Experience of islet isolation without neutral protease supplementation

We have reported improved islet isolation outcomes using a new digestion protocol where the pancreas is perfused only with collagenase, and neutral protease (NP) is administered during the digestion phase. Since the inception of this protocol, we have had some cases where administration of NP was not required. Our new protocol was utilized in 94 islet isolations. The timing of adding NP was dependent on the progression of digestion but in 10 cases the progression was rapid and most islets in the assessment samples were free from the exocrine tissue. As a result NP was not added at all for these isolations (no-NP group). In the remaining 84 isolations, NP was added during digestion phase (control group). Pancreata in the each group were digested with a similar collagenase dose. Digestion time was shorter in the no-NP (15.0 +/- 1.8 vs. 19.5 +/- 0.6 min, p = 0.004). At post-digestion, the no-NP had fewer trapped islets (10.9 +/- 2.8 vs. 28.1 +/- 2.4%, p = 0.009). Post-purification islet yield was similar (355 +/- 45 x 10(3) vs. 318 +/- 17 x 10(3) IE, p = 0.29). Five preparations in the no-NP were used for clinical transplantation, leading to a 64.3 +/- 15.2% reduction in insulin usage. Interestingly, cold ischemia time was longer in the no-NP (10.3 +/- 0.9 vs. 7.9 +/- 0.4 h, p = 0.04). One particular collagenase lot having the highest NP activity contamination was used in 7 isolations in the no-NP. Our experience indicates that supplementation of collagenase with NP is not always necessary for effective isolation. Cold ischemia time and NP contamination should be evaluated for optimal NP dosage.