Korean Diabetes Journal 2004;28(2):75-87.
Published online April 1, 2004.
Development of Adult Porcine Islet Isolation Method for Xenotransplantation.
Sung Rae Kim, Kun Ho Yoon, Hyuk Sang Kwon, Sun Hee Suh, Seung Hyun Ko, Jung Min Lee, Soon Jib Yoo, Yoo Bae Ahn, Ki Ho Song, Hyun Shik Son, Moo Il Kang, Bong Yun Cha, Kwang Woo Lee, Ho Young Son, Sung Koo Kang
Division of Endocrinology and Metabolism, Department of Internal Medicine, The Catholic University of Korea, Seoul, Korea.
Abstract
BACKGROUND
AND PURPOSE: Xenotransplantation using porcine islet cells might be an alternative to allotransplantation, which has been limited due to the lack of donors. Various researches using porcine islet cells have been performed in foreign countries; however, they have never been studies in Korea. Therefore, the purpose of this study was to explore the possibility of thise new treatment for cases of diabetes by establishing of improved islet isolation skill. METHODS: The pancreas and islets were extracted from pigs weighing around 100kg. To establish an islet isolation method, the islet yield, purity and the distribution size of the isolated islets were step wise compared in various ways, and then the superior method adopted. To determine the conveyance method after organ extraction, the conveyance method of pouring collagenase P was compared with the conveyance method of injecting Custidol. For digestion, the mechanical shaking and static incubation methods were also compared. To isolate islets from the digested pancreata, isolation methods were analyzed using 3 and 4 layers' Ficoll. The islet yield was appraised after their isolation using the optimized islet isolation method. To assess the results of the islet isolation, appraised the purity and the survival rates of cells, the insulin secretion resulting from the glucose stimulation test was examined. RESULTS: The method of injecting 4degrees C Custidol was effective for the conveyance and storage of the isolated pancreas in comparison with an injection of collagenase P(3465+/-1488 IEQ/g pancreas vs. 48+/-1.7 IEQ/g pancreas, p<0.01). The digestion method was superior to the mechanical shaking method at keeping a stable condition(3465+/-1488 IEQ/g pancreas vs. 1265+/-141.4 IEQ/g pancreas, p<0.01). Ficoll isolation using 3 layers gave the same results as using 4 layers. The average weights of the isolate Pancreatic islets was 23.8+/-3.3g. The numbers of islets per gram was 3465+/-1488.2(IEQ), with a the purity of 86.3+/-2.0%, and a survival rate of over 95%. The insulin secretion caused by glucose stimulation substantially increased in concentration from 24 to 72 hours(24hr: 5mM 3.12mU/mL --< 20mM 6.79mU/mL(2.17 fold), 72hr: 5mM 2.38mU/mL --< 9.93mU/mL(4.17fold))
Key Words: Porcine pancreas, Xenotransplantation, Islet isolation
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