IMMEDIATE REVERSAL OF DIABETES IN PRIMATES FOLLOWING INTRAPORTAL TRANSPLANTATION OF PORCINE ISLETS PURIFIED ON A NEW HISTIDINE-LACTOBIONATE-IODIXANOL GRADIENT

Abstract 856

Information on islet xenotransplantation in relevant preclinical models is limited. One of the first issues to be addressed is whether porcine islets transplanted into primates are subject to hyperacute rejection. Previous work by others has emphasized the importance of density gradient media. To assure that technical factors do not compromise interpretation of early graft function in this model we sought to refine density gradient media such that immediate function of purified and cultured islets is consistently achieved in diabetic nude mouse recipients. Recently, we have shown large-scale isopycnic islet purification utilizing University of Wisconsin solution with iodixanol (UWI) facilitates immediate single-donor pig islet allograft function. However, histidine-lactobionate (HL) solution was shown to be superior to UW for cold storage of purified islets. Therefore, we developed new density gradient media based on HL and iodixanol (HLI). The purpose of this study is to compare HLI to UWI-gradient media and examine whether porcine islets purified with newly designed gradient media reverse diabetes promptly in nude mice and rhesus monkeys.

Methods. Porcine islets were isolated by the automated method. Free islets were separated from non-islet tissue utilizing continuous UWI or HLI gradients on a Cobe 2991 cell separator. After a 48-hr culture period, islet viability was assessed by a trypan blue uptake test and functional integrity in vivo was evaluated by renal subcapsular transplantation of 2,000 islet equivalents (IEQ) into diabetic nude mice. HLI-purified and 48-hr cultured islets (20,000 IEQ/kg) were transplanted intraportally into streptozotocin-diabetic rhesus monkeys.

Results. The percent recovery and purity of HLI purified islets were significantly higher compared to UWI gradient (100.0 ± 5.8% vs. 81.8 ± 5.0% p<0.05 and 93.0 ± 0.6 vs. 89.3 ± 2.8% p<0.03), respectively. Using HLI, culture recovery was significantly higher (73.9 ± 4.1% and 57.3 ± 2.5%, respectively (p<0.01)) and viability using trypan blue uptake test was also significantly higher (99.4 ± 0.1% vs. 96.8 ± 0.5% respectively (p<0.001)). All nude mice recipients of HLI-purified islets became normoglycemic within one day (n=8) in contrast to recipients of UWI islets (2.3 ± 0.5 days, p<0.02). Diabetes was reversed in 5 of 6 rhesus monkeys within 24 hrs after transplantation of HLI islets.

Conclusions. HLI gradients recovered viable porcine islets perfectly. Islets purified by HLI reverse diabetes promptly both in nude mice and rhesus monkeys and seem therefore adequate to be used in ongoing studies designed to clarify the mechanisms underlying islet xenograft rejection in primates.