| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2006: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2005: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Research Abstract |
It is important to distinguish living cells from dead ones in cell culture area, especially in regenerate medicine field including cell therapy, since those cells are usually in short supply and consequently the ex-vivo culture process should be operated strictly. Conventional methods for distinguishing a living from dead cell usually require labeling of the cell with a dye, which spoils the culture. For a living cell, cell activities should be accompanied by biochemical reactions and materials movements across the cell membrane, i.e., certain materials move into the cell (e.g., nutrients and oxygen) and out of the cell (metabolic products and CO2). The materials movements generate change in concentration gradients near the cell membrane. The change of the concentration gradient induces a change in refractive index gradient, which in turn generates change in deflection angle of the probe beam passing through a vicinity of the cell membrane. However, when the cell is dead, no or little
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material movement of a across the cell membrane occurs. Accordingly, no or little change in deflection angle of the probe beam is generated. Therefore, whether a cell is alive or dead can be diagnosed by determining and analyzing deflection signal of the probe beam.
In this study, we developed a simple non-invasive method for diagnosing whether a cell is dead or alive with a probe beam. Human hepatic cell lines HepG2 are cultured. The culture dish was placed on a platform with a micro-stage in a microscope for detection of beam deflection. A probe beam from a diode laser was introduced into the microscope. The probe beam was reflected inside the microscope, and then focused to a vicinity of cell membrane of a cultured lung cell by an objective lens. The deflection signal was monitored for about 5 min. Then, the culture dish was moved by the micro-stage and the probe beam was focused to vicinity of another cell. This operation was repeated. After deflection signals from 17-20 cells were obtained in 2 hours, trypan blue was added into the culture dish and the conventional trypan blue exclusion method was used for determination of viability of the cells. Finally, deflection signals from dead cells only were determined for comparison with living cells. Judgment on whether a cell is dead or alive from the deflection signal agreed with the conventional decision.
Capillary electrophoresis was used for identification of the difference in the components of cell cultures containing living and dead cells, respectively. Furthermore, in-capillary preconcentration methods for capillary electrophoresis of determinations of trace components have been developed. Less
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