A New Method to Value Efficiency of Enzyme Blends for Pancreatic Tissue Digestion M. Salamone, G. Seidita, A. Cuttitta, S. Rigogliuso, S. Mazzola, F. Bertuzzi, and G. Ghersi ABSTRACT Islet transplantation, since the 1990s, has been an example of human cell therapy. Nevertheless, the islet isolation procedure is not completely standardized; in fact, 50% of islet procedures do not eventuate in transplantation due both to the variability of a donor’s pancreas and to the unpredictable efficiency of an enzymatic blend. The enzymes used in pancreas isolation to digest several substrates are extracted from Clostridium histolyticum. In particular, they have strong collagenolytic activity compared with verte- brate collagenases. However, several impediments persist in human islet isolation success, probably owing to the variable composition and concentration of collagenases employed during the digestion phase. For islet isolation processes, neutral proteases play important roles. However, they should be considered to be double-edged swords, contributing to tissue dissociation but, sometimes, decreasing islet yield through fragmentation, break- down, and inactivation. Protease activities cannot be preciously adjusted in a narrow range, there is no approach to determine the optimal dosage and composition of enzymes for extraction of human islets from the pancreas. At this time, available data on commercial enzymatic activity are not sufficient to predict their efficiency for pancreas digestion; consequently, it is difficult to select enzyme batches. For these reasons, we sought to generate an innovative evaluation assay to select enzymes useful for isolation procedures of pancreatic islets. O PTIMIZATION OF isolation techniques is a prereq- uisite for successful transplantation to replace -cell function. However, organ digestion is not a fully standard- ized procedure. Although many modifications have been proposed for human pancreas digestion, the overall number of isolated islets are similar in different publications. 1–4 It seems to be necessary to continuously propose modifica- tions of the isolation methods to counterbalance unpredict- able variability in success over the years. Indeed, many data have shown that this problem is due to variable enzyme efficacy. 5–7 Collagenase shows different batch-to-batch and vial-to-vial characteristics owing to the not completely standardized procedures of enzyme extraction and purifi- cation as well as to enzyme instability. 8 The first conse- quence is that isolation procedures must be adapted to the variable enzyme characteristics. The components of the enzymatic blends that are cur- rently available more known now than in the past. Two classes isoforms (CI and CII) of collagenase are the main components in commercially available preparations 9 : CII is the most efficient at digesting exocrine tissue, whereas CI increases the digestion rate. 10,11 In addition, neutral pro- teases are also needed in the early phase of digestion. 12,13 Therefore, an appropriate mix of CI and CII as well as neutral proteases has been proposed to optimize islet isolation. 14 An additional consequence of enzyme variability is that it is necessary to experiment with an enzyme batch many times before determining its suitability for isola- tion. In fact, at the moment, available data on enzymatic From the IAMC-CNR (M.S., A.C., S.M.), U.O. Capo Granitola, Mazara del Vallo, Trapani, Italy; the Dipartimento di Biopatologia e Biotecnologie Mediche e Forensi (G.S.) and the Dipartimento di Biologia Cellulare e dello Sviluppo (M.S., S.R., G.G.), Università degli Studi di Palermo, Palermo, Italy; and the Ospedale Ni- guarda Cà Granda (F.B.), Milan, Italy. Address reprint requests to Monica Salamone, IAMC-CNR, U.O. Capo Granitola, via Luigi Vaccara 61, 91026 Mazara del Vallo, Trapani, Italy; E-mail: monica.salamone@irma.pa.cnr.it © 2010 by Elsevier Inc. All rights reserved. 0041-1345/–see front matter 360 Park Avenue South, New York, NY 10010-1710 doi:10.1016/j.transproceed.2010.05.107 Transplantation Proceedings, 42, 2043–2048 (2010) 2043