Co-axial printing of growth factor-laden microspheres for pancreatic islet transplantation

Type I diabetes is an autoimmune disease affecting millions of people in the world. It occurs when the pancreas cannot produce insulin, resulting in episodes of hyperglycaemia that can lead to heart attacks, renal failure, or death. The main cause is the auto destruction of beta cells that produce insulin, located in the pancreatic islets (or islets of Langerhans). Current treatments include insulin injections that decrease the blood glucose level. However, it can sometimes generate hypoglycaemia or insulin resistance on the patients. Bioprinting allows controlled engineering of pancreatic islets with hydrogel scaffolds and transplanting them into the patients. Nevertheless, immunotolerance of the grafted constructs has yet to be achieved. Currently, the islets are implanted together with immunosuppressors to avoid the rejection, but these affect the functionality of the beta cells. Co-transplanting regulatory T cells (Tregs) that regulate the autoimmune response could be the solution to immune rejection. Thus, co-axial extrusion printing is a promising approach, as it allows printing two types of bioinks. Pancreatic islets can be printed in the core of the structure and Tregs in the shell, protecting the islets. This project was mainly focused on the development of the bioink for the shell. The ink consists of a hydrogel that promotes cell growth and allows bioprinting (2% alginate/7.5% gelatin methacrylolyl (GelMA)/3.5% gelatin), and growth factors for Treg functionality (IL-2). The growth factors were encapsulated in GelMA microspheres for a sustained release inside the ink. The release rate of IL-2 was studied, as well as the ink properties and printability

Encapsulation of Human Natural and Induced Regulatory T-Cells in IL-2 and CCL1 Supplemented Alginate-GelMA Hydrogel for 3D Bioprinting

2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Regulatory T-cells (Tregs) are important modulators of the immune system through their intrinsic suppressive functions. Systemic adoptive transfer of ex vivo expanded Tregs has been extensively investigated for allogeneic transplantation. Due to the time-consuming and costly expansion protocols of Tregs, more targeted approaches could be beneficial. The encapsulation of human natural and induced Tregs for localized immunosuppression is described for the first time. Tregs encapsulated in alginate-gelatin methacryloyl hydrogel remain viable, phenotypically stable, functional, and confined in the structure. Supplementation of the hydrogel with the Treg-specific bioactive factors interleukin-2 and chemokine ligand 1 improves Treg viability, suppressive phenotype, and function, and attracts to the structure CCR8+ T-cells enriched with anti-inflammatory subpopulations, including Tregs, from human peripheral blood. Furthermore, these findings are applicable to 3D bioprinting. Co-axial printing of murine pancreatic islets with human natural and induced Tregs protects the islets from xenoresponse upon co-culture with human peripheral blood mononuclear cells. This establishes the co-encapsulation of Tregs by co-axial 3D bioprinting as a valid option for providing local immune protection to allogeneic cellular transplants such as pancreatic islets

Encapsulation of Human Natural and Induced Regulatory T-Cells in IL-2 and CCL1 Supplemented Alginate-GelMA Hydrogel for 3D Bioprinting

2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Regulatory T-cells (Tregs) are important modulators of the immune system through their intrinsic suppressive functions. Systemic adoptive transfer of ex vivo expanded Tregs has been extensively investigated for allogeneic transplantation. Due to the time-consuming and costly expansion protocols of Tregs, more targeted approaches could be beneficial. The encapsulation of human natural and induced Tregs for localized immunosuppression is described for the first time. Tregs encapsulated in alginate-gelatin methacryloyl hydrogel remain viable, phenotypically stable, functional, and confined in the structure. Supplementation of the hydrogel with the Treg-specific bioactive factors interleukin-2 and chemokine ligand 1 improves Treg viability, suppressive phenotype, and function, and attracts to the structure CCR8+ T-cells enriched with anti-inflammatory subpopulations, including Tregs, from human peripheral blood. Furthermore, these findings are applicable to 3D bioprinting. Co-axial printing of murine pancreatic islets with human natural and induced Tregs protects the islets from xenoresponse upon co-culture with human peripheral blood mononuclear cells. This establishes the co-encapsulation of Tregs by co-axial 3D bioprinting as a valid option for providing local immune protection to allogeneic cellular transplants such as pancreatic islets

