Biomedical subjects

W Cui

Publications and source records attributed to W Cui.

93 records · Page 6Linked to original sources

PubMedDate not supplied

Improved large-scale isolation of breeder porcine islets: possibility of harvesting from nonheart-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 conventional isolation procedure. At a slaughterhouse we first selected a breeder pig over 1.5 years old (and over 200 kg in weight) with warm ischemic time (WIT) of 15 +/- 2 minutes as nonheart-beating donors. Then, we made a special enzymic mixture that consisted of collagenase S-1 (260 U/mg, NittaZelatin, Japan), collagenase P (1.86 U/ml Lyo Boehringer-Mannheim, USA), DNase (Sigma, St. Louis, Mo), Disparse (NittaZelatin, Japan), and protease inhibitor (Sigma). Third, this mixture was injected very gently into the pancreatic duct at the time of pancreatic harvesting. To prevent overdigestion of the pancreas, the mixture was first cooled to less than 10 degrees C. Fourth, during the warm digestion of pancreas, the pancreas with the enzymic mixture was quietly put in a water bath at 37 degrees C without mechanical shaking. Fifth, we purified the islets with a COBE 2991 cell processor by the Dextran 70 gradient method, because Dextran 70 is very cheap and has the same purification effect as the Ficoll gradient. The results of 10 consecutive breeder porcine islet isolations are reported. The total yield of isolations of islets over 50 microm in the longest diameter after staining with 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%. Glucose stimulation during in vitro incubation induced significant insulin release from isolated breeder porcine islets. In two of the diabetic rats receiving encapsulated islets grafts using a mesh-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 islets without immunosuppression. In conclusion, we succeeded in a more efficient and less-expensive isolation of a large amount of adult porcine islets from a nonheart-beating donor.

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PubMedDate not supplied

Effect of a new immunosuppressive agent, FTY720, on survival of islet allografts.

A newly developed immunosuppressant, FTY720, has a unique mechanism that is quite different from those of conventional immunosuppressants, and is presumed to be mediated through decreases in the number of peripheral lymphocytes, especially helper T cells. This study was performed to ascertain whether this innovative drug could prolong islet allograft survival. The donors were inbred Lewis rats and the recipients were ACI rats rendered hyperglycemic with intravenous streptozotocin. In the study group, FTY720 dissolved in distilled water was orally administered at a dose of 5 mg/kg to the recipient ACI rats 1 day before and on the day of grafting. In the control group, only distilled water was orally administered to the recipient ACI rats on the day before and the day of grafting. Two thousand islets were transplanted into the portal vein of the recipient rats in the study and control groups immediately after isolation. The graft survival time in the study group was significantly longer than that in the control group, indicating that FTY720 retains a potent effect on the prolongation of islet allograft survival. FTY720 could become a useful immunosuppressant for future clinical islet allotransplantation.

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PubMedDate not supplied

Novel method for isolation of adult porcine pancreatic islets with two-stage digestion procedure.

It is particularly difficult to isolate porcine islets (PI). Experience suggests that the success rate of porcine islet isolation (PII) is probably considerably influenced by the distension and digestion of the pancreas. In this study, we divided the digestion procedure into two stages and developed a new enzyme solution to improve both the distension and digestion procedures. As a result, we established a novel and stable method of large-scale adult porcine islet isolation (APII). The harvested pancreata of 2-year-old pigs weighing over 200 kg (n = 18) were distended by introducing our new enzyme solution gently and slowly through the pancreatic ducts. Two-stage digestion (cold, then warm) was then performed by first placing the distended pancreata on ice for 2 h to cause diffusion of the enzyme solution around the islets, and then by incubating the pancreata in a water bath at 37 degrees C for 45 min without shaking. The islets were purified by a COBE 2991 cell processor on dextran T70 discontinuous density gradients. Histological study was performed on porcine pancreata sampled after 0, 15, 30, and 45 min of the second stage, and stained with H&E stain. Next, islet equivalent was calculated. Static incubation study was performed by stimulating the islets with 3.3 and 16.7 mM glucose in Krebs' Ringer bicarbonate buffer (KRBB) solution at 37 degrees C for 1 h, and finally the insulin released was measured. The dilated acinar cells septa around the islets were observed at time 0. Destruction of the acinar cells around the islets by warm digestion was recognized at 15 and 30 min, and destroyed and separated acinar cells present around the islets at 45 min. During the entire course of the warm digestion, the islets remained intact. The number of isolated islets was 291,667 +/- 240,452 IEQ/pancreas (n = 14) and 3,294 +/- 2199 IEQ/g of pancreatic tissue. The purity of recovered porcine islets was over 90%. The concentration of the insulin secreted by 10,000 IEQ islets selected at random was 83.9 +/- 13.4 microU/dish/h in response to 3.3 mM glucose and 104.1 +/- 12.9 microU/dish/h in response to 16.7 mM glucose (n = 20). A success rate of approximately 80% was attained with APII. We demonstrated that this increase in the success rate was due to the improved distension and digestion provided by this method. This two-stage APII method with its new enzyme solution may facilitate the future use of porcine islets in clinical xenotransplantation trials.

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