Technical Improvement of Human Pancreatic Islet Isolation

Technical Improvement of Human Pancreatic Islet Isolation M. Goto, T.M. Eich, M. Stahle, A. Malmborg, M. Engkvist, and O. Korsgren ABSTRACT Introduction. A key factor for successful islet isolation is to place the optimal amount of enzyme into the pancreatic ducts prior to starting digestion of pancreatic glands. To improve this procedure, we introduced novel techniques to identify and repair tissue damage resulting in leakage of collagenase solution. Materials and Methods. One hundred twelve standardized consecutive islet isolations were for the effects of dye and glue on islet yield, islet function using a perifusion assay, and the possibility of clinical transplantation. One group of pancreata (n ⫽ 26) obtained en bloc together with duodenum were carefully detached with ligation of accessory ducts in an isolation unit (WPD group), whereas the pancreata were dissected from the duodenum in the operating room in the other 86 isolations. In 28 of 86 isolations, whole glands were used (WP group), while only the body and tail area were applied in the remaining 58 isolations (PP group). Results. Both dye and glue effectively prevented leakage of collagenase from the gland. Both islet yield and success rate were higher with these tools without adverse effects on islet function or collagenase activity. The success rate of isolations and islet yield were significantly higher in the WPD group (P ⫽ .02 and .001, respectively). Conclusions. Dye and glue may be useful tools to improve human islet isolation procedures. In addition, the use of the whole pancreas further improves the outcome.

D

ESPITE THE BREAKTHROUGH introduced by the Edmonton protocol, islet transplantation still has a major drawback: in most cases islets from one donor are insufficient to cure diabetic patients.1 A key factor for successful islet isolation is to place the optimal amount of enzyme into the pancreatic ducts prior to starting digestion of the pancreatic glands.2,3 To improve this procedure, we introduced novel techniques to identify and repair tissue damage resulting in leakage of collagenase solution. Furthermore, three dissection procedures were compared in terms of complete digestion of the glands. MATERIALS AND METHODS One hundred twelve standardized consecutive islet isolations were evaluated. Dye solutions (Metyltioninklorid) and tissue glue (Indermil) were applied to detect leakage and repair damaged parenchyma. The effects of dye and glue were evaluated in terms of islet yield, islet function using the perifusion assay, and the possibility to use these preparations for clinical transplantation. One group of pancreata (n ⫽ 26) obtained en bloc together with the duodenum were carefully detached with ligation of accessory ducts in an isolation unit (whole pancreas with duodenum: WPD group),

whereas the pancreata were dissected from the duodenum in the operating room in the other 86 isolations. In 28 of the 86 isolations, whole glands were used (whole pancreas: WP group), while only the body and tail area were obtained for the remaining 58 isolations (partial pancreas: PP group).

RESULTS

Both dye and glue effectively prevented leakage of collagenase from the gland. Despite having been applied to only From the Department of Radiology, Oncology and Clinical Immunology, Division of Clinical Immunology, the Rudbeck Laboratory, University Hospital, Uppsala, Sweden. This study was supported by grants from the Swedish Medical Research Council (16P-13568 and 16X-12219), the Åke Wiberg Foundation, the Nordic Insulin Fund, the Torsten and Ragnar Soderbergs Foundation, the Ernfors Family Fund, Barn Diabetes Fonden, the Swedish Diabetes Association, the Juvenile Diabetes Foundation International, the Knut and Alice Wallenberg Foundation, and the Novo Nordic Foundation. Address reprint requests to Masafumi Goto, Division of Clinical Immunology, The Rudbeck Laboratory C11, SE-751 85 Uppsala, Sweden. E-mail: [email protected]

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0041-1345/05/$–see front matter doi:10.1016/j.transproceed.2005.01.074

Transplantation Proceedings, 37, 1313–1314 (2005)

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Table 1. Effect of the Use of Dye and Glue, and Dissection Procedure on the Outcome of Isolation

Dye ⫹ (n ⫽ 23) ⫺ (n ⫽ 89) P Glue ⫹ (n ⫽ 6) ⫺ (n ⫽ 106) P Dissection procedure PP (n ⫽ 58) WP (n ⫽ 28) WPD (n ⫽ 26) P

Success rate (%)

IEQs/g

34.8 23.6 .27

4207 ⫾ 451 3469 ⫾ 342 .02

50.0 22.3 .18

4479 ⫾ 809 3579 ⫾ 299 .14

22.4 (13/58) 14.3 (4/28) 46.2 (12/26) .02

3954 ⫾ 464 1946 ⫾ 212 4685 ⫾ 481 .001

IEQs, islet equivalents; PP, dissection using partial pancreas; WP, dissection using whole pancreas; WPD, dissection using whole pancreas with duodenum.

damaged pancreata, both islet yield and success rates were higher when these tools were used (dye: IEQs/g, 4207 ⫾ 451 vs 3469 ⫾ 342 [P ⫽ .02]; success rate, 34.8% vs 23.6% [P ⫽ .27]; glue: IEQs/g, 4479 ⫾ 809 vs 3579 ⫾ 299 [P ⫽ 0.14];

success rate, 50.0% vs 22.3% [P ⫽ .18]; Table 1). No adverse effects on islet function or collagenase activity were observed. The success rate of isolations and islet yields were significantly higher among the WPD group (P ⫽ .02 and .001, respectively; Table 1). When the WPD group was combined with the use of dye and glue, the success rate significantly increased (50.0% vs 21.3%, P ⫽ .02). DISCUSSION

Dye and glue are useful tools to improve the islet isolation procedure. In addition, the use of the whole pancreas may further improve outcomes. REFERENCES 1. Ryan EA, Lakey JR, Rajotte RV, et al: Clinical outcomes and insulin secretion after islet transplantation with the Edmonton protocol. Diabetes 50:710, 2001 2. Lakey JR, Warnock GL, Shapiro AM, et al: Intraductal collagenase delivery into the human pancreas using syringe loading or controlled perfusion. Cell Transplant 8:285, 1999 3. Shapiro AM, Hao E, Rajotte RV, et al: Stationary digestion after intraductal collagenase improves islet recovery in the rat. Transplant Proc 27:3378, 1995