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Culturing Free-Floating and Fibrin-Embedded Islets with Endothelial Cells: Effects on Insulin Secretion and Apoptosis

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Abstract

There is a need to develop methods to culture isolated endocrine pancreatic islets in vitro, as their capacity to secrete insulin typically declines following isolation and they usually undergo apoptosis. In this study, the effects on insulin secretion and apoptosis were tested for two co-culture systems of endocrine porcine islets (1) embedded in fibrin with porcine liver microvascular endothelial cells (PLMEC) and (2) seeded on PLMEC monolayers. The addition of endothelial cells in fibrin resulted in better preserved islet integrity, higher insulin secretion over 7 days, improved glucose-stimulated insulin secretion (GSIS), less loss of insulin-expressing cells over 7 days, and reduced apoptosis, the latter only for 2 days. Seeding islets on PLMEC monolayers had marginal improvement of the insulin secretion over 7 days. It improved GSIS for 2 days, but not over 7 days, as it did not affect the loss of insulin-expressing cells over 7 days. When compared to freely-floating islets in tissue culture polystyrene, the apoptosis level of islets seeded on PLMEC monolayers was on par and slightly decreased over 2 and 7 days, respectively. For both islets co-seeded with PLMEC in fibrin and those cultured on PLMEC monolayers, insulin secretion decreased over 7 days.

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Abbreviations

BSA:

Bovine serum albumin

CMRL:

Connaught Medical Research Laboratories

DAPI:

4′,6-Diamidino-2-phenylindole

ECM:

Extracellular matrix

ELISA:

Enzyme-linked immunosorbent assay

GSIS:

Glucose stimulated insulin secretion

HBSS:

Hank’s balanced salt solution

IEQ:

Islet equivalent

Penstrep:

Penicillin–streptomycin

PI:

Porcine islet

PLMEC:

Porcine liver microvascular endothelial cell

RPMI:

Roswell Park Memorial Institute

TCPS:

Tissue culture polystyrene

TUNEL:

Terminal deoxynucleotide transferase dUTP Nick End Labeling

References

  1. Andrades, P., C. Asiedu, C. Rodriguez, K. J. Goodwin, J. McCarn, and J. M. Thomas. Subcutaneous pancreatic islet transplantation using fibrin glue as a carrier. Transplant. Proc. 39:191–192, 2007.

    Article  Google Scholar 

  2. Ballian, N., and F. C. Brunicardi. Islet vasculature as a regulator of endocrine pancreas function. World J. Surg. 31:705–714, 2007.

    Article  Google Scholar 

  3. Barkai, U., G. C. Weir, C. K. Colton, B. Ludwig, S. R. Bornstein, M. D. Brendel, T. Neufeld, C. Bremer, A. Leon, Y. Evron, K. Yavriyants, D. Azarov, B. Zimermann, S. Maimon, N. Shabtay, M. Balyura, T. Rozenshtein, P. Vardi, K. Bloch, P. de Vos, and A. Rotem. Enhanced oxygen supply improves islet viability in a new bioartificial pancreas. Cell Transplant. 22:1463–1476, 2013.

    Article  Google Scholar 

  4. Beattie, G. M., A. M. P. Montgomery, A. D. Lopez, E. Hao, B. Perez, M. L. Just, J. R. T. Lakey, M. E. Hart, and A. Hayek. A novel approach to increase human islet cell mass while preserving β-cell function. Diabetes 51:3435–3439, 2002.

    Article  Google Scholar 

  5. Buchwald, P. FEM-based oxygen consumption and cell viability models for avascular pancreatic islets. Theor. Biol. Med. Modell. 6:5, 2009.

    Article  Google Scholar 

  6. Daoud, J. T., M. S. Petropavlovskaia, J. M. Patapas, C. E. Degrandpré, R. W. DiRaddo, L. Rosenberg, and M. Tabrizian. Long-term in vitro human pancreatic islet culture using three-dimensional microfabricated scaffolds. Biomaterials 32:1536–1542, 2011.

    Article  Google Scholar 

  7. Daoud, J., M. Petropavlovskaia, L. Rosenberg, and M. Tabrizian. The effect of extracellular matrix components on the preservation of human islet function in vitro. Biomaterials 31:1676–1682, 2010.

