Periodic Reporting for period 2 - ENLIGHT (ENable LIGHT- and synthetic biology-driven volumetric bioprinting of functional human tissues)
Reporting period: 2022-05-01 to 2023-10-31
The ENLIGHT project aims at developing a novel light-based 3D bioprinting technology for the generation of complex engineered human tissue with a high level of mimicry of native tissue functionality. As proof of concept, ENLIGHT will produce a centimeter-cube scale, advanced, vascularized analogue of the endocrine pancreas, that exhibit physiological-like ability to modulate glucose metabolism. These engineered tissues will be investigated as in vitro platforms for the testing and development of novel anti-diabetes drugs, and also in a proof-of-concept in vivo assay, as transplantable graft to treat diabetes.
To achieve this goal, ENLIGHT will:
- Develop a novel, highly efficient route to generate multiple subsets of endocrine pancreatic cells from stem cells/induced pluripotent stem cells
- Develop materials able to substitute the native pancreas extracellular matrix, to allow to nurture the engineered stem cells in 3D
- Develop a novel, ultra-fast volumetric bioprinting technique to sculpt these cells and materials into large-sized pancreatic organoids
- Enable the long term culture of such 3D organoids in a perfusion system.
- Investigate the potential of these organoids as drug testing platform and develop a strategy for their use a transplantable cell therapy.
To achieve this goal, ENLIGHT will:
- Develop a novel, highly efficient route to generate multiple subsets of endocrine pancreatic cells from stem cells/induced pluripotent stem cells
- Develop materials able to substitute the native pancreas extracellular matrix, to allow to nurture the engineered stem cells in 3D
- Develop a novel, ultra-fast volumetric bioprinting technique to sculpt these cells and materials into large-sized pancreatic organoids
- Enable the long term culture of such 3D organoids in a perfusion system.
- Investigate the potential of these organoids as drug testing platform and develop a strategy for their use a transplantable cell therapy.
Within this first and second reporting period, the main research activities have been focused on designing and completing the building blocks (engineered cells, biomaterials and 3D printing set-ups), that are needed to create (bioprint) the pancreatic tissue constructs to be used as drug testing platforms and implantable grafts. In particular, we have developed two new synthetic biology-based strategies that enable the activation of cell responses upon exposure to light stimuli. As a proof-of-concept of the functionality of these networks, production and release of insulin in cell lines was demonstrated. This knowledge is now being translated towards application in the expression of factors needed to differentiate iPS cells into pancreatic cells. We have developed lentiviral vectors to stably engineer iPSC with these constructs. In addition, we developed a robust method to generate pancreatic islets from iPSC using a precise array of soluble factors. These stem cell-derived islets are rich in beta-, alpha- and delta-like cells, and respond to glucose stimuli with proportional insulin secretion, and are already in use in our cultures involving bioprinted constructs.
In parallel, several gelatin-based hydrogels formulations have been developed, and a base design that allows the formation of 3D clusters of engineered beta-like cells, to preserve the identity and function of iPSC-derived pancreatic islets as well as the formation of interconnected capillary networks from a co-culture of endothelial HUVEC cells and mesenchymal stromal cells has been defined. The material can be shaped via volumetric printing, and their functionality via the embedding of ECM matrix components. To sculpt these cell-laden materials, a new volumetric, tomographic 3D printing technology has been developed, enabling the rapid fabrication of centimeter scale constructs in less than 30 seconds. Several strategies have been developed to ensure the encapsulation of high cell densities contextually to a high shape fidelity and printing resolution. Light-based printing can be impaired by opaque media and scattering caused by the cells in the printable hydrogel., Data-driven scattering correction by frequency boosting allows to fabricate unobstructed vasculature models in preliminary experiments at 4 million cells/mL. In addition, refractive-index matching with biocompatible contrast agents improves print resolution in preliminary experiments in presence of 10 million cells/mL. Biomaterial design and software can be used to compensate for scattering and achieve high resolution in cell-laden hydrogels. . We also demonstrate multiple strategies to produce geometrically complex multi-material prints, which comprise also spatial patterning of multiple cell types.
