Effect of Trypsin Inhibitor on Islet Isolation From Fresh and Cold Preserved Rat Pancreas W.-T. Lu, J.R.T. Lakey, J.-H. Juang, B.R.-S. Hsu, and R.V. Rajotte O BTAINING more islets has limited the progress of human islet transplantation. Multiple factors, includ- ing ischemia time, different types of collagenase, distension degree applied to the pancreas, mechanic trauma, and variable digestion might be involved in the low islet recov- ery rate from a cadaveric pancreas. 1–3 Among these factors, the collagenase digestion phase is thought to be the most crucial issue because of the between batch variation. 4 In addition to the variable content of exogenous proteases in different collagenase blends, it is more attractive to inves- tigate the variable activity of endogenous proteases re- leased by pancreatic acinar cells during different digestion conditions. 5 Wolters et al, described a positive effect (48 %) of a collagenase solution supplemented with 10% bovine serum albumin as a protease inhibitor to increase rat islet yield. 6 Two studies reported that pefabloc reduces trypsin activity leading to a higher yield in pig islet isola- tion. 7,8 However, one study suggested that trypsin could be used to isolate islets from the rat pancreas. 9 Soybean trypsin inhibitor (STI) inhibits the activity of trypsin derived from different species. 10 To test the effects of trypsin inhibitor on islet yield, we checked islet number, size distribution, and viability in the presence of different concentrations of STI added during collagenase digestion of rat pancreata. MATERIALS AND METHODS Animals Eighty Lewis male rats, weight 200 to 250 g, were anesthetized with intraperitoneal somnotol. Islet Isolation Pancreatic islets of Langerhans were isolated using the standard technique of collagenase digestion and discontinuous Ficoll gradi- ent purification. Briefly, the common bile duct was cannulated with a PE50 tube, and the pancreas distended with 10 mL chilled University of Wisconsin (UW) solution or Hanks’ balanced salt solution (HBSS; GIBCO), containing glucose (100 mg/dL), peni- cillin (100 U/mL), and streptomycin (100 g/mL). The pancreas was carefully dissected from surrounding tissues and placed in cooled HBSS (supplemented as above) either for digestion or for exposure to UW solution for 6 to 8 hours cold preservation. Before digestion, each of five pancreata was cut into 1 to 2 mm, 3 fragments then digested immediately under five different conditions: group A (n  10 for fresh and cold preserved pancreas, respectively): 2 mg/mL type V collagenase (Sigma, ST Louis, Mo) without soybean trypsin inhibitor (STI); Group B (n  10 for fresh and cold preserved pancreas, respectively) mixed with 1 mmol/L STI; Group C(n  10 for fresh and cold preserved pancreas, respectively) mixed with 2 mmol/L STI; and group D (n  10 for fresh and cold preserved pancreas, respectively) mixed with 4 mmol/L STI. After digestion, the islets were filtered through a 220-m nylon screen, then purified by Ficoll density gradient centrifugation. Cell Count, Size and Morphology The hand-picked islets were counted under a stereomicroscope. After removing 100 islets for the static study and 150 islets for culture, the remaining islets were stained with dithizone, and the mean diameters of all islets smaller than 75 m, were recorded in 50-m increments for calculation of islet equivalent (IEQ) yield according to the international procedure. 6 Viability One hundred fifty islets (100 to 200 m in diameter) were put into the CMRL culture medium supplemented with 10 % (vol/vol) fetal calf serum (FCS, Gibco, Grand Island, NY), 10 mmol/L HEPES, 100 U/mL penicillin and 100 g/mL streptomycin for culture (37°C, 5 % CO 2 , 95 % air for 36 to 48 hrs). For testing viability before and 36 to 48 hours after culture, 25 islets (100 to 200 m in diameter) were hand-picked and incubated in 1.5 mL RPMI medium supple- mented with 2 mmol/L L-glutamine, 0.5 % BSA and either 2.8 or 20 mmol/L glucose solution for 2 hours. The insulin concentration of the medium was measured using an ELISA (Boehinger Mann- heim). Statistical Analysis Results are expressed as mean values and standard errors of the mean (X  S.E). For comparisons among the three groups, differences were analyzed by one-way ANOVA. P  .05 was considered to be a significant difference. From the Division of Endocrinology and Metabolism, Chang Gung Memorial Hospital, Taoyuan, Taiwan and the Department of Surgery-Medicine Institute, University of Alberta, Alberta, Canada. Supported by grants from the Chang-Gung Medical Research Program (CMRP 1090). Address reprint request to Dr. Wen-Tsoung Lu, Division of Endocrinology and Metabolism, Department of Internal Medi- cine, 5 Fu-Shin St., Kweishan, Taoyuan, Taiwan. 0041-1345/03/$–see front matter © 2003 by Elsevier Science Inc. doi:10.1016/S0041-1345(02)03947-7 360 Park Avenue South, New York, NY 10010-1710 488 Transplantation Proceedings, 35, 488 – 489 (2003)