Highly efficient isolation of porcine islets of Langerhans for xenotransplantation: numbers, purity, yield and in vitro function.

UNLABELLED

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 Liberase PI and digestion-filtration. Average islet size was detected by dithizone staining of tissue sections prior to isolation; only organs with an average islet size > or = 200 microns were used. Density gradient purification with OptiPrep 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 IEQ/g) and old retired breeders (5,182 +/- 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 IEQ/g) 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 degrees C) prior to perifusion studies had no detrimental effect on insulin release. Long-term culture over 11 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.