Wlodek Lopaczynski, Virginia Chung, Troy Moore, Jaime Guidry, Linda Merritt, Mark Cosentino and Kathi Shea. BBI Biotech Research Laboratories, 217 Perry Parkway, Gaithersburg, Maryland.

PBMCs are typically stored in the vapor phase of liquid nitrogen (<-150oC). It is quite possible that cryopreserved samples would frequently be exposed to GTTW when retrieval of specimens is required, especially from large active repository collections. We have determined the rate and the extent to which frozen samples will warm when removed from a liquid nitrogen freezer. A rack containing mock samples was removed and laid on top of the open freezer. Using our computerized freezer monitoring system, we showed that the mock samples’ temperature, after only 5 minutes in this position, had increased by between 14oC to 55oC, which equaled an average temperature of –114oC. This increase was well above the GTTW (-132oC).

Given these dramatic temperature effects observed with mock samples, we cycled viably cryopreserved PBMCs through the GTTW to observe the effects on apoptosis. Using a programmable rate-controlled freezer, we exposed PBMC samples to one complete cycle of temperature change (–150oC to –114oC) for 10 min and then back to –150oC. Next, we exposed aliquots to multiple temperature cycles (10, 20 and 40). All samples were then evaluated for the occurrence of apoptosis using Hoechst staining. There was no significant difference in the number of positively stained cells (% apoptotic nuclei) from PBMCs stored at -150oC compared to those that underwent 1, 10, 20 and 40 multiple temperature cycles.

Consequently, we decided to examine this issue by incubating frozen PBMC samples at –114oC for a longer time (e.g., 48 hours) and then returning them to –150oC. Apoptosis values were significantly higher from PBMC samples held at –114oC than in PBMCs stored for the duration of the experiment at -150oC.

Our data illustrate that the quality of cryopreserved samples was negatively affected when exposed to a warmer temperature for 48 hours. We conclude that apoptosis occurred due to temperature fluctuations around the GTTW (–132oC). Similar findings were obtained when PBMCs where stored at –80oC for 48 hours.