Investigation of the Influence of Cell Density of Human Fibroblasts Cryopreserved Inside Collagen Sponges at Various Cooling Rates

The influence of cell density of cells cryopreserved inside a collagen matrix at various cooling rates was investigated. 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 to imitate biological tissue (artificial tissue). Different cell densities for the artificial tissue were used, from 10⁵ to 10⁷ cells/cm³. Four artificial tissues were first stacked in a test chamber, frozen at a cooling rate of 0.3 to 50°C/min in a solution of Dulbecco's Modified Eagle Medium, 20% Fetal Bovine Serum and 10% dimethylsulfoxide, kept frozen below – 185°C for 2 hours, and then finally thawed. Membrane integrity of fibroblasts using a trypan blue exclusion assay was evaluated as an index for post-thaw cellular viability. Results show that with increasing cell density, the post-thaw membrane integrity decreased. Therefore, in the cryopreservation of biological tissue, it seems high cell density is one factor which causes a decline in viability.