In pancreatic islets isolation for cell therapy the major enzymes used areobtained from Clostridium hystoliticum: class I and class II collagenases.They are used in a defined tissue dissociation enzyme mixture together withneutral protease (Dispase) or thermolysin (Thermostable Neutral Protease).However, just to now, people working in islets production found variableoutcomes in isolation procedures mainly due to large variability in enzymaticblend composition and efficacy. Using electrophoresis and gelatin zymographyapproaches together with densitometry evaluation assays we comparedthe composition, stability and auto-digestion processes of C.hystoliticum collagenases, Neutral protease and Thermolysin from Rochesand Serva. Moreover, we have assessed: –the stability of enzymes when theyare 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 compositionof the different enzymatic blend enzymes analyzed. Furthermore,heterogeneity is observed among different batch enzymes; we found severalmore proteins and/or fragments compare to HPLC profiles published byvendors. In gelatin zymographyes several digestive bands were catalytic,showing very high complex degradative patterns, in part active even incondition of calcium deprivation. Additionally, in neutral protease fromServa (and not in Thermolysin) contaminants with gelatinolytic activitieswere detected. An auto-digestive/inactivation processes of enzymes occurredat different working temperatures, reduced by lowering temperature up to25 C. These data taken together strongly imply a not controlled digestiveprocesses due to several contaminants in enzyme blends and to autocatalyticprocesses. Moreover, the presence of low molecular weight gelatine/degradative activities obstacles the possibility to control islet digestion due toaspecific catalytic activities. Generation of recombinant collagenases probablycould be of help to overcome the variability in the extractive processes.
|Numero di pagine||6|
|Stato di pubblicazione||Published - 2010|
All Science Journal Classification (ASJC) codes