Summary
Recent progress in islet isolation from the pancreas of large mammals including man, accentuated the need for the development of precise and reproducible techniques to assess islet yield. In this report both quantitative and qualitative criteria for islet isolation assessment were discussed, the main topics being the determination of number, volume, purity, morphologic integrity andin vitro andin vivo function tests of the final islet preparations. It has been recommended that dithizone should be used as a specific stain for immediate detection of islet tissue making it possible to estimate both the total number of islets (dividing them into classes of 50 µ diameter range increments) and the purity of the final preparation. Appropriate morphological assessment should include confirmation of islet identification, assessment of the morphological integrity and of the purity of the islet preparation. The use of fluorometric inclusion and exclusion dyes together have been suggested as a viability assay to simultaneously quantitate the proportion of cells that are intact or damaged. Perifusion of islets with glucose provides a dynamic profile of glucose-mediated insulin release and of the ability of the cells to down regulate insulin secretion after the glycemic challenge is interrupted. Although perifusion data provides a useful guide to islet viability the quantity and kinetics of insulin release do not necessarily predict islet performance after implantation. Therefore, the ultimate test of islet viability is their function after transplantation into a diabetic recipient. For this reason,in vivo models of transplantation of an aliquot of the final islet preparation into diabetic nude (athymic) rodents have been suggested. We hope that these general guidelines will be of assistance to standardize the assessment of islet isolations, making it possible to better interpret and compare procedures from different centers.
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References
Alejandro R., Cutfield R. G., Shienvold F. L., Polonsky K. S., Noel J., Olson L., Lillberger J., Miller J., Mintz D. H.: Natural history of intrahepatic canine islet cell autografts — J. clin. Invest.78, 1339–1348, 1986.
Andersson A., Borg H., Groth C. G., Gunnarsson R., Hellerstrom C., Lundgren G., Westman J., Ostman J.: Survival of isolated human islets of Langerhans maintained in tissue culture — J. clin. Invest.57, 1295–1301, 1976.
Ashcroft S. J. H., Bassett J. M., Randle P. J.: Isolation of human pancreatic islets capable of releasing insulin and metabolizing glucose in vitro — Lanceti, 888–889, 1971.
Ballinger W. F., Lacy P. E.: Transplantation of intact pancreatic islets in rats — Surgery72, 175–186, 1972.
Bank H. L.: Assessment of islet cell viability using fluorescent dyes — Diabetologia30, 812–817, 1987.
Bank H. L.: Rapid assessment of islet cell viability with acridine orange and propidium iodide — In Vitro24, 266–272, 1988.
Brelje T. C., Sharp D. W., Sorenson R. L.: Three-dimensional imaging of intact isolated islets of Langerhans with cofocal microscopy — Diabetes38, 808–814, 1989.
Buschard K., Rygaard J.: Restitution of streptozotocin induced diabetes mellitus in nude mice with pancreatic graft from the rat — Acta path. microbiol. scand. (Sect. C)84, 221–226, 1976.
Evans M. G., Rajotte R. V., Warnock G. L., Kneteman N. M.: Viability studies of cryopreserved isolated canine islets of Langerhans — Trans. Proc.21, 3368–3370, 1989.
Festing M. F. W.: Athymic nude rats. In:Gershwin E., Merchant B. (Eds): Immunological Defects in Laboratory Animals. Vol. 1. Plenum Co., 1981; pp. 12–24.
Gray D. W. R., McShane P., Grant A., Morris P. J.: A method for isolation of islets of Langerhans from the human pancreas — Diabetes33, 1055–1061, 1984.
Gray D. W. R., Morris P. J.: The use of fluorescein diacetate and etheidum bromide as a viability stain for isolated islets of Langerhans — Stain. Technol.62, 379–381, 1987.
Hedeskov C. J.: Mechanism of glucose-induces insulin secretion — Physiol. Rev.60, 442–509, 1980.
Kneteman N. M., Rajotte R. V.: Isolation and cryopreservation of human pancreatic islets — Trans. Proc.18, 182–185, 1986.
Lacy P. E.: Beta cell secretion — from the standpoint of a pathobiologist — Diabetes19, 895–905, 1970.
Lacy P. E., Finke E. H., Conant S., Naber S.: Long-term perfusion of isolated rat islets in vitro — Diabetes25, 484–493, 1976.
Lacy P. E., Kostianovsky M.: Method for the isolation of intact islets of Langerhans from the rat pancreas — Diabetes16, 35–39, 1967.
Lake S. P., Chamberlain J., Bassett P. D., London N. J., Walczak K., Bell P. R. F., James R. F. L.: Successful reversal of diabetes in nude rats by transplantation of isolated adult human islets of Langerhans — Diabetes38, 146–151, 1989.
Lake S. P., Chamberlain J., Husken P., Bell P. R. F., James R. F. L.: In vivo assessment of isolated pancreatic viability using the streptozotocin-induced diabetic nude rat — Diabetologia31, 390–394, 1988.
Latif Z. A., Noel J., Alejandro R.: A simple method of staining fresh and cultured islets — Transplantation45, 827–830, 1988.
Lundgren G., Andersson A., Borg H., Buschard K., Groth C. G., Gunnarsson R., Hellerstrom C., Petersson B., Ostman J.: Structural and functional integrity of human islets of Langerhans maintained in tissue culture for 1–3 weeks — Trans. Proc.19, 237–240, 1977.
