STUDY OF EFFECTS OF HIGH FREQUENCY VIBRATION ON PROLIFERATION OF HUMAN PRIMARY CELLS AND ON MOUSE CELL LINES WITH xCELLigenceTM SYSTEM

INTRODUCTION In recent years, vibrating platforms have become increasingly available and used at sports and rehabilitation institutes. In fact, Bosco’s studies regarding whole body vibration (WBV) demonstrated efficacy of this treatment on muscles [1,2], so it’s very widespread in fitness centre and in sportsman training. On the other hand, WBV is also used in clinical therapy and medical rehabilitation for bone diseases, such as osteoporosis and others bone and muscle senile diseases [3,4]. Moreover, intracellular metabolic pathways are influenced by the excitation of cytoskeleton components by mechanical cell deformation [5]. Consequently, it’s useful to investigate these effects on in vitro cells cultures. In particular, we focused on the effects of the stimuli on proliferation of established human primary cell lines and mouse cell lines. We used human bone marrow stromal cells (hBMSC), freshly derived, and SAOS-2 cells, an osteogenic human cell line and C2C12 and L6C11 as mouse cell lines. To evaluate rate of proliferation of cells we used xCELLigence™ sytem, that monitors cellular events in real time without the incorporation of labels. The RTCA SP system measures electrical impedance across interdigitated micro-electrodes integrated on the bottom of 96-multiwell culture plate. A dimensionless parameter termed Cell Index (CI) is derived as a relative change in measured electrical impedance to represent cell status, in our case cell proliferation. We treated hBMSC, SAOS-2, C2C12 and L6C11 cells for 1h/day for 1 week at 30 Hz and we evaluated proliferation levels of treated cells against not treated control cells. Results demonstrated that high frequency vibration (HFV) slows down proliferation of cells, as indicated by graphics below. MATERIALS AND METHODS The device to produce the vibrating stimuli to in vitro cells cultures is composed by an eccentric, voltage-controlled motor effecting a displacement of 11 mm and working between 1 Hz and 120 Hz, and by a plate bound to the motor that allows to stimulate dishes for cells. Cells were stimulated with high frequency vibration (HFV) at 30 Hz for 1 week, for 1h/day. During this week Real Time Cell Analyzer (RTCA) SP was used to supervise proliferation level of each type of cells. The RTCA SP station (Fig. 2) is located inside a tissue culture incubator and it is capable of switching on any of the wells on the 96-multiwell plate. For each cell type (in dodecuplicate), we seeded 5000 cells/cm2 (980 cells/well) in 180 μl of DMEM 10% FCS in E-Plate 96. Cells proliferation was monitored for 1 week, both for control and treated multiwells, with an impedance measurement every minute. Results from the cell-sensor impedance were expressed as an arbitrary units called Cell Index (CI). In this experiment we tested effects of HFV on proliferation of human and mouse cell lines using xCELLigence™ system to evaluate every minute changing in cells growth. As results demonstrate, treated cells shown a proliferation rate lower than control cells (Fig. 2, 3, 4, 5), in fact mechanical vibration seems to decelerate cell growth. In Fig.2, 3, 4 and 5 proliferation curves of each cell type are represented with red curves that symbolize dynamic cultures and blue curves that represente static ones. In every graphics both curves start from the same point (25 hours), because we waited for 1 day that cells adhered completely to multiwells, and they finished after 1 week of analysis. Despite differences of curves due to cell type distinctions, we can see that dynamic curves are always lower than static curves for all the duration of the treatment with HFV. Probably, mechanical vibration enhances differentiation and fusion of cells rather than proliferation and growth. This experiment represent a preliminary investigation of the effect of HFV at cellular level. Moreover it can establish the starting point for further studies on effects of this stimuli on cells and tissues.