Beneficial effect of recombinant rC1rC2 collagenases on human islet function: Efficacy of low-dose enzymes on pancreas digestion and yield.

A high number of human islets can be isolated by using modern purified tissue dissociation enzymes; however, this requires the use of >20 Wunsch units (WU)/g of pancreas for digestion. Attempts to reduce this dose have resulted in pancreas underdigestion and poor islet recovery but improved islet function. In this study, we achieved a high number of functional islets using a low dose of recombinant collagenase enzyme mixture (RCEM-1200 WU rC2 and 10 million collagen-degrading activity [CDA] U of rC1 containing about 209 mg of collagenase to digest a 100-g pancreas). The collagenase dose used in these isolations is about 42% of the natural collagenase enzyme mixture (NCEM) dose commonly used to digest a 100-g pancreas. Low-dose RCEM was efficient in digesting entire pancreases to obtain higher yield (5535 ± 830 and 2582 ± 925 islet equivalent/g, P < .05) and less undigested tissue (16.7 ± 5% and 37.8 ± 3%, P < .05) compared with low-dose NCEM (12WU/g). Additionally, low-dose RCEM islets retained better morphology (confirmed with scanning electron microscopy) and higher in vitro basal insulin release (2391 ± 1342 and 1778 ± 978 µU/mL; P < .05) compared with standard-dose NCEM. Nude mouse bioassay demonstrated better islet function for low-dose RCEM (area under the curve [AUC] 24 968) compared with low-dose (AUC-38 225) or standard-dose NCEM (AUC-38 685), P < .05. This is the first report indicating that islet function can be improved by using low-dose rC1rC2 (RCEM).

Effectiveness of different molecular forms of C. histolyticum class I collagenase to recover islets.

One factor that may contribute to variability between different lots of purified collagenase to recover islets is the molecular form of C. histolyticum class I (C1) collagenase used in the isolation procedure. Two different enzyme mixtures containing C1, class II (C2) collagenase and BP Protease were compared for their effectiveness to recover islets from split adult porcine pancreas. The same enzyme activities per g trimmed tissue were used for all isolations with the only difference being the mass of C1 required to achieve 25,000 collagen degradation activity U/g tissue. The results show no differences in performance of the two enzyme mixtures. The only significant difference is 19 fold more truncated C1 was required to achieve the same result as intact C1.

Optimizing Porcine Islet Isolation to Markedly Reduce Enzyme Consumption Without Sacrificing Islet Yield or Function.

BACKGROUND: Human allogeneic islet transplantation for treatment of type 1 diabetes provides numerous clinical benefits, such as fewer episodes of hypoglycemic unawareness and tighter control of blood glucose levels. Availability of human pancreas for clinical and research use, however, is severely limited. Porcine pancreas offers an abundant source of tissue for optimization of islet isolation methodology and future clinical transplantation, thereby increasing patient access to this potentially lifesaving procedure. METHODS: Porcine islet isolations were performed using varying amounts of collagenase (7.5, 3.75, or 2.5 Wunsch units per gram tissue) and neutral protease activity (12 000, 6000, or 4000 neutral protease units per gram tissue) and perfusion volumes (1.7 or 0.85 mL/g tissue) to assess their effects on isolation outcomes. Retention of dissociative enzymes within the pancreas during perfusion and digestion was evaluated, along with distribution of the perfusion solution within the tissue. RESULTS: Reducing enzyme usage by as much as 67% and perfusion volume by 50% led to equally successful islet isolation outcomes when compared with the control group (48 ± 7% of tissue digested and 1088 ± 299 islet equivalents per gram of pancreas vs 47 ± 11% and 1080 ± 512, respectively). Using margin-marking dye in the perfusion solution to visualize enzyme distribution demonstrated that increasing perfusion volume did not improve tissue infiltration. CONCLUSIONS: Current protocols for porcine islet isolation consume excessive amounts of dissociative enzymes, elevating cost and limiting research and development. These data demonstrate that islet isolation protocols can be optimized to significantly reduce enzyme usage while maintaining yield and function and thus accelerating progress toward clinical application.

Identifying Effective Enzyme Activity Targets for Recombinant Class I and Class II Collagenase for Successful Human Islet Isolation.

