Calcium: A Crucial Potentiator for Efficient Enzyme Digestion of the Human Pancreas.

BACKGROUND: Effective digestive enzymes are crucial for successful islet isolation. Supplemental proteases are essential because they synergize with collagenase for effective pancreatic digestion. The activity of these enzymes is critically dependent on the presence of Ca2+ ions at a concentration of 5-10 mM. The present study aimed to determine the Ca2+ concentration during human islet isolation and to ascertain whether the addition of supplementary Ca2+ is required to maintain an optimal Ca2+ concentration during the various phases of the islet isolation process. METHODS: Human islets were isolated according to standard methods and isolation parameters. Islet quality control and the number of isolations fulfilling standard transplantation criteria were evaluated. Ca2+ was determined by using standard clinical chemistry routines. Islet isolation was performed with or without addition of supplementary Ca2+ to reach a Ca2+ of 5 mM. RESULTS: Ca2+ concentration was markedly reduced in bicarbonate-based buffers, especially if additional bicarbonate was used to adjust the pH as recommended by the Clinical Islet Transplantation Consortium. A major reduction in Ca2+ concentration was also observed during pancreatic enzyme perfusion, digestion, and harvest. Additional Ca2+ supplementation of media used for dissolving the enzymes and during digestion, perfusion, and harvest was necessary in order to obtain the concentration recommended for optimal enzyme activity and efficient liberation of a large number of islets from the human pancreas. CONCLUSIONS: Ca2+ is to a large extent consumed during clinical islet isolation, and in the absence of supplementation, the concentration fell below that recommended for optimal enzyme activity. Ca2+ supplementation of the media used during human pancreas digestion is necessary to maintain the concentration recommended for optimal enzyme activity. Addition of Ca2+ to the enzyme blend has been implemented in the standard isolation protocols in the Nordic Network for Clinical Islet Transplantation.

Quantifying the Effects of Different Neutral Proteases on Human Islet Integrity.

Efficient islet release from the pancreas requires the combination of collagenase, neutral protease (cNP), or thermolysin (TL). Recently, it has been shown that clostripain (CP) may also contribute to efficient islet release from the human pancreas. The aim of this study was to evaluate the impact of these proteases on human islet integrity in a prospective approach. Islets were isolated from the pancreas of 10 brain-dead human organ donors. Purified islets were precultured for 3 to 4 d at 37 °C to ensure that preparations were cleared of predamaged islets, and only integral islets were subjected to 90 min of incubation at 37 °C in Hank's balanced salt solution supplemented with cNP, TL, or CP. The protease concentrations were calculated for a pancreas of 100 g trimmed weight utilizing 120 dimethyl-casein units of cNP, 70,000 caseinase units of TL, or 200 benzoyl-l-arginine-ethyl-ester units of CP (1×). These activities were then increased both 5× and 10×. After subsequent 24-h culture in enzyme-free culture medium, treated islets were assessed and normalized to sham-treated controls. Compared with controls and CP, islet yield was significantly reduced by using the 5× activity of cNP and TL, inducing also fragmentation and DNA release. Viability significantly decreased not until adding the 1× activity of cNP, 5× activity of TL, or 10× activity of CP. Although mitochondrial function was significantly lowered by 1× cNP and 5× TL, CP did not affect mitochondria at any concentration. cNP- and TL-incubated islets significantly lost intracellular insulin already at 1× activity, while the 10× activity of CP had to be added to observe a similar effect. cNP and TL have a similar toxic potency regarding islet integrity. CP also induces adverse effects on islets, but the toxic threshold is generally higher. We hypothesize that CP can serve as supplementary protease to minimize cNP or TL activity for efficient pancreas digestion.