Comparison of islet isolation result and clinical applicability according to GMP-grade collagenase enzyme blend in adult porcine islet isolation and culture
| العنوان: | Comparison of islet isolation result and clinical applicability according to GMP-grade collagenase enzyme blend in adult porcine islet isolation and culture |
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| المؤلفون: | Hyunil Kim, Michael Alexander, Joohyun Shim, Kyungmin Kwak, Yongjin Lee, Hyoung-Joo Kim, Jun-Hyeong Kim, Kimyung Choi, Jonathan R. T. Lakey, Nayoung Ko, Jae-Kyung Park |
| المصدر: | Xenotransplantation, vol 28, iss 4 Xenotransplantation |
| بيانات النشر: | eScholarship, University of California, 2021. |
| سنة النشر: | 2021 |
| مصطلحات موضوعية: | 0301 basic medicine, digestion enzymes, endocrine system, endocrine system diseases, Swine, type 1 diabetes, medicine.medical_treatment, Xenotransplantation, Immunology, Transplantation, Heterologous, Clinical Sciences, Islets of Langerhans Transplantation, Cell Separation, islet yield, 030230 surgery, Biology, Andrology, 03 medical and health sciences, Tissue culture, Islets of Langerhans, 0302 clinical medicine, In vivo, medicine, adult porcine islets, islet isolation, 5.4 Surgery, Animals, Collagenases, Pancreas, Metabolic and endocrine, geography, Transplantation, Heterologous, geography.geographical_feature_category, Insulin, Diabetes, Original Articles, Islet, collagenase, 030104 developmental biology, Collagenase, Pancreatic islet transplantation, Original Article, Surgery, medicine.drug |
| الوصف: | Author(s): Kwak, Kyungmin; Park, Jae-Kyung; Shim, Joohyun; Ko, Nayoung; Kim, Hyoung-Joo; Lee, Yongjin; Kim, Jun-Hyeong; Alexander, Michael; Lakey, Jonathan RT; Kim, Hyunil; Choi, Kimyung | Abstract: BackgroundPorcine islet xenotransplantation is a promising treatment for type 1 diabetes as an alternative to human pancreatic islet transplantation and long-term insulin therapy. Several research groups have explored porcine islets as an alternative to the inconsistent and chronic shortage of pancreases from human organ donors. Studies have confirmed successful transplant of porcine islets into non-human primate models of diabetes; however, in most cases, they require more than one adult porcine donor to achieve sufficient viable islet mass for sustained function. The importance of GMP-grade reagents includes the following: specific enzymes utilized in the pancreatic isolation process were identified as a key factor in successful human clinical islet transplantation trials using cadaveric islets. As xenotransplantation clinical research progresses, isolation reagents and digestion enzymes play a key role in the consistency of the product and ultimately the outcome of the islet xenotransplant. In this study, we evaluated several commercially available enzyme blends that have been used for islet isolation. We evaluated their impact on islet isolation yield and subsequent islet function as part of our plan to bring xenotransplantation into clinical xenotransplantation trials.MethodsAdult porcine islets were isolated from 16 to 17-month-old Yucatan miniature pigs following standard rapid procurement. Pigs weighed on average 48.71n±n2.85nkg, and the produced pancreases were 39.51n±n1.80 grams (meann±nSEM). After ductal cannulation, we evaluated both GMP-grade enzymes (Collagenase AF-1 GMP grade and Liberase MTF C/T GMP grade) and compared with standard non-GMP enzyme blend (Collagenase P). Islet quality control assessments including islet yield, islet size (IEQ), membrane integrity (acridine orange/propidium iodide), and functional viability (GSIS) were evaluated in triplicate on day 1 post-islet isolation culture.ResultsIslet yield was highest in the group of adult pigs where Collagenase AF-1 GMP grade was utilized. The mean islet yield was 16n586n±n1391nIEQ/g vs 8302n±n986nIEQ/g from pancreases isolated using unpurified crude Collagenase P. The mean islet size was higher in Collagenase AF-1 GMP grade with neutral protease than in Collagenase P and Liberase MTF C/T GMP grade. We observed no significant difference between the experimental groups, but in vitro islet function after overnight tissue culture was significantly higher in Collagenase AF-1 GMP grade with neutral protease and Liberase MTF C/T GMP grade than the crude control enzyme group. As expected, the GMP-grade enzyme has significantly lower endotoxin levels than the crude control enzyme group when measured.ConclusionsThis study validates the importance of using specifically blended GMP grade for adult pig islet isolation for xenotransplantation trials and the ability to isolate a sufficient number of viable islets from one adult pig to provide a sufficient number for islets for a clinical islet transplantation. GMP-grade enzymes are highly efficient in increasing islet yield, size, viability, and function at a lower and acceptable endotoxin level. Ongoing research transplants these islets into animal models of diabetes to validate in vivo function. Also, these defined and reproducible techniques using GMP-grade enzymes allow for continuance of our plan to advance to xenotransplantation of isolated pig islets for the treatment of type 1 diabetes. |
| وصف الملف: | application/pdf |
| الوصول الحر: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::50df463e8358fe89740486805ccdf347Test https://escholarship.org/uc/item/5sz810j3Test |
| حقوق: | OPEN |
| رقم الانضمام: | edsair.doi.dedup.....50df463e8358fe89740486805ccdf347 |
| قاعدة البيانات: | OpenAIRE |
| الوصف غير متاح. |