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Effect of University of Wisconsin solution on the rat islet isolation
Effect of University of Wisconsin Solution on the Rat Islet Isolation W.-T. Lu, B.R.S. Hsu, and J.H. Juang
T
O PROTECT solid organs and possibly cells from injury before transplantation, the cytoprotectant University of Wisconsin (UW) solution, has shown advantages for clinical transplantation. Many centers have used chilled UW solution prevent ischemic injury in a human pancreas before islet isolation. In addition to the ischemic insult, many other forms of injury may occur to islet cells during the isolation process. UW solution was shown to increase canine islet yield and purity during the process of dispersion and purification.1 Furthermore, studies reporting intraductal administration of collagenase in UW solution at the time of harvesting or after 1-hour cold preservation suggest significantly improved porcine islet isolation.2,3 However, the suspension of collagenase in UW solution at the time of distension and digestion of the pancreas produced inferior results in the number and size of isolated human islets.4 To assess the effect of UW replacing Hanks’ balanced salt solution (HBSS) solution during the period of distension, procurement, and digestion of pancreas, we compared the yield and function of islets isolated from nonpreserved rat pancreata. MATERIALS AND METHODS Animals Forty Lewis male rats, weight 200 to 250 g, were anesthetized with intraperitoneally amytal.
Islet Isolation Pancreatic islets of Langerhans were isolated using the two steps of digestion and discontinuous Ficoll gradient purification. Briefly, the common bile duct was cannulated with a PE50 tube, and the pancreas was distended with 10 mL chilled UW solution or HBSS (GIBCO), containing glucose (100 mg/dL), penicillin (100 U/mL), and streptomycin (100 g/mL). The pancreas was carefully dissected from surrounding tissue and placed in cooled HBSS (supplemented as above). After removing residual tissue, each of four pancreata was cut into fragments of 1 to 2 mm3, then digested immediately in UW or HBSS solution with 2 mg/mL type V collagenase (Sigma, St Louis, Mo). For a single isolation, four pancreata were divided into four groups: group A: pancreas was distended with UW and digested with collagenase in UW solution; group B: pancreas distended with HBSS solution and digested with collagenase in UW solution; group C: pancreas distended with UW solution and digested with collagenase in HBSS solution; and group D: pancreas distended with HBSS solution and digested with collagenase in HBSS solution. After digestion, the islets were purified by Ficoll density gradient centrifugation. 0041-1345/03/$–see front matter doi:10.1016/S0041-1345(02)03948-9 490
Cell Count, Size, and Morphology The handed-pick islets were counted under a stereomicroscope. After taking out 100 islets for a 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.
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 (Gibco, Grand Island, NY), 10 mmol/L HEPES, 100 U/mL penicillin, and 100 g/mL streptomycin for culture (37°C, 5% CO2, 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 supplemented with 2 mmol/L L-glutamin, 0.5% bovine serum albumin and either 2.8 or 20 mmol/L glucose solution for 2 hours. The insulin concentration of medium was measured by method of ELISA (Boehinger Mannheim).
Statistical Analysis Results were expressed as mean values with standard error of the mean (X ⫾ SE). For comparisons among the four groups, differences were analyzed by one-way ANOVA. P ⬍ .05 was considered to be a significant difference.
RESULTS
In each situation of isolation, the warm and cold ischemic time of pancreas was less than 1 hour. The mean (⫾ S.E.) islet numbers were 490 ⫾ 17, 491 ⫾ 19, 512 ⫾ 20, 509 ⫾ 12 (P ⫽ .703) and the IEQs were 405.4 ⫾ 14, 398.9 ⫾ 13.1, 394 ⫾ 22.9, 430 ⫾ 15.7 (P ⫽ .384) in groups A, B, C and D, respectively. There were no significant differences in islet yields among the four groups. Before islet culture, the insulin responses to 2.8 glucose solution were 130 ⫾ 11.3, 114 ⫾ 7.2, 115.2 ⫾ 7.1 and 124.2 ⫾ 9.1 U/L, respectively. When using 20 mmol/L glucose solution instead of 2.8 From the Division of Endocrinology and Metabolism, Chang Gung Memorial Hospital, Taoyuan, Taiwan. Supported by grants from the Chang-Gung Medical Research Program (CMRP 1090). Address reprint requests to Dr. Wen-Tsoung Lu, Division of Endocrinology and Metabolism, Department of Internal Medicine, 5 Fu-Shin St., Kweishan, Taoyuan, Taiwan. © 2003 by Elsevier Science Inc. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 35, 490 – 491 (2003)
EFFECT OF UNIVERSITY OF WISCONSIN SOLUTION
mmol/L glucose solution, the insulin responses were 2238.5 ⫾ 108.9, 2177.2 ⫾ 152.4, 2174.8 ⫾ 165.8 and 2177.0 ⫾ 170.9 U/L for groups A, B, C, and D, respectively. After 2-hour culture, the insulin response to 2.8 mmol/L were 172.5 ⫾ 11.8, 183.8 ⫾ 10.2, 178.2 ⫾ 17.1, and 174.5 ⫾ 9.9 U/L for groups A, B, C, and D, respectively. To 20 mmol/L glucose solution, the insulin response were 1797.3 ⫾ 106.6, 1801.2 ⫾ 130.1, 1822.3 ⫾ 106.7, and 1828 ⫾ 85.1 U/L for groups A, B, C, and D, respectively. There were no significant differences in the insulin response to 2.8 or 20 mmol/L glucose solution in this static study. CONCLUSION AND DISCUSSION
