Abstract
The effects of temperature dependency of cooling rate arising in the thickness direction during freezing on post-thaw cell viability in oriented cell monolayers simulating thick tissue implants of about 20 mm in thickness was investigated. Human dermal fibroblasts were cultured on a grooved substrate of 24 × 24 mm for 24 hours; a test sample of an oriented cell monolayer was prepared. This sample was placed perpendicularly in an experimental container, frozen from the bottom at 0.2, 0.3 or 0.5°C/min from 4 to -40°C, cooled to below -180°C, and then thawed. Post-thaw cell viability was evaluated. These conditions were designated as variation conditions. To determine the cooling rate in each part, temperature was measured during freezing in three regions corresponding to the thickness direction; 4, 12 and 20 mm from the lower end of the substrate. Furthermore, post-thaw cell viability of samples frozen with level placement and constant cooling rates was evaluated for comparison; these conditions were known as constant conditions. As a result, the cooling rate of each part had a convex curve as a function of temperature, and maximum deviation of the cooling rate between each region and the control region increased with distance from the lower end of the sample. Cell viability, in contrast to the decline in average cooling rate from -7 to -40°C under variation conditions, was much lower than that under constant conditions. Thus, the temperature dependency of cooling rate arising in the thickness direction affected post-thaw cell viability in thick tissue implants.
