Abstract
The effect of area cell density on the post-thaw viability of cells in cryopreserved monolayer cells was studied. Human fibroblasts were two-dimensionally cultured for 1 day in a culture dish (Φ35mm). Different cell densities for the artificial tissue were used, from 2×(10)^3 to 5×(10)^4 cells/(cm)^2. Cultured monolayer cells was first stacked in a test chamber (Φ35×12mm), then frozen at a cooling rate of 0.3 to 30℃/min from 4 to-80℃ in a solution of Dulbecco's Modified Eagle Medium, 20% Fetal Bovine Serum, and 10% dimethylsulfoxide, then cooled down to -160℃ and lower, and finally thawed. Monolayer cells were suspended using trypsin. Post-thaw viability of fibroblasts was evaluated by using a trypan blue exclusion assay. Results show that with increasing area cell density, the post-thaw viability decreased, and contact of the cells was observed. Contact of the cells may induce intracellular freezing or give mechanical stress to a cell during freezing, and therefore the post-thaw viability seems to decrease.
