MOMOSE N Toyama Prefectural University, Faculty of Engineering, Assistant Professor, 工学部, 助手 (80239590)
TADA Y Kanazawa University, Graduate school of Natural Science and Technology, Assistan, 自然科学研究科, 助手 (20179708)
TAKIMOTO A Kanazawa University, Faculty of Engineering, Professor, 工学部, 教授 (20019780)
|Budget Amount *help
¥6,300,000 (Direct Cost : ¥6,300,000)
Fiscal Year 1994 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 1993 : ¥1,600,000 (Direct Cost : ¥1,600,000)
Fiscal Year 1992 : ¥4,000,000 (Direct Cost : ¥4,000,000)
During freezing, the extra-and intra-cellular ice formation, osmotic water permeation through cell membrane, deformation of cell and other behaviors occur at microscale and they bring serious injuries connecting to the life-and-death of living cells. These micro behaviors occurred on at least cell size level seem to be due to the physicochemical complexity of biological materials. Water is chemically restrained in the states of aqueous solution and colloidal solution, and those are physically confined in small spaces or compartmentalized by the cross-linked network structures.
Firstly, simplified physicochemical model was proposed and the extra- and intra-cellular ice formation, osmotic water permeation through cell membrane and other microscale behavior durig the freezing of biological cell were discussed as a function of temperature considering the effect of cryoprotectant. Extending the simplified model proposed for the cell element, the freezing of the biological substances was anal
yzed to get basic understanding for developing the cryopreservation technique.
Furthermore, the freezing and thawing experiments were conducted to pursue the relation between the micro-behavior of biological cell and the injury during freezing and thawing. As a sample of biological cells, protoplasts isolated from cultured wheat cells were selectively used. As the results of microscopic observation using a cold stage whose cooling and warming velocities were controlled, the recovery of cell by water influx due to osmotic pressure difference, and the fusion of intracellular ice were clarified with warming velocity. The thawing injuries such as rupture and swell of the cells were detected, and it was found that the osmotic stress acting on the cell membrane causes these injuries. The survival of cells were also inspected by dye-exclusion test using Evans Blue. The results suggested the rapid temperature-rising is more harmful for slow-freezed cell, but also useful for rapid-freezed cell in which small ice formed.