| Budget Amount *help |
¥3,900,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥300,000)
Fiscal Year 2007: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
Fiscal Year 2006: ¥2,600,000 (Direct Cost: ¥2,600,000)
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| Research Abstract |
The purpose of this project was to investigate the influence of cellular orientation and contact state with high cell density on post-thaw viability of cells in cryopreserved biological tissue. The following four themes were carried out : 1) The survival distribution of cells by the fluorescence image after cryopreservation of the monolayer culture cell without the cell orientation which imitated biological tissue was investigated ; 2) The orientation control of the monolayer cell was established, the sample was frozen with changing the freezing direction, and the viability was evaluated ; 3) The relationship between the contact cause of high cell density with slow cooling in cell suspension and the viability was investigated ; 4) The cryopreservation state of the artificial tissue with rapid cooling by the thermophysical property was evaluated.
Human dermal fibroblast was used for all the experiments. The sample of monolayer cell with orientation control of cells was cultured on groove
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processing cover slip for two days (0.2 to 5×10^5 cells/cm^2, it is variable about the freezing direction by an inclination), the sample without orientation control of cells was cultured in the culture dish, in the themes 1) and 2). The sample was frozen in the temperature of 4 to 80℃ at a cooling rate of 0.1 to 10℃/min, it thawed after kept frozen below-185℃. Then two-dimensional survival distribution and cellular viability were investigated from digital image by fluorescence double dyeing of Calcein-AM and DAPI. On the same cooling conditions as the theme 1), the cell suspension(10^6 cells/cm^3) was frozen with avoidance of cell contact by using a high-polymer substance (existence), and post-thaw viability was evaluated in the theme 3). The effective thermal diffusivity in the cryopreserved cell of the sample artificial tissue which cultured the cell for two days in collagen sponge (20 mm in diameter and 1 mm in thickness, 10^6 and 10^7 cells/cm^3) at a cooling rate of 30 to 700℃/min was estimated, and it compared with post-thaw viability in the theme 4).
As a result, close-packed dead cells were locally observed in non-oriented samples after cryopreservation from the theme 1). The orientation control of cells in a monolayer cell is established ; and it was found that the difference in the freezing direction affects the viability after cryopreservation rather than in cellular orientation from the theme 2). The decreased viability by the contact following high density of the cell under slow cooling was clarified from the theme 3). In the rapid freezing of artificial tissue, it was suggested that a cell density did not participate in the decreased viability by an intracellular freezing from the theme 4). Thus it is suggested that the increase in the contact opportunity of cells causes the decrease of cellular viability after cryopreservation of biological tissue by slow cooling rate. Less
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