Three healthy female individuals, between 20 and 50 years, participated in this study, with written informed consent. They underwent elective liposuction procedures at the Plastic Surgery outpatient clinic in Hospital da Plástica de Mato Grosso do Sul, Campo Grande, MS. The study was submitted to the Research Ethics Committee Involving Human Beings at the Universidade Federal do Mato Grosso do Sul and approved under process number 3.070.576.

The adipose tissue was isolated by the abdominal liposuction procedure performed according to Pesarini et al. (2017). For the processing of each of the three liposuction samples and isolation of ADSCs, collagenase was used for enzymatic digestion, as described by Markarian et al. (2014) with modifications. The culture medium was Dulbecco´s Modified Eagle´s Medium (DMEM Sigma®; catalog number D5523-10L) - low glucose, 10 mM HEPES supplemented with 10% fetal bovine serum (Gibco™ catalog number 10091148) and 1% antibiotic (Penicillin/Streptomycin, Sigma®, A5955). ADSCs until 5^th passage were used in the experiments, because according to Adami et al. (2021), this is the limit passage to use the ADSCs to human application, since cells do not suffer any morfological change until this moment. Thus, this conception was also adopted in our laboratory routine.

The cell surface markers of the extracted cells were examined using a flow cytometer (FACScalibur - Becton Dickinson, San Diego, CA). A total of 2.5×10⁵ ADSCs in 5^th passage were dissociated in trypsin, centrifuged and incubated during 30 min. at 4°C using the following antibody cell markers: CD105, CD90, MHC II and CD45 (Pharmingen BD, San DIEGO, CA. USA). The cells were analyzed in 10⁴ events using the 488nm laser FACScalibur (Becton Dickinson, San Diego, CA) in the CellQuest software. The MDI 2.8 software was used to generate the histograms.

For these assays, 1x10⁵ cells/well (3^rd passage) were seeded in 6-well plates. After cell adhesion (24 hours), the DMEM was discarded, and specific media (adipogenic, chondrogenic and osteogenic) were added for differentiation, as described by Schweich et al. (2017). Their multipotency was also analyzed. To confirm the differentiations, Oil Red (adipogenic), Red Alizarin (osteogenic) and Alcian Blue (chondrogenic) dyes were used. The procedures were performed in triplicates.

The ADSCs (5th passage) were trypsinized as previously described (Markarian et al., 2014), and the comet test was performed according to Singh, McCoy, Tice, and Schneider (1988), with modifications proposed by Navarro et al. (2014). An aliquot (40 µL) was resuspended in low-melting 0.75% (120 µL) agarose (Invitrogen Co, Carlsbad, CA) at 37°C, and distributed on slides previously coated with normal melting point agarose 1,5% (Invitrogen Co, Carlsbad, CA), covered with a coverslip. The slides were immediately placed in the refrigerator (4ºC) for 20 minutes. After the solidification of the agarose, the coverslips were removed and the slides were immersed in a lysis solution (2.25 M NaCl, 90 mM EDTA, 9 mM Tris-HCl, 10% DMSO and 1% Triton X-100), being kept in the dark at 4ºC, for 2 or 24 hours, according to each protocol. After cell lysis, the slides remained in an electrophoresis vat, covered with alkaline buffer (pH 12 or ≥ 13, specific to each protocol), for 20 minutes (NaOH 10N, EDTA titriplex 200 mM) for DNA denaturation. Electrophoresis (300 mA and 25 V) was performed with the same buffer for 20 minutes more. Subsequently, the slides were neutralized with 0.4 M Tris, pH 7.5, for 15 minutes (3 times of 5 minutes each), and fixed in absolute ethanol for 10 minutes, then, left to dry overnight at room temperature and stored until analysis.

The material was stained (100 µl of ethidium bromide, 20 mg mL^-1) and the nucleoids were photographed under a fluorescence microscope (Leica, DMi8), with 200X magnification. Subsequently, more than 200 nucleoids were analyzed per repetition by the CometScore 2.0.0.38 TriTek program (CometScore Comet Scoring Software, 2004), and the tail moment parameter (% DNA in the tail multiplied by the length of the tail) was evaluated (Livak & Schmittgen, 2001).

