Abstract: The limited availability of human-donors makes the search for alternative islet sources mandatory for future developments in pancreatic islet transplantation. In this study, we report on the massive isolation of bovine islets of proven in vitro and in vivo viability. The islets were prepared by collagenase digestion, sequential filtrations, and density-gradient purification by modifying a technique previously developed in our laboratory for the porcine pancreas. The prepurification yield was 2,743 +/- 78 islet equivalents (IE)/g pancreas (mean +/- SE), with a postpurification recovery of 78.7 +/- 2.2%. Purity ranged from 80 to 90%. The histological and immunocytochemical studies demonstrated the identity and integrity of the islets with well-preserved insulin-, glucagon-, and somatostatin-containing cells. The morphological integrity of cultured bovine islets was demonstrated for up to 4 weeks from isolation. Insulin secretion from freshly isolated islets was similar at 3.3 mmol/I glucose (0.36 +/- 0.06 pmol . IE(-1) . min(-1)) and at 14 mmol/I glucose (0.42 +/- 0.00 pmol . IE(-1) . min(-1)), and it increased significantly (P < 0.01) at 25 mmol/I glucose (1.44 +/- 0.12 pmol . IE(-1) . min(-1)). Arginine, theophylline, and propionic acid increased insulin secretion from freshly isolated islets at 3.3 and 14 mmol/I glucose, but not at 25 mmol/I glucose. Islets cultured at 37 degrees C in CMRL 1066 culture medium for at least 10 days were shown to become responsive to a lower glucose concentration, as demonstrated by the significant increase of insulin release in response to 14 mmol/I glucose, when compared with basal secretion. This recovered responsivity to glucose was maintained after 4 weeks of culture. Reversal of hyperglycemia was obtained in nude mice with streptozotocin-induced diabetes, after transplantation of similar to 1,500 bovine islets under the kidney capsule. Retrieval of the islet-bearing kidneys 28 days after implantation caused a return to hyperglycemia. Taken together, these results support the use of bovine islets as an alternative and readily available source of pancreatic endocrine tissue transplantation studies.
MASSIVE ISOLATION, MORPHOLOGICAL AND FUNCTIONAL-CHARACTERIZATION, AND XENOTRANSPLANTATION OF BOVINE PANCREATIC-ISLETS
MARCHETTI, PIERO;MASIELLO, PELLEGRINO;
1995-01-01
Abstract
Abstract: The limited availability of human-donors makes the search for alternative islet sources mandatory for future developments in pancreatic islet transplantation. In this study, we report on the massive isolation of bovine islets of proven in vitro and in vivo viability. The islets were prepared by collagenase digestion, sequential filtrations, and density-gradient purification by modifying a technique previously developed in our laboratory for the porcine pancreas. The prepurification yield was 2,743 +/- 78 islet equivalents (IE)/g pancreas (mean +/- SE), with a postpurification recovery of 78.7 +/- 2.2%. Purity ranged from 80 to 90%. The histological and immunocytochemical studies demonstrated the identity and integrity of the islets with well-preserved insulin-, glucagon-, and somatostatin-containing cells. The morphological integrity of cultured bovine islets was demonstrated for up to 4 weeks from isolation. Insulin secretion from freshly isolated islets was similar at 3.3 mmol/I glucose (0.36 +/- 0.06 pmol . IE(-1) . min(-1)) and at 14 mmol/I glucose (0.42 +/- 0.00 pmol . IE(-1) . min(-1)), and it increased significantly (P < 0.01) at 25 mmol/I glucose (1.44 +/- 0.12 pmol . IE(-1) . min(-1)). Arginine, theophylline, and propionic acid increased insulin secretion from freshly isolated islets at 3.3 and 14 mmol/I glucose, but not at 25 mmol/I glucose. Islets cultured at 37 degrees C in CMRL 1066 culture medium for at least 10 days were shown to become responsive to a lower glucose concentration, as demonstrated by the significant increase of insulin release in response to 14 mmol/I glucose, when compared with basal secretion. This recovered responsivity to glucose was maintained after 4 weeks of culture. Reversal of hyperglycemia was obtained in nude mice with streptozotocin-induced diabetes, after transplantation of similar to 1,500 bovine islets under the kidney capsule. Retrieval of the islet-bearing kidneys 28 days after implantation caused a return to hyperglycemia. Taken together, these results support the use of bovine islets as an alternative and readily available source of pancreatic endocrine tissue transplantation studies.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
