Viability of Densely Cultured Artificial Tissue in Collagen Matrix after Cryopreservation

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  • 1443 凍結保存を目的とした高密度培養人工組織の解凍後細胞生存率の評価

Abstract

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 collagen sponge (Φ20×1mm) as an extracellular matrix to imitate biological tissue (artificial tissue). 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 slow or fast cooling rate (either 1 or 50℃/min) in a solution of Dulbecco's Modified Eagle Medium, 20% Fetal Bovine Serum, and 10% dimethylsulfoxide, then kept frozen at -196℃ for 2 hours, and finally thawed. Collagen matrix of artificial tissue was resolved by using collagenase. Post-thaw viability of fibroblasts was evaluated by using a trypan blue exclusion assay. The experiments were prepeated, and then the latent heat of artificial tissue (3×3×1mm) during the freeze-thaw 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 constant. With increasing cell density at the slow cooling rate, temperature of the peak latent heat decreased and and the accumulated fraction of latent heat significantly increased, possibly leading to intracellular freezing. Moreover, when the cell density was high, dehydration was obstructed, and thus the number of fibroblasts exhibiting intracellular freezing increased, which in turn led to an increase in latent heat. Therefore, the post-thaw viability decreased as the number of cells exhibiting intracellular freezing increased.

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