a The AFCs and CVCs in primary culture and subculture are indicated. amniotic fluid cells were characterized as mesenchymal cells that share most cell surface markers and have a similar response to colchicine. Colchicine did not DCPLA-ME induce a decline in cell viability at low concentrations but suppressed cell proliferation by arresting the cell cycle in the G2/M phase and increased the risk of tetraploid generation in a small subset of cases. Conclusions Our study revealed the results of a colchicine-induced toxicity test in prenatal cells and decided the anti-mitotic biologically functional dose and manner of administration that might reduce the risk of tetraploid generation. Value)Value) /th /thead ?AFC 146, XY2 VS. 200 VS. 15 ( 0.05)0 VS. 19 ( 0.05)?AFC 246, XX10 VS. 893 VS. 49 ( 0.05)8 VS. 65 ( 0.05)?AFC 346, XX0 VS. 154 VS. 22 ( 0.05)2 VS. 25 ( 0.05)?AFC 446, XY2 VS. 253 VS. 29 ( 0.05)10 VS. 19 (0.014)?CVC 146, XY1 VS. 783 VS. 87 ( 0.05)0 VS. 90 ( 0.05)?CVC DCPLA-ME 246, XX3 VS. 805 VS. 97 ( 0.05)2 VS. 61 ( 0.05) Open in a separate DCPLA-ME window Significant data are in strong Results Isolation, Rabbit polyclonal to Caspase 3.This gene encodes a protein which is a member of the cysteine-aspartic acid protease (caspase) family.Sequential activation of caspases culture and characteristics of prenatal cells After the initial culture, cells (amniotic fluid) and tissues (chorionic villus) were managed in culture medium for 7?days, and spindle-shaped fibroblast-like cells appeared. Then, the cells were fed fresh medium for 3?days during their growth, and they formed main colonies with unclear edges (Fig.?1a). The colonies were detached into single cells by trypsin and re-seeded into the culture flask for the subculture. The morphology of the CVCs and AFCs was homogeneous after three generations of subculture (Fig. ?(Fig.1a).1a). The cell surface markers were recognized by circulation cytometry and then utilized for the colchicine-induced toxicity study. The CVCs and AFCs were identified as one kind of mesenchymal cell with shared markers: they were positive for CD29, CD44 and CD73 and were unfavorable for CD14, CD34 and CD45, but the CVCs and AFCs experienced different levels of CD105 expression (Fig. ?(Fig.11b). Open in a separate window Fig. 1 The isolation and characteristics of AFCs and CVCs. a The AFCs and CVCs in main culture and subculture are indicated. b The surface markers of the subcultured AFCs and CVCs are indicated. The peak area in reddish represent unfavorable markers, and the black represents markers detected in the cells. The number in the plot indicates the ratio of each positive marker Colchicine affects cell viability in a time- and dose-dependent manner To evaluate the colchicine-induced toxicity in prenatal cells, we recorded the cell morphology and conducted cell viability analysis. The CCK-8 assay was utilized for the cell viability evaluation. The CVCs and AFCs displayed different sensitivity of colchicine, with the CVCs more easily induced by colchicine treatment to undergo cell death than the AFCs. For the dose-dependence test, the prenatal cells were treated for 3?h, and there were no significant changes in AFC morphology or cell viability with increasing concentrations of colchicine (from 0 to 2.4?g/ml), while 1.2?g/ml and 2.4?g/ml of colchicine induced significant decline in CVC viability (Fig.?2a, b and c). For the time-dependence test, the prenatal cells were treated with 0.15?g/ml colchicine, and a significant decline in cell viability was found for both AFCs (after 24?h) and CVCs (after 12?h) (Fig.?3a, b and c). Furthermore, we used flow cytometry to determine the colchicine-induced cell death ratio of the AFCs, as determined by cell viability. Even though cell viability did not switch after a 3?h treatment with 0.15?g/ml colchicine, there was a significant increase in the ratio of double-positive annexin V and propidium iodide (PI) cells compared with the control group. However, there was no significant switch between 3?h and 24?h of treatment (Fig. ?(Fig.33d). Open in a separate window Fig. 2 The dose-dependence of colchicine-induced toxicity in the AFCs and CVCs. The cell morphology (a) and cell viability (b for AFCs and c for CVCs) indicated for the AFCs and CVCs treated with.