Patient samples
The adipose tissues were obtained at Vinmec International Hospital between 2018 and 2019 and cryopreserved in the biobank after the donors signed the informed consent form. The expression of surface markers was retrospectively analyzed on 42 ADMSC samples isolated in PS (Pan-Biotech, Germany), of which 31 patients participated in a previous study [34]. This study enrolled patients with sexual functional deficiency. The other 11 samples were obtained from healthy donors. We performed a prospective study on seven samples, of which donors agreed to donate their biological materials for the current research, to investigate the cellular properties of ADMSCs cultured in different xeno-free and serum-free MSC expansion media (Suppl. Figure 1). Sample collection and data analysis were approved by the Ethics Committee of Vinmec Healthcare System and carried out in accordance with the Declaration of Helsinki.
Isolation, culture, and cryopreservation of ADMSCs
ADMSCs were isolated as previously described [35]. Briefly, adipose tissues were harvested from the donor’s lower abdomen under general anesthesia. Then adipose tissue was minced into small pieces and digested using 200 unit/ml Collagenase type I solution (Gibco, Denmark) supplemented with 200 g/l Human Albumin (Baxter, USA) at 37°C with agitation 300 rpm/min for 60 min. The mixture was centrifuged at 1500 rpm/min for 10 min to collect the stromal vascular fraction in the pellet. Finally, the cells were resuspended in PS (Pan-Biotech, Germany) supplemented with 1% Penicillin/Streptomycin (Gibco, USA). Cells were incubated at 37oC and 5% CO2 in a humidified atmosphere with medium exchange every three days. When reached 80-90% confluence, the cells were harvested using TrypLE™ Select Enzyme (Gibco, USA) and counted after staining with trypan blue (Life Technologies, USA).
Cells at passage 0 were cryopreserved in the Cryostor solution (Stemcell Technologies, Canada). All the cryovials were kept at -80oC for no more than seven days and transferred to BioStoreTM III Cryo (Brooks Life Sciences, USA) for long-term storage. After four months of cryopreservation, the cells were thawed rapidly at 37oC and maintained in PS or SM (Miltenyi Biotec, Germany) to evaluate cell growth, phenotype, and functional properties.
Growth curve
To determine the proliferative capability of ADMSCs, cells were seeded with the density of 5,000 cells/cm2 in T25 flasks (Nunc) and cultured up to passage 7 as previously described [35]. Experiments were performed in triplicate for each donor. Population doubling (PD) was calculated using the formula: PD = 3.32 x log (a/b), in which a is the number of harvested cells and b is the seeding density. Finally, the population doubling time (PDT) was calculated as t/n, where t is the days (or hours) of culture, n is the number of PD reached during a passage. The cell morphology was visualized under an invert phase-contrast microscope (Eclipse Ti-S/DS-Fi2-L3, Japan).
Colony-forming unit (CFU) assay
Cells were seeded in triplicate in a six-well dish at the concentration of 20 cells/cm2 and cultured in the respective media (PS or SM) for 10 to 14 days as previously described [35]. Cells were fixed in Methanol (Merck, Germany) and stained with Giemsa solution (Merck, Germany), both for 5 minutes at room temperature. Colonies formed on each dish were counted.
Flow cytometry analysis
ADMSCs harvested from passage 3 and 7 were assessed for surface marker expression by flow cytometry. Cells were resuspended in PBS (Gibco, USA) supplemented with 1% human serum (Pan-Biotech, Germany). The cells were incubated using the Human MSC Analysis kit (Becton Dickinson, USA) according to the manufacturer’s instruction. To examine the expression of negative surface markers individually, cells were stained with the following antibodies: CD34 Monoclonal Antibody (581) FITC (Invitrogen, USA), CD11b Monoclonal Antibody (ICRF44) PE (Invitrogen, USA), CD19 Monoclonal Antibody (HIB19) PerCP-Cyanine 5.5 (Invitrogen, USA), CD45 Monoclonal Antibody (HI30) APC (Invitrogen, USA), Anti HLA-DR-FITC (Beckman Coulter, France). The samples were analyzed using BD Canton II (Becton Dickinson, USA). At least 20,000 cells per run were acquired. The data analysis was performed using the FlowJo software (Becton Dickinson, USA).
