THE ADVANTAGE OF INSULIN TREATMENT IN RAT ISLET TRANSPLANTATION
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P1180 Aims: In the initial days after transplantation hyperglycemia has been related to increasedβcell death and reducedβcell mass, suggesting that transitory hyperglycemia could have a positive effect on transplanted islets. The aim of this study is to identify the insulin treatment that can reduce βcell death after syngeneic islet transplantation. And to test whether insulin treatment to recipient before and after transplantation can save the number of transplanted islet. Methods: Male Lewis rats aged 8-10weeks were used as donors and recipients of transplantation. Islets were isolated by collagenase digestion and histopaque separation method and hand-picked under a dissecting microscope. Islets were cultured for 5days and transplanted into the renal subcapsular space of streptozotocin induced diabetic rats. Three groups of recipient were studied. Group1 (n=6): no insulin treatment; Group2 (n=6): insulin treatment from day 7 before transplantation to transplantation day; Group3(n=6): insulin treatment from day 7 before transplantation to day 7 after transplantation. In each group, 250 islets and 500 islets were transplanted. Blood glucose were measured 28 day after transplantation. Nephrectomy was performed on day 28 and graft was examined in histology (hematoxylin and eosin stain) and immunohistochemistory (insulin stain). Results: In each group, recipient rats transplanted 500 islets maintained normoglycemia throughout the initial 14days. Islet grafts were nice looking and well vascularized. Histologically, these grafts contained abundantβcell. On the other hand, in group3 recipient rats transplanted 250 islets maintained normoglycemia and grafts contained enoughβcell, but failed in that of group1 and group2. Conclusions: Insulin-induced normoglycemia in the initial 7days after islet transplantation could maintain the islets graft in good condition. As a result, we could reduce transplanted islets (250 islets) to maintain normoglycemia.Keywords:
Islet cell transplantation
The enzymatic dissociation of acinar tissue by collagenase is a substantial step in the isolation of pancreatic islets. Although essential collagenase components have been purified, the variability in the activity of different batches limits long-term reproducibility of isolation success. The utilization of purified recombinant proteases would solve this problem. In the present study, pancreases from multiorgan donors were dissociated by means of digestion-filtration using either Liberase HI (n = 51) or a recombinant collagenase blend (n = 25). No significant differences were found regarding islet yield before and after purification, the percent of exocrine-attached islets, and final purity. However, the ratio between islet equivalents and islet numbers indicated a lesser fragmentation in islets isolated with recombinant collagenase (P < 0.01). In contrast, viability was slightly higher in islets isolated with Liberase (92.3 ± 0.8 vs. 85.6 ± 2.9%; P < 0.05). Insulin release during static glucose incubation was not different between experimental groups. Islet transplantation into diabetic nude mice resulted in sustained normoglycemia in either group until the graft was removed. These results demonstrated that viable human islets can be isolated using recombinant collagenase. Final optimization of this enzyme blend would offer continuous reproducibility of isolation success.
Microbial collagenase
Interstitial collagenase
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Objective To observe the effect of treating diabetic mice with microcapsulated rat islet cell transplantation.Methods Diabetic mice were randomly divided into 3 groups:control group,no-microencapsulated islet cells transplantation group and microencapsulated islet cells transplantation group.Normal saline,pared rat islet cells and microcapsulated islet cells were respectively transplantated into abdominal cavity of three groups of diabetic mice.Results The isolated islet cells had a good reaction for glucose stimulation.Both the microcapsulated islet cell transplantation and non-microcapsulated islet cell transplantation could be decreased the high blood glucose level,and the former one kept longer.Conclusion It is believed that microcapsulated islet cell transplantation exerts good effect on diabetic mice and the microcapsules have good immuno-isolating function.