Mandel T. E., Georgiou H. M.: Xenotransplantation of human fetal islets in nude mice — Trans. Proc.16, 849–850, 1984.
Millard P. R., Reece-Smith H., Smart Y. C., McShane P., Morris P. J.: Observations on rat collagenase-separated islets and long-term composite islet allografts — Brit. J. exp. Pathol.65, 745–751, 1984.
Morgan C. R., Lazarow A.: Immunoassay of insulin: antibody system. Plasma insulin levels of normal, subdiabetic and diabetic rats — Diabetes12, 115–126, 1963.
Moskalewski S.: Isolation and culture of islet of Langerhans of the guinea pig — Gen. comp. Endocrinol.5, 342–353, 1985.
Petkov P., Hahn H. J., Galabova R., Zielger M.: Investigations on islets of Langerhans in vitro. Ultrastructure and insulin secretion of isolated rat islets after different digestion with collagenase — Acta histochem. (Jena)51, 50–60, 1984.
Povlsen C. O., Skakkebaek N. E., Rygaard J., Jensen G.: Heterotransplantation of human foetal organs to the mouse mutant nude — Nature (Lond.)248, 247–249, 1974.
Pretlow II T. G., Pretlow T. P.: Evaluation of data, problems, and general approach. In:Pretlow II T. G., Pretlow T. P. (Eds): Cell Separation: Methods and Selected Applications. Vol 1. Academic Press, New York, London, 1982; pp. 31–40.
Rajotte R. V., Warnock G. L., Kneteman N. M., Erickson C., Ellis D.: Optimizing cryopreservation of isolated islets — Trans. Proc.21, 2638–2640, 1989.
Ricordi C., Finke E. H., Lacy P. E.: A method for the mass isolation of islets from the adult pig pancreas — Diabetes35, 649–653, 1986.
Ricordi C., Kneteman N. M., Scharp D. W., Lacy P. E.: Transplantation of cryopreserved human pancreatic islets into diabetic nude mice — World J. Surgery12, 861–864, 1988.
Ricordi C., Lacy P. E., Finke E. H., Olack B. J., Scharp D. W.: Automated method for isolation of human pancreatic islets — Diabetes37, 413–420, 1988.
Ricordi C., Scharp D. W., Lacy P. E.: Reversal of diabetes in nude mouse after transplantation of fresh and 7-day cultured (24 degrees C) human pancreatic islets — Transplantation45, 994–996, 1988.
Scharp D. W., Lacy P. E., Finke E., Olack B.: Low temperature culture of human islets isolated by the distension method and purified with Ficoll and Percoll gradients — Surgery102, 869–879, 1987.
Schwartz B. D., Traverso W.: Morphological changes in pancreatic fragments prepared for transplantation by collagenase treatment — Transplantation38, 273–280, 1984.
Schwedes U., Kaul S., Wdowinski J., Klempa I., Bastert G., Usadel K. H.: The use of human fetal pancreas for transplantation: experimental and clinical results — Hormone metabol. Res.13, 87–90, 1983.
Sutherland D. E. R., Matas A. J., Steffes M. W., Najarian J. S.: Infant human pancreas: a potential source of islet tissue for transplantation — Diabetes25, 1123–1128, 1976.
Sutherland D. E. R., Steffes M. W., Bauer G. E., McManus D., Noe B. P., Najarian J. S.: Isolation of human and porcine islets of Langerhans and islet transplantation in pigs — J. surg. Res.16, 102–111, 1974.
Syed Ali S., Syed Ali M. M., Hering B. J., Bretzel R. G., Federlin K.: Morphological studies on bovine islets isolated by digestion filtration — Cell Tiss. Res. (In press).
Tuch B. E., Jones A., Turtle J. R.: Maturation of response to human fetal pancreatic explants to glucose — Diabetologia28, 28–31, 1985.
Tuch B. E., Ng A. B. P., Jones A., Turtle J. R.: Histologic differentiation of human fetal pancreatic explants transplanted into nude mice — Diabetes33, 1180–1187, 1984.
Usadel K. H., Schwedes U., Bastert G., Steinau U., Klempa I., Fassbinder W., Schoffling K.: Transplantation of human fetal pancreas. Experience in thymus aplastic mice and rats and in a diabetic patient — Diabetes29 (Suppl. 1), 74–79, 1980.
Warnock G. L., Gray D. W. R., McShane P., Peters M., Morris P. J.: Survival of cryopreserved isolated adult human pancreatic islets of Langerhans — Transplantation44, 75–82, 1987.
Warnock G. L., Ellis D., Rajotte R. V., Dawidson I., Baekkeskov S., Edebjerg J.: Studies of the isolation and viability of human islets of Langerhans — Transplantation45, 957–963, 1988.
Warnock G. L., Rajotte R. V., Evans M. G., Ellis D. K., DeGroot T., Dawidson I.: Isolation of islets of Langerhans following cold storage of human pancreas — Trans. Proc.19, 3466–3468, 1987.
Zimny M. L., Blackard W. G.: The surface of isolated pancreatic islet cells — Cell Tiss. Res.164, 467–471, 1975.
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Ricordi, C., Gray, D.W.R., Hering, B.J. et al. Islet isolation assessment in man and large animals. Acta diabet. lat 27, 185–195 (1990). https://doi.org/10.1007/BF02581331
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DOI: https://doi.org/10.1007/BF02581331