3D bioprinting of pancreatic islets
PhD student: Sarah Duin
Supervisor at TUD: Barbara Ludwig, Michael Gelinsky
Supervisor at KCL: Shanta Persaud
Start date: 01.01.2016 Date of defense: 17.12.2020 Dr. rer. medic.
To prevent long-term complications in T1D, donor islets can be transplanted; this requires life-long strict immunosuppressive regimens though. Encapsulation of islets can circumvent immunosuppression but upscaling is difficult. The aim of this thesis was to develop a strategy for the plotting of scalable semipermeable macroporous scaffolds with a large surface-to-volume ratio, containing viable and functional pancreatic islets.
The hydrogel used for bioprinting in this thesis was a blend of 3% alginate (Alg) with 9% methylcellulose (MC) as a thickener to create a plottable paste. The existing blend had to be adapted for the use of highly purified, i.e. non-immunogenic (“clinical-grade”) alginate instead of the cell-compatible but less stringently purified (“research-grade”) alginate used previously. Stability was tested as well as permeability for glucose and insulin was analysed with uptake/ release assays and in a diffusion chamber system developed for this thesis. For pancreatic islets, which as cell clusters are more sensitive to shear stress than single cells, a workflow for the incorporation into the highly viscous blend was successfully developed. Plotting of islets was performed with adult murine islets as well as neonatal porcine islet-like clusters (NICC) which were tested for distribution, viability, apoptosis, and presence of hormones with stainings. Functional response was analysed via glucose-stimulated insulin release assays (GSIR) by detection of insulin in the supernatant released under hypoglycaemic (3.3mM glucose) or hyperglycaemic (16.4mM glucose) conditions.
Pastes prepared with the different alginates showed a comparable viscosity suitable for the plotting of stable structures. Clinical-grade scaffolds had a slightly reduced crosslinking density which was attributed to differences in the M:G-ratio. Scaffolds crosslinked with 70mM SrCl2 remained stable over 21d. Over time, release of crosslinking ions is sustained and independent of alginate-type, but dependent on the type of medium used. In permeability analyses overall kinetics of diffusion observed in the chamber system followed the normal course of diffusion through hydrogels.The permeability for glucose was comparable between the all tested materials. The permeability for insulin needs to be verified in further experiments due to binding to the diffusion chamber and a lack of stability of gel discs.
Metabolically active murine islets containing insulin were distributed homogeneously throughout the scaffolds. Viability was 70-80%, comparable to control islets in suspension culture, over a total of 14d. DNA-content in plotted scaffolds was strongly reduced over a total of 21d. In all islets observed over 7d a limited amount of apoptotic nuclei was present. Number of apoptotic nuclei was not significantly different between control and plotted islets. Overall, during incorporation into the blend and plotting the morphology and viability of islets were retained. The pancreatic hormones insulin and glucagon were detected in the appropriate ratio and locations. Stimulation of murine islets was verified and showed a lower total amount of insulin released from plotted than from control islets but a comparable stimulation index (SI) calculated from GSIR on day 4 and 7 of culture. Furthermore, it could be shown that when islets are exposed to successive low-high-low glucose stimulation, release of insulin corresponded to glucose concentration, showing that plotted islets truly sense external glucose concentrations and react accordingly. In summary, while total release of insulin was lower from plotted than from control islets, functionality was retained in plotted scaffolds.
For validation of the results from adult murine islets the more sensitive NICC were used in a preliminary study. Metabolically active NICC containing insulin were distributed homogeneously throughout the scaffolds. Viability of NICC was comparatively low (~60%) without any significant changes over 21d, but equal to control NICC. All plotted NICC retained some viability and number of apoptotic nuclei increased significantly over 7d for plotted but not control NICC. The pancreatic hormones insulin, glucagon and somatostatin were detected in a low number of cells scattered randomly throughout the cell clusters over 7d. Functional response was not impacted by the incorporation into the material nor the plotting. With amplification with a GLP-1 analogue, NICC showed a functional response which was comparable between plotted and control samples. The SI decreased over the time of observation and resulted in an SI<2 after 14 and 21d of culture in plotted and control samples respectively.
The adapted Alg/MC blend showed sufficient stability and permeability for the plotting of islets. The feasibility of 3D plotting of viable and functional pancreatic islets with the proposed hydrogel blend was demonstrated in a proof-of-concept study with adult murine islets. Preliminary results indicate that the adapted blend is also an appropriate basis for the plotting of NICC. Further characterisation and material adaptation to support viability and maturation of NICC in vitro will be analysed in a subsequent study.
Publications:
Development of a clay based bioink for 3D cell printing for skeletal application. T. Ahlfeld, G. Cidonio, D. Kilian, S. Duin, A.R. Akkineni, J.I. Dawson, S. Yang, A. Lode, R.O.C. Oreffo, M. Gelinsky. Biofabrication. 2017;9:034103.
Coacervation-Mediated Combinatorial Synthesis of Biomatrices for Stem Cell Culture and Directed Differentiation. R. Wieduwild, R. Wetzel, D. Husman, S. Bauer, I. El-Sayed, S. Duin, P. Murawala, A.K. Thomas, M. Wobus, M. Bornhauser, Y. Zhang. Adv Mater. 2018;30:e1706100.
3D Bioprinting of Functional Islets of Langerhans in an Alginate/Methylcellulose Hydrogel Blend. S. Duin, K. Schutz, T. Ahlfeld, S. Lehmann, A. Lode, B. Ludwig, M. Gelinsky. Adv Healthc Mater. 2019;8:e1801631.
Investigating the effect of sterilisation methods on the physical properties and cytocompatibility of methyl cellulose used in combination with alginate for 3D-bioplotting of chondrocytes. E. Hodder*, S. Duin*, D. Kilian, T. Ahlfeld, J. Seidel, C. Nachtigall, P. Bush, D. Covill, M. Gelinsky, A. Lode. J Mater Sci Mater Med. 2019;30:10.
Methylcellulose–a versatile printing material that enables biofabrication of tissue equivalents with high shape fidelity. T. Ahlfeld, V. Guduric, S. Duin, A. Akkineni, K. Schütz, D. Kilian, J. Emmermacher, N. Cubo-Mateo, S. Dani, M.v. Witzleben. Biomaterials Science. 2020;8:2102-2110.
The Secretome of Hypoxia Conditioned hMSC Loaded in a Central Depot Induces Chemotaxis and Angiogenesis in a Biomimetic Mineralized Collagen Bone Replacement Material. M. Quade, P. Munch, A. Lode, S. Duin, C. Vater, A. Gabrielyan, A. Rosen-Wolff, M. Gelinsky. Adv Healthc Mater. 2020;9:e1901426.