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Chapter 5 Co-Culture of Young Porcine Islets with Liver Microvascular Endothelial Cells in Fibrin Improves Insulin Secretion and Reduces Apoptosis

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Chapter 5 Co-Culture of Young Porcine Islets with Liver Microvascular Endothelial Cells in Fibrin Improves Insulin Secretion and Reduces Apoptosis UNIVERSITE DE SHERBROOKE Faculte de genie Departement de genie chimique et de genie biotechnologique DEVELOPPEMENT ET VALIDATION DE SURFACES ET D’ECHAFAUDAGES PROPICES AU DEVELOPPEMENT ET AU MAINTIEN DES FONCTIONS DE CELLULES PANCREATIQUES BETA TOWARDS THE DEVELOPMENT AND VALIDATION OF BIOMATERIAL SURFACES AND SCAFFOLDS SUITABLE FOR PANCREATIC BETA­ CELL DEVELOPMENT AND FUNCTION These de doctorat Specialite: genie chimique Evan Aiozie DUBIEL Jury : Patrick VERMETTE (directeur) Jonathan LAKEY Steven PARASKEVAS Marie-Josee BOUCHER Marcel LACROIX (rapporteur) Sherbrooke (Quebec) Canada Novembre 2012 Library and Archives Bibliotheque et Canada Archives Canada Published Heritage Direction du 1+1 Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington Ottawa ON K1A0N4 Ottawa ON K1A 0N4 Canada Canada Your file Votre reference ISBN: 978-0-494-93268-1 Our file Notre reference ISBN: 978-0-494-93268-1 NOTICE: AVIS: The author has granted a non­ L'auteur a accorde une licence non exclusive exclusive license allowing Library and permettant a la Bibliotheque et Archives Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par I'lnternet, preter, telecommunication or on the Internet, distribuer et vendre des theses partout dans le loan, distrbute and sell theses monde, a des fins commerciales ou autres, sur worldwide, for commercial or non­ support microforme, papier, electronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats. The author retains copyright L'auteur conserve la propriete du droit d'auteur ownership and moral rights in this et des droits moraux qui protege cette these. Ni thesis. Neither the thesis nor la these ni des extraits substantiels de celle-ci substantial extracts from it may be ne doivent etre imprimes ou autrement printed or otherwise reproduced reproduits sans son autorisation. without the author's permission. In compliance with the Canadian Conformement a la loi canadienne sur la Privacy Act some supporting forms protection de la vie privee, quelques may have been removed from this formulaires secondaires ont ete enleves de thesis. cette these. While these forms may be included Bien que ces formulaires aient inclus dans in the document page count, their la pagination, il n'y aura aucun contenu removal does not represent any loss manquant. of content from the thesis. Canada To my siblings: Erin, Nathan, Matthew, and Kerrie RESUME Le diabete mellitus de type I est une maladie de plus en plus abondante. Cette demiere est caracterisee par la destruction auto-immunitaire des ilots de Langerhans incluant les cellules de type p qui produisent de l’insuline dans le pancreas endocrinien. Une option de traitement pour les patients atteints de cette maladie est notamment une greffe des ilots de Langerhans. Ce traitement est limits du au nombre restreint de donneurs d’organes et aussi a la perte de fonctionnalite des ilots suite a la greffe. Les etudes effectuees tout au long de cette these ont pour optique d’adresser ces contraintes par le biais de la science des biomateriaux. La these debute avec un survol details des concepts de base et des complexites associes aux interactions de type cellules et surfaces trouvees dans la litterature. II s’agit specifiquement des interactions physiques et chimiques, des systemes experimentaux ainsi que des caracterisations et modifications associes aux interactions entre cellules et surfaces. La premiere etude de nature experimentale examine la morphogenese des cellules progenitrices ductales (PANC-1 cell line) vers des ilots qui produisent des agregats semblables a des ilots (ILA). Le tout est fait sur des surfaces de carboxymethyl dextrane (CMD) sur lesquelles le RGD est greffe via un lien covalent. L’expression des marqueurs d’lLAs (cytokeratin-19, Ki67, et E-cadherin) qui peuvent etre associes k un changement de phenotype de ces cellules a ete evaluee ainsi que la secretion et 1’expression de l’insuline. La seconde etude de nature experimentale a pour optique 1* immobilisation de la fibronectine (FN) sur les memes surfaces de CMD mentionnees auparavant sur lesquelles des cellules ayant un phenotype fi ( INS-1 cell line) ont prolifere. Lors du processus d’immobilisation, plusieurs solutions ont ete etudiees. L’immobilisation de la fibronectine sur des surfaces de CMD a ete validee par la spectrometrie de photoelectrons induits par rayons X. Le mecanisme d’immobilisation a ete determine par imagerie et mesures de force par microscopie a force atomique, la spectroscopie de dichroisme circulaire ainsi que par la diffusion dynamique