Abstract
To determine the effects of procedural modifications, 23 human islet isolations were analyzed. Isolations were divided into two groups based on the enzyme used. The influence of Liberase™, with an improved method of mechanical disassociation of pancreas, was compared to an automated method using Sevac collagenase. Pancreases were processed within 10 h of cross clamping. Following ductal injection of the enzyme, tissue was placed in the digestion chamber for disassociation. Purification was accomplished using a COBE 2991 cell processor and continuous gradients of Hypaque EuroFicoll.Isolations in Group I (Sevac) had an average yield of 138,602 ± 128,364 islet equivalents (IE) (2083 ± 1679 IE/g) with a purity of 85 ± 11%. Group II (Liberase) showed an average yield of 389,586 ± 191,161 IE (5,958 ± 3,083 IE/g) with a purity of 90 ± 6.8%. Viability was confirmed by fluorescein diacetate and propidium iodide staining, static incubations, and perifusions. In conclusion, the combination of the enzyme blend, Liberase, and a more gentle system of disassociation has proven to be a more productive method of islet isolation with higher purity than the previously published methods.
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