Islet isolation success for clinical islet transplantation critically depends on digestive enzyme variables such as enzyme activities (1–3), quantity (2), and ratio of specific components (1, 2, 4–11). Furthermore, enzyme localization in native pancreas tissue may play a role (12, 13). Commercial enzymes represent the greatest reagent costs for human islet isolation, yet little is known about their uptake into the pancreas. Absorption of commercially available digestive enzymes within a donor pancreas is postulated to have a major impact on the outcome of islet isolation. To assess enzyme dynamics on samples obtained during the initial 20 min of enzyme retrograde ductal perfusion into the human pancreas, measurements of total protein as well as the degradation of N-3-([2-furyl]-acryloyl)-Leu-Gly-Pro-Ala (FALGPA) (14), described herein as class II collagenolytic-like activity (C2LA), and Nα-benzoyl-L-arginine-ethyl ester hydrochloride (BAEE) as a measure of tryptic-like activity (TLA), were performed.
All pancreases were intact with no abnormal morphology: age (yr), body mass index (kg/m2), islet equivalents per gram pancreas, percent islet purity, and transplanted yes/no (Y/N) were 45, 33.6, 1953, 41%, and Y for pancreas 1; 46, N/A, 3975, 38%, and Y for pancreas 2; 62, 34, 718, 17%, and N for pancreas 3; 56, 23.7, 2446, 68%, and Y for pancreas 4; and 63, 22.7, 1537, 29%, and N for pancreas 5. Collagenase and thermolysin enzymes (Vitacyte, Indianapolis, IN; lot numbers BHA-110919 and TH-101020 for pancreas 1, BHA-120511 and TH-120607 for pancreases 2–4, and BHA-120511 and TH-110130 for pancreas 5) were dissolved in standard enzyme buffer (15). Supplemental enzyme buffer was added as required during infusion into the pancreatic duct (total volumes for pancreases 1–5 were 120, 140, 160, 156, and 135 mL, respectively), and the Clinical Islet Transplantation Consortium recommendations for perfusion pressure were followed. Before the perfusion start and at 5-min intervals (20 min total, the 5-min sample was not collected from pancreases 4 and 5), 500 μL samples were collected and filtrated through a cellulose filter (0.45 μm; Steriltech, Kent, WA) to avoid tissue and cell contamination on subsequent measurements of enzyme activities and protein concentrations. The C2LA and TLA measurements were determined in quadruplicate at 25°C at 0.1 mM FALGPA (Sigma-Aldrich, Stockholm, Sweden) and 0.5 mM BAEE (Sigma), respectively, with a Hitachi U-2910 spectrophotometer. For C2LA and TLA, 1 U was defined as the conversion of 1 μmol FALGPA or BAEE substrate, respectively, to product per minute. Protein concentrations were determined in quadruplicate by Bradford assay with bovine serum albumin standards (Sigma) (16, 17). Values are presented as mean±standard deviation.
The changes in total circulating levels of C2LA and TLA correspond to the amount of enzymes absorbed by the pancreas during the 20 min of sustained ductal infusion. The dynamics of C2LA and TLA had diverging patterns, with decreases in C2LA (Fig. 1A) and stable or increasing TLA (Fig. 1B). TLA increased by 56±20 U after 20 min, likely indicating release of TLA enzymes from the organ.
The C2LA (Fig. 1A) from the start of the perfusion phase was 1077±143 U. After 10 min, C2LA activities decreased by 35%±12%, indicating it as the major period of enzyme absorption, with little benefit from increased time of perfusion. Alarmingly, even after the completion of the 20 min of recirculation of enzymes into the pancreatic duct, no additional C2LA activity was absorbed within the pancreas. Hence, a vast amount of collagenase remains in the liquid phase with little effect on the digestion of the pancreas, that is, a pancreatic biopsy taken before ductal perfusion of enzymes and subsequently placed in the digestion chamber remains macroscopically intact, whereas the pancreas containing the enzyme blends is efficiently digested. By the end of perfusion, circulating protein levels were similar or increased (Fig. 1C), reflecting low enzyme digestion activity at 8°C to 14°C.