    Article  Google Scholar 

  8. Dichmann, D. S., C. Rescan, U. Frandsen, and P. Serup. Unspecific labeling of pancreatic islets by antisera against fibroblast growth factors and their receptors. J. Histochem. Cytochem. 51:397–400, 2003.

    Article  Google Scholar 

  9. Dubiel, E. A., C. Kuehn, R. Wang, and P. Vermette. In vitro morphogenesis of PANC-1 cells into islet-like aggregates using RGD-covered dextran derivative surfaces. Colloids Surf. B 89:117–125, 2012.

    Article  Google Scholar 

  10. Dubiel, E. A., Y. Martin, and P. Vermette. Bridging the gap between physicochemistry and interpretation prevalent in cell − surface interactions. Chem. Rev. 111:2900–2936, 2011.

    Article  Google Scholar 

  11. Eberhard, D., M. Kragl, and E. Lammert. Giving and taking : endothelial and β-cells in the islets of Langerhans. Trends Endocrinol. Metab. 21:457–463, 2010.

    Article  Google Scholar 

  12. Ellis, C. E., E. Suuronen, T. Yeung, K. Seeberger, and G. S. Korbutt. Bioengineering a highly vascularized matrix for the ectopic transplantation of islets. Islets 5:216–225, 2013.

    Article  Google Scholar 

  13. Froud, T., C. Ricordi, D. A. Baidal, M. M. Hafiz, G. Ponte, P. Cure, A. Pileggi, R. Poggioli, H. Ichii, A. Khan, J. V. Ferreira, A. Pugliese, V. V. Esquenazi, N. S. Kenyon, and R. Alejandro. Islet transplantation in type 1 diabetes mellitus using cultured islets and steroid-free immunosuppression: Miami experience. Am. J. Transplant. 5:2037–2046, 2005.

    Article  Google Scholar 

  14. Holdcraft, R. W., L. S. Gazda, L. Circle, H. Adkins, S. G. Harbeck, E. B. Meyer, M. A. Bautista, P. C. Martis, M. A. Laramore, H. V. Vinerean, R. D. Hall, and B. H. Smith. Enhancement of in vitro and in vivo function of agarose encapsulated porcine islets by changes in the islet microenvironment. Cell Transplant. 2014. doi:10.3727/096368913x667033.

  15. Jindal, R. M., R. A. Sidner, H. B. McDaniel, M. S. Johnson, and S. E. Fineberg. Intraportal vs kidney subcapsular site for human pancreatic islet transplantation. Transplant. Proc. 30:398–399, 1998.

    Article  Google Scholar 

  16. Johansson, U., I. Rasmusson, S. P. Niclou, N. Forslund, L. Gustavsson, B. Nilsson, O. Korsgren, and P. U. Magnusson. Formation of composite endothelial cell–mesenchymal stem cell islets. Diabetes 57:2393–2401, 2008.

    Article  Google Scholar 

  17. Jun, Y., A. R. Kang, J. S. Lee, G. S. Jeong, J. Ju, D. Y. Lee, and S.-H. Lee. 3D co-culturing model of primary pancreatic islets and hepatocytes in hybrid spheroid to overcome pancreatic cell shortage. Biomaterials 34:3784–3794, 2013.

    Article  Google Scholar 

  18. Lamb, M., K. Laugenour, O. Liang, M. Alexander, C. E. Foster, and J. R. T. Lakey. In vitro maturation of viable islets from partially digested young pig pancreas. Cell Transplant. 23:263–272, 2014.

    Article  Google Scholar 

  19. Lammert, E., O. Cleaver, and D. Melton. Induction of pancreatic differentiation by signals from blood vessels. Science 294:564–567, 2001.

    Article  Google Scholar 

  20. Lee, R. H., J. Carter, G. L. Szot, A. Posselt, and P. Stock. Human albumin preserves islet mass and function better than whole serum during pretransplantation islet culture. Transplant. Proc. 40:384–386, 2008.

    Article  Google Scholar 

  21. Lehmann, R., R. A. Zuellig, P. Kugelmeier, P. B. Baenninger, W. Moritz, A. Perren, P.-A. Clavien, M. Weber, and G. A. Spinas. Superiority of small islets in human islet transplantation. Diabetes 56:594–603, 2007.

