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. 1996 May 1;97(9):2119–2129. doi: 10.1172/JCI118649

Large scale isolation, growth, and function of porcine neonatal islet cells.

G S Korbutt 1, J F Elliott 1, Z Ao 1, D K Smith 1, G L Warnock 1, R V Rajotte 1
PMCID: PMC507287  PMID: 8621802

Abstract

Based upon existing methods of isolating fetal porcine islet tissue, a simple, reliable procedure was developed for the preparation of porcine neonatal islet cell aggregates with a reproducible and defined cellular composition. After 9 d of in vitro culture, tissue from one neonatal pig pancreas yielded approximately 50,000 islet cell aggregates, consisting of primarily epithelial cells (57%) and pancreatic endocrine cells (35%). During the culture period, the total beta cell mass decreased initially, but subsequently increased 1.5-fold between days 3 and 9. Transplantation of grafts consisting of 3 x 10(5) beta cells (1,000 aggregated) under the kidney capsule of alloxan-diabetic nude mice corrected hyperglycemia in 75% (10/13) of the animals, whereas, 100% (20/20) of recipients implanted with 6 x 10(5) beta cells (2,000 aggregates) achieved euglycemia within 8 wk posttransplantation. Nephrectomy of the graft bearing kidney at 14 wk posttransplantation resulted in hyperglycemia in all recipients, and examination of the grafts revealed the presence of numerous well-granulated insulin- and glucagon-containing cells. The cellular insulin content of these grafts was 20 to 30-fold higher than at the time of transplantation. These results indicate that the neonatal porcine pancrease can be used as a source of large numbers of viable islet cells, which have the potential for growth both in vitro and in vivo, and exhibit the metabolic capacity to correct diabetes in nude mice.

