Islet-like cell clusters: viability, cell types, and subretinal transplantation in pancreatectomized cats

Abstract

We investigated a method for isolating sufficient feline islets of Langerhans to restore normoglycaemia following transplantation into the subretinal space of pancreatectomized cats. Collagenase digestate of feline pancreas was maintained in serum-free tissue culture medium for 1-9 days. Viability of islet-like cell clusters (ICC) was assessed with ethidium bromide and fluorescein diacetate staining; cell types were identified immunohistochemically. After nine days, the ICC were transplanted. We estimated viable ICC in tissue culture at nine days as 3800 +/- 2000 (mean +/- SD) per pancreas. While numbers of beta cells decreased over time in culture, ductal cells increased. Bromodeoxyuridine labelling showed no proliferation of beta cells but extensive proliferation of ductal cells. Subretinal transplants of cultured ICC in the diabetic cats maintained normoglycaemia for up to 12 days, while they provoked massive lymphocyte infiltration indicating rejection. Islet transplantation into the feline subretinal space temporarily restored normoglycaemia. Our current method of culture achieved sufficient reduction of acinar cells but an insufficient yield of insulin-producing cells.

Assessment of six different collagenase-based methods to isolate feline pancreatic islets

“…Isolation of pancreatic islets is mandatory in order to characterize in detail the pathogenesis of islet amyloid deposition and β-cell dysfunction and loss in diabetic cats. Isolation of feline islets has been described in only one report (Maeno et al, 2006). In this study, collagenase digestion led to the recovery of isolated islets that were surrounded by residual exocrine pancreas.…”

“…In this study, collagenase digestion led to the recovery of isolated islets that were surrounded by residual exocrine pancreas. It was suggested that feline islets are difficult to separate completely from the exocrine pancreas because they are delimited by very little peri-islet matrix (Maeno et al, 2006). We also sought to isolate islets from healthy cats using the same collagenase-based protocol and achieved similar disappointing results (Zini et al, in press).…”

Laser Microdissection of Pancreatic Islets Allows for Quantitative Real-Time PCR Detection of Islet-Specific Gene Expression in Healthy and Diabetic Cats

“…gene expression profiling. Feline islets have been considered particularly difficult to isolate with standard collagenase protocols due to a lack of peri-islet matrix, and previous isolation attempts have resulted in islets surrounded by a rim of exocrine tissue [10,11,24]. It is advantageous to minimize the amount of exocrine tissue contamination when performing gene expression studies on pancreatic islets.…”

“…It is difficult to isolate feline islets without contamination with exocrine tissue, and it has been suggested that feline islets are particularly difficult to isolate with collagenase digestion because they are delimited with very little peri-islet matrix [10,11]. This leads to either damaged islets, or islets coated with acinar cells (referred to in the literature as islet-like cell clusters) [11,12], which either way compromise the results obtained from gene expression studies.…”

“…This leads to either damaged islets, or islets coated with acinar cells (referred to in the literature as islet-like cell clusters) [11,12], which either way compromise the results obtained from gene expression studies. The use of the Laser Microdissection (LMD) technique can eliminate some of the problems inherent in the use of collagenase-isolated islets [13].…”