| Budget Amount *help |
¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 2000: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1999: ¥2,800,000 (Direct Cost: ¥2,800,000)
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| Research Abstract |
Cryosurgery is the use of freezing to destroy abnormal tissue, which is then left in situ to be sloughed or resorbed by the body. Despite a number of advantage of cryosurgery over traditional surgical tissue removal, few cases are reported in Japan recently because of unexpected low cure rate in early days in the past. Since the real-time monitoring of frozen region, that was the critical problem in the past, is now capable by using modern imaging method, one of the most important problems to make cryosurgery popular is to develop appropreate cryosurgical protocols. Theoretical and scientific background that provides a methodology to assess the protocol is the study on the mechansm of freezing damage in cell level. Recent studies show that during slow freezing of cells, in addition to chemical damage, the cells may be also injured by ice crystal compression induced mechanical damage. The goal of this study is to develop a quantitative understanding of cell destruction by deformation.
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new experimental procedure was developed to study the relation between the cell viability and deformation. In this method a drop of cell suspension with micro-glass beads with a precisely known diameter is placed on a glass plate, and the top cover slip is pushed toward the bottom glass substrate. A specially made stage is used to keep the two glass surfaces parallel during the compression process and to ensure that the procedures is done at a precisely controlled temperatures. The glass beads, which are randomly distributed throughout the solution, behave as spacers with precisely known dimensions. The cells become deformed between two flat surfaces and the distance between these surfaces is known from the diameter of the glass beads. The glass plates with the compressed cells are placed under the ocular path of microscope and viewed. The viability of the deformed cells is measured with a trypan blue dye exclusion assay and is defined by the ratio between the total number of cells and those that have taken up the dye.
Experiments were performed with a human prostatic adenocarcinoma cell line PC-3. The cell is almost spherical with mean diameter of about 18 micron. The viability of the deformed cells was measured at 0 ℃ and 23 ℃, and six different nominal gaps : 32.3, 16.2, 11.4, 8.8, 5.9, 3.5 microns. The data from the 32.3 micron gap experiments served for controls. The relation between measured viability and gap size for 0 ℃ and 23 ℃ are identical. While more than 80 % of cells survive deformation in a gap of 11.4 micron, 40 to 50 % are destructed at 5.9 micron and 90 % are destroyed when the gap size is reduced to 3.5 micron. These results show that about half of cells are destroyed when cells are squeezed to 30 % of their original diameter. If uniform expansion of cell membrane is assumed, this corresponds to 50 % increase in the cell membrane surface area. The fact that the viability at 0 ℃ and 23 ℃ is essentially identical is extremely interesting. At 23 ℃ the cells are above the cell membrane lipid phase transition temperature and at 0 ℃ they are below the lipid phase transition temperature. The observation that the deformed cell viability is independent on temperature may therefore suggest that deformation damage is not related to the mechanical properties of the lipid membrane. Therefore a possible mechanism of damage during freezing cell compression may be related to the deformation of the cytoskelton. Less
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