Islet cell transplantation
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Digestion
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Isolation
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Microbial collagenase
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Optimal human islet isolation requires the delivery of bacterial collagenase to the pancreatic islet-exocrine interface. However, we have previously demonstrated the presence of collagenase within human islets immediately following intraductal collagenase administration. This potentially has significant implications for patient safety. The present study aimed to determine if collagenase becomes internalized into islets during the isolation procedure and if it remains within the islet postisolation. Islet samples were taken at various stages throughout 14 clinical human islet isolations: during digest collection, following University of Wisconsin solution incubation, immediately postisolation, and after 24 h of culture. Samples were embedded in agar, cryosectioned, and then assessed by immunolabeling for collagenase and insulin. Immunoreactivity for collagenase was not observed in isolated islets in any preparation. Collagenase labeling was detected in one sample taken at the digest collection phase in one islet preparation only. No collagenase-specific labeling was seen in islets sampled at any of the other time points in any of the 14 islet preparations. Collagenase that enters islets during intraductal administration is washed out of the islets during the collection phase of the isolation process and thus does not remain in islets after isolation. This observation alleviates some of the important safety concerns that collagenase remains within islet grafts.
Pancreatic Islets
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A delivery of collagenase at the islet-exocrine interface is crucial for successful human islet isolation. In this study, we investigated how the ductal preservation method at the procurement site affected collagenase distribution. At first, we analyzed human islet isolation data among groups using Serva collagenase with or without ductal injection (DI) or using new Liberase MTF with DI. Then, to assess the distribution of collagenase, human pancreata were classified into two groups: without DI (no DI, n = 5) and with DI at the procurement site (DI, n = 5). Collagenase with 1% marking dye was perfused in the same manner as in our clinical isolation. The distension of the pancreas and the microscopic distribution of the dyed collagenase in pancreas sections were examined. For microscopic analysis, islets were counted and classified into three criteria: unreached, dye didn't reach the islet surface; surface, dye resided on the surface of the islet but not inside; and inside, dye was found inside the islet. As a result, DI groups substantially improved islet yields. In addition, Liberase MTF with DI significantly improved efficacy of pancreas digestion. All pancreata were well distended macroscopically. However, microscopically, the majority of islets in the no DI group were untouched by the dyed collagenase. Ductal preservation substantially improved dyed collagenase delivery on the surface of islets. In conclusion, delivery of collagenase on the surface of islets was unexpectedly insufficient without DI, which was substantially improved by DI. Thus, ductal preservation is a potent method to improve collagenase delivery and islet yields.
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Islet cell transplantation
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Isolation following a good manufacturing practice-compliant, human islet product requires development of a robust islet isolation procedure where effective limits of key reagents are known. The enzymes used for islet isolation are critical but little is known about the doses of class I and class II collagenase required for successful islet isolation.We used a factorial approach to evaluate the effect of high and low target activities of recombinant class I (rC1) and class II (rC2) collagenase on human islet yield. Consequently, 4 different enzyme formulations with divergent C1:C2 collagenase mass ratios were assessed, each supplemented with the same dose of neutral protease. Both split pancreas and whole pancreas models were used to test enzyme targets (n = 20). Islet yield/g pancreas was compared with historical enzymes (n = 42).Varying the Wunsch (rC2) and collagen degradation activity (CDA, rC1) target dose, and consequently the C1:C2 mass ratio, had no significant effect on tissue digestion. Digestions using higher doses of Wunsch and CDA resulted in comparable islet yields to those obtained with 60% and 50% of those activities, respectively. Factorial analysis revealed no significant main effect of Wunsch activity or CDA for any parameter measured. Aggregate results from 4 different collagenase formulations gave 44% higher islet yield (>5000 islet equivalents/g) in the body/tail of the pancreas (n = 12) when compared with those from the same segment using a standard natural collagenase/protease mixture (n = 6). Additionally, islet yields greater than 5000 islet equivalents/g pancreas were also obtained in whole human pancreas.A broader C1:C2 ratio can be used for human islet isolation than has been used in the past. Recombinant collagenase is an effective replacement for the natural enzyme and we have determined that high islet yield can be obtained even with low doses of rC1:rC2, which is beneficial for the survival of islets.
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