At the same time, mechanical characteristics of cells (particularly their stiffness) can be used as the measure of their intact structure. Measurements of the mechanical characteristics of cells can be performed in vivo within a short period of time using AFM. In view of the above, the main objective of this study was to determine the mechanical characteristics of mesenchymal stem cells when cultured TGF beta inhibitor in the presence of silica and silica-boron nanoparticles. Methods Isolation of mesenchymal

stem cells and their cultivation conditions In order to obtain the primary culture, a method of enzymatic processing of the stromal vascular fraction isolation from human lipoaspirates was used [17, 18]. The obtained cells were cultivated in α-MEM medium (MP Biomedicals, Santa Ana, CA, USA) with 2 mM of glutamine (PanEco, Moscow, Russia), 100 IU/mL of penicillin, 100 μ/mL of streptomycin (PanEco), and 10% fetal bovine serum (Hyclone, Logan, UT, USA) added to the culture. The cell seeding density was 3 × 103 cells/cm2. Standard cultivation was performed at 37°C and under 5% CO2 using a CO2 cultivator (Sanyo, Moriguchi, Osaka, Japan). The cells of passages 3 to 5 were used for the experiments. Silica (Si) and silica-boron (SiB) NPs were added to the culture medium at the same concentration of 100 μg/mL. Cultivations were performed for 1 and 24

h. Nanoparticles were prepared at the Prokhorov selleck compound General Physics Institute RAS by the method described in detail previously [19]. Evaluation of mesenchymal stem cell viability The proportion of AnV + cells (early apoptosis), AnV+/PI + cells (post-apoptotic CB-839 necrosis), and PI + cells (necrosis) was determined using

an Annexin V-FITC/PI kit (Beckman Coulter, Brea, CA, USA) and Epic XL flow cytofluorimeter (Beckman Coulter) in strict accordance with the standard procedure stated in the manufacturer’s manual. At least 10,000 events were analyzed. Atomic force microscopy Atomic force DNA ligase microscopy (AFM) is a useful tool for studying cell mechanics [20, 21]. Measurements of transversal stiffness in this study were conducted using a Solver P47-Pro instrument (NT-MDT, Moscow, Russia), in accordance with a technique which has previously been described in detail [22]. For each cantilever, the stiffness (N/m) was adjusted using the resonance position. When working in liquid, soft cantilevers were used with the stiffness coefficient of approximately 0.01 N/m. The contact mode was applied to record the force curves. The radius of curvature (r c) of the tips of all cantilevers used was assumed to be of 10 nm. Mechanical characteristics of cells were determined by obtaining the calibration force curve on the glass first in order to calculate the coefficient, which converts cantilever deflection expressed in units of current into units of distance-a (m/A).