Review of vitreous islet cryopreservation: Some practical issues and their resolution

Abstract

Transplantation of pancreatic islets for the treatment of diabetes mellitus is widely anticipated to eventually provide a cure once a means for preventing rejection is found without reliance upon global immunosuppression. Long-term storage of islets is crucial for the organization of transplantation, islet banking, tissue matching, organ sharing, immuno-manipulation and multiple donor transplantation. Existing methods of cryopreservation involving freezing are known to be suboptimal providing only about 50% survival. The development of techniques for ice-free cryopreservation of mammalian tissues using both natural and synthetic ice blocking molecules, and the process of vitrification (formation of a glass as opposed to crystalline ice) has been a focus of research during recent years. These approaches have established in other tissues that vitrification can markedly improve survival by circumventing ice-induced injury. Here we review some of the underlying issues that impact the vitrification approach to islet cryopreservation and describe some initial studies to apply these new technologies to the long-term storage of pancreatic islets. These studies were designed to optimize both the pre-vitrification hypothermic exposure conditions using newly developed media and to compare new techniques for ice-free cryopreservation with conventional freezing protocols. Some practical constraints and feasible resolutions are discussed. Eventually the optimized techniques will be applied to clinical allografts and xenografts or genetically-modified islets designed to overcome immune responses in the diabetic host.

Keywords: cryoprotectants; islet banking; islet cryopreservation; islet vitrification; pancreatic islets; rat islets; vitrification.

Figure 1

Plots of normalized cell volume showing the transient volume changes in islets during cryoprotectant addition (steps 1–3) and removal (steps 4–8) for the vitrification solutions VS55 and DP6-1,3 CHD. The protocols for both vitrification solutions do not exceed osmotic tolerance limits for islets of 0.6 V/V_iso during CPA addition and 1.53 V/V_iso during removal.

Figure 2

Metabolic activity of isolated rat islets normalized to untreated controls after either 2 hr or 24 hr recovery following exposure to combinations of the VS55 or DP6-1,3 CHD CPA cocktails prepared in either EuroCollins (EC) or Unisol-UHK vehicle solutions.

Figure 3

Islet tolerance to CPA exposure without subsequent sub-zero cooling and vitrification. Data is plotted as mean (±SEM) normalized stimulated insulin secretion after either 2 hr or 24 hr recovery.

Figure 4

Gross morphology of isolated rat islets showing their comparative integrity following cryopreservation and compared with untreated control islets (A). Islets were cryopreserved using either the conventional freezing/thawing method (B) or vitrification in DP6-1,3 CHD (C) or VS55 (D).

Figure 5

Functional recovery of batches of rat islets following vitrification in either VS55 or DP6-1,3 CHD media compared with conventional freezing and thawing in 2 M DMSO (CRYO). Islet function is shown as the mean (±SEM) stimulation index normalized to untreated control islets. Batches of islets were assessed for stimulated insulin secretion either within 2 hr of rewarming to 37°C or after 24 hr in culture.