Highly purified versus filtered crude collagenase: comparable human islet isolation outcomes

Abstract

This study was designed to retrospectively compare the impact of crude Sigma V collagenase (Sigma V, n = 52) with high-purified Serva NB1 collagenase (Serva NB1, n = 42) on human islet isolation outcomes. A three-step filtration was applied to the crude Sigma V to remove endotoxin contamination and impurities; in addition, this process was used as a lot prescreening tool. Isolation outcomes were determined by digestion efficacy, islet yields, purity, viability, glucose-stimulated insulin release, and endotoxin content. The digestion efficacy between Sigma V and Serva NB1 was statistically significant (Sigma V: 64.71% vs. Serva NB1: 69.71%, p = 0.0014). However, the islet yields were similar (Sigma V: 23422.58 vs. Serva NB1: 271097 IEq, p = 0.23) between groups. There was no significant purity difference observed in fractions with purities greater than 75%. Viability (Sigma V: 93.3% vs. Serva NB1: 94.8%, p = 0.061) and stimulation indexes (Sigma V: 3.41 vs. Serva NB1: 2.74, p = 0.187) were also similar between the two groups. The impact of cold ischemia and age on the isolation outcome in the Sigma V group was comparable to the Serva NB1 group. The endotoxin content of the final products in the filtered Sigma V group was significantly less than that in the high-purified Serva NB1 group (0.022 vs. 0.052 EU/ml, p = 0.003). Additionally, in the Sigma V group there was minimal lot to lot variation and no significant loss of enzymatic activity after filtration. These findings indicate that the use of Sigma V or other crude enzyme blends for research pancreata is warranted to reduce isolation costs and increase the amount of islets available for critical islet research. These findings also validate the need for a systematic enzyme analysis to resolve these inconsistencies in overall enzyme quality once and for all.

Figure 1. Purification outcomes of human pancreata

(A) Purified islet distribution over a density gradient of 1.066-1.078 g/cm³. (B) Tissue volume collected on continuous gradient. (C) Purity of top fraction. (D) Purification recovery rate. Data are expressed as mean ± SE and analyzed by unpaired student t-test by two-tail distribution.

Figure 2. The size characteristics of isolated human islets

(A) Size distribution. (B) Ratio of actual islet number vs. IEq. Data are expressed as mean ± SE and analyzed by unpaired student t-test by two-tail distribution. IEq=islet equivalent.

Figure 3. Impact of age and cold ischemia on the isolation outcomes of Sigma V

(A) Age impact on isolation outcomes of Sigma V. (B) CIT impact on isolation outcomes of Sigma V. Data are expressed as mean ± SE and analyzed by unpaired student t-test by two-tail distribution. CIT=cold ischemia time. IEq=islet equivalent.