A new method to value efficiency of enzyme blends for pancreas tissue digestion

Gregorio Seidita, Giulio Ghersi, Monica Salamone, Salvatrice Rigogliuso, Angela Cuttitta, Monica Salamone, Salvatore Mazzola, Bertuzzi

Risultato della ricerca: Article

10 Citazioni (Scopus)

Abstract

In pancreatic islets isolation for cell therapy the major enzymes used are obtained from Clostridium hystoliticum: class I and class II collagenases. They are used in a defined tissue dissociation enzyme mixture together with neutral protease (Dispase) or thermolysin (Thermostable Neutral Protease). However, just to now, people working in islets production found variable outcomes in isolation procedures mainly due to large variability in enzymatic blend composition and efficacy. Using electrophoresis and gelatin zymography approaches together with densitometry evaluation assays we compared the composition, stability and auto-digestion processes of C. hystoliticum collagenases, Neutral protease and Thermolysin from Roches and Serva. Moreover, we have assessed: –the stability of enzymes when they are in solution at different temperatures (-20 C; 0 C and room temperature); –their digestive activity when applied at different working temperatures (25 C; 30 C; 37 C and 42 C). Our results shown a heterogeneous composition of the different enzymatic blend enzymes analyzed. Furthermore, heterogeneity is observed among different batch enzymes; we found several more proteins and/or fragments compare to HPLC profiles published by vendors. In gelatin zymographyes several digestive bands were catalytic, showing very high complex degradative patterns, in part active even in condition of calcium deprivation. Additionally, in neutral protease from Serva (and not in Thermolysin) contaminants with gelatinolytic activities were detected. An auto-digestive/inactivation processes of enzymes occurred at different working temperatures, reduced by lowering temperature up to 25 C. These data taken together strongly imply a not controlled digestive processes due to several contaminants in enzyme blends and to autocatalytic processes. Moreover, the presence of low molecular weight gelatine/ degradative activities obstacles the possibility to control islet digestion due to aspecific catalytic activities. Generation of recombinant collagenases probably could be of help to overcome the variability in the extractive processes.
Lingua originaleEnglish
pagine (da-a)2043-2048
Numero di pagine6
RivistaTransplantation Proceedings
Volume42
Stato di pubblicazionePublished - 2010

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Pancreas
Digestion
Thermolysin
Collagenases
Peptide Hydrolases
Enzymes
Temperature
Gelatin
Islets of Langerhans
Enzyme Stability
Clostridium
Densitometry
Cell- and Tissue-Based Therapy
Electrophoresis
Molecular Weight
High Pressure Liquid Chromatography
Calcium
Proteins

All Science Journal Classification (ASJC) codes

  • Surgery
  • Transplantation

Cita questo

A new method to value efficiency of enzyme blends for pancreas tissue digestion. / Seidita, Gregorio; Ghersi, Giulio; Salamone, Monica; Rigogliuso, Salvatrice; Cuttitta, Angela; Salamone, Monica; Mazzola, Salvatore; Bertuzzi.

In: Transplantation Proceedings, Vol. 42, 2010, pag. 2043-2048.

Risultato della ricerca: Article

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abstract = "In pancreatic islets isolation for cell therapy the major enzymes used are obtained from Clostridium hystoliticum: class I and class II collagenases. They are used in a defined tissue dissociation enzyme mixture together with neutral protease (Dispase) or thermolysin (Thermostable Neutral Protease). However, just to now, people working in islets production found variable outcomes in isolation procedures mainly due to large variability in enzymatic blend composition and efficacy. Using electrophoresis and gelatin zymography approaches together with densitometry evaluation assays we compared the composition, stability and auto-digestion processes of C. hystoliticum collagenases, Neutral protease and Thermolysin from Roches and Serva. Moreover, we have assessed: –the stability of enzymes when they are in solution at different temperatures (-20 C; 0 C and room temperature); –their digestive activity when applied at different working temperatures (25 C; 30 C; 37 C and 42 C). Our results shown a heterogeneous composition of the different enzymatic blend enzymes analyzed. Furthermore, heterogeneity is observed among different batch enzymes; we found several more proteins and/or fragments compare to HPLC profiles published by vendors. In gelatin zymographyes several digestive bands were catalytic, showing very high complex degradative patterns, in part active even in condition of calcium deprivation. Additionally, in neutral protease from Serva (and not in Thermolysin) contaminants with gelatinolytic activities were detected. An auto-digestive/inactivation processes of enzymes occurred at different working temperatures, reduced by lowering temperature up to 25 C. These data taken together strongly imply a not controlled digestive processes due to several contaminants in enzyme blends and to autocatalytic processes. Moreover, the presence of low molecular weight gelatine/ degradative activities obstacles the possibility to control islet digestion due to aspecific catalytic activities. Generation of recombinant collagenases probably could be of help to overcome the variability in the extractive processes.",
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AU - Seidita, Gregorio