    Article  Google Scholar 

  22. Lim, J.-Y., B.-H. Min, B.-G. Kim, H.-J. Han, S.-J. Kim, C.-W. Kim, S–. S. Han, and J.-S. Shin. A fibrin gel carrier system for islet transplantation into kidney subcapsule. Acta Diabetol. 46:243–248, 2009.

    Article  Google Scholar 

  23. Linn, T., K. Schneider, H. P. Hammes, K. T. Preissner, H. Brandhorst, E. Morgenstern, F. Kiefer, and R. G. Bretzel. Angiogenic capacity of endothelial cells in islets of Langerhans. FASEB J. 17:881–883, 2003.

    Google Scholar 

  24. London, N. J., S. M. Swift, and H. A. Clayton. Isolation, culture and functional evaluation of islets of Langerhans. Diabetes Metab. 24:200–207, 1998.

    Google Scholar 

  25. Lyle, D. B., J. C. Shallcross, and J. J. Langone. Sensitivity of insulin production from encapsulated islets to endotoxin-stimulated macrophage inflammatory mediators. J. Biomed. Mater. Res., Part A 91A:1221–1238, 2009.

    Article  Google Scholar 

  26. Maillard, E., M. T. Juszczak, A. Clark, S. J. Hughes, D. R. W. Gray, and P. R. V. Johnson. Perfluorodecalin-enriched fibrin matrix for human islet culture. Biomaterials 32:9282–9289, 2011.

    Article  Google Scholar 

  27. Merani, S., C. Toso, J. Emamaullee, and A. M. J. Shapiro. Optimal implantation site for pancreatic islet transplantation. Br. J. Surg. 95:1449–1461, 2008.

    Article  Google Scholar 

  28. Paget, M. B., H. E. Murray, C. J. Bailey, P. R. Flatt, and R. Downing. Rotational co-culture of clonal β-cells with endothelial cells: effect of PPAR-γ agonism in vitro on insulin and VEGF secretion. Diabetes. Obes. Metab. 13:662–668, 2011.

    Article  Google Scholar 

  29. Pan, X., W. Xue, Y. Li, X. Feng, X. Tian, and C. Ding. Islet graft survival and function: concomitant culture and transplantation with vascular endothelial cells in diabetic rats. Transplantation 92:1208–1214, 2011.

    Article  Google Scholar 

  30. Papas, K. K., A. Pisania, H. Wu, G. C. Weir, and C. K. Colton. A stirred microchamber for oxygen consumption rate measurements with pancreatic islets. Biotechnol. Bioeng. 98:1071–1082, 2007.

    Article  Google Scholar 

  31. Paraskevas, S., D. Maysinger, R. Wang, W. P. Duguid, and L. Rosenberg. Cell loss in isolated human islets occurs by apoptosis. Pancreas 20:270–276, 2000.

    Article  Google Scholar 

  32. Scuteri, A., E. Donzelli, V. Rodriguez-Menendez, M. Ravasi, M. Monfrini, B. Bonandrini, M. Figliuzzi, A. Remuzzi, and G. Tredici. A double mechanism for the mesenchymal stem cells’ positive effect on pancreatic islets. PLoS ONE 9:e84309, 2014.

    Article  Google Scholar 

  33. Shaikh, F. M., A. Callanan, E. G. Kavanagh, P. E. Burke, P. A. Grace, and T. M. McGloughlin. Fibrin: a natural biodegradable scaffold in vascular tissue engineering. Cells Tissues Organs 188:333–346, 2008.

    Article  Google Scholar 

  34. Shkilnyy, A., J. Dubois, G. Sabra, J. Sharp, S. Gagnon, P. Proulx, and P. Vermette. Bioreactor controlled by PI algorithm and operated with a perfusion chamber to support endothelial cell survival and proliferation. Biotechnol. Bioeng. 109:1305–1313, 2012.

    Article  Google Scholar 

  35. Shkilnyy, A., P. Proulx, J. Sharp, M. Lepage, and P. Vermette. Diffusion of rhodamine B and bovine serum albumin in fibrin gels seeded with primary endothelial cells. Colloids Surf. B 93:202–207, 2012.

    Article  Google Scholar 

  36. Su, J., B.-H. Hu, W. L. Lowe, Jr, D. B. Kaufman, and P. B. Messersmith. Anti-inflammatory peptide-functionalized hydrogels for insulin-secreting cell encapsulation. Biomaterials 31:308–314, 2010.

