| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2000: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1999: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Research Abstract |
In this study, I clarified the effect of cell density on the post-thaw viability of cells in cryopreserved artificial tissue (Imitation of the inside of the hybrid artificial organ), and whether microencapsulated cells (one of the hybrid artificial organ) have an advantage over suspended cells in cryopreservation.
Firstly, human fibroblasts were three-dimensionally cultured for 2 days in a collagen sponge (20 mm in diameter and 1 mm in thickness) as an extracellular matrix. Different cell densities for the artificial tissue were used, from 10^4 to 10^7 cells/cm^3. Four artificial tissues were first stacked in a test chamber, then frozen at a cooling rate of 0.3 to 50℃/min in a solution (culture medium) containing 10% dimethylsulfoxide, then kept frozen at -196℃ for 2 hours, and finally thawed. After dissolving the collagen matrix, post-thaw viability of fibroblasts was evaluated by using a trypan blue exclusion assay. Results show that with increasing cell density, the post-thaw viability decreased, and the most suitable cooling rate for high viability shifted to the low cooling rate side. When the cell density was high, cell-to-cell contact or an obstruction to dehydration seemed to induce intracellular freezing.
Secondly, rat pheochromocytoma (PC12) cells were microencapsulated in alginate-polylysine-alginate membranes. Microencapsulated PC12 cells were frozen with differential scanning calorimetry at a cooling rate of 0.5 to 10℃/min over 4 to -80℃, and their latent heat was measured. The post-thaw viability was evaluated with dopamine release. As a result, latent heat of encapsulated cells was lower than that of suspended cells at 0.5 and 1℃/min. The post-thaw viability of microencapsulated PC12 cells was improved at 0.5 and 1℃/min compared with that of suspended cells. Therefore, in microencapsulated PC12 cells, achievement of intra-capsules unfrozen condition during freezing leads to reducing the solution effect and improving the post-thaw viability.
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