Abstract
The aim of this study was to develop an optimal isolation technique for neonatal rat islets of Langerhans, to perform functional evaluation in vitro, to evaluate immunohistochemically isolated rat islets and fetal rat pancreata after a variable period of culture, and to study growth potentials by means of autoradiography. The islets were isolated using minor modifications of standard procedures including collagenase and DNase. Islets were separated on a discontinuous Percoll gradient. The maximum yield of islets amounted to 240 per pancreas. Fetal pancreata from rats were cultured under similar conditions as neonatal islets to compare their insulin secretory capacity after different periods of culture. The insulin secretion increased gradually, and isolated islets achieved a similar secretion potential to adult rat islets. The mitotic activity of both islets and fetal pancreata was confirmed using tritiated thymidine. The isolation procedure was found suitable for producing well-functioning islets, which could be kept in culture for a period of about 1 month without deterioration in their insulin secretory capacity. The gradual increase in insulin secretory capacity of islets and fetal pancreata was due, in part, to hyperplasia and not just hypertrophia. Autoradiographical evaluation revealed a high mitotic activity after culture, in particular of fetal pancreata. Fetal pancreata cultured for about 10 days showed a phenomenon of budding endocrine cells at the organ surface. A high mitotic activity was found in these buds.
Similar content being viewed by others
References
Moskalewski S, Isolation and culture of islets of Langerhans from the guinea pig. Gen Comp Endocrinol 5:342–353, 1965
Noel J, Rabinovitch A, Olson L, Kyriakides G, Miller J, Mintz DH, A method for large-scale, high-yield isolation of canine pancreatic islets of Langerhans. Metabolism 31:184–187, 1982
Gray DWR, McShane P, Grant A, Morris PJ, A method for isolation of islets of Langerhans from the human pancreas. Diabetes 33:1055–1061, 1984
Hegre OD, Marshall S, Schute BA, et al, Nonenzymatic in vitro isolation of perinatal islets of Langerhans. In Vitro 19:611–620, 1983
Hellerström C, Lewis NJ, Borg H, Johnson R, Freinkel N, Method for large-scale isolation of pancreatic islets by tissue culture of fetal rat pancreas. Diabetes 28:769–776, 1979
McEvoy RC, Hegre OD, Syngeneic transplantation of fetal rat pancreas. III. Effect of insulin treatment on the growth and differentiation of the pancreatic implants after reversal of diabetes. Diabetes 28:141–146, 1979
Mandel TE, Hoffman L, Collier S, Organ cultured fetal pancreas: a source of islets for transplantation in diabetic mice. Transplant Proc 13:143–147, 1981
Simeonovic CJ, Wilson JD, Hegre OD, Sun L, Lafferty KJ, Reversal of diabetes by proislet isotransplantation. Transplant Proc 17:1728–1730, 1985
Yderstraede KB, Starklint H, Steinbruchel D, Jorgensen TW, Gotfredsen CF, Fetal rat pancreas transplantation in BB rats: immunohistochemical and functional evaluation. Virchows Arch [B] 64:13–19, 1993
Heding L, Determination of total serum insulin (IRI) in insulintreated diabetic patients. Diabetologia 8:260–266, 1972
Rogers AW, Techniques of autoradiography. Elsevier, New York, 1969
Buitrago A, Gylfe E, Henriksson C, Pertoft H, Rapid isolation of pancreatic islets from collagenase digested pancreas by sedimentation through Percoll at unit gravity. Biochem Biophys Res Commun 79:823–828, 1977
Brunstedt J, Rapid isolation of functionally intact pancreatic islets from mice and rats by Percoll gradient centrifugation. Diabete Metab 6:87–89, 1980
Vonen B, Florholmen J, Giaever AK, Burhol PG, A methodological study of discontinous Percoll gradient separation of pancreatic islet from rats. Scand J Clin Lab Invest 47:415–420, 1987
Wolters GH, Vos-Scheperkeuter GH, van Deijnen JH, van Schilfgaarde R, An analysis of the role of collagenase and protease in the enzymatic dissociation of the rat pancreas for islet isolation. Diabetologia 35:735–742, 1992
Matas AJ, Sutherland DER, Steffes MW, Najarian JS, Islet transplantation using neonatal rat pancreata: quantitative studies. J Surg Res 20:143–147, 1976
Dudek RW, Kawabe T, Brinn JE, O'Brien K, Poole MC, Morgan CR, Glucose affects in vitro maturation of fetal rat islets. Endocrinology 114:582–587, 1984
Hayek A, Lopez AD, Beattie M, Decrease in the number of neonatal islets required for successful transplantation by strict metabolic control of diabetic rats. Transplantation 45:940–942, 1988
Sodoyez-Goffaux FR, Sodoyez JD, de Vos CJ, Insulin secretion and metabolism during the perinatal period in the rat. Evidence for a placental role in fetal hyperinsulinism. J Clin Invest 63:1095–1102, 1979
Mandel TE, Hoffman L, Collier S, Carter WM, Koulmanda M, Organ culture of fetal mouse and fetal human pancreatic islets for allografting. Diabetes 31 (Suppl 4):39–47, 1982
Hayek A, Beattie GM, Intrapancreatic islet transplantation in experimental diabetes in the rat. Metabolism 41:1367–1369, 1992
Serie JR, Cooper HN, Kemmer KA, Hegre OD, Growth of neonatal islet transplants in the spontaneously diabetic BB/Wor rat. Diabetes 41:1122–1129, 1992
Hegre OD, Serie JR, Enriquez AJ, Weinhaus AJ, Ketchum RJ, Sueppel KL, Growth of neonatal islet graft following transplantation to the BB/Wor rat. Horm Metab Res Suppl 25:142–147, 1990
Wohlrab F, Schmidt S, Kloting I, Wilke B, Cossel L, Ducto-insular proliferation of beta-cells after syngeneic islet transplantation into the spleen of streptozotocin-diabetic Lewis rats. Int J Pancreatol 5:77–83, 1989
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Yderstræde, K.B., Flindt-Egebak, P. Neonatal rat islets of Langerhans and fetal rat pancreas Isolation, immunohistochemical, functional, and autoradiographic evaluation. Acta Diabetol 32, 95–101 (1995). https://doi.org/10.1007/BF00569565
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00569565