In vitro stability of insulin-loaded erythrocytes after freezing storage

In order to clarify the utility of resealed erythrocytes as a cell carrier of insulin the freezing stabilities of loaded cells encapsulating two different amounts of insulin (about 0.1 and 7.0 IU/ml of packed cells) were investigated in an anti-freezing reagent containing a high concentration of glycerol at -80 degrees C. After storing for 1 week under the above condition the preparation was thawed at 37 degrees C. Although the osmotic resistance of the loaded cells was lowered more than that of intact cells, the freezing and thawing treatment did not change the resistance of the loaded cells to osmotic shock. Recoveries of hemoglobin from loaded cells with a low insulin content (0.1 IU/ml of cells) and the cell volume after freezing and thawing were similar to those from intact cells and the cells resealed without insulin. However, the recovery of insulin from the cells with a high insulin content (7.0 IU/ml of cells) was the same as that from one with a low insulin content (about 16%). In the in vitro stability experiment at 37 degrees C the insulin content in the loaded cells decreased according to a biexponential function. The considerable fraction of the loaded cells were lysed by freezing and thawing. In the in vitro stability experiment the decline curve of insulin in the cells showed apparently monoexponential kinetics. From these results, it was established that insulin-loaded erythrocytes may be stored in a high concentration of glycerol under freezing at -80 degrees C, although a partial decrease in the stability of cells was observed after thawing.