Abstract
Aims/hypothesisThere are potential advantages to the low-temperature (−196°C) banking of isolated islets, including the maintenance of viable islets for future research. We therefore assessed the in vitro and in vivo function of islets cryopreserved for nearly 20 years.MethodsHuman islets were cryopreserved from 1991 to 2001 and thawed between 2012 and 2014. These were characterised by immunostaining, patch-clamp electrophysiology, insulin secretion, transcriptome analysis and transplantation into a streptozotocin (STZ)-induced mouse model of diabetes.ResultsThe cryopreservation time was 17.6 ± 0.4 years (n = 43). The thawed islets stained positive with dithizone, contained insulin-positive and glucagon-positive cells, and displayed levels of apoptosis and transcriptome profiles similar to those of freshly isolated islets, although their insulin content was lower. The cryopreserved beta cells possessed ion channels and exocytotic responses identical to those of freshly isolated beta cells. Cells from a subset of five donors demonstrated similar perifusion insulin secretion profiles pre- and post-cryopreservation. The transplantation of cryopreserved islets into the diabetic mice improved their glucose tolerance but did not completely normalise their blood glucose levels. Circulating human insulin and insulin-positive grafts were detectable at 10 weeks post-transplantation.Conclusions/interpretationWe have demonstrated the potential for long-term banking of human islets for research, which could enable the use of tissue from a large number of donors with future technologies to gain new insight into diabetes.
Highlights
The ability to preserve human islets for an extended period of time has several benefits
Immunofluorescence staining revealed the expression of both insulin-positive beta cells and glucagon-positive alpha cells (Fig. 1c), which appeared to be similar in their distribution to previous reports [33]
Cryopreserved beta cells retain ion channel function and exocytotic responsiveness A critical component of beta cell function is the electrical activity mediated by the ion channels, leading to Ca2+ influx and the exocytosis of insulin-containing granules [38, 39]
Summary
The ability to preserve human islets for an extended period of time has several benefits. These may include increasing the practicality of islet transplantation for type 1 diabetes by improving the prospects for pre-transplant testing, simultaneous multiple donor transplants and simplified logistics. The successful storage of functional islets has not been achieved beyond 2 years—far too short a time for successful long-term biobanking. Culturing islets extends their viable lifespan to a certain degree, but long-term maintenance of the phenotype has proved problematic [3, 4]. The transplantation of a mixture of fresh and cryopreserved islets into patients with type 1 diabetes receiving kidney transplants demonstrated long-term graft function, with one patient becoming independent of insulin for 2.5 years [13, 14]
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