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Although human islet transplantation has been established as valid and safe treatment for patients with type 1 diabetes, the utilization rates of human pancreases for clinical islet transplantation are still limited and substantially determined by the quality and composition of collagenase blends. While function and integrity of collagenase has been extensively investigated, information is still lacking about the most suitable supplementary neutral proteases. The present study compared islet isolation outcome after pancreas digestion by means of collagenase used alone or supplemented with either neutral protease (NP), clostripain (CP), or both proteases. Decent amounts of islet equivalents (IEQ) were isolated using collagenase alone (3090 +/- 550 IEQ/g), or in combination with NP (2340 +/- 450 IEQ/g) or CP (2740 +/- 280 IEQ/g). Nevertheless, the proportion of undigested tissue was higher after using collagenase alone (21.1 +/- 1.1%, P < 0.05) compared with addition of NP (13.3 +/- 2.2%) or CP plus NP (13.7 +/- 2.6%). Likewise, the percentage of embedded islets was highest using collagenase only (13 +/- 2%) and lowest adding NP plus CP (4 +/- 1%, P < 0.01). The latter combination resulted in lowest post-culture overall survival (42.7 +/- 3.9%), while highest survival was observed after supplementation with CP (74.5 +/- 4.8%, P < 0.01). An insulin response toward glucose challenge was present in all experimental groups, but the stimulation index was significantly decreased using collagenase plus NP (2.0 +/- 0.12) compared with supplementation with CP (3.16 +/- 0.4, P < 0.001). This study demonstrates for the first time that it is possible to isolate significant numbers of human islets combining collagenase only with CP. The supplementation with CP is an effective means to substantially reduce NP activity, which significantly decreases survival and viability after culture. This will facilitate the manufacturing of enzyme blends with less harmful characteris

Although human islet transplantation has been established as valid and safe treatment for patients with type 1 diabetes, the utilization rates of human pancreases for clinical islet transplantation are still limited and substantially determined by the quality and composition of collagenase blends. While function and integrity of collagenase has been extensively investigated, information is still lacking about the most suitable supplementary neutral proteases. The present study compared islet isolation outcome after pancreas digestion by means of collagenase used alone or supplemented with either neutral protease (NP), clostripain (CP), or both proteases. Decent amounts of islet equivalents (IEQ) were isolated using collagenase alone (3090 +/- 550 IEQ/g), or in combination with NP (2340 +/- 450 IEQ/g) or CP (2740 +/- 280 IEQ/g). Nevertheless, the proportion of undigested tissue was higher after using collagenase alone (21.1 +/- 1.1%, P < 0.05) compared with addition of NP (13.3 +/- 2.2%) or CP plus NP (13.7 +/- 2.6%). Likewise, the percentage of embedded islets was highest using collagenase only (13 +/- 2%) and lowest adding NP plus CP (4 +/- 1%, P < 0.01). The latter combination resulted in lowest post-culture overall survival (42.7 +/- 3.9%), while highest survival was observed after supplementation with CP (74.5 +/- 4.8%, P < 0.01). An insulin response toward glucose challenge was present in all experimental groups, but the stimulation index was significantly decreased using collagenase plus NP (2.0 +/- 0.12) compared with supplementation with CP (3.16 +/- 0.4, P < 0.001). This study demonstrates for the first time that it is possible to isolate significant numbers of human islets combining collagenase only with CP. The supplementation with CP is an effective means to substantially reduce NP activity, which significantly decreases survival and viability after culture. This will facilitate the manufacturing of enzyme blends with less harmful characteris