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Selected References

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  1. Alumets J., Håkanson R., Sundler F. Ontogeny of endocrine cells in porcine gut and pancreas. An immunocytochemical study. Gastroenterology. 1983 Dec;85(6):1359–1372. [PubMed] [Google Scholar]
  2. Archer F. J. Monolayer culture of neonatal pig pancreatic islet cells. Diabetologia. 1983 Mar;24(3):185–190. doi: 10.1007/BF00250159. [DOI] [PubMed] [Google Scholar]
  3. Asplund K. Dynamics of insulin release from the foetal and neonatal rat pancreas. Eur J Clin Invest. 1973 Jul;3(4):338–344. doi: 10.1111/j.1365-2362.1973.tb00360.x. [DOI] [PubMed] [Google Scholar]
  4. Asplund K., Westman S., Hellerström C. Glucose stimulation of insulin secretion from the isolated pancreas of foetal and newborn rats. Diabetologia. 1969 Aug;5(4):260–262. doi: 10.1007/BF01212095. [DOI] [PubMed] [Google Scholar]
  5. Britt L. D., Stojeba P. C., Scharp C. R., Greider M. H., Scharp D. W. Neonatal pig pseudo-islets. A product of selective aggregation. Diabetes. 1981 Jul;30(7):580–583. doi: 10.2337/diab.30.7.580. [DOI] [PubMed] [Google Scholar]
  6. Clark S. A., Chick W. L. Islet cell culture in defined serum-free medium. Endocrinology. 1990 Apr;126(4):1895–1903. doi: 10.1210/endo-126-4-1895. [DOI] [PubMed] [Google Scholar]
  7. Davalli A. M., Ogawa Y., Scaglia L., Wu Y. J., Hollister J., Bonner-Weir S., Weir G. C. Function, mass, and replication of porcine and rat islets transplanted into diabetic nude mice. Diabetes. 1995 Jan;44(1):104–111. doi: 10.2337/diab.44.1.104. [DOI] [PubMed] [Google Scholar]
  8. Gray D. W., McShane P., Grant A., Morris P. J. A method for isolation of islets of Langerhans from the human pancreas. Diabetes. 1984 Nov;33(11):1055–1061. doi: 10.2337/diab.33.11.1055. [DOI] [PubMed] [Google Scholar]
  9. Groth C. G., Korsgren O., Tibell A., Tollemar J., Möller E., Bolinder J., Ostman J., Reinholt F. P., Hellerström C., Andersson A. Transplantation of porcine fetal pancreas to diabetic patients. Lancet. 1994 Nov 19;344(8934):1402–1404. doi: 10.1016/s0140-6736(94)90570-3. [DOI] [PubMed] [Google Scholar]
  10. Hellerström C., Swenne I. Functional maturation and proliferation of fetal pancreatic beta-cells. Diabetes. 1991 Dec;40 (Suppl 2):89–93. doi: 10.2337/diab.40.2.s89. [DOI] [PubMed] [Google Scholar]
  11. Hole R. L., Pian-Smith M. C., Sharp G. W. Development of the biphasic response to glucose in fetal and neonatal rat pancreas. Am J Physiol. 1988 Feb;254(2 Pt 1):E167–E174. doi: 10.1152/ajpendo.1988.254.2.E167. [DOI] [PubMed] [Google Scholar]
  12. Kirchhof N., Hering B. J., Geiss V., Federlin K., Bretzel R. G. Evidence for breed-dependent differences in porcine islets of Langerhans. Transplant Proc. 1994 Apr;26(2):616–617. [PubMed] [Google Scholar]
  13. Korbutt G. S., Pipeleers D. G. Cold storage of rat pancreas before purification of islet beta-cells. Diabetes. 1992 Mar;41(3):299–307. doi: 10.2337/diab.41.3.299. [DOI] [PubMed] [Google Scholar]
  14. Korbutt G. S., Pipeleers D. G. Rat pancreas preparation for cold storage and subsequent islet cell isolation. Transplantation. 1993 Sep;56(3):500–503. doi: 10.1097/00007890-199309000-00002. [DOI] [PubMed] [Google Scholar]
  15. Korsgren O., Andersson A., Sandler S. Pretreatment of fetal porcine pancreas in culture with nicotinamide accelerates reversal of diabetes after transplantation to nude mice. Surgery. 1993 Feb;113(2):205–214. [PubMed] [Google Scholar]
  16. Korsgren O., Jansson L., Eizirik D., Andersson A. Functional and morphological differentiation of fetal porcine islet-like cell clusters after transplantation into nude mice. Diabetologia. 1991 Jun;34(6):379–386. doi: 10.1007/BF00403174. [DOI] [PubMed] [Google Scholar]