High-resolution lithographic biofabrication of hydrogels with complex microchannels from low-temperature-soluble gelatin bioresins

Biofabrication via light-based 3D printing offers superior resolution and ability to generate free-form architectures, compared to conventional extrusion technologies. While extensive efforts in the design of new hydrogel bioinks lead to major advances in extrusion methods, the accessibility of lithographic bioprinting is still hampered by a limited choice of cell-friendly resins. Herein, we report the development of a novel set of photoresponsive bioresins derived from ichthyic-origin gelatin, designed to print high-resolution hydrogel constructs with embedded convoluted networks of vessel-mimetic channels. Unlike mammalian gelatins, these materials display thermal stability as pre-hydrogel solutions at room temperature, ideal for bioprinting on any easily-accessible lithographic printer. Norbornene- and methacryloyl-modification of the gelatin backbone, combined with a ruthenium-based visible light photoinitiator and new coccine as a cytocompatible photoabsorber, allowed to print structures resolving single-pixel features (∼50 ​μm) with high shape fidelity, even when using low stiffness gels, ideal for cell encapsulation (1-2 ​kPa). Moreover, aqueous two-phase emulsion bioresins allowed to modulate the permeability of the printed hydrogel bulk. Bioprinted mesenchymal stromal cells displayed high functionality over a month of culture, and underwent multi-lineage differentiation while colonizing the bioresin bulk with tissue-specific neo-deposited extracellular matrix. Importantly, printed hydrogels embedding complex channels with perfusable lumen (diameter <200 ​μm) were obtained, replicating anatomical 3D networks with out-of-plane branches (i.e. brain vessels) that cannot otherwise be reproduced by extrusion bioprinting. This versatile bioresin platform opens new avenues for the widespread adoption of lithographic biofabrication, and for bioprinting complex channel-laden constructs with envisioned applications in regenerative medicine and hydrogel-based organ-on-a-chip devices

High-resolution lithographic biofabrication of hydrogels with complex microchannels from low-temperature-soluble gelatin bioresins

Biofabrication via light-based 3D printing offers superior resolution and ability to generate free-form architectures, compared to conventional extrusion technologies. While extensive efforts in the design of new hydrogel bioinks lead to major advances in extrusion methods, the accessibility of lithographic bioprinting is still hampered by a limited choice of cell-friendly resins. Herein, we report the development of a novel set of photoresponsive bioresins derived from ichthyic-origin gelatin, designed to print high-resolution hydrogel constructs with embedded convoluted networks of vessel-mimetic channels. Unlike mammalian gelatins, these materials display thermal stability as pre-hydrogel solutions at room temperature, ideal for bioprinting on any easily-accessible lithographic printer. Norbornene- and methacryloyl-modification of the gelatin backbone, combined with a ruthenium-based visible light photoinitiator and new coccine as a cytocompatible photoabsorber, allowed to print structures resolving single-pixel features (∼50 ​μm) with high shape fidelity, even when using low stiffness gels, ideal for cell encapsulation (1-2 ​kPa). Moreover, aqueous two-phase emulsion bioresins allowed to modulate the permeability of the printed hydrogel bulk. Bioprinted mesenchymal stromal cells displayed high functionality over a month of culture, and underwent multi-lineage differentiation while colonizing the bioresin bulk with tissue-specific neo-deposited extracellular matrix. Importantly, printed hydrogels embedding complex channels with perfusable lumen (diameter <200 ​μm) were obtained, replicating anatomical 3D networks with out-of-plane branches (i.e. brain vessels) that cannot otherwise be reproduced by extrusion bioprinting. This versatile bioresin platform opens new avenues for the widespread adoption of lithographic biofabrication, and for bioprinting complex channel-laden constructs with envisioned applications in regenerative medicine and hydrogel-based organ-on-a-chip devices