Effect of the cooling rate on insulin release from frozen-thawed In-111 cells

Cultured cells derived from hamster insulinoma (In-111 R1 cells) were placed in 1.4 M dimethyl sulfoxide (Me2SO)-containing RPMI 1640 at 20 degrees C for 20 min. They were frozen to -40 degrees C at a cooling rate of 1.0 or 0.5 degrees C/min, subsequently to -80 degrees C at 3 degrees C/min with a programmable freezer. After being maintained at -80 degrees C, they were rapidly thawed to 37 degrees C. Thawed cells were washed with 0.75 M sucrose for removal of Me2SO. Recovered cells were cultured in 2 ml of RPMI 1640 with 1.3 mM theophylline under a gas phase of 95% air -5% CO2 at 37 degrees C for 2 days. In both cooling rates, frozen-thawed cells discharged more insulin than the thawed in the absence of theophylline. However, this released insulin level was higher in the cells frozen at a cooling rate of 0.5 degrees C/min than that at 1.0 degrees C/min. Moreover, insulin released from frozen-thawed hamster insulinoma cells increased significantly with the addition of 1.3 mM theophylline. Considering that the higher insulin release level at 11.1 mM glucose alone might indicate cellular damage, it is suggested that the cooling rate of 1 degree C/min may be better for cryopreservation of the dispersed cells under the present protocol for the assessment of the function of insulin release.