| Budget Amount *help |
¥9,500,000 (Direct Cost: ¥9,500,000)
Fiscal Year 1991: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1990: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1989: ¥7,700,000 (Direct Cost: ¥7,700,000)
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| Research Abstract |
The human neuroblastoma cell line, KP-N-RT, was used. The medium was supplemented with 10 % fetal bovine serum. Minivitafiber unit (Grace Japan, Amicon) was chosen in this study. Cells were introduced into the hollow-fiber unit and allowed to deposit and reattach for a few hours, and then the intracapillary space (ICS was begun to be perfused. The perfusion of the extracapillary space ECS) was started 24 hours after inoculation. A peristaltic pump was used to maintain the flow rate of 25 ml/min in ICS and 8 ml/hr in ECS. Phosphorus-31 nuclear magnetic resonance ( ^<31>P-NMR) spectra were obtained on CSIII (GE) operating at 81 MHz for phosphorus. The spectral conditions were a 60ﾟ pulse, a 2.0 recycle time. Starling 14 days after inoculation of the culture unit, prominent spectra could be obtained by 2000 scans on intact cells aerated by continuous medium perfusion 37ﾟC. Peaks were phosphomonoesters (PME), inorganic phosphate (Pi), phosphopdiesters (PDE), phosphocreatine (PCr), gamma -adennosine triphosphate (ATP), alpha -ATP and beta -ATP.
The ^<31P>-NMR spectra of human neuroblastoma cells were obtained on intact cells cultured in hollow fiber. The spectra were dominated by phosphomonoester peaks. The perfusion system was developed to achieve the high cell density and maintain viable intact cells for NMR study.
This system would be useful for study of energy metabolism and phospholipid metabolism in culture cells.
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