Periodic Reporting for period 3 - ENLIGHT (ENable LIGHT- and synthetic biology-driven volumetric bioprinting of functional human tissues)
Période du rapport: 2023-11-01 au 2025-04-30
The ENLIGHT project aimed 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 focused on producing a centimeter-cube scale, advanced analogue of the endocrine pancreas, that exhibit physiological-like ability to modulate glucose metabolism. These engineered tissues were 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 tackle diabetes.
To achieve this goal, ENLIGHT focused on:
- Developing a novel, highly efficient route to generate multiple subsets of endocrine pancreatic cells from stem cells/induced pluripotent stem cells (iPSC). The ENLIGHT team successfully established a protocol to generate iPSC-derived pancreatic islets, which could be combined and maintained functional also when bioprinted and encapsulated in 3D hydrogel systems. Morevoer, the ENLIGHT team developed several optogenetics and synthetic biology tools to engineer different cell types (cell lines) into acquiring the ability to produce insulin.
- Developing materials able to substitute the native pancreas extracellular matrix, to allow to nurture the engineered stem cells in 3D. The ENLIGHT team developed a novel library of gelatin-based materials, which can be successfully used in different bioprinting applications, and that sustain iPSC-islet function over multiple weeks of culture. These materials are now commercially available via consortium partners.
- Developing a novel, ultra-fast volumetric bioprinting technique to sculpt these cells and materials into large-sized pancreatic organoids. We developed a novel multi-wavelength volumetric bioprinter. This device is now commercially available through our consortium partner.
- Enabling the long term culture of 3D pancreatic organoids in a perfusion system. We established a multi-islet construct with controlled flow properties and porosity, which can be kept in culture in dedicated perfusion chambers for over a month.
- Investigating the potential of these organoids as drug testing platform and develop a strategy for their use a transplantable cell therapy. The bioprinted organoids are responsive to anti-diabetic drugs and allow to test their effect in vitro. Engineered cells were tested in vivo, showing the ability to restore euglycemia in diabetic rodents. Moreover, transplanted stem-cell derivied islets (non-bioprinted) are also responsive to glucose stimulation. Further studies are necessary to improve and validate the in vivo function of the whole bioprinted organoids.
To achieve this goal, ENLIGHT focused on:
- Developing a novel, highly efficient route to generate multiple subsets of endocrine pancreatic cells from stem cells/induced pluripotent stem cells (iPSC). The ENLIGHT team successfully established a protocol to generate iPSC-derived pancreatic islets, which could be combined and maintained functional also when bioprinted and encapsulated in 3D hydrogel systems. Morevoer, the ENLIGHT team developed several optogenetics and synthetic biology tools to engineer different cell types (cell lines) into acquiring the ability to produce insulin.
- Developing materials able to substitute the native pancreas extracellular matrix, to allow to nurture the engineered stem cells in 3D. The ENLIGHT team developed a novel library of gelatin-based materials, which can be successfully used in different bioprinting applications, and that sustain iPSC-islet function over multiple weeks of culture. These materials are now commercially available via consortium partners.
- Developing a novel, ultra-fast volumetric bioprinting technique to sculpt these cells and materials into large-sized pancreatic organoids. We developed a novel multi-wavelength volumetric bioprinter. This device is now commercially available through our consortium partner.
- Enabling the long term culture of 3D pancreatic organoids in a perfusion system. We established a multi-islet construct with controlled flow properties and porosity, which can be kept in culture in dedicated perfusion chambers for over a month.
- Investigating the potential of these organoids as drug testing platform and develop a strategy for their use a transplantable cell therapy. The bioprinted organoids are responsive to anti-diabetic drugs and allow to test their effect in vitro. Engineered cells were tested in vivo, showing the ability to restore euglycemia in diabetic rodents. Moreover, transplanted stem-cell derivied islets (non-bioprinted) are also responsive to glucose stimulation. Further studies are necessary to improve and validate the in vivo function of the whole bioprinted organoids.
Within the course of the project, the team first 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. 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.
Combining this knowledge and technologies, we built 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, culture up to 28 days, with nearly 100% viability has been performed. This system allows for simulating systemic flow, and to subject the islets to different type of metabolic and chemical challenges, such as fluctuations of glucose concentrations, as well as to the addition of anti-diabetic drugs (i.e. GLP-1 analogs) and toxic compounds. The system is a promising platform for advanced in vitro drug testing to reduce animal experimentation. In addition, we also investigated the therapeutic potential of both iPSC islets and of cell lines designed to act as beta cells. The engineered cells were able to restore glicemic control in diabetic mice.
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.
Combining this knowledge and technologies, we built 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, culture up to 28 days, with nearly 100% viability has been performed. This system allows for simulating systemic flow, and to subject the islets to different type of metabolic and chemical challenges, such as fluctuations of glucose concentrations, as well as to the addition of anti-diabetic drugs (i.e. GLP-1 analogs) and toxic compounds. The system is a promising platform for advanced in vitro drug testing to reduce animal experimentation. In addition, we also investigated the therapeutic potential of both iPSC islets and of cell lines designed to act as beta cells. The engineered cells were able to restore glicemic control in diabetic mice.
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 its innovative research efforts, ENLIGHT resulted 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 volumetric 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). Notably, 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.