health of Vietnam.

Patient follow-up and treatment

During the trial, patients with diabetes, dyslipidemia, and obesity continued to take their medications. While adjusting the drug dose limited to the maximum dosage, any changes to the dose were used simultaneously with the experiment treatment and were consulted by a doctor to ensure patient safety.

Patients in the study underwent a total of 5 evaluation visits: visit to participate in the study (V1, D-44 ± 7), visit to harvest and culture cells (V2, D-30 ± 14), first MSC transplant (V3, D0), second MSC transplant (V4, D90 ± 7), and last visit/end of study visit (V5, D180 ± 14). In V1, patients were screened and selected for study. In V2, selected patients were liposuctioned, and their cells were cultured. First, 100 million autologous AD-MSC were i.v transfused in V3, followed by a second transfusion in V4. The monitoring and evaluation were performed in V5 (Fig. 1).

Fig. 1MSC preparation and administration

Autologous MSCs from adipose tissue were harvested, cultured, cryopreserved, and quality checked according to the institute’s standard operating procedure (DNA International General Hospital Joint Stock Company, Ho Chi Minh City, Vietnam). Briefly, MSCs were isolated from the patient’s adipose tissue after lipoaspiration by collagenase (code N0002778, AMSBIO, MA, USA). MSCs were expanded in an MSC culture medium kit (ADSCCult I, Stem Cell Institute, Vietnam) to passage 2 or 3 and then frozen in liquid nitrogen for later infusion. Prior to storage or transfusion, the MSCs were subjected to quality control tests as described in Table 1, including mycoplasma (MycoAlert™ PLUS, code: LT07-703, Lonza, Switzerland), sterility (Fluid Thioglycollate Medium and Soybean-Casein Digest Medium, Merck Millipore, Germany), endotoxin < 0.5 EU/ml (Kinetic-QCL™ Kinetic Chromogenic LAL Assay, code: 50-650U, Lonza), cell survival > 90% (Trypan Blue staining count), and marker expression for MSC identification [21] (BD Stemflow™ Human MSC Analysis Kit, code: 562245, BD Biosciences, NJ, USA). All patients were given 2 intravenous autologous infusions on day 0 (D0) and day 90 (D90). A total of 100 million single cells were suspended in 0.9% sterile saline solution and were given intravenously at a 5-ml/min rate for 45 min. The safety variables were monitored for the whole duration of the study.

Table 1 Adipose tissue-derived mesenchymal stem cell qualityOutcomesSafety

Safety assessments included adverse events (AEs) and serious adverse events (SAEs) during the entire study follow-up period. AEs are also classified separately, including patients who stopped or withdrew from the study. Frequency of AEs and serious SAEs within 24 h after stem cell transplant was measured. This study used the National Institutes of Health’s (NIH) AE/SAE assessment and recording guideline document, Common Terminology Criteria for Adverse Events CTCAE version 5.0, which accompanies the study.

Clinical tests used to evaluate the safety include basic hematologic and biochemical tests performed at a local laboratory. Changes in vital signs and physical examination over time were not analyzed separately from the study protocol. In cases where these changes met the criteria of an AE/SAE, the changes were analyzed jointly with the AE/SAEs.

Efficiency Cytokine measurements

The serum concentrations of IL-1α/β, TNF − α, IL-2, IL-6, IL-8, VEGF, IFN-γ, IL-4, IL-10, MCP-1, and EGF were measured by a cytokine growth factor array kit (cat EV3513, Randox, UK) at Cho Ray Hospital, Ho Chi Minh City, Vietnam.

Absolute changes in proinflammatory cytokines (IL-1α/β, TNF-α, IL-2, IL-6, IL-8, VEGF, IFN-γ) and anti-inflammatory cytokines (IL-4, IL-10, MCP-1, EGF) after transplantation of AD-MSC at D90 and D180 were compared to the time before study D-44.

Ratio of proinflammatory cytokines and anti-inflammatory cytokines after treatment at D90 and D180 was compared with pretreatment levels (IL-4/IL-10, IL-1β/IL-10, IL-6/IL-10, IL-2/IL-10, IL-6/IL-10, IL-1β/EGF).

Statistical analysis

The study screened 21 patients and included 12 patients in the study. No imputation for missing variables was performed since the data set was complete for the 12 patients before research and at day 90 and day 180 that included the measurement of all 12 cytokines.

Continuous data are presented with descriptive statistics (e.g., mean and standard deviation, median, range, Q1, Q3). The 95% CI of a mean, a ratio, or the difference between two means or two ratios will be calculated as appropriate. The charts show the average value of the cytokines with their standard error (SE) and visualized by the GraphPad Software.

The Kolmogorov-Smirnov and Shapiro-Wilks tests determined whether the data followed a normal distribution. In this study, Fisher’s exact test was used to compare the two ratios. With quantitative testing values, according to the nonstandard distribution assumption, the nonparametric Wilcoxon signed-rank test was used for comparison. Differences were considered significant at p < 0.05 unless otherwise stated.

The statistical analyses were performed with the SAS® System 9.4 software (SAS Institute Inc., Cary, NC).