A fully functioning set-up for the sterile perfusion of geometrically complex, centimetre scale constructs printed from hydrogels displaying low mechanical properties was designed and tested. Perfusion culture of endothelial cells seeded in bioprinted channels was achieved. Bioprinting of iPSC derived islet organoids (obtained by chemical differentiation) is possible, and to date, culture up to 21 days, with nearly 100% viability has been performed.
Moreover, the dissemination framework for ENLIGHT was established in the first six months of the project. This included the website, logo, and social media platforms, along with a data management plan. The ENLIGHT partners were active in dissemination and communication efforts, including press releases by international media outlets, the development of project videos, presentations at virtual, global scientific conferences, and the publishing of peer-reviewed publications. In the first 6 months, the project management structure and consortium guidelines were established and distributed to the consortium via the PM Handbook. The project management strategy has thus far been sufficient to maintain lines of communication between partners and to ensure that any potential issues are addressed early and proactively.
In parallel, several gelatin-based hydrogels formulations have been developed, and a base design that allows the formation of 3D clusters of engineered beta-like cells, to preserve the identity and function of iPSC-derived pancreatic islets as well as the formation of interconnected capillary networks from a co-culture of endothelial HUVEC cells and mesenchymal stromal cells has been defined. The material can be shaped via volumetric printing, and their functionality via the embedding of ECM matrix components. To sculpt these cell-laden materials, a new volumetric, tomographic 3D printing technology has been developed, enabling the rapid fabrication of centimeter scale constructs in less than 30 seconds. Several strategies have been developed to ensure the encapsulation of high cell densities contextually to a high shape fidelity and printing resolution. Light-based printing can be impaired by opaque media and scattering caused by the cells in the printable hydrogel., Data-driven scattering correction by frequency boosting allows to fabricate unobstructed vasculature models in preliminary experiments at 4 million cells/mL. In addition, refractive-index matching with biocompatible contrast agents improves print resolution in preliminary experiments in presence of 10 million cells/mL. Biomaterial design and software can be used to compensate for scattering and achieve high resolution in cell-laden hydrogels. . We also demonstrate multiple strategies to produce geometrically complex multi-material prints, which comprise also spatial patterning of multiple cell types.
A fully functioning set-up for the sterile perfusion of geometrically complex, centimetre scale constructs printed from hydrogels displaying low mechanical properties was designed and tested. Perfusion culture of endothelial cells seeded in bioprinted channels was achieved. Bioprinting of iPSC derived islet organoids (obtained by chemical differentiation) is possible, and to date, culture up to 21 days, with nearly 100% viability has been performed.
Moreover, the dissemination framework for ENLIGHT was established in the first six months of the project. This included the website, logo, and social media platforms, along with a data management plan. The ENLIGHT partners were active in dissemination and communication efforts, including press releases by international media outlets, the development of project videos, presentations at virtual, global scientific conferences, and the publishing of peer-reviewed publications. In the first 6 months, the project management structure and consortium guidelines were established and distributed to the consortium via the PM Handbook. The project management strategy has thus far been sufficient to maintain lines of communication between partners and to ensure that any potential issues are addressed early and proactively.
In its innovative research efforts, ENLIGHT is expected to result in the generation of i) a new and highly efficient protocol for the generation of pancreatic cells (specifically alpha and beta cells), a ii) a versatile array of hydrogels as printable inks and 3D, chemically defined cell culture matrices, a iii) new commercially marketable bioprinter, and iv) bioengineered organoids with application in diabetes research and therapy. The unique combination of native-like physiological architecture, centimeter-scale size, survival and functionality over long culture time offered by these bioengineering organoids, constitutes a considerable progress over the existing, simplified models used in diabetes research (explanted islets and 2D tissue cultures). In addition, two a new multiwavelength volumetric 3D printer and gelatin-based biomaterials for tissue culture and bioprinting that were developed in this project are now commercialized by project partners. The constructs produced in ENLIGHT will have direct impact for pharmaceutical and biotechnology industries and laboratories, increasing the competitivity of these sectors, and provide new tools to accelerate the development of anti-diabetes therapies.