Our data indicate that there is no significant difference in islet yield or function by replacing HBSS with UW for distension and digestion of the nonstored rat pancreas. As we know, the pancreas should be well distended before collagenase digestion to obtain a good yield. There are two methods to distend and digest the pancreas. Direct flushing of collagenase into the pancreatic duct achieves greater yield but less purity. On the other hand, adding collagenase to digest the pancreas after distension of the pancreas with medium obtained a lower yield but greater purity. In this study, we separated distension of pancreas from digestion of pancreas to see the effects of UW. Although a UW flush has the advantage of achieving a better yield when the pancreas was stored for more than 6 hours,5 using UW solution to distend the nonstored pancreas was not helpful
491
when the ischemic time was less than 1 hour. During the whole process of canine islet isolation, UW solution has proved helpful in the phases of dispersion and purification.1 In addition, UW exhibited cytoprotective effects when collagenase dissolved in UW and intraductally flushed into the rat or dog pancreas after 24 and 48 hours of cold storage.6 However, there are controversies about the effects when collagenase is added to UW solution to increase islet yield in porcine, and humans.2– 4 In our study, UW solution exhibited no additional benefit or harm in the digestion phase of a nonstored rat pancreas. In conclusion, UW solution may helpful to protect islets from injury during prolonged cold ischemic periods but showed no additional benefit for pancreas distension or collagenase digestion of a nonstored rat pancreas.
REFERENCES 1. Van der Burg MPM, Guicherit OR, Frolich M, et al: Cell Transplantation 3:315, 1994 2. White SA, Hughes DP, Contractor HH, et al: Transplant Proc 27:3366, 1995 3. Van der Burg MPM, Basir I, Zwaan RP, et al: Transplant Proc 30:360, 1998 4. Casanova D, Xenos E, Lloveras JJ, et al: Transplant Proc 26:588, 1994 5. Kneteman NM, Degroot TJ, Warnock GL, et al: Transplant Proc 22:541, 1990 6. Munn SR, Kaufman DB, Field MJ, et al: Transplant Proc 22:2635, 1989
T
O PROTECT solid organs and possibly cells from injury before transplantation, the cytoprotectant University of Wisconsin (UW) solution, has shown advantages for clinical transplantation. Many centers have used chilled UW solution prevent ischemic injury in a human pancreas before islet isolation. In addition to the ischemic insult, many other forms of injury may occur to islet cells during the isolation process. UW solution was shown to increase canine islet yield and purity during the process of dispersion and purification.1 Furthermore, studies reporting intraductal administration of collagenase in UW solution at the time of harvesting or after 1-hour cold preservation suggest significantly improved porcine islet isolation.2,3 However, the suspension of collagenase in UW solution at the time of distension and digestion of the pancreas produced inferior results in the number and size of isolated human islets.4 To assess the effect of UW replacing Hanks’ balanced salt solution (HBSS) solution during the period of distension, procurement, and digestion of pancreas, we compared the yield and function of islets isolated from nonpreserved rat pancreata. MATERIALS AND METHODS Animals Forty Lewis male rats, weight 200 to 250 g, were anesthetized with intraperitoneally amytal.
Islet Isolation Pancreatic islets of Langerhans were isolated using the two steps of digestion and discontinuous Ficoll gradient purification. Briefly, the common bile duct was cannulated with a PE50 tube, and the pancreas was distended with 10 mL chilled UW solution or HBSS (GIBCO), containing glucose (100 mg/dL), penicillin (100 U/mL), and streptomycin (100 g/mL). The pancreas was carefully dissected from surrounding tissue and placed in cooled HBSS (supplemented as above). After removing residual tissue, each of four pancreata was cut into fragments of 1 to 2 mm3, then digested immediately in UW or HBSS solution with 2 mg/mL type V collagenase (Sigma, St Louis, Mo). For a single isolation, four pancreata were divided into four groups: group A: pancreas was distended with UW and digested with collagenase in UW solution; group B: pancreas distended with HBSS solution and digested with collagenase in UW solution; group C: pancreas distended with UW solution and digested with collagenase in HBSS solution; and group D: pancreas distended with HBSS solution and digested with collagenase in HBSS solution. After digestion, the islets were purified by Ficoll density gradient centrifugation. 0041-1345/03/$–see front matter doi:10.1016/S0041-1345(02)03948-9 490
Cell Count, Size, and Morphology The handed-pick islets were counted under a stereomicroscope. After taking out 100 islets for a 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.
