INFLUENCE OF NEUTRAL PROTEASE ACTIVITY ON HUMAN ISLET ISOLATION OUTCOME

Abstract

O455 Aims: The enzymatic dissociation of pancreatic tissue by collagenase is a key step in the islet isolation procedure. Previous investigations in the rat clearly showed that neutral protease activity is a critical component regarding the efficient release of islets from the acinar tissue and viability of isolated islets. However, no information is available about the effect of neutral protease (NP) activity on human islet isolation. The aim of the present study was therefore to compare the efficiency of different concentrations of NP combined with purified collagenase on human islet isolation outcome. Methods: Islets were isolated by digestion-filtration from UW-perfused pancreata (76±4 g) obtained from adult (51±2 years) and slightly obese (26±1 BMI) multiorgan donors after a cold ischemia time of 9±1 hours. Following intraductal collagenase distension pancreata were digested at 37°C by means of 20 U/g Collagenase NB 1 (SERVA) supplemented either with 2.6 (n=10) or 4.5% (n=10) of Neutral Protease NB (SERVA). Prior to and subsequent to purification on a Ficoll-Na-diatrizoate gradient islet yield was determined as islet equivalents (IEQ). Viability (trypan-blue exclusion) and glucose stimulation index during static incubation (20 vs 2.8 mM glucose) were assessed after overnight culture. Results: Regarding donor variables no significant differences were found between experimental groups. Increasing concentration of NP from 2.6 to 4.5% resulted in a significant reduction of undigested tissue from 22±2 to 17±2 g (P<0.05) remaining in the chamber. This corresponds to a significant increase of the digested tissue volume (26±2 vs 40±3 mL, P<0.01) which correlates with a significant increase of total islet yield prepurification from 459800±22900 to 587600±69000 IEQ (P<0.05). The difference in islet yields after utilization of 2.6 or 4.5% NP was diminished after purification (345700±31200 vs 391500±35400 IEQ, NS). Nevertheless, quality control revealed a significant reduction of glucose stimulation index by increasing NP concentration from 2.6 to 4.5% (4.8±1.1 vs 2.1±0.5, P<0.05). Viability (100±1 vs 95±3%, P<0.05) and purity (70±6 vs 49±5 %, P<0.05) were significantly affected by enhanced NP concentration as well. Conclusions Although increased neutral protease activity seems to improve islet release from the adult human pancreas by promoting enzymatic tissue dissociation, it significantly decreases islet in vitro function and viability. The reduction of purity indicates severe damage of acinar tissue by enhancing NP activity presumably affecting islet integrity as well.