This study was approved by the Ethics Committee of The Third Affiliated Hospital of Guangzhou Medical University (Approval number: 2017-001). Six pregnant patients signed informed consent and were told that the diagnostic sample would be used for research. Four pregnant women at 18-21 weeks gestation were undergoing amniocentesis, and two at 12 weeks gestation were undergoing chorionic villus sampling. After the cytogenetic diagnoses were completed, the four AFC lines and two CVC lines were used in this study.
The clinical samples were 8ml amniotic fluid or 100g chorionic villus tissue for each patient. For primary culture, amniotic fluid was centrifuged in 300G and chorionic villus tissue was cut into approximately 1 mm2 tissue masses. Then, cell precipitates (or the tissue masses) were resuspended in 4 ml amnioMAX-C100 medium (Cat#12558011, Invitrogen Life Technologies, Carlsbad, CA, USA) and seeded into two 25-cm2 culture flasks where they were maintained for 7-9 days at 37°C in 5% CO2. Cells were detached by 0.05% trypsin (Cat#25300054, GIBCO, Invitrogen China Limited, Shanghai, China) for subculture. Clinical cytogenetic diagnosis was performed on passage 1. AFCs and CVCs were subcultured in complete MEM medium (Cat#12571071, GIBCO) supplemented with 20% foetal calf serum (Cat#10100, GIBCO) and 100 U/ml of penicillin and streptomycin (Cat#10378016, Invitrogen). Cells from passages 3-5 were used for the toxicity assays of colchicine (Cat# S2284, Selleck Chemicals, Houston, TX, USA). Colchicine was dissolved in MEM medium, for dose-dependent, cells were treated by 0, 0.15, 0.3, 0.6, 1.2 and 2.4μg/ml colchicine for 3 hours; for time-dependent, cells were treated by 0.15μg/ml colchicine for 0, 12, 24, 48 and 72 hours. As colchicine diluted in MEM, untreated group was control.
The P1 subcultured cells were treated with 0.15 μg/ml colchicine for 3 h, detached by 0.05% trypsin, subjected to hypotonic treatment with 0.02% sodium citrate and 2% potassium chloride and fixed in a mixture of methanol and acetic acid in 3:1 (volume to volume). The fix cells were dripped on cold slides and stained with Giemsa for metaphase chromosome analysis that was conducted with an Ikaros system (Carl Zeiss AG, Oberkochen, Germany).
Cell viability assay
A total of 5000 cells were counted in each well of 96-well plates, with triplicate sets for each group. After treatment with different drug concentrations at specific time points, the cell viability was assessed using a commercial CCK-8 (Cell Counting Kit-8) assay (Cat#C0039, Beyotime Biotechnology, Shanghai, China).
Cell surface marker assays
The cells were detached by trypsin and stained by the following fluorescence-conjugated antibodies: CD14-APC, CD34-PE, CD45-FITC, CD105-FITC, CD29-PE, CD44-FITC and CD73-APC. The population analysis was conducted on an Attune NxT flow cytometer (Invitrogen).
Cell cycle assay
Drug-treated cells were detached by trypsin and fixed in 70% ethanol overnight. The fixed cells were assayed using a cell cycle and apoptosis analysis kit (Cat#C1052, Beyotime Biotechnology) on a flow cytometer. The fluorescence intensity was used to distinguish different phases of the cell cycle.
Cell apoptosis assay
1×106 cells were seeded in to a 100 cm2 dish overnight for attachment. Drug treatment was accessed by change medium supplemented with 0.15 μg/ml colchicine. After drug treatment, the cells were stained with an annexin V-FITC apoptosis detection kit (Cat#C1062M, Beyotime Biotechnology) and counted by a flow cytometer for analysis. The cells in early apoptosis were the annexin V-positive and PI (propidium iodide)-negative cells, and the cells in late apoptosis were the annexin V- and PI-positive cells.
The cell viability was expressed as the mean ± SD, and the percentage of the total number of cells in metaphase was expressed in a scatter diagram. The number of diploid and tetraploid cells in each analysis are presented in the table1. For two groups, the data were statistically analysed by paired Student's t-tests; for different concentrations of colchicine and different time points, the data were statistically analysed by ANOVA, and a p-value of <0.05 was considered statistically signiﬁcant. A chi-square test was used to compare the proportion of tetraploid cells in the control group and in the drug-treated groups during each metaphase analysis. A p-value of <0.05 was considered statistically signiﬁcant for either group.