Modified procedure of a direct in vitro exposure system for mammalian cells to whole cigarette smoke

Summary In vitro biological studies on cigarette smoke have usually been made using either cigarette smoke condensate – obtained by trapping the particulate phase of smoke on a filter, or soluble smoke components – obtained by trapping cigarette smoke in buffer solution. However, these approaches may not truly reflect the physical and chemical condition of freshly generated smoke. Clearly it is important to be able to evaluate the biological effects of fresh smoke on mammalian cells for a better understanding of the potential effects of smoking. The CULTEX™ technology is a new experimental system for cultivation and exposure techniques enhanced the efficiency of in vitro studies, and allows direct exposure of cells intermittently at the air/liquid interface with ultrafine particles, gases, or mixtures of both which fixedly flows. The CULTEX™ technology has therefore been modified to evaluate the biological effects of whole cigarette smoke in an in vitro system. The exposure system design was based on a combination of the sedimentation procedure and the CULTEX™ cultivation technique. After freshly generated smoke was delivered onto cells, the flow was shut off and the medium was slowly removed. In this manner, cells were exposed to both the vapor and particulate phase of smoke efficiently. Cells were maintained in the liquid medium except during the exposure period to maintain the culture conditions and to protect the cells from both the influence of puff pressure and the airflow, which served to remove residual cigarette smoke. The medium was changed at every puff of smoke and so effectively eliminating the possibility of any effects caused by accumulation of soluble cigarette smoke components into the medium. This cycle was repeated and cells were exposed to freshly generated cigarette smoke intermittently. The direct in vitro exposure system was modified to measure the effects of whole cigarette smoke on mammalian cells. It was confirmed that this system is effective for in vitro studies of cigarette smoking by dose-response relationship and reproducibility, discrimination of vapor phase and whole smoke, and the evaluation on the vapor phase components.