  17. Korsgren O., Sandler S., Landström A. S., Jansson L., Andersson A. Large-scale production of fetal porcine pancreatic isletlike cell clusters. An experimental tool for studies of islet cell differentiation and xenotransplantation. Transplantation. 1988 Mar;45(3):509–514. doi: 10.1097/00007890-198803000-00001. [DOI] [PubMed] [Google Scholar]
  18. Ling Z., Hannaert J. C., Pipeleers D. Effect of nutrients, hormones and serum on survival of rat islet beta cells in culture. Diabetologia. 1994 Jan;37(1):15–21. doi: 10.1007/BF00428772. [DOI] [PubMed] [Google Scholar]
  19. Liu X. M., Federlin K. F., Bretzel R. G., Hering B. J., Brendel M. D. Persistent reversal of diabetes by transplantation of fetal pig proislets into nude mice. Diabetes. 1991 Jul;40(7):858–866. doi: 10.2337/diab.40.7.858. [DOI] [PubMed] [Google Scholar]
  20. Marchetti P., Giannarelli R., Cosimi S., Masiello P., Coppelli A., Viacava P., Navalesi R. Massive isolation, morphological and functional characterization, and xenotransplantation of bovine pancreatic islets. Diabetes. 1995 Apr;44(4):375–381. doi: 10.2337/diab.44.4.375. [DOI] [PubMed] [Google Scholar]
  21. Otonkoski T., Beattie G. M., Mally M. I., Ricordi C., Hayek A. Nicotinamide is a potent inducer of endocrine differentiation in cultured human fetal pancreatic cells. J Clin Invest. 1993 Sep;92(3):1459–1466. doi: 10.1172/JCI116723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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 Jun;20(6):654–663. doi: 10.1007/BF00257436. [DOI] [PubMed] [Google Scholar]
  23. Pipeleers D. G., Pipeleers-Marichal M., Hannaert J. C., Berghmans M., In't Veld P. A., Rozing J., Van de Winkel M., Gepts W. Transplantation of purified islet cells in diabetic rats. I. Standardization of islet cell grafts. Diabetes. 1991 Jul;40(7):908–919. doi: 10.2337/diab.40.7.908. [DOI] [PubMed] [Google Scholar]
  24. Pipeleers D. G., in't Veld P. A., Van de Winkel M., Maes E., Schuit F. C., Gepts W. A new in vitro model for the study of pancreatic A and B cells. Endocrinology. 1985 Sep;117(3):806–816. doi: 10.1210/endo-117-3-806. [DOI] [PubMed] [Google Scholar]
  25. Rabinovitch A., Blondel B., Murray T., Mintz D. H. Cyclic adenosine-3',5'-monophosphate stimulates islet B cell replication in neonatal rat pancreatic monolayer cultures. J Clin Invest. 1980 Nov;66(5):1065–1071. doi: 10.1172/JCI109935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Rhoten W. B. Insulin secretory dynamics during development of rat pancreas. Am J Physiol. 1980 Jul;239(1):E57–E63. doi: 10.1152/ajpendo.1980.239.1.E57. [DOI] [PubMed] [Google Scholar]
  27. Ricordi C., Lacy P. E., Finke E. H., Olack B. J., Scharp D. W. Automated method for isolation of human pancreatic islets. Diabetes. 1988 Apr;37(4):413–420. doi: 10.2337/diab.37.4.413. [DOI] [PubMed] [Google Scholar]
  28. Ricordi C. Quantitative and qualitative standards for islet isolation assessment in humans and large mammals. Pancreas. 1991 Mar;6(2):242–244. doi: 10.1097/00006676-199103000-00018. [DOI] [PubMed] [Google Scholar]
  29. Ricordi C., Socci C., Davalli A. M., Staudacher C., Baro P., Vertova A., Sassi I., Gavazzi F., Pozza G., Di Carlo V. Isolation of the elusive pig islet. Surgery. 1990 Jun;107(6):688–694. [PubMed] [Google Scholar]
  30. Ricordi C., Tzakis A. G., Carroll P. B., Zeng Y. J., Rilo H. L., Alejandro R., Shapiro A., Fung J. J., Demetris A. J., Mintz D. H. Human islet isolation and allotransplantation in 22 consecutive cases. Transplantation. 1992 Feb;53(2):407–414. doi: 10.1097/00007890-199202010-00027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sandler S., Andersson A. Long-term effects of exposure of pancreatic islets to nicotinamide in vitro on DNA synthesis, metabolism and B-cell function. Diabetologia. 1986 Mar;29(3):199–202. doi: 10.1007/BF02427093. [DOI] [PubMed] [Google Scholar]