Although human islet transplantation has been established as valid and safe treatment for patients with type 1 diabetes, the utilization rates of human pancreases for clinical islet transplantation are still limited and substantially determined by the quality and composition of collagenase blends. While function and integrity of collagenase has been extensively investigated, information is still lacking about the most suitable supplementary neutral proteases. The present study compared islet isolation outcome after pancreas digestion by means of collagenase used alone or supplemented with either neutral protease (NP), clostripain (CP), or both proteases. Decent amounts of islet equivalents (IEQ) were isolated using collagenase alone (3090 +/- 550 IEQ/g), or in combination with NP (2340 +/- 450 IEQ/g) or CP (2740 +/- 280 IEQ/g). Nevertheless, the proportion of undigested tissue was higher after using collagenase alone (21.1 +/- 1.1%, P < 0.05) compared with addition of NP (13.3 +/- 2.2%) or CP plus NP (13.7 +/- 2.6%). Likewise, the percentage of embedded islets was highest using collagenase only (13 +/- 2%) and lowest adding NP plus CP (4 +/- 1%, P < 0.01). The latter combination resulted in lowest post-culture overall survival (42.7 +/- 3.9%), while highest survival was observed after supplementation with CP (74.5 +/- 4.8%, P < 0.01). An insulin response toward glucose challenge was present in all experimental groups, but the stimulation index was significantly decreased using collagenase plus NP (2.0 +/- 0.12) compared with supplementation with CP (3.16 +/- 0.4, P < 0.001). This study demonstrates for the first time that it is possible to isolate significant numbers of human islets combining collagenase only with CP. The supplementation with CP is an effective means to substantially reduce NP activity, which significantly decreases survival and viability after culture. This will facilitate the manufacturing of enzyme blends with less harmful characteris

Although human islet transplantation has been established as valid and safe treatment for patients with type 1 diabetes, the utilization rates of human pancreases for clinical islet transplantation are still limited and substantially determined by the quality and composition of collagenase blends. While function and integrity of collagenase has been extensively investigated, information is still lacking about the most suitable supplementary neutral proteases. The present study compared islet isolation outcome after pancreas digestion by means of collagenase used alone or supplemented with either neutral protease (NP), clostripain (CP), or both proteases. Decent amounts of islet equivalents (IEQ) were isolated using collagenase alone (3090 +/- 550 IEQ/g), or in combination with NP (2340 +/- 450 IEQ/g) or CP (2740 +/- 280 IEQ/g). Nevertheless, the proportion of undigested tissue was higher after using collagenase alone (21.1 +/- 1.1%, P < 0.05) compared with addition of NP (13.3 +/- 2.2%) or CP plus NP (13.7 +/- 2.6%). Likewise, the percentage of embedded islets was highest using collagenase only (13 +/- 2%) and lowest adding NP plus CP (4 +/- 1%, P < 0.01). The latter combination resulted in lowest post-culture overall survival (42.7 +/- 3.9%), while highest survival was observed after supplementation with CP (74.5 +/- 4.8%, P < 0.01). An insulin response toward glucose challenge was present in all experimental groups, but the stimulation index was significantly decreased using collagenase plus NP (2.0 +/- 0.12) compared with supplementation with CP (3.16 +/- 0.4, P < 0.001). This study demonstrates for the first time that it is possible to isolate significant numbers of human islets combining collagenase only with CP. The supplementation with CP is an effective means to substantially reduce NP activity, which significantly decreases survival and viability after culture. This will facilitate the manufacturing of enzyme blends with less harmful characteris

Although human islet transplantation has been established as valid and safe treatment for patients with type 1 diabetes, the utilization rates of human pancreases for clinical islet transplantation are still limited and substantially determined by the quality and composition of collagenase blends. While function and integrity of collagenase has been extensively investigated, information is still lacking about the most suitable supplementary neutral proteases. The present study compared islet isolation outcome after pancreas digestion by means of collagenase used alone or supplemented with either neutral protease (NP), clostripain (CP), or both proteases. Decent amounts of islet equivalents (IEQ) were isolated using collagenase alone (3090 +/- 550 IEQ/g), or in combination with NP (2340 +/- 450 IEQ/g) or CP (2740 +/- 280 IEQ/g). Nevertheless, the proportion of undigested tissue was higher after using collagenase alone (21.1 +/- 1.1%, P < 0.05) compared with addition of NP (13.3 +/- 2.2%) or CP plus NP (13.7 +/- 2.6%). Likewise, the percentage of embedded islets was highest using collagenase only (13 +/- 2%) and lowest adding NP plus CP (4 +/- 1%, P < 0.01). The latter combination resulted in lowest post-culture overall survival (42.7 +/- 3.9%), while highest survival was observed after supplementation with CP (74.5 +/- 4.8%, P < 0.01). An insulin response toward glucose challenge was present in all experimental groups, but the stimulation index was significantly decreased using collagenase plus NP (2.0 +/- 0.12) compared with supplementation with CP (3.16 +/- 0.4, P < 0.001). This study demonstrates for the first time that it is possible to isolate significant numbers of human islets combining collagenase only with CP. The supplementation with CP is an effective means to substantially reduce NP activity, which significantly decreases survival and viability after culture. This will facilitate the manufacturing of enzyme blends with less harmful characteris

