Manning Fox et al. (2015) present in vivo and in vitro human islet cell functionality data from cryopreserved biosamples stored for up to 20 years.1 The results show the potential value of historical biosamples for research and clinical treatment from biobanked human pancreatic tissue preparations.
For research into type 1 diabetes mellitus and onward translation into clinical practice, researchers need to characterize human islet cell function in general and beta cell performance in particular. Although it is possible to harvest islets, having sufficient numbers for consistent and valid experimental design is problematic since both long-term banking and maintenance in culture have proven difficult. Maintaining cell phenotype beyond 60 days in vitro is not possible, and insulin secretion functionality is lost at day 28. In addition to research, clinical scientists note the ongoing success of transplantation models for managing type 1 diabetes; development of efficient storage protocols to maintain harvested human islet cells will be imperative for future programs.
Manning Fox et al. describe the first steps in this process, reporting functionality and viability testing in human islets banked between 1991 and 2001 (median age = 17.6 ± 0.4 years) in the University of Alberta’s cryopreservation collection. They note that since the biosamples are stored in fetal bovine serum (FBS) under liquid nitrogen, this collection is suitable only for research purposes.
The team thawed the islet cells between 2012 and 2014, bringing them rapidly from −196°C to 4°C at 150°C/minute before removing the dimethyl sulfoxide cryoprotectant by serial dilutions and a sucrose gradient. With this methodology, Manning Fox et al. hoped to limit osmotic damage. Following thawing, the research team cultured the cells overnight in CMRL 1066 medium supplemented with 0.5% bovine serum albumin, among other additives. They followed this with 24 hours in Dulbecco’s Modified Eagle Medium plus 10% FBS.
Comparing the thawed human islet samples with freshly harvested cells, the researchers examined morphology, physiology and viability to characterize cryopreserved human islet function. Immunostaining showed no alteration in cell morphology, with no increase in apoptosis. Compared to the freshly harvested cells, banked cells did show a reduction in insulin content; however, secretion profiles were similar. Further characterization using patch-clamp electrophysiology and transcriptome analysis showed only minor differences between banked and fresh cells.
Next, the researchers examined banked islet cell functionality in vivo by transplanting the thawed biosamples into a streptozotocin-induced mouse diabetes mellitus model. They found that transplantation improved oral glucose tolerance testing in recipient animals, although they did not achieve normoglycemia. At 10 weeks post-transplantation, Manning Fox et al. could detect circulating human insulin in the mice and found insulin-positive grafts at the surgical sites.
The researchers conclude that even after almost 20 years in cryopreservation, human islet cells are still viable and capable of secreting insulin. Moreover, they retain in vivo functionality upon transplantation. Although they show some variability in insulin response compared to the freshly harvested islet controls, Manning Fox et al. consider that cryopreserved human islet cells are a valid source of material for clinical researchers. Furthermore, since research methodology and techniques advance rapidly with a concurrent reduction in experimental costs, historical samples such as these increase in value, thus proving the argument for extensive and ongoing biobanking for clinical research.
Don’t throw anything away!
1. Manning Fox, J.E., et al. (2015) “Human islet function following 20 years of cryogenic biobanking,” Diabetologia, 58(7) (pp.1503–1512), doi: 10.1007/s00125-015-3598-4.