Comparison of Neutral Proteases and Collagenase Class I as Essential Enzymes for Human Islet Isolation

Abstract

BackgroundEfficient islet isolation requires synergistic interaction between collagenase class I (CI) and class II (CII). The CI degradation alters the ratio between CI and CII and is responsible for batch-to-batch variations. This study compares the role of neutral protease (NP) plus clostripain (CP) with CI as essential enzymes for human islet isolation.MethodsHuman islets were isolated using 4 different enzyme mixtures composed of CII plus either intact (CI-115) or degraded CI (CI-100). Blends were administered either with or without NP/CP. Purified islets were cultured for 3 to 4 days before islet quality assessment.ResultsWhereas using intact CI-115 without NP/CP did not significantly reduce islet yield (3429 ± 631 vs 3087 ± 970 islet equivalent/g, nonsignificant), administration of degraded CI-100 without NP/CP decreased islet yield from 3501 ± 580 to 1312 ± 244 islet equivalent/g (P < 0.01), doubled the amount of undigested tissue from 11.8 ± 1.6 to 24.4 ± 1.2% (P < 0.01) and triplicated the percentage of trapped islets from 7.7 ± 2.8 to 22.5 ± 3.6% (P < 0.05). Islet yield did not vary between supplemented CI-115 and CI-100, but was increased using CI-115 when NP/CP was omitted (P < 0.05). A trend toward higher viability and increased secretory insulin response was noted in both CI-100 and CI-115 when NP/CP was not added.ConclusionsThis study suggests that NP/CP can compensate reduced CI activity. Future attempts to optimize enzyme blends should consider the possibility to increase the proportion of collagenase CI to reduce the need for potentially harmful NPs.

Enzyme Development for Human Islet Isolation: Five Decades of Progress or Stagnation?

“…Previous studies in the rat identified CC-II as the most relevant isoform for pancreas digestion when compared with CC-I [47,59]. In agreement with the observations of the Groningen group [60], we found that human pancreases are efficiently digested even in the absence of CC-I as long as sufficient activities of supplementary neutral proteases are present [61,62]. Nevertheless, for optimal collagenase function, a certain amount of CC-I has to be present within the enzyme blend.…”

“…In addition to observations made in the rat [46,68], we described the detrimental effect of increased CNP activities on integrity and viability of isolated human islets [69]. In fact, it seems to be possible to isolate human islets using only a marginal amount of CNP [62,70]. These observations raise the question of whether the identification of an effective replacement for clostridial CNP could improve the quality and functional potency of isolated islets.…”

Testing of a New Collagenase Blend for Pancreatic Islet Isolation Produced by &lt;i&gt;Clostridium histolyticum&lt;/i&gt;

“…Study by Brandhorst et al [12] showed that neither C1 nor C2 alone were able to dissociate pancreatic tissue. Rapid and complete loss of peri-islet ECM depends on both collagenase isoform and is best achieved with assistance of other proteolytic enzymes [10] [12] [13]. This conclusion was also confirmed in our study, where the best results were achieved with a batch from Group 3 containing intact and fragmented collagenase in combination with neutral protease (NP) and clostripain.…”

“…This conclusion was also confirmed in our study, where the best results were achieved with a batch from Group 3 containing intact and fragmented collagenase in combination with neutral protease (NP) and clostripain. Previously published studies [11] [13] showed that NP and clostripain can compensate reduced C1 activity; however the results of our study revealed successful islet isolations with batches without the presence of collagenase. Potentially deteriorate influence of NP on islet viability was not fully confirmed in our study except decreased vitality and lover glucose stimulated insulin secretion 24 hours after isolation in Group 2 but not in Group 3.…”

Evolution of Enzyme Requirements for Human Islet Isolation

“…A recent report showed intact C1 was more effective in islet recovery than truncated C1 when enzyme mixtures contained C2 but no supplemental protease [69]. In contrast, there was no difference in islet recoveries when the enzyme mixtures above contained supplemental neutral protease activity.…”