For the different study groups, the lysis solution was the same. However, the lysis time varied (2 hours or 24 hours). To detect DNA damage, for each of these times, two types of electrophoresis buffer were evaluated: one with pH 12 (Villela et al., 2007) and the other with pH ≥ 13 (Tice et al., 2000). Thus, the analyzed groups were: Protocol 1 - lysis time of 2 hours and electrophoresis buffer solution with pH 12; Protocol 2 - 24-hour lysis time and pH 12 electrophoresis buffer solution; Protocol 3 - lysis time of 2 hours and electrophoresis buffer solution with pH ≥ 13; and Protocol 4 - 24-hour lysis time and electrophoresis buffer solution with pH ≥ 13 (Figure 1).

Figure 1. Tested protocols for detecting DNA damage in adipose-derived stem cells.

For validation of the test, three independent samples of ADSCs were treated with doxorubicin (DXR) (Libbs®, catalog number 734079) at a concentration of 5 µM and pH 13 for 3 hours. The slides of this positive control were submitted to lysis and electrophoresis together with the slides of the study protocols.

For the evaluation of gene expression, 2 x 10⁵ ADSCs/well (5th passage), were seeded in 6-well cultivation plates, and freshly extracted ADSCs (passage 0) were used as a negative control. The cells were collected and the total RNA was extracted by an in-house method. For this purpose, it was used a lysis buffer based on guanidine thiocyanate (Guanidine thiocyanate 5 moles L^-1, Tris Hydrochloride (HCL) 11.2 g L^-1 (pH 6.4), ethylenediaminetetraacetic acid (EDTA) NaOH 7, 43 g L^-1, Triton X-100 7.8 mL). For each 200 µL of cell suspension, 200 µL of lysis buffer (1: 1) was added. After homogenization during 10 minutes, 50 µL of magnetic bead (Nuclisens®, easyMag®, bioMérieux®, catalog number 280133) was added, with subsequent rest for 10 minutes. Impurities (cell debris) were removed by two washes with lysis buffer (2.5M NaCl, 100 mM EDTA titriplex, 10 mM Tris (pH 10), 1% Triton X-100 and 10% dimethyl sulfoxide (DMSO). The excess of guanidine thiocyanate and salts were removed with two washes of 70% alcohol. The beads were washed with PA acetone, heated for one minute at 60ºC in Dry Bath Heat & Cool Control™ dry bath equipment (DB-HC, Loccus®), and the genetic material was eluted with 30 µL of TE solution (NucliSENS®, catalog number 280132). After extraction, the treatment was performed with RQ1 RNase-Free DNase (Promega®, catalog number M6101,) according to the manufacturer's specifications. After the DNA degradation step, the quality of the extracted material was analyzed using: (I) the A260/A280 and A230/A260 quotient in a NanoVue™ Plus spectrophotometer (GE Healthcare - Life Sciences®); and (II) the denaturing agarose gel integrity (1%). Only samples with a quotient between 1.8 to 2.1 were used for the synthesis of complementary DNA (cDNA).

The cDNA synthesis was performed in a T100™ thermocycler (Thermal Cycler, Bio-Rad ™) using 250 ng of RNA in the GoScript™ Reverse Transcriptase kit (Promega®, catalog number A5001) following the manufacturer's protocol, in which 5µL GoScript™ 5X Buffer, 3.8 mM MgCl2, 0.5 mM DNTPs, 20 RNAse inhibitor units and 1 µL of GoScript™ Reverse were used. The mixture was incubated for 5 minutes at 25^oC, followed by 60 minutes at 42^oC and 15 minutes at 70^oC.

The quality of the reverse transcription step was analyzed by the quotient A260/A280 and A230/A260 in a spectrophotometer NanoVue™ Plus (GE Healthcare - Life Sciences®), and only the samples with a quotient between 1.8 and 2.1 were admitted. Quantification of the cDNA was performed on the same equipment.

The qPCR reactions were performed in triplicates in the Rotor Gene® device (Qiagen). The analyzed genes are described in Table 1. The genes involved in the DNA damage and repair process were tested. For the reaction, it was added 10 µL of GoTaq® master mix, 2 pmol of each oligonucleotide, and 500 ng of cDNA and ribonuclease-free water q.s.p. (Promega®, catalog number A6002), totaling 20 µL. The mixture was submitted to 95ºC for 5 minutes and 40 cycles of 95ºC for 2 seconds and 60ºC for 30 seconds. At the end, the melting curve was performed to assess the specificity of the formed products. The β-actin (ACTB) gene was used as a normalizer (housekeeping), and the results were analyzed using Rotor Gene® (Qiagen) v2.3.1.

