Improved method for the isolation and purification of human islets of Langerhans using Liberase (TM) enzyme blend

To determine the effects of procedural modifications, 23 human islet isolat ions were analyzed. Isolations were divided into two groups based on the en zyme used. The influence of Liberase(TM), with an improved method of mechan ical 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 digesti on chamber for disassociation. Purification was accomplished using a COBE 2 991 cell processor and continuous gradients of Hypaque EuroFicoll. Isolatio ns in Group I(Sevac) had an average yield of 138,602 +/- 125,364 islet equi valents (IE) (2083 +/- 1679 IE/g) with a purity of 85 +/- 11%. Group II (Li berase) showed an average yield of 389,586 +/- 191,161 IE (5,958 +/- 3,081 IE/g) with a purity of 90 +/- 6.8%. Viability was confirmed by fluorescein diacetate and propidium iodide staining, static incubations, and perifusion s. In conclusion, the combination of the enzyme blend, Liberase, and a more gent le system of disassociation has proven to be a more productive method of islet isolation with higher purity than the previously published method s. (C) American Society for Histocompatibility and Immunogenetics, 1999 Pub lished by Elsevier Science Inc.