To check viability, cells were suspended in PBS (Gibco, USA) supplemented with 1% human serum (Pan-Biotech, Germany) and stained with 7-AAD Staining Solution (Miltenyi, Germany) following the manufacturer’s instruction. For the cell cycle analysis, cells were fixed with eBioscience™ Intracellular Fixation & Permeabilization Buffer Set (Invitrogen/Thermo Fisher Scientific, USA) and stained with 7-AAD Staining Solution (Miltenyi, Germany) as previously described [36]. At least 20,000 cells were analyzed per run using BD Canton II (Becton Dickinson, USA) and the FlowJo software (Becton Dickinson, USA).
MSCs differentiation assays
MSCs were differentiated into adipogenic, chondrogenic and osteogenic lineages using the StemPro® Adipogenesis Differentiation Kit, StemPro™ Chondrogenesis Differentiation Kit, StemPro™ Osteogenesis Differentiation Kit (Gibco, USA) in duplicates as previously described [35]. For this purpose, cells were cultured in either differentiation media or MSC culture media as the control for 14 days. They were then fixed by using 4% PFA (Merck, Germany) for 30 minutes before staining with the Oil Red O (Merck, Germany) solution to detect lipid droplets, the Alcian Blue (Sigma Aldrich) solution to find the presence of proteoglycan and Alizarin Red S (Merck, Germany) solution to mark calcium deposition. The staining was observed under an inverted microscope equipped with a camera (Olympus Corporation, Tokyo, Japan).
To perform quantitative analysis, ADMSCs were cultured in the differentiation media for 14 days. Cells of the same passage were used as the undifferentiated control. Total RNA was extracted using RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer’s instruction. cDNA synthesis was carried out using the SuperScript™ IV First-Strand Synthesis System (Invitrogen/Thermo Fisher Scientific, USA), and qPCR was performed using the 7500 Fast Real-Time PCR System and PerfeCTa SYBR Green SuperMix according to the manufacturer’s instructions. The expression of the following genes was analyzed: PPARγ and Leptin for adipogenesis, ALP and PTH-R for osteogenesis, SOX9 for chondrogenesis and GAPDH for the reference gene. Primer sequences are presented in the supplementary information.
Senescence assay
ADMSCs were seeded on six-well plates and cultured in the PS or SM media to obtain passage 3 and passage 7 cultures. Cell senescence was analyzed using Senescence Cells Histochemical Staining Kit (Sigma Aldrich, USA) following the manufacturer’s instruction as previously described [37]. Briefly, cells were fixed and stained with Staining Mixture containing X-gal substrate overnight after reaching 70-80% confluence. Senescent cells with β-galactosidase activity were stained blue. The total number of cells was examined using DAPI (Abcam, UK) counting. Cell images were observed and captured by a IX73 microscope and cellSens software (Olympus, Japan). Cell counting was performed using Image J software (version 1.46r). The percentage of senescent cells was calculated by dividing the total number of DAPI-stained cells by the number of blue-stained cells.
Karyotype
To evaluate the karyotype of ADMSCs after prolonged culture in vitro, passage 3 and 7 cells were checked for karyotype as previously described [26]. When the cells reached 80% confluence, they were incubated in KaryoMAX® Colcemid™ 10 µl/ ml (Gibco, USA), diluted to 1:1000, at 37 °C in a 5% CO2 incubator for 30 min. Cells were detached from the flask by incubating in 2 ml of Trypsin EDTA 0.25% (Gibco, USA) at 37oC within 1 to 2 minutes and then in a pre-warmed hypotonic solution (KCl 0.56%) for 30 minutes. Cells were washed three times with Carnoy’s solution (3:1 v/v absolute ethanol: glacial acetic acid) before being dropped on 3-4 clean slides to spread metaphase. The slides were incubated at 60°C overnight and stained the day after using G-banding technique. Metaphase was captured by MetaSystems, Carl Zeizz and analyzed by Ikaros software. Twelve to twenty metaphase spreads were analyzed for each sample by an experienced technician and validated by a medical genetic scientist. Karyogram is 400 band according to ISCN (An International System for Human Cytogenomic Nomenclature) 2016 standard [38].
Statistical analysis
All experiments in this study were performed in triplicates if not otherwise indicated. Data are present as mean ± SD if not otherwise indicated. Student t-test was used for a normally distributed population, while Mann–Whitney U test was employed in nonparametric distribution. A p-value below 0.05 was considered statistically significant. Differences among multiple groups were statistically analyzed using one‐way ANOVA and Tukey's multiple comparisons test. Univariate analyses were conducted using Spearman’s rank correlation and Kruskal–Wallis test. Statistical analyses were performed using the R software version 3.6.1 by a statistician [39]. Plots were prepared using the software GraphPad Prism 5.0 (GraphPad Software, La Jolla, CA).