| Budget Amount *help |
¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 1996: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1995: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Research Abstract |
1. Improved large-scale isolation of breeder porcine islets - Possibility of harvesting from non-heart-beating donor -
To establish a large-scale isolation procedure for adult porcine islets usable as a donor source for xenotransplantation and as a model of human islet isolation. we improved several characteristics of the coventional isolation procedure. At a slaughterhouse we first selected breeder pig over 1.5 years old (and over 200 kg in weight) whose warm ischemic time (WIT) were 1.5*2 minutes as non-heart-beating donors. Then, we made a special enzymic mixture whose maincontents were collagenase S-1 (260 U/mg, NittaZelatin, Japan), collagenase P (1.86 U/ml Lyo Boehringer-Mannheim. U.S.A.), DNase (Sigma. U.S.A.). Disparse (NittaZelatin, Japan), Protease inhibitor (Sigma, USA). Neat, this mixture was injected very gently into the pancreatic duct at the time of pancretic harvesting. To prevent overdigestion of the pancreas, the mixture was first cooled to less than 10ﾟC.Fourth by, we
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purified the islets with a COBE 2991 cell processor by the Dextran 70 gradient method. because Dextran 7O is very cheap and has the same purification effect as the Ficoll gradient. The results often consecutive breeder porcine islet isolations are reported. The total yield of isolations of islets over 50 mu m in the longest diameter after staining wish Dithizone (DTZ) was 85,900 * 19,954 islets. 291,667 * 240,452 IEQ (2,900*2,324 IEQ/g). The purity of the isolated islets was very high : 90.2 * 3.8%. Glncose stimulation during in vitro incubation induced signiliesnt insulin release from isolated breeder porcine islets. In two of the diabetic rats receiving encapsulated islets grafts using am esh-reinforced polyvinyl alcohol hydrogel bag (MRPB), a prominent reduction in serum glucose levels (less than 200 mg/dl) persisted for 13 and 19 days. respectively, after intraperitoneal xenotransplantation of isolated breeder porcine islets without immunosuppression. In conclusion, we succeeded in efficiently and cheaply isolating a large amount of adult porcine islets from a non-heart-beating donor.
2. Effectiveness of acidic oxidative potential water in preventing bacterial infection in islet transplantation
"Acidic oxidative potential water" (also termed "electrolyzed strong acid solution", "function water") which was developed in Japan, is a strong acid formed on the anode in the electrolysis of water containing a small amount of sodium chloride. It has these physical properties : pH, from 2.3 to 2.7 ; oxidative-reduction potential, from 1,000 to 1,100 mV ; dissolved chlorine. from 30 to 40 ppm ; and dissolved oxygen. from 10 to 30 ppm. Because of these properties, acidic oxidative potential water has strong bactericidal effects on all bacteria including methicillin-resistant staphylococcus oureus (MRSA), viruscs including HIV, HBV, HCV, CMV, and fungi as a result of the action the active oxygen and active chlorine that it contains. We conducted this study to evaluate the effect of acidic oxidative potential water irrigation on bacterial contamination on the harvesting of porcine pancreata from slaughterhouses for islet xenofransplantation by counting the number of pancreatic surface bacteria using the Dip-slide method, and on the results of islet culture ; and to evaluate the direct effect on isolated islets when it is used to prevent bacterial contamination by the static incubation test and by morphorogical examination. Direct irrigation of the pancreas by acidic oxidative potential water was found to very effective by prevent bacterial contamination, but direct inigation of isolated islets slightly decreased their viability and function. Less
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