Automated Islet Isolation From Human Pancreas

Abstract

An automated method for the isolation of human pancreatic islets is described. The procedure meets the following requirements: 1) minimal traumatic action on the islets, 2) continuous digestion in which the islets that are progressively liberated can be saved from further enzymatic action, 3) minimal human intervention in the digestion process, and 4) high yield and purity of the isolated islets. After purification of Ficoll gradients, an average of 2279 islets/g pancreas was obtained, with an average purity of 79% islets. The average volume and average insulin content of the final islet preparation were 348 mm3 and 93.4 U, respectively. The islets were morphologically intact with a normal degree of beta-cell granulation, responded to glucose stimulation with a fivefold increase of insulin secretion over basal levels, and produced normoglycemia after transplantation into diabetic mice. The procedure is being used in the second phase of clinical trials of islet transplantation in patients with insulin-dependent diabetes mellitus.

Factors Influencing the Loss of β-Cell Mass in Islet Transplantation

“…The development of the "automated method" for with recent modifications of the Edmonton Protocol, 1-year insulin independence rates are approximately 70-high-yield islet isolation by Ricordi in 1989 was a major turning point, and a subsequent series of surgical pa-80% after completed transplants (60). Similar results have been reported by the Miami-Baylor collaboration, tients achieved 50% rates of insulin independence with single-donor islet infusions after cluster islet-liver transwhere 1-year insulin independence rates of 90% have been achieved in a series of more than 30 patients (16, plants for abdominal malignancies, albeit in the setting of nonautoimmune diabetes (56,74). Groups in Giessen, 23,26,54).…”

“…The islets are then purified over a sepathophysiological stress results in the failure of transries of gradients and maintained in culture until transplanted islets to become established (5). plantation (55,56). This process is extremely stressful to the islets, and the β-cells specifically are very sensitive HYPOXIA DURING ISOLATION and easily lost.…”

“…Unlike models, examination of islet grafts in normoglycemic resolid organ transplantation, where the donor tissue is cipients has shown that a majority of the islet tissue is delicately handled and maintained at 4°C until being restill apoptotic (8). Taken collectively, the fact that a maperfused in a recipient, a pancreas selected for islet jority of the islet mass is lost rapidly following transtransplantation must be perfused with digestive enzyme, plantation into normoglycemic, syngeneic, or immunomechanically digested, and shaken for up to 30 min at compromised recipients strongly suggests that a significant 37°C (37,56,73). The islets are then purified over a sepathophysiological stress results in the failure of transries of gradients and maintained in culture until transplanted islets to become established (5).…”

Chromatin-bound mitogen-activated protein kinases transmit dynamic signals in transcription complexes in β-cells

“…Islets were isolated from human cadaveric pancreata according to methods described in ref. 38 at the cGMP Islet Cell Processing Laboratory, Baylor Regional Transplant Institute. After procurement, pancreata were preserved by using a combination of University of Wisconsin solution and oxygenated perfluorodecalin (two-layer method) before islet isolation (39,40).…”

Stable transplantation results of magnetically retracted islets: a novel method

“…those using the fluorescence-activated cell sorter, or immunomagnetic beads for the removal of exocrine tissue [11,12,13,14,15,16,17]. Two methods to obtain purified rat islets are currently in common use.…”