    Article  Google Scholar 

  37. van Hinsbergh, V. W. M., A. Collen, and P. Koolwijk. Role of fibrin matrix in angiogenesis. Ann. N. Y. Acad. Sci. 936:426–437, 2001.

    Article  Google Scholar 

  38. Walpita, D., T. Hasaka, J. Spoonamore, A. Vetere, K. K. Takane, D. Fomina-Yadlin, N. Fiaschi-Taesch, A. Shamji, P. A. Clemons, A. F. Stewart, S. L. Schreiber, and B. K. Wagner. A human islet cell culture system for high-throughput screening. J. Biomol. Screen. 17:509–518, 2012.

    Article  Google Scholar 

  39. Wang, R. N., and L. Rosenberg. Maintenance of beta-cell function and survival following islet isolation requires re-establishment of the islet-matrix relationship. J. Endocrinol. 163:181–190, 1999.

    Article  Google Scholar 

  40. Weber, L. M., and K. S. Anseth. Hydrogel encapsulation environments functionalized with extracellular matrix interactions increase islet insulin secretion. Matrix Biol. 27:667–673, 2008.

    Article  Google Scholar 

  41. Weber, L. M., K. N. Hayda, K. Haskins, and K. S. Anseth. The effects of cell–matrix interactions on encapsulated β-cell function within hydrogels functionalized with matrix-derived adhesive peptides. Biomaterials 28:3004–3011, 2007.

    Article  Google Scholar 

  42. Wittig, C., M. W. Laschke, C. Scheuer, and M. D. Menger. Incorporation of bone marrow cells in pancreatic pseudoislets improves posttransplant vascularization and endocrine function. PLoS ONE 8:e69975, 2013.

    Article  Google Scholar 

  43. Yamada, S., M. Shimada, T. Utsunomiya, T. Ikemoto, Y. Saito, Y. Morine, S. Imura, H. Mori, Y. Arakawa, M. Kanamoto, and S. Iwahashi. Trophic effect of adipose tissue-derived stem cells on porcine islet cells. J. Surg. Res. 187:667–672, 2014.

    Article  Google Scholar 

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Acknowledgments

This project was supported by Université de Sherbrooke, NSERC through a Discovery Grant (Patrick Vermette, grant #250296-07) and through the Department of Surgery, University of California Irvine. We are also grateful to Jocelyne Ayotte and Michael Alexander for their technical assistance.

Conflict of interest

Evan A. Dubiel, Jonathan R.T. Lakey, Morgan W. Lamb, and Patrick Vermette declare that they have no conflict of interest associated with this study.

Ethical Standards

All animal procedures including but not limited to monitoring, surgery and euthanasia were performed with the approval from the University of California Institutional Animal Care and Use Committee (IACUC Protocol #2008-2823; Approval Period 09/28/2012–09/27/2013) at the University of California, Irvine. All efforts were made to reduce the quantity of animals required for this study. No human studies were carried out by the authors for this article.

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Authors and Affiliations

  1. Laboratoire de bio-ingénierie et de biophysique de l’Université de Sherbrooke, Department of Chemical and Biotechnological Engineering, Université de Sherbrooke, 2500, boulevard de l’Université, Sherbrooke, QC, J1K 2R1, Canada

    Evan A. Dubiel & Patrick Vermette

  2. Research Centre on Aging, Institut universitaire de gériatrie de Sherbrooke, 1036, rue Belvédère Sud, Sherbrooke, QC, J1H 4C4, Canada

    Evan A. Dubiel & Patrick Vermette

  3. Department of Surgery and Biomedical Engineering, University of California, Irvine, 333 City Boulevard West, Suite 700, Orange, CA, 92868, USA

    Jonathan R. T. Lakey & Morgan W. Lamb

Authors
  1. Evan A. Dubiel
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  2. Jonathan R. T. Lakey
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  4. Patrick Vermette
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Corresponding author

Correspondence to Patrick Vermette.

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Associate Editor Tejal Desai oversaw the review of this article.

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Dubiel, E.A., Lakey, J.R.T., Lamb, M.W. et al. Culturing Free-Floating and Fibrin-Embedded Islets with Endothelial Cells: Effects on Insulin Secretion and Apoptosis. Cel. Mol. Bioeng. 7, 243–253 (2014). https://doi.org/10.1007/s12195-014-0332-0

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