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 degrees C to ensure that preparations were cleared of predamaged islets, and only integral islets were subjected to 90 min of incubation at 37 degrees 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-Larginine- ethyl-ester units of CP (1x). These activities were then increased both 5 x and 10 x. 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 x activity of cNP and TL, inducing also fragmentation and DNA release. Viability significantly decreased not until adding the 1 x activity of cNP, 5 x activity of TL, or 10 x activity of CP. Although mitochondrial function was significantly lowered by 1 x cNP and 5 x TL, CP did not affect mitochondria at any concentration. cNP-and TL-incubated islets significantly lost intracellular insulin already at 1 x activity, while the 10 x 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.

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 degrees C to ensure that preparations were cleared of predamaged islets, and only integral islets were subjected to 90 min of incubation at 37 degrees 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-Larginine- ethyl-ester units of CP (1x). These activities were then increased both 5 x and 10 x. 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 x activity of cNP and TL, inducing also fragmentation and DNA release. Viability significantly decreased not until adding the 1 x activity of cNP, 5 x activity of TL, or 10 x activity of CP. Although mitochondrial function was significantly lowered by 1 x cNP and 5 x TL, CP did not affect mitochondria at any concentration. cNP-and TL-incubated islets significantly lost intracellular insulin already at 1 x activity, while the 10 x 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.

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 degrees C to ensure that preparations were cleared of predamaged islets, and only integral islets were subjected to 90 min of incubation at 37 degrees 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-Larginine- ethyl-ester units of CP (1x). These activities were then increased both 5 x and 10 x. 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 x activity of cNP and TL, inducing also fragmentation and DNA release. Viability significantly decreased not until adding the 1 x activity of cNP, 5 x activity of TL, or 10 x activity of CP. Although mitochondrial function was significantly lowered by 1 x cNP and 5 x TL, CP did not affect mitochondria at any concentration. cNP-and TL-incubated islets significantly lost intracellular insulin already at 1 x activity, while the 10 x 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.

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 degrees C to ensure that preparations were cleared of predamaged islets, and only integral islets were subjected to 90 min of incubation at 37 degrees 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-Larginine- ethyl-ester units of CP (1x). These activities were then increased both 5 x and 10 x. 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 x activity of cNP and TL, inducing also fragmentation and DNA release. Viability significantly decreased not until adding the 1 x activity of cNP, 5 x activity of TL, or 10 x activity of CP. Although mitochondrial function was significantly lowered by 1 x cNP and 5 x TL, CP did not affect mitochondria at any concentration. cNP-and TL-incubated islets significantly lost intracellular insulin already at 1 x activity, while the 10 x 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.

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 degrees C to ensure that preparations were cleared of predamaged islets, and only integral islets were subjected to 90 min of incubation at 37 degrees 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-Larginine- ethyl-ester units of CP (1x). These activities were then increased both 5 x and 10 x. 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 x activity of cNP and TL, inducing also fragmentation and DNA release. Viability significantly decreased not until adding the 1 x activity of cNP, 5 x activity of TL, or 10 x activity of CP. Although mitochondrial function was significantly lowered by 1 x cNP and 5 x TL, CP did not affect mitochondria at any concentration. cNP-and TL-incubated islets significantly lost intracellular insulin already at 1 x activity, while the 10 x 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.