Development of a cryopreservation procedure employing a freezer bag for pancreatic islets using a newly developed cryoprotectant.

摘要

One of the most important requirements for success in clinical islet transplantation is the use of a large number of viable donor islets. To achieve this, the ability to cryopreserve islets and to establish an islet bank are critical. Previously, we developed a two-step cryopreservation procedure with freezing tubes utilizing low and high concentrations of dimethyl sulfoxide (DMSO) and using a fully automated cryomachine for human pancreatic islets and porcine islet-like cell clusters (ICCs). Based on these experiments, we developed a simple and efficient cryopreservation procedure of a freezer bag for isolated islets using a fully automated computer-controlled cryomachine with a newly developed cryoprotectant consisting of ethylene glycol (EG) instead of DMSO for decreasing injury of the islets by freezing. A 250 ml Cryocyte blood freezer bag and our newly developed cryoprotectant containing ethylene glycol (EG) were used in the freezing procedure. The islets were frozen by a fully automated computer-controlled cryomachine (GE 9,000) with our original program of slow cooling. Nucleation occurred at -8 degrees C, and the frozen islets were stored at -196 degrees C in a liquid nitrogen tank. The frozen-stored islets were subsequently rapidly thawed in a 37 degrees C water bath and cultured before viability testing. In vitro function, the stimulation index of insulin release during the static incubation test for rat islets cryopreserved in a freezer bag vs. nonfrozen islets as control, was 2.13 +/- 0.42 and 2.02 +/- 0.38 (94.8% compared with control), respectively (n = 5, p = NS). The islet recovery compared with the nonfrozen control group was 85% (n = 5) in insulin content. When 1000 rat islets cryopreserved in a freezer bag were transplanted into the renal capsule of diabetic athymic mice, all the mice became normoglycemic within 7 days from transplantation. Before nephrectomy, the intravenous glucose torelance test (IVGTT) was performed. The fractional decay constant of the glucose level (K value) of the frozen-thawed group was 0.42 +/- 0.06%/min. A histological study of renal subcapsular grafts demonstrated the morphological integrity of the islets. These results demonstrate the utility of our cryopreservation procedure of a freezer bag for isolated islets using a fully automated computer-controlled cryomachine with a newly developed cryoprotectant for the maintenance of viability and function of frozen-stored islets both in culture and after transplantation. Cryopreservation using freezer bags with the new cryoprotectant is an effective and simple method for making an islet bank for clinical trials of islet transplantation.