Highly Efficient Isolation of Porcine Islets of Langerhans for Xenotransplantation: Numbers, Purity, Yieldand In Vitro Fundion
Mareike Krickhahn, Thomas Meyer, Christoph Buhler, Arnolf Thiede, Karin Ulrichs
Ann Transplant 2001; 6(3): 48-56
Xenogeneic transplantation of porcine islets of Langerhans is regarded as a potential future treatment for diabetes mellitus. Despite considerable biotechnological progress, however, it is still very difficult and often unreliable to isolate sufficient numbers of highly purified. intact islets from the porcine pancreas with good in vitro function. Objective of this study was to describe an efficient and reliable method to isolate sufficient numbers of highly purified islets of Langerhans with good in vitro function from adult as well as from young hybrid pigs. Methods: Islets were isolated from the pancreas of young (4-6 months) hybrid pigs and old (2-3 years) retired breeders using LiberasePI and digestion-filtration. Average islet size was detected by dithizone staining of tissue sections prior to isolation; only organs with an average islet size ~ 200pm were used. Density gradient purification with OptiPrep TM was performed in a COBE 2991 cell processor. Viability was investigated using fluorescence staining. Perifusion studies were carried out to asses in vitro function of isolated islets. Results: Islets were successfully isolated from young hybrid pigs (3.671 :!::598 IEQlg) and old retired breeders (5. I82:!::545 IEQ/g). After purification islet purity was 92% for retired breeders and 87% for young hybrid pigs. Yield after purification was still not satisfactory: 64% for retired breeders (3,209:!::444 IEQlg) and 44% for young hybrid pigs (1,669:!:: 386 IEQ/g). Viability of isolated islets was 80-95%. Perifusion studies of porcine islets showed sufficient insulin release upon glucose challenge; however, the level of insulin release depended on the density of islets within the perifusion chamber. Low temperature culture (24°C) prior to perifusion studies had no detrimental effect on insulin release. Long-term culture over II days was followed by a dramatic loss of islet function. Conclusions: If xenograft rejection can be overcome and the risk of xenosis can be minimised. sufficient numbers of purified porcine islets with good in vitro function can be isolated to serve as a potential source for islet transplantation in diabetic patients.
Keywords: Xenotransplantation, islet of langerhans, pancreas. pig, Swine, Isolation, enzymatic digestion