The effect of cell density on the post-thaw viability of cells in cryopreserved artificial tissue was studied. Human fibroblasts were three-dimensionally cultured for 2 days in a collagen sponge (ϕ20×1mm) as an extracellular matrix to imitate biological tissue (artificial tissue). Different cell densities for the artificial tissue were used, from 104 to 107 cells/cm3. Four artificial tissues were first stacked in a test chamber, then frozen at a slow or fast cooling rate (either 1 or 50°C/min) in a solution of Dulbecco’s Modified Eagle Medium, 20% Fetal Bovine Serum, and 10% dimethylsulfoxide, then kept frozen at −196°C for 2 hours, and finally thawed. The collagen matrix of the artificial tissue was dissolved using collagenase. Post-thaw viability of fibroblasts was evaluated by using a trypan blue exclusion assay. The experiments were prepared, and then the latent heat of artificial tissue (3.5×3.5×1mm) during the freezing process was measured by using a differential scanning calorimeter. Results show that with increasing cell density, the post-thaw viability decreased, whereas the latent heat was almost independent of cell density. With increasing cell density at the slow cooling rate, the degree of supercooling of the intracellular solution increased with decreasing temperature, possibly leading to intracellular freezing. Moreover, when the cell density was high, cell-to-cell contact or an obstruction to dehydration seemed to induce intracellular freezing. Therefore, the post-thaw viability seems to decrease as the number of cells exhibiting intracellular freezing increased.

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