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 (Gibco, Grand Island, NY), 10 mmol/L HEPES, 100 U/mL penicillin, and 100 g/mL streptomycin for culture (37°C, 5% CO2, 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 supplemented with 2 mmol/L L-glutamin, 0.5% bovine serum albumin and either 2.8 or 20 mmol/L glucose solution for 2 hours. The insulin concentration of medium was measured by method of ELISA (Boehinger Mannheim).
Statistical Analysis Results were expressed as mean values with standard error of the mean (X ⫾ SE). For comparisons among the four groups, differences were analyzed by one-way ANOVA. P ⬍ .05 was considered to be a significant difference.
RESULTS
In each situation of isolation, the warm and cold ischemic time of pancreas was less than 1 hour. The mean (⫾ S.E.) islet numbers were 490 ⫾ 17, 491 ⫾ 19, 512 ⫾ 20, 509 ⫾ 12 (P ⫽ .703) and the IEQs were 405.4 ⫾ 14, 398.9 ⫾ 13.1, 394 ⫾ 22.9, 430 ⫾ 15.7 (P ⫽ .384) in groups A, B, C and D, respectively. There were no significant differences in islet yields among the four groups. Before islet culture, the insulin responses to 2.8 glucose solution were 130 ⫾ 11.3, 114 ⫾ 7.2, 115.2 ⫾ 7.1 and 124.2 ⫾ 9.1 U/L, respectively. When using 20 mmol/L glucose solution instead of 2.8 From the Division of Endocrinology and Metabolism, Chang Gung Memorial Hospital, Taoyuan, Taiwan. Supported by grants from the Chang-Gung Medical Research Program (CMRP 1090). Address reprint requests to Dr. Wen-Tsoung Lu, Division of Endocrinology and Metabolism, Department of Internal Medicine, 5 Fu-Shin St., Kweishan, Taoyuan, Taiwan. © 2003 by Elsevier Science Inc. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 35, 490 – 491 (2003)
EFFECT OF UNIVERSITY OF WISCONSIN SOLUTION
mmol/L glucose solution, the insulin responses were 2238.5 ⫾ 108.9, 2177.2 ⫾ 152.4, 2174.8 ⫾ 165.8 and 2177.0 ⫾ 170.9 U/L for groups A, B, C, and D, respectively. After 2-hour culture, the insulin response to 2.8 mmol/L were 172.5 ⫾ 11.8, 183.8 ⫾ 10.2, 178.2 ⫾ 17.1, and 174.5 ⫾ 9.9 U/L for groups A, B, C, and D, respectively. To 20 mmol/L glucose solution, the insulin response were 1797.3 ⫾ 106.6, 1801.2 ⫾ 130.1, 1822.3 ⫾ 106.7, and 1828 ⫾ 85.1 U/L for groups A, B, C, and D, respectively. There were no significant differences in the insulin response to 2.8 or 20 mmol/L glucose solution in this static study. CONCLUSION AND DISCUSSION
Our data indicate that there is no significant difference in islet yield or function by replacing HBSS with UW for distension and digestion of the nonstored rat pancreas. As we know, the pancreas should be well distended before collagenase digestion to obtain a good yield. There are two methods to distend and digest the pancreas. Direct flushing of collagenase into the pancreatic duct achieves greater yield but less purity. On the other hand, adding collagenase to digest the pancreas after distension of the pancreas with medium obtained a lower yield but greater purity. In this study, we separated distension of pancreas from digestion of pancreas to see the effects of UW. Although a UW flush has the advantage of achieving a better yield when the pancreas was stored for more than 6 hours,5 using UW solution to distend the nonstored pancreas was not helpful
491
when the ischemic time was less than 1 hour. During the whole process of canine islet isolation, UW solution has proved helpful in the phases of dispersion and purification.1 In addition, UW exhibited cytoprotective effects when collagenase dissolved in UW and intraductally flushed into the rat or dog pancreas after 24 and 48 hours of cold storage.6 However, there are controversies about the effects when collagenase is added to UW solution to increase islet yield in porcine, and humans.2– 4 In our study, UW solution exhibited no additional benefit or harm in the digestion phase of a nonstored rat pancreas. In conclusion, UW solution may helpful to protect islets from injury during prolonged cold ischemic periods but showed no additional benefit for pancreas distension or collagenase digestion of a nonstored rat pancreas.
REFERENCES 1. Van der Burg MPM, Guicherit OR, Frolich M, et al: Cell Transplantation 3:315, 1994 2. White SA, Hughes DP, Contractor HH, et al: Transplant Proc 27:3366, 1995 3. Van der Burg MPM, Basir I, Zwaan RP, et al: Transplant Proc 30:360, 1998 4. Casanova D, Xenos E, Lloveras JJ, et al: Transplant Proc 26:588, 1994 5. Kneteman NM, Degroot TJ, Warnock GL, et al: Transplant Proc 22:541, 1990 6. Munn SR, Kaufman DB, Field MJ, et al: Transplant Proc 22:2635, 1989