UNLABELLED: Isolation following a good manufacturing practice-compliant, human islet product requires development of a robust islet isolation procedure where effective limits of key reagents are known. The enzymes used for islet isolation are critical but little is known about the doses of class I and class II collagenase required for successful islet isolation. METHODS: We used a factorial approach to evaluate the effect of high and low target activities of recombinant class I (rC1) and class II (rC2) collagenase on human islet yield. Consequently, 4 different enzyme formulations with divergent C1:C2 collagenase mass ratios were assessed, each supplemented with the same dose of neutral protease. Both split pancreas and whole pancreas models were used to test enzyme targets (n = 20). Islet yield/g pancreas was compared with historical enzymes (n = 42). RESULTS: Varying the Wunsch (rC2) and collagen degradation activity (CDA, rC1) target dose, and consequently the C1:C2 mass ratio, had no significant effect on tissue digestion. Digestions using higher doses of Wunsch and CDA resulted in comparable islet yields to those obtained with 60% and 50% of those activities, respectively. Factorial analysis revealed no significant main effect of Wunsch activity or CDA for any parameter measured. Aggregate results from 4 different collagenase formulations gave 44% higher islet yield (>5000 islet equivalents/g) in the body/tail of the pancreas (n = 12) when compared with those from the same segment using a standard natural collagenase/protease mixture (n = 6). Additionally, islet yields greater than 5000 islet equivalents/g pancreas were also obtained in whole human pancreas. CONCLUSIONS: A broader C1:C2 ratio can be used for human islet isolation than has been used in the past. Recombinant collagenase is an effective replacement for the natural enzyme and we have determined that high islet yield can be obtained even with low doses of rC1:rC2, which is beneficial for the survival of islets.

Development and application of purified tissue dissociation enzyme mixtures for human hepatocyte isolation.

Human hepatocyte transplantation is gaining acceptance for the treatment of liver diseases. However, the reagents used to isolate hepatocytes from liver tissue are not standardized and show lot-to-lot variability in enzyme activity and endotoxin contamination. For clinical application, highly purified reagents are preferable to crude digest preparations. A purified tissue dissociating enzyme (TDE) preparation (CIzyme(TM) purified enzymes) was developed based on the enzyme compositions found in a superior lot of collagenase previously used by our group for human hepatocyte isolation. The performance of this enzyme preparation was compared to collagenase type XI on 110 liver cases by assessing hepatocyte yield, viability, and seven other functional assays that included plating efficiency, basal and induced CYP450 activities, phase II conjugation activity, and ammonia metabolism. No statistically significant difference was observed between these TDEs when they were used to isolate hepatocytes from liver resections or organ donor tissue on 54 hepatocyte isolations with type XI enzyme and 56 isolations using CIzyme(TM). These results show that a highly purified and defined TDE preparation can be formulated that provides excellent performance with respect to viability, yield, and functional activity of the isolated cells. In addition to reproducible formulation, these purified enzyme products have only 2-3% of the endotoxin of crude enzyme preparations. These results show that purified enzymes such as CIzyme(TM) will be a safe and effective for the isolation of human hepatocytes for clinical transplants.

Tissue dissociation enzymes for isolating human islets for transplantation: factors to consider in setting enzyme acceptance criteria.

Tissue dissociation enzymes are critical reagents that affect the yield and quality of human pancreatic islets required for islet transplantation. The United States Food and Drug Administration's oversight of this procedure recommends laboratories to set acceptance criteria for enzymes used in the manufacture of islet products for transplantation. Currently, many laboratories base this selection on personal experience because biochemical analysis is not predictive of success of the islet isolation procedure. This review identifies the challenges of correlating results from enzyme biochemical analysis to their effectiveness in human islet isolation and suggests a path forward to address these challenges to improve control of the islet manufacturing process.

Successful human islet isolation and transplantation indicating the importance of class 1 collagenase and collagen degradation activity assay.

BACKGROUND: Purified tissue dissociation enzymes (TDEs) are critical to successful human islet isolation required for clinical transplantation, but little is known about the characteristics of the key enzymes-class I (C1) and class II (C2) collagenase from Clostridium histolyticum-used in these procedures. Here, we show the differences between the C1 collagenase found in purified collagenase products manufactured by three suppliers and the impact of differences in C1 between two suppliers on human islet yield. METHODS: Collagenase from Roche, Serva/Nordmark (Uetersen, Germany), and VitaCyte (Indianapolis, IN) were analyzed by analytical high-performance liquid chromatography and collagen degradation activity (CDA), an assay that preferentially detects intact C1 collagenase. Human islet isolations were performed using current standard practices. RESULTS: These studies showed that the highest amount of intact C1 that correlated with a high specific CDA (CDA unit per milligram of protein). The highest specific CDA was found in VitaCyte product followed by the Roche and Serva/Nordmark products. The products of VitaCyte were used successfully for human islet isolation (n=14) with an average final islet yield obtained was 419,100+/-150,900 islet equivalent number (IEQ) (4147+/-1759 IEQ/g pancreas). Four of these preparations were used successfully in clinical transplantation procedures. These TDEs gave significantly better results when compared with earlier data where 27 isolations were performed using Serva NB1 collagenase and NB neutral protease where the final islet yield was 217,500+/-152,400 IEQ (2134+/-1524 IEQ/g pancreas). CONCLUSIONS: These data indicate the importance of intact C1 and the use of the appropriate analytical assays to correlate biochemical characteristics of TDEs to islet quality and yield.