ABSTRACT
Human mesenchymal stem cells (hMSCs) are multipotent cells used in cell therapy research. One of the problems involving hMSCs is the possibility of genetic instability during in vitro expansion required to obtain a suitable number of cells for clinical applications. The cytokinesis-block micronucleus (CBMN) assay measures genetic instability by analyzing the presence of micronucleus (MN), nucleoplasmic bridges (NPBs), and nuclear buds (NBUDs) in binucleated cells. The present study describes modifications in the CBMN assay methodology to analyze genetic instability in hMSCs isolated from the umbilical vein and in vitro expanded. The best protocol to achieve binucleated hMSCs with preserved cytoplasm was as follows: cytochalasin B concentration (4.0 µg/mL), use of hypotonic treatment (3 min), and the fixative solution (9 methanol:1 acetic acid). These adaptations were reproduced in three hMSC primary cell cultures and also in XP4PA and A549 cell lines. The frequency of hMSCs treated with mitomycin-C presenting MN was lower than that with other nuclear alterations, indicating that the hMSCs contain mechanisms to avoid a high level of chromosomal breaks. However, a high frequency of cells with NPBs was detected and spontaneous anaphase bridges under normal hMSC in vitro culture were observed. Considering that anaphase bridges are characteristic alterations in tumor cells, the CBMN assay is indicated as an important tool associated with other genetic analyses in order to ensure the safe clinical use of hMSCs in cell therapy.
Subject(s)
Cytokinesis/drug effects , Genomic Instability , Mesenchymal Stem Cells/physiology , Biomarkers/metabolism , Cell Differentiation , Cell Line, Tumor , Cell Nucleus , Cell Shape , Cytochalasin B/pharmacology , Female , Humans , Infant, Newborn , Male , Micronucleus Tests/methods , Middle Aged , Primary Cell CultureABSTRACT
Titanium (Ti) is currently the most widely used material for the manufacture of orthopedic and dental implants. Changes in the surface of commercial pure Ti (cp Ti) can determine the functional response of cells, and is therefore a critical factor for the success of the implant. However, the genotoxicity of titanium surfaces has been poorly studied. Thus, the purpose of this study was to evaluate the genotoxic potential of a new titanium surface developed by plasma treatment using argon-ion bombardment and compare it with an untreated titanium surface. Accordingly, comet assay, analysis of chromosomal aberrations (CAs), and Cytokinesis Block Micronucleus (CBMN) assay were carried out, using CHO-K1 (Chinese hamster ovary) cells grown on both titanium surfaces. Our results show that the untreated titanium surface caused a significant increase in % tail moment, in the number of cells with CAs, tetraploidy, micronucleus frequency, and other nuclear alterations when compared with the negative control and with the plasma-treated titanium surface. This difference may be attributed to increased surface roughness and changes in titanium oxide layer thickness.
