Follow-Up Study of the Revascularization Process of Cryopreserved Islets of Langerhans

Abstract

Cryopreservation of islets of Langerhans is a necessary procedure since human pancreatic islet transplantation has become a reality for the clinical treatment of Type I, insulin-dependent diabetes mellitus. Although successful cryopreservation of rodent and human islets is a well-established technique for islet storage after isolation and purification, little is known about the influence of the freeze–thaw procedure on the islets’ potential to induce angiogenesis and revascularization, a major process necessary for the viability of grafted cells. In this study, the revascularization process of cryopreserved islets transplanted in the liver and in the renal subcapsular space of diabetic and nondiabetic rats is analyzed by a double indirect immunofluorescence technique. Frozen–thawed pancreatic islets were cooled slowly to −40°C, stored at −196°C, and thawed rapidly. Lewis rats were grafted with either Lewis (isografts) or Wistar (allografts) overnight-cultured and frozen–thawed islets obtained by collagenase digestion. Rats were killed different days after implantation, and the livers and kidneys bearing the grafted islets were snap-frozen and immunohistochemically stained with a double immunofluorescence technique using a rabbit anti-factor VIII antiserum (which labels endothelial cells) and a guinea pig anti-insulin antibody. Overnight-cultured islet grafts completed revascularization by Days 4–7 after transplantation, as shown by the detection of endothelial cells within and surrounding the islets. The identical staining pattern of revascularization was observed in islets frozen–thawed before transplantation. It is concluded that islet cryopreservation is a suitable technique for long-term storage prior to transplantation since it does not interfere with the neovascularization process of islet grafts.