Figure 1.

Schematic diagrams to show (A) research outline and (B) two vitrification procedures employed. (A) Functional revivability of cryopreserved rat pancreatic islets is evaluated by both in vitro and in vivo assays. Pancreatic islets are isolated by liberase digestion from Brown-Norway (BN) donor rats, and cultured for 24 h to remove debris damaged by the enzymatic digestion. Islets with a diameter of 101–200 µm are vitrified, stored in liquid nitrogen (LN₂), and warmed-up. The post-warm islets are stained with fluorescent diacetate (FDA) and propidium iodide (PI) for estimating the in vitro viability, or are subjected to glucose-stimulated insulin secretion (GSIS) assay for evaluating their insulin secretion ability. Furthermore, the post-warm islets are transplanted beneath the kidney capsule of diabetes model BN rats (Day-0), and blood glucose levels of the recipients are monitored. Recipient rats achieved normoglycemia are subjected to an intraperitoneal glucose tolerance test (IPGTT) on Day-30 and -60. The cured recipients are nephrectomized on Day-70 and checked whether they can regain the diabetic hyperglycemia. Extracted grafts are sectioned and stained with hematoxylin/eosin (H&E) for histological evaluation. (B) Rat islets are vitrified-warmed using either an NM device or an SF sponge disc. Islets are exposed for 3 min to equilibration solution (ES) containing the membrane-permeable cryoprotective agents (CPAs) at low concentrations, and then loaded on NM or SF device in vitrification solution (VS) containing the permeable CPAs at high concentrations and the non-permeable disaccharide, sucrose. Absorption by a paper towel is used to minimize the VS volume surrounding the islets in both procedures for islet vitrification. Within 70 sec, islets on the NM device can be cooled by direct immersion into LN₂ (minimum volume cooling [MVC] protocol), while islets on the SF sponge disc are cooled by placing on an aluminum boat floating on LN₂ (solid surface vitrification [SSV] protocol). After rapid warming and stepwise CPA dilutions, islets are retrieved from the cryodevice and kept for 2 h in culture medium (CM)