Abstract
Flow cytometry has been employed as a method to study homogeneity of isolated islet subpopulations. After collagenase digestion of rat pancreas and elutriation of tissue fragments, islets were isolated and dissociated, and cells were analyzed and sorted according to their low forward angle light scattering properties by using automated flow cytometry. A standardized procedure was developed for the preparation of rat islet cell grafts for purification of islet cells. In this process, after collagenase digestion of pancreas, islets were isolated, dissociated, identification by dithizone method and then with enzymatic procedure by DNase and trypsin, the islet cells changed into single cells and beta cells were identified by immunofluorescence method and then assayed by flow cytometry. Methods have been developed for the preparation of suspension of viable rat pancreatic islet cells and their analysis and sorting in the fluorescence activated cell sorter (FACC IV, Becton Dickinson, Sunnyvale, Ca). Flow cytometry of these cells indicated that there were 91% of beta cells in cell suspension. Most of the exocrine particles were lost during digestion. Purified endocrine islet cell grafts were prepared by pure beta-cells, without endocrine non-beta cells. The purified aggregates were devoid of endocrine non-beta cells and damaged cells.
Key Words: Islet cells, Isolation, Purification, Flow cytometry
Full Text
The Full Text of this article is available as a PDF (175.9 KB).
References
- 1.Lacy P.E., Greider M.H. Anatomy and ultra structural organization of pancreatic islets. In: DeGroot L. J., editor. Endocrinology. New York: Grune and Stratton; 1979. pp. 907–919. [Google Scholar]
- 2.Pipeleers D.G., Pipeleers Marichal M.A. A method for the purification of single A, B and D cells and for the isolation of coupled cells from isolated rat Islets. Diabetologia. 1981;20:654–663. doi: 10.1007/BF00257436. [DOI] [PubMed] [Google Scholar]
- 3.Rabinovitch A., Russel T. Preparation of rat islet B-Cell enriched fractions by light-scatter flow cytometry. Diabetes. 1981;31:939–943. doi: 10.2337/diabetes.31.11.939. [DOI] [PubMed] [Google Scholar]
- 4.Gray D.W.R., Morris P.J. Developments in isolated pancreatic Islet transplantation. Transplantation. 1987;43(3):321–31. doi: 10.1097/00007890-198703000-00001. [DOI] [PubMed] [Google Scholar]
- 5.Pipeleers D.G., Pipeleers Marichal M., Hannaert J.C., Berghmans M., Veld P.A.I., Rozing J., et al. Transplantation of purified Islet Cells in diabetic Rats. Diabetes. 1991;40:908–919. doi: 10.2337/diabetes.40.7.908. [DOI] [PubMed] [Google Scholar]
- 6.Nielsen D.A., Lernmark A., Berelowitz M., Bloom G.D., Steiner D.F. Sorting of pancreatic islet cell subpopulations by light scattering using a fluorescence-activated cell sorter. Diabetes. 1982;31:229–306. doi: 10.2337/diabetes.31.4.299. [DOI] [PubMed] [Google Scholar]
- 7.The diabetes controlcomplications trial research group. The effect of Intensive treatment of diabetes on the development and progression of long-Term complications in insulin-dependent diabetes mellitus. New Engl J Med. 1993;329(14):977–985. doi: 10.1056/NEJM199309303291401. [DOI] [PubMed] [Google Scholar]
- 8.Lacy P.E., Kostianovsky M., Louis S. Method for the isolation of intact islets of Langerhans from the rat pancreas. Diabetes. 1967;16(1):35–39. doi: 10.2337/diab.16.1.35. [DOI] [PubMed] [Google Scholar]
- 9.Pipeleers D.G., Veld P.A., Winkel M.V.D., Maes E., Schuit F.C., Gepts W. A new in vitro model for the study of pancreatic A and B cells. The Endocrine Society. 1985;117:806–16. doi: 10.1210/endo-117-3-806. [DOI] [PubMed] [Google Scholar]
- 10.Kuo W.N., Hodgins D.S., Kou J.F. Adenylate cyclase in islets of Langerhans: Isolation of islets and regulation of adenylate cyclase activity by various Hormones and agents. J Biol Chem. 1973;248:2705–2715. [PubMed] [Google Scholar]
- 11.Olack B.J., Swanson C.J., Howard T.K., Mohanakumar T. Improved Method for the isolation and purification of human islets of Langerhans using Liberase TM enzyme blend. Human Immunology. 1999;60:1303–1309. doi: 10.1016/S0198-8859(99)00118-4. [DOI] [PubMed] [Google Scholar]
- 12.Winkel M.V.D., Maes E., Pipeleers D. Islet cell analysis and purification by Light scatter and auto fluorescence. Biochemical and biophysical research Communications. 1982;170:525–532. doi: 10.1016/0006-291x(82)91523-6. [DOI] [PubMed] [Google Scholar]
- 13.Rabinovitch A., Russell T., Shienvold F., Noel J., Files N., Patel Y., Ingram M. Preparation of rat islet B-cell-enriched fractions by light-scatter flow Cytometry. Diabetes. 1982;31:939–943. doi: 10.2337/diabetes.31.11.939. [DOI] [PubMed] [Google Scholar]
- 14.Winkel M.V.D., Pipeleers D. Auto fluorescence activated cell sorting of Pancreatic Islet cells: purification of insulin-containing b-cells according to Glucose-induced changes in cellular redox state. Biochemical and biophysical Research Communications. 1983;114(2):835–842. doi: 10.1016/0006-291X(83)90857-4. [DOI] [PubMed] [Google Scholar]
- 15.Bock T., Pakkenberg B. Buschard. Increased islet volume but unchanged Islet number in ob/ob mice. Diabetes. 2003;52:1716–1722. doi: 10.2337/diabetes.52.7.1716. [DOI] [PubMed] [Google Scholar]
- 16.Alexander von Mach M., Schlosser J., Weiland M., Feilen P.J. Cryopreservation of islets of Langerhans: optimization of protocols using rat Pancreatic tissue. EXCLI Journal. 2003;2:6–21. [Google Scholar]
- 17.Thomas Titus, Lionel Badet, Derek WR Gray. Islet cell transplantation for insulin-dependent diabetes mellitus: perspectives from the present and prospects for the future. Expert Reviews in Molecular Medicine. 6 September 2000; http://www.expertreviews.org/00001861h.htm page 1–27. [DOI] [PubMed]