Isolation of neonatal porcine islet tissue and transplantation into diabetic mice. A methodological evaluation

Abstract

Diabetes mellitus type I can be a disabling disease with a high risk of complications (i.e. neuropathy, atherosclerosis and nephropathy). Furthermore, the treatment with insulin can be complicated with severe episodes of hypoglycaemia.

As diabetes type 1 is caused by an autoimmune destruction of the islets of Langerhans, replacement of these islets could be the treatment of choice for this disease provided the immunological process is halted. Centres world wide perform islet allotransplantation (i.e. transplantation within species) as an experimental treatment of diabetes (Ricordi et al. 1992, Secchi et al. 1997, Warnock et al. 1992, Birkeland et al. 1995). However the results are poor with only 8 % of cases having more than one year of insulin independence after transplantation (international islet transplant registry, 1999).

Islet transplantation faces two major obstacles : rejection and shortage of human donors. Rejection is avoided by immunosuppression and in experimental models by immunoprotection of the grafts with micro-encapsulation or larger encapsulating devices (Lanza et al. 1995, Lacy et al. 1991, Monaco et al. 1991). Shortage of human donors has necessitated the search for alternative donors, and in this context the pig has drawn most attention, because the physiology of the pig is comparable to that of humans. Indeed, pig insulin has been successfully used for decades in the treatment of human diabetics. Pigs breed fast, have large litters and can be bred under standardised conditions, which is important, as factors such as strain and age influence the outcome of porcine islet isolations (Socci et al. 1990, Heiser et al. 1994).

By small modifications of the automated method developed for human islet isolation, it is possible to isolate large numbers of adult porcine islets (Ricordi et al. 1990). These islets are well functioning in vivo after transplantation to rodents. However, unlike human islets, the adult porcine islets have a very thin peri-insular capsule and are therefore very fragile disintegrating easily in overnight culture (van Deijnen et al. 1992, Warnock G.L. et al. 1995).

Islet tissue from foetuses possess a potential for growth, and has been extensively examined in the rat (Yderstraede et al. 1995), pig (Korsgren et al. 1988) and humans (Tuch et al. 1991). Foetal porcine islet tissue has even been transplanted to eight diabetic humans with histological signs of graft survival after three weeks, and porcine C-peptide production detectable up to eight months after transplantation, but without any detectable metabolic improvement in the patients (Groth et al. 1993).

Recently, a method has been developed for the isolation of islet tissue from neonatal pigs (Korbutt et al. 1996). The neonatal islet-like cell clusters (NICC’s) can be isolated in large numbers, and are able to differentiate and maybe even proliferate in vitro and in vivo.

The purpose of this study is to describe the methods for isolation, transplantation and in vivo evaluation of porcine neonatal islet-like cell clusters. Focus is on the transplantation procedure, but aspects of isolation and functional outcome in vitro and in vivo are also included.