de la lumiere. De plus, la croissance des cellules de type INS-1 et la secretion d’insuline ont ete evaluees. La demiere etude de nature experimentale visait l’etude de la coculture des cellules endotheliales et des ilots de pore dans un gel de fibrine. L’effet de la presence des cellules endotheliales sur la production d’insuline des ilots a ete evalue. De plus, l’apoptose cellulaire en coculture a ete evaluee et comparee aux cultures simples. Mots cl6s: biomateriaux; forces de surface et intermoleculaires; modifications de surface; surfaces anti-adherentes; adsorption de proteines; diabete; ilots de Langerhans; secretion d ’insuline ABSTRACT Type I diabetes mellitus is a disease of increasing abundance, characterized by the auto-immune destruction of insulin producing p-cells comprising islets of Langerhans in the endocrine pancreas. A treatment option for patients with severe type I diabetes mellitus is the surgical transplantation of islets of Langerghans. This treatment is limited because of pancreas donor scarcity and the progressive loss of islet graft function after both islet isolation from the donor pancreas and transplantation. The present studies within this thesis aim to address these issues by means of biomaterials science. It begins with a detailed broad review of general concepts and complexities associated with cell - surface interaction studies. More specifically physical & chemical interactions, experimental systems, and surface modification & characterization associated with cell - surface interactions are discussed. The first experimental work studies the morphogenesis of ductal-like progenitor cells (i.e., PANC-1 cell line) to insulin expressing and producing islet-like aggregates (ILA) on well-defined low-fouling carboxymethyl-dextran (CMD) surfaces upon which the ligand arginine-glycine-asparic acid is covalently grafted. ILA expression patterns of markers (i.e., cytokeratin-19, Ki67, and E-cadherin) typically associated with a change PANC-1 phenotype were assessed along with insulin expression and secretion. The second experimental study involves the immobilization of the protein fibronectin (FN) onto low-fouling CMD surfaces upon where cells of a P-cell-like phenotype (i.e., INS-1 cell line) were cultured. Various solutions were used during the immobilization process. The successful immobilization of FN on CMD was assessed via X-ray photoelectron spectroscopy and the mechanisms of immobilization were assessed via atomic force microscopy imaging & force measurements, circular dichroism spectroscopy, and photon correlation spectroscopy. INS-1 cell growth and insulin secretion were also assessed. The final experimental work studied the effect of the co-culture of endothelial cells with isolated porcine islets in fibrin gel. The effect endothelial cells had on the capacity of islets to secrete insulin was accessed. In addition, cellular apoptosis was accessed with co-cultures and compared to cultures without endothelial cells. Keywords: Biomaterials; Surface and Intermolecular Forces; Surface Modification; Low- Fouling; Protein Adsorption; Diabetes; Islets o f Langerhans; Insulin Secretion ACKNOWLEGEMENTS First and foremost, I would like to thank my mentor and research director Patrick Vermette for the opportunity to perform the boundless scientific research that enlivens me. The past years have been a pleasure and I look forward to our future work together. I would also like to thank Dr. Rennian Wang and her research group at the University of Western Ontario for providing me with their laboratory and knowledge that gave me the training necessary to perform much of my lab work. I am also grateful to Dr. Steven Paraskevas and Craig Hasilo at McGill University for providing me with a generous quantity of isolated islets of Langerhans that was used to access the feasibility of my final experimental study. A very special thank you goes to Dr. Jonathan Lakey, Morgan Lamb, and their entire laboratory for their continuous and unrestrictive supply of islets throughout my final experimental study. Such work would not have been performed without such generosity. I would also like to thank Mathew Evans for his assistance in the French language. Finally, I would like to thank all my colleagues over the years at the Universite de Sherbrooke for providing a pleasant work environment and members of the jury (i.e., Dr. Jonathan Lakey, Dr. Steven Paraskevas, Dr. Marie-Josee Boucher, and Dr. Marcel Lacroix) for providing their insights and for their longevity in reading a particularly lengthy thesis. TABLE OF CONTENTS RESUME................................................................................................................................................ i ABSTRACT...........................................................................................................................................ii
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