  32. Sandler S., Welsh M., Andersson A. Streptozotocin-induced impairment of islet B-cell metabolism and its prevention by a hydroxyl radical scavenger and inhibitors of poly(ADP-ribose) synthetase. Acta Pharmacol Toxicol (Copenh) 1983 Nov;53(5):392–400. doi: 10.1111/j.1600-0773.1983.tb03440.x. [DOI] [PubMed] [Google Scholar]
  33. Scharp D. W., Lacy P. E., Santiago J. V., McCullough C. S., Weide L. G., Falqui L., Marchetti P., Gingerich R. L., Jaffe A. S., Cryer P. E. Insulin independence after islet transplantation into type I diabetic patient. Diabetes. 1990 Apr;39(4):515–518. doi: 10.2337/diab.39.4.515. [DOI] [PubMed] [Google Scholar]
  34. Schein P. S., Cooney D. A., Vernon M. L. The use of nicotinamide to modify the toxicity of streptozotocin diabetes without loss of antitumor activity. Cancer Res. 1967 Dec;27(12):2324–2332. [PubMed] [Google Scholar]
  35. Socci C., Falqui L., Davalli A. M., Ricordi C., Braghi S., Bertuzzi F., Maffi P., Secchi A., Gavazzi F., Freschi M. Fresh human islet transplantation to replace pancreatic endocrine function in type 1 diabetic patients. Report of six cases. Acta Diabetol. 1991;28(2):151–157. doi: 10.1007/BF00579718. [DOI] [PubMed] [Google Scholar]
  36. Socci C., Ricordi C., Davalli A. M., Staudacher C., Baro P., Vertova A., Freschi M., Gavazzi F., Braghi S., Pozza G. Selection of donors significantly improves pig islet isolation yield. Horm Metab Res Suppl. 1990;25:32–34. [PubMed] [Google Scholar]
  37. Swenne I., Bone A. J., Howell S. L., Hellerström C. Effects of glucose and amino acids on the biosynthesis of DNA and insulin in fetal rat islets maintained in tissue culture. Diabetes. 1980 Sep;29(9):686–692. doi: 10.2337/diab.29.9.686. [DOI] [PubMed] [Google Scholar]
  38. Tuch B. E., Jones A., Turtle J. R. Maturation of the response of human fetal pancreatic explants to glucose. Diabetologia. 1985 Jan;28(1):28–31. doi: 10.1007/BF00276996. [DOI] [PubMed] [Google Scholar]
  39. Warnock G. L., Ellis D., Rajotte R. V., Dawidson I., Baekkeskov S., Egebjerg J. Studies of the isolation and viability of human islets of Langerhans. Transplantation. 1988 May;45(5):957–963. doi: 10.1097/00007890-198805000-00024. [DOI] [PubMed] [Google Scholar]
  40. Warnock G. L., Kneteman N. M., Ryan E. A., Rabinovitch A., Rajotte R. V. Long-term follow-up after transplantation of insulin-producing pancreatic islets into patients with type 1 (insulin-dependent) diabetes mellitus. Diabetologia. 1992 Jan;35(1):89–95. doi: 10.1007/BF00400857. [DOI] [PubMed] [Google Scholar]
  41. Warnock G. L., Kneteman N. M., Ryan E., Seelis R. E., Rabinovitch A., Rajotte R. V. Normoglycaemia after transplantation of freshly isolated and cryopreserved pancreatic islets in type 1 (insulin-dependent) diabetes mellitus. Diabetologia. 1991 Jan;34(1):55–58. doi: 10.1007/BF00404026. [DOI] [PubMed] [Google Scholar]
  42. Yamada K., Nonaka K., Hanafusa T., Miyazaki A., Toyoshima H., Tarui S. Preventive and therapeutic effects of large-dose nicotinamide injections on diabetes associated with insulitis. An observation in nonobese diabetic (NOD) mice. Diabetes. 1982 Sep;31(9):749–753. doi: 10.2337/diab.31.9.749. [DOI] [PubMed] [Google Scholar]
  43. van Deijnen J. H., Hulstaert C. E., Wolters G. H., van Schilfgaarde R. Significance of the peri-insular extracellular matrix for islet isolation from the pancreas of rat, dog, pig, and man. Cell Tissue Res. 1992 Jan;267(1):139–146. doi: 10.1007/BF00318700. [DOI] [PubMed] [Google Scholar]

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