Strategies to improve encapsulated islet survival and oxygenation

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CANADIAN JOURNAL OF DIABETES

D-0752 strategies to improve encapsulated islet survival and oxygenation S. Bilodeau1, S.K. Tam2, J. Dusseault1, G. Langlois1, J.P. Hallé1 1 Hôpital Maisonneuve-Rosemont, Centre de recherche, Montreal, Canada 2 École Polytechnique de Montréal, Institut de génie biomédical, Montreal, Canada The microencapsulation of pancreatic islets is a promising treatment for type I diabetes. Although microcapsule biocompatibility has improved, islet cell survival is still problematic. During islet isolation, the extracellular matrix (ECM) around the islet is destroyed, which negatively affects islet function and survival. Also, smaller islets have been shown to have better function and survival. Since encapsulated islets are not re-vascularized, their oxygen supply is entirely dependent upon diffusion. The use of dispersed islet cell re-aggregates of smaller diameter now allows the investigation of microcapsules of further reduced size. aim: The aim of this study is to improve microencapsulated islet cell survival, using three approaches: restoring the ECM loss, reducing the microcapsule diameter from 300 µm to 100 µm and reducing the islet diameter, using islet cell re-aggregates. It is known that dissociated islet cells have the capacity re-assemble in clusters that have the same properties of a normal islet. The first steps were to 1) characterize ECM in islets, after isolation and encapsulation, 2) produce islet cell re-aggregates and 3) evaluate the feasibility of producing 100-150 µm diameter microcapsules. Methods: The presence of ECM molecules (collagen I and IV, fibronectin and laminin) was evaluated by immunohistochemistry in islets that were cultured for different time periods, comparing encapsulated to non-encapsulated islets. Secondly, islet-like clusters were formed after dissociation of islets with trypsin and rotational incubation during 5-6 days. Different conditions were tested to achieve an optimal size. Finally, alginate-poly-L-lysine microcapsules were produced using an electrostatic pulse generator and different fabrication parameters were tested in order to achieve specific capsule diameters. results: Collagen IV and laminin were found to be present in whole pancreases used as a control, but only a minimal amount was found in isolated islets for all of the tested conditions. The other proteins under study were present for all conditions(fibronectin, collagen I). Isletlike clusters were produced and encapsulated in small microcapsules. We were able to produce microcapsules with a diameter of 132,32 ± 6,29 µm by extruding alginate through a 30G needle and reducing the alginate flow rate from 3,5mL/min to 0,3mL/min. Discussion/conclusion: This data suggests that replacing collagen IV and/or laminin in the re-aggregates might be useful. We also showed the feasibility of producing APA microcapsules of approximately 130 µm and of encapsulating islet cell re-aggregates within these microcapsules. Further research will be conducted to determine whether the use of this system improves oxygen supply to islets and islet survival. Transplantation - islet and pancreas No conflict of interest D-0754 Preservation of human islet function in vitro through EMc reestablishment D. Jamal1, M. Petropavlovskaia2, L. Rosenberg2, M. Tabrizian1 1 McGill University, Biomedical Engineering, Montreal, Canada 2 Montreal General Hospital, Surgical Research, Montreal, Canada Pancreatic islet transplantation requires successful isolation and in vitro survival; however, studies have shown that islet isolation exposes the islet to a variety of cellular stresses, destroys the basement membrane (BM) and disrupts the cell-matrix relationship, leading to apoptosis. In the current state of islet regeneration research, we are still unable to reemulate the ECM conditions of the pancreatic islet and restore the BM which is lost during isolation. The rationale behind this research study

is to identify factors responsible for islet preservation and survival in vitro. This will lend to emulate the basement membrane of native islet tissue through proper cell-substrate interactions. This study has shed light into important factors that promote human islet adhesion, morphology, survival and functionality in vitro. A direct relationship was observed between surface roughness and wettability of the modified protein surfaces. Also, the AFM analysis showed a homogeneous distribution of the proteins on their respective surfaces. Fibronectin, followed by laminin, possessed the greatest contact angle and surface roughness which directly correlate with hydrophobicity. Adhesion studies showed that collagen I, collagen IV, and fibronectin surfaces were the most effective in inducing adhesion at levels of over 50%. Also, islets cultured on these surfaces showed high metabolic activity and viability. Islet morphology was maintained best on fibronectin surfaces, while being most compromised on collagen I due to islet spreading and monolayer formation. However, the strongest adhesion occurred on collagen I, followed by collagen IV and fibronectin. Furthermore, glucose-induced insulin release was optimal for fibronectin cultured islets, in contrast to its ECM counterparts. Islet gene expression of insulin, glucagon, somatostatin, pancreatic polypeptide and PDX1 were also elevated relative to islets cultured on BSA-control surfaces. This investigation into appropriate surfaces for islet adhesion and viability provide valuable insight into the optimal conditions to ensure islet survival in vitro. More importantly, this forms the basis for further experiments into investigating the effects of geometry of the surfaces on adhesion, viability, and differentiation; as well as the implementation of a favourable microenvironment for isolated pancreatic islets. Transplantation - islet and pancreas No conflict of interest D-0755 Understanding the biocompatibility of microcapsules designed for islet cell transplantation S.K. Tam1, J. Dusseault2, G. Langlois2, S. Bilodeau2, L.H. Yahia1, J.P. Hallé2 École Polytechnique de Montréal, Institut de génie biomédical, Montréal, Canada 2 Hôpital Maisonneuve-Rosemont, Centre de recherche, Montréal, Canada

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The microencapsulation and transplantation of islet cells is a very promising approach to effectively treat insulin-dependent diabetes mellitus. To encapsulate islets, most research groups use alginate as the base material and then incorporate a polycation into the capsule membrane. Problematic: The biocompatibility of microcapsules designed for islet transplantation remains difficult to control, predict and reproduce. This has the effect of limiting the long-term function of the graft. aim: The purpose of this work is to explain the biological response to alginate-based microcapsules in terms of their fabrication design and surface properties. Methods: Microcapsules are made using different alginates (Alg I or Alg II) and have either no polycation, poly-L-lysine (PLL), or poly-L-ornithine (PLO) incorporated into the membranes. Alg I is composed of 37% guluronic acid (G) while Alg II is composed of 58% G, as confirmed by nuclear magnetic resonance. To evaluate their biocompatibility in vivo, microcapsules are implanted into the peritoneal cavity of C56BL/6 mice for 2 days and then immune cell adhesion to the explanted capsules is measured using a semiquantitative scoring system with a scale of 0 to 2. Films that recreate the microcapsule structure are analyzed in terms of chemical composition using x-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy. Film hydrophilicity is evaluated using various methods to measure the contact angle at the water/air/surface interface. results: In the absence of a polycation, Alg I capsules were almost free from cellular adhesion after 2 days implantation (score 0.05 ± 0.01) while Alg II capsules were often covered with immune cells