Ischaemia impairs organ quality during preservation in a time‐dependent manner, due to a lack of oxygen supply. Its impact on pancreas and islet transplantation outcome has been demonstrated by a correlation between cold ischaemia time and poor islet isolation efficiency. Our goal in the present study was to improve pancreas and islet quality using a novel natural oxygen carrier (M101, 2 g/L), which has been proven safe and efficient in other clinical applications, including kidney transplantation, and for several pre‐clinical transplantation models. When M101 was added to the preservation solution of rat pancreas during ischaemia, a decrease in oxidative stress (ROS), necrosis (HMGB1), and cellular stress pathway (p38 MAPK)activity was observed. Freshly isolated islets had improved function when M101 was injected in the pancreas. Additionally, human pancreases exposed to M101 for 3 hours had an increase in complex 1 mitochondrial activity, as well as activation of AKT activity, a cell survival marker. Insulin secretion was also up‐regulated for isolated islets. In summary, these results demonstrate a positive effect of the oxygen carrier M101 on rat and human pancreas during preservation, with an overall improvement in post‐isolation islet quality.
Background: The emergent field of 3D bioprinting has the potential to overcome hypoxia and lack of immunoprotection, two major limitations of islet transplantation in encapsulation systems. When transitioning tissue engineered constructs from bench to bedside, several design parameters must be considered, including tissue geometry, islet density, and oxygen tension in different transplantation sites. To address this challenging multifactorial optimization process, we have designed an in vitro flow device that can be used to evaluate bioprinted tissue performance in vitro under different flow, oxygen and tissue geometry conditions. The aim of this work was to assess the function and viability of single 3D bioprinted core-shell fibres containing pseudo-islets or human islets in a novel perfusion device designed to accommodate bioprinted tissues. Methods: The perfusion device was designed using computer-aided design modelling. Computational fluid dynamics (CFD) was used to simulate flow and compute input parameters that would ensure laminar, uniform flow. Pseudo-islets were formed after aggregation of MIN6 cells in AggrewellTM culture plates during 48h. Pseudo-islets and human islets were 3D bioprinted in alginate using an RX1 bioprinter (AspectBiosystems, Vancouver, CA). Free or encapsulated pseudo-islets or human islets were cultured in static conditions as controls. Pseudo-islet function was determined using a glucose stimulation insulin secretion (GSIS) assay in static or perfused conditions. Viability of human islets was evaluated using Calcein AM/Ethidium Homodimer staining. Results: Based on our computational models, by setting the inlet flow speed to 1.0 cm/s, we can achieve physiological flow velocities within the device. This inlet flow speed is also expected to generate smooth, undisturbed streamlines and laminar flow. After a 24h culture in the perfusion device, we detected increased insulin secretion of pseudo-islets in fibres in response to high glucose stimulation (ratio of secreted to total insulin of 0.27% after 15 min at high glucose vs 0.13% after 15 min at low glucose). Human islets bioprinted in fibres showed higher cell viability compared to free human islets after a 48h culture (95% of viability in fibres cultured in the perfusion device compared to 80% viability for free islets). As these results were obtained from a single human pancreas donor, further studies are needed to assess the reproducibility and statistical significance of these observations. Conclusion: These promising preliminary results suggest that (1) the flow device we designed can be used to evaluate the performance of 3D bioprinted pancreatic tissue and (2) pseudo-islets and human islets can be safely bioprinted in core-shell fibres. The platform can be used to streamline the characterization and optimize the configuration in vitro of promising artificial tissues at human-scale.
High pancreatic islet sensitivity to hypoxia is an important issue in the field of pancreatic islet transplantation. A promising strategy to improve islet oxygenation in hypoxic conditions is to leverage the properties of hemoglobin as a natural carrier of oxygen. Studies using human or bovine hemoglobin have failed to demonstrate efficacy, probably due to the molecule being unstable in the absence of protective erythrocytes. Recently, marine worm hemoglobins have been shown to be more stable and to possess higher oxygen carrier potential, with 156 oxygen binding sites per molecule compared to four in humans. Previous studies have shown the beneficial effects of two marine worm hemoglobins, M101 and M201, on nonhuman pancreatic islets. However, their effects on human islets have not been tested or compared. In this study, we assessed the impact of both molecules during human islet culture in vitro under hypoxic conditions. Human islets were exposed to both molecules for 24 h in high islet density-induced hypoxia [600 islet equivalents (IEQ)/cm²]. M101 and M201 reduced the release of hypoxic (VEGF) and apoptotic (cyt c) markers in the medium after 24-h culture. Human islet function or viability was improved in vitro in the presence of these oxygen carriers. Thus, the utilization of M101 or M201 could be a safe and easy way to improve human islet oxygenation and survival in hypoxic conditions as observed during islet culture prior to transplantation or islet encapsulation.
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