Previous publications have focused on characterizing analytic enzyme data, for example, protein composition (1, 2, 11), integrity (3, 4), and initial activities added to the pancreas (6, 10). The approach applied here provides new insights into the absorption of enzyme activities in the human pancreas during islet isolation. Surprisingly, an ordinary human pancreas seems to be saturated already after 10 min of low-temperature enzyme perfusion and absorbs only about a third of total C2LA units of collagenase. The presented findings imply that the amount and administration of enzymes utilized for human islet isolation can be dramatically optimized. Enzyme activities can be monitored to identify specific, dynamic variables, potentially leading to optimal application of costly commercial enzymes to increase the yield and quality of isolated human islets.
Andrew S. Friberg
Division of Clinical Immunology
Department of Immunology
Genetics and Pathology
1. Antonioli B, Fermo I, Cainarca S, et al.. Characterization of collagenase blend enzymes for human islet transplantation. Transplantation
2007; 84: 1568.
2. Barnett MJ, Zhai X, LeGatt DF, et al.. Quantitative assessment of collagenase blends for human islet isolation. Transplantation
2005; 80: 723.
3. Balamurugan AN, Breite AG, Anazawa T, et al.. Successful human islet isolation and transplantation indicating the importance of class 1 collagenase and collagen degradation activity assay. Transplantation
2010; 89: 954.
4. Brandhorst H, Asif S, Andersson K, et al.. The effect of truncated collagenase class I isomers on human islet isolation outcome. Transplantation
2010; 90: 334.
5. Brandhorst H, Brendel MD, Eckhard M, et al.. Influence of neutral protease activity on human islet isolation outcome. Transplant Proc
2005; 37: 241.
6. Kin T, O’Gorman D, Senior P, et al.. Experience of islet isolation without neutral protease supplementation. Islets
2010; 2: 278.
7. Kin T, Zhai X, O’Gorman D, et al.. Detrimental effect of excessive collagenase class II on human islet isolation outcome. Transpl Int
2008; 21: 1059.
8. Kin T, Zhai X, Murdoch TB, et al.. Enhancing the success of human islet isolation through optimization and characterization of pancreas dissociation enzyme. Am J Transplant
2007; 7: 1233.
9. Wolters GH, Vos-Scheperkeuter GH, van Deijnen JH, et al.. An analysis of the role of collagenase and protease in the enzymatic dissociation of the rat pancreas for islet isolation. Diabetologia
1992; 35: 735.
10. Brandhorst H, Friberg A, Andersson HH, et al.. The importance of tryptic-like activity in purified enzyme blends for efficient islet isolation. Transplantation
2009; 87: 370.
11. Balamurugan AN, Loganathan G, Bellin MD, et al.. A new enzyme mixture to increase the yield and transplant rate of autologous and allogeneic human islet products. Transplantation
2012; 93: 693.
12. Cross SE, Hughes SJ, Partridge CJ, et al.. Collagenase penetrates human pancreatic islets following standard intraductal administration. Transplantation
2008; 86: 907.
13. Balamurugan AN, He J, Guo F, et al.. Harmful delayed effects of exogenous isolation enzymes on isolated human islets: relevance to clinical transplantation. Am J Transplant
2005; 5: 2671.
14. Van Wart HE, Steinbrink DR. A continuous spectrophotometric assay for Clostridium histolyticum
collagenase. Anal Biochem
1981; 113: 356.
15. Goto M, Eich TM, Felldin M, et al.. Refinement of the automated method for human islet isolation and presentation of a closed system for in vitro islet culture. Transplantation
2004; 78: 1367.
16. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem
1976; 72: 248.
17. Stoscheck CM. Quantitation of protein. Methods Enzymol
1990; 182: 50.