Table 1. Sequence of primers (5′– 3′) used in real-time PCR reactions.

Legend - ACTB: β-actin gene (ACTB); ATM: serine/threonine kinase gene; ATR: serine/threonine-protein kinase gene; GADD45: damage-inducible gene; P21: P21 protein; TP53: Transformation-related protein 53.

The statistical analysis of comets was performed by ANOVA/Tukey, and results p ≤ 0.05 were considered statistically significant (GraphPad InStat). The statistical analysis of the data obtained in the RT-qPCR was performed using the REST program, and the difference was considered significant when the level of relative expression was equal to/or less than -2, or equal to/or greater 2.

Undifferentiated cells (control) (Figure 1A) and confirmation of adipogenic, osteogenic and chondrogenic differentiation were observed by the morphological changes of the ADSCs (Figure 2). Calcium deposits stained with Alizarin red confirmed the osteogenic differentiation (Figure 2B). The lipid vacuoles stained with Oil Red O confirmed the adipogenic differentiation (Figure 2C). The extracellular matrix rich in glycosaminoglycans stained with Alcian Blue proved the chondrogenic differentiation (Figure 2D).

ADSCs expressed CD105 and CD90 markers, and did not express CD34 and CD133. Thus, the analysis of this immunophenotypic profile demonstrates that these are mesenchymal stem cells (Figure 2E).

Figure 2. Characterization of multipotency and immunophenotyping of ADSCs. Photomicrographs of cultures of undifferentiated ADSCs with fibroblastoid aspect (A); adipogenic differentiation confirmed by the coloring of Oil Red (B); osteogenic differentiation confirmed by Alizarin Red staining (C); and chondrogenic differentiation confirmed by the coloration of Alcian Blue (D) (400x magnification). Immunophenotypic profile of ADSCs that expressed the CD90 and CD105 markers and did not express the CD38 and CD133 markers (E).

The comet assay demonstrated that, regardless of the lysis time and pH of the electrophoresis buffer solution, the MSC did not show any DNA damage (p > 0.05) (Figure 3A-E). Treatment with DXR demonstrated that the comet assay was properly performed and there was damage to the DNA (p < 0.001) of the ADSCs (Figure 3A and 3F).

Figure 3. Evaluation of genotoxicity (tail moment) in ADSCs using 4 comet assay protocols. (A) Box plot showing the mean and standard deviation of the frequency of DNA damage for each of the comet protocols and for the positive control (Doxorubicin - 5µM); (B) nucleoid of ADSCs submitted to protocol 1 (lysis time of 2 hours and electrophoresis in pH 12 buffer), (C) nucleoid of ADSCs submitted to protocol 2 (lysis time of 24 hours and electrophoresis in pH 12 buffer), (D) nucleoid of ADSCs submitted to protocol 3 (lysis time of 2 hours and electrophoresis in buffer pH ≥ 13); (E) nucleoid of ADSCs submitted to protocol 4 (lysis time of 24 hours and electrophoresis in pH ≥ 13 buffer); (F) Nucleoid of ADSCs treated with Doxorubicin and submitted to electrophoresis at pH ≥ 13. Different letters indicate statistically significant differences (p < 0.05, ANOVA/Tukey).

The results of the gene expression assay, performed by qPCR, demonstrated that there was no significant increase (p > 0.05) in the expression of the analyzed genes. The ATM gene was expressed at 1.54x, ATR at 1.32x, CDKN1A at 1.48x, TP53 at 1.71 and GADD45A at 1.97x (Figure 4).

Figure 4. Gene expression assay by qPCR. The analyzed genes are involved in the DNA damage and repair process. As control, ADSCs obtained immediately after the extraction process were used (cells that were not subjected to the cell culture process). The test sample, on the other hand, was composed of ADSCs, which were in the fifth cultivation pass. The ACTB gene was used as a normalizer. There was no significant difference in the expression of the analyzed genes (p < 0.05, REST).

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