AU - Ghersi, Giulio

AU - Salamone, Monica

AU - Rigogliuso, Salvatrice

AU - Cuttitta, Angela

AU - Salamone, Monica

AU - Mazzola, Salvatore

AU - Bertuzzi, null

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N2 - In pancreatic islets isolation for cell therapy the major enzymes used are obtained from Clostridium hystoliticum: class I and class II collagenases. They are used in a defined tissue dissociation enzyme mixture together with neutral protease (Dispase) or thermolysin (Thermostable Neutral Protease). However, just to now, people working in islets production found variable outcomes in isolation procedures mainly due to large variability in enzymatic blend composition and efficacy. Using electrophoresis and gelatin zymography approaches together with densitometry evaluation assays we compared the composition, stability and auto-digestion processes of C. hystoliticum collagenases, Neutral protease and Thermolysin from Roches and Serva. Moreover, we have assessed: –the stability of enzymes when they are in solution at different temperatures (-20 C; 0 C and room temperature); –their digestive activity when applied at different working temperatures (25 C; 30 C; 37 C and 42 C). Our results shown a heterogeneous composition of the different enzymatic blend enzymes analyzed. Furthermore, heterogeneity is observed among different batch enzymes; we found several more proteins and/or fragments compare to HPLC profiles published by vendors. In gelatin zymographyes several digestive bands were catalytic, showing very high complex degradative patterns, in part active even in condition of calcium deprivation. Additionally, in neutral protease from Serva (and not in Thermolysin) contaminants with gelatinolytic activities were detected. An auto-digestive/inactivation processes of enzymes occurred at different working temperatures, reduced by lowering temperature up to 25 C. These data taken together strongly imply a not controlled digestive processes due to several contaminants in enzyme blends and to autocatalytic processes. Moreover, the presence of low molecular weight gelatine/ degradative activities obstacles the possibility to control islet digestion due to aspecific catalytic activities. Generation of recombinant collagenases probably could be of help to overcome the variability in the extractive processes.

AB - In pancreatic islets isolation for cell therapy the major enzymes used are obtained from Clostridium hystoliticum: class I and class II collagenases. They are used in a defined tissue dissociation enzyme mixture together with neutral protease (Dispase) or thermolysin (Thermostable Neutral Protease). However, just to now, people working in islets production found variable outcomes in isolation procedures mainly due to large variability in enzymatic blend composition and efficacy. Using electrophoresis and gelatin zymography approaches together with densitometry evaluation assays we compared the composition, stability and auto-digestion processes of C. hystoliticum collagenases, Neutral protease and Thermolysin from Roches and Serva. Moreover, we have assessed: –the stability of enzymes when they are in solution at different temperatures (-20 C; 0 C and room temperature); –their digestive activity when applied at different working temperatures (25 C; 30 C; 37 C and 42 C). Our results shown a heterogeneous composition of the different enzymatic blend enzymes analyzed. Furthermore, heterogeneity is observed among different batch enzymes; we found several more proteins and/or fragments compare to HPLC profiles published by vendors. In gelatin zymographyes several digestive bands were catalytic, showing very high complex degradative patterns, in part active even in condition of calcium deprivation. Additionally, in neutral protease from Serva (and not in Thermolysin) contaminants with gelatinolytic activities were detected. An auto-digestive/inactivation processes of enzymes occurred at different working temperatures, reduced by lowering temperature up to 25 C. These data taken together strongly imply a not controlled digestive processes due to several contaminants in enzyme blends and to autocatalytic processes. Moreover, the presence of low molecular weight gelatine/ degradative activities obstacles the possibility to control islet digestion due to aspecific catalytic activities. Generation of recombinant collagenases probably could be of help to overcome the variability in the extractive processes.

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JO - Transplantation Proceedings

JF - Transplantation Proceedings

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