Periodic Reporting for period 2 - ORGANTRANS (Controlled Organoids transplantation as enabler for regenerative medicine translation)
Reporting period: 2021-07-01 to 2023-06-30
End-stage liver failure is a major healthcare challenge. Liver diseases account for approximately 2 million deaths per year worldwide. Liver transplantation is the most effective way to re-establish liver function for various diseases including acute liver failure or liver malignancies. Currently, less than 10% of global transplantation needs are met and the gap between patients on transplant waiting lists and available donor organs is steadily increasing.
ORGANTRANS proposes a disruptive alternative to donor organs for patients with chronic or end-stage liver diseases who have still to isolate autologous liver stem cells. Driven by a need of leading European transplant centers, ORGANTRANS is tackling current obstacles for liver regenerative medicine by combining advanced know-how in cell biology, biomaterials, bioengineering, automation, standardization and clinical translation.
ORGANTRANS aims to develop a disruptive alternative to donor organs for patients with chronic end-stage liver diseases, through the following elements / specific objectives:
1. Cell source for tissue engineering
2. Genomic and proteomic profiling of standardized spheroids
3. Spheroid sorting
4. Hydrogel bioink
5. Bioprinting platform
6. Vascularization architecture
7. Bioreactor for liver construct maturation
8. In vitro testing
9. In vivo testing of the liver tissue transplant
ORGANTRANS proposes a disruptive alternative to donor organs for patients with chronic or end-stage liver diseases who have still to isolate autologous liver stem cells. Driven by a need of leading European transplant centers, ORGANTRANS is tackling current obstacles for liver regenerative medicine by combining advanced know-how in cell biology, biomaterials, bioengineering, automation, standardization and clinical translation.
ORGANTRANS aims to develop a disruptive alternative to donor organs for patients with chronic end-stage liver diseases, through the following elements / specific objectives:
1. Cell source for tissue engineering
2. Genomic and proteomic profiling of standardized spheroids
3. Spheroid sorting
4. Hydrogel bioink
5. Bioprinting platform
6. Vascularization architecture
7. Bioreactor for liver construct maturation
8. In vitro testing
9. In vivo testing of the liver tissue transplant
ORGANTRANS achievements based on the objectives are:
• Obective 1 (O1) and Objective 2 (O2)
Six liver organoid lines from three male and three female donors were established and characterised on the genetic and protein level. Results indicate that the cells are stable, can be differentiated into the hepatocyte lineage and are therefore a viable cell source for clinical application.
Generation of clinical grade liver spheroid in the Sphericalplate 5D and characterization of these spheroids at the genomic and proteomic level. Moreover, an in-house culture media was developed and GMP training modules were given to consortium members to bring the ORGANTRANS research towards clinical applications.
• O3
Spheroid sorting was achieved with a robotic strategy, enabling the sorting of ~20’000 spheroids (2% of bad spheroids) in less than 2 hrs in a sterile environment, ensuring viability of the sorted spheroids. A microfluidic sorting strategy was developed for higher throughput.
• O4
A fully synthetic PEG-based bioink was developed that can crosslink upon mixing directly during printing with the use of a microfluidic chip. The bioink is 3D printed with sacrificial channels obtained with gelatin embedded with HUVECs to support the formation of a vascular structure. Full size (10x10x5 mm3) constructs were successfully 3D printed using a combination of nanocellulose and gelatin as sacrificial material to initially support the scaffold during printing and culture. The hydrogel formulation was optimized to promote spheroid and endothelial cells sprouting during maturation.
• O5
New technology modules dedicated to liver spheroid printing were developed, functional prototypes were fabricated and tested on the new Biofabrication Platform. Those modules include a spheroid compatible and user-friendly printhead, an environment control and software platform with algorithmic assistance enabling complex tissue design and process.
• O6
Experiments were performed to define and optimize hydrogel cross-linking and gelation time, cell densities (vascular endothelial and mural cells), and culture conditions compatible with the growth of a 3-dimensional vascular network structure.
• O7
A bioreactor for liver construct maturation was designed, to hold up to 6 printed bioconstructs and enable continuous and unidirectional perfusion of the tissue in a dedicated closed and sterile chamber. The system was designed to meet specific requirements from the tissue biology and biomaterial perspective, ensuring compatibility with the bioprinter.
• O8
Assays to assess hepatocyte and cholangiocyte maturation and functionality were developed that can be applied on the liver constructs.
• O9
Protocols and experimental set-ups were prepared for in vivo analysis of the tissue constructs. Ethical approval for those animal experiments was obtained.
• Obective 1 (O1) and Objective 2 (O2)
Six liver organoid lines from three male and three female donors were established and characterised on the genetic and protein level. Results indicate that the cells are stable, can be differentiated into the hepatocyte lineage and are therefore a viable cell source for clinical application.
Generation of clinical grade liver spheroid in the Sphericalplate 5D and characterization of these spheroids at the genomic and proteomic level. Moreover, an in-house culture media was developed and GMP training modules were given to consortium members to bring the ORGANTRANS research towards clinical applications.
• O3
Spheroid sorting was achieved with a robotic strategy, enabling the sorting of ~20’000 spheroids (2% of bad spheroids) in less than 2 hrs in a sterile environment, ensuring viability of the sorted spheroids. A microfluidic sorting strategy was developed for higher throughput.
• O4
A fully synthetic PEG-based bioink was developed that can crosslink upon mixing directly during printing with the use of a microfluidic chip. The bioink is 3D printed with sacrificial channels obtained with gelatin embedded with HUVECs to support the formation of a vascular structure. Full size (10x10x5 mm3) constructs were successfully 3D printed using a combination of nanocellulose and gelatin as sacrificial material to initially support the scaffold during printing and culture. The hydrogel formulation was optimized to promote spheroid and endothelial cells sprouting during maturation.
• O5
New technology modules dedicated to liver spheroid printing were developed, functional prototypes were fabricated and tested on the new Biofabrication Platform. Those modules include a spheroid compatible and user-friendly printhead, an environment control and software platform with algorithmic assistance enabling complex tissue design and process.
• O6
Experiments were performed to define and optimize hydrogel cross-linking and gelation time, cell densities (vascular endothelial and mural cells), and culture conditions compatible with the growth of a 3-dimensional vascular network structure.
• O7
A bioreactor for liver construct maturation was designed, to hold up to 6 printed bioconstructs and enable continuous and unidirectional perfusion of the tissue in a dedicated closed and sterile chamber. The system was designed to meet specific requirements from the tissue biology and biomaterial perspective, ensuring compatibility with the bioprinter.
• O8
Assays to assess hepatocyte and cholangiocyte maturation and functionality were developed that can be applied on the liver constructs.
• O9
Protocols and experimental set-ups were prepared for in vivo analysis of the tissue constructs. Ethical approval for those animal experiments was obtained.
The consortium partners are bringing the main state-of the art in the following:
1. Spheroid building blocks and architecture
To translate the spheroid application into the clinic at a later stage, a detailed cGMP roadmap was developed and a company specialized in cGMP-media development was appointed.
CELLS: Hepatocytes/ cholangiocytes issued from adult stem cells with Supporting mesenchymal cells, and endothelial cells in a concentration of~100'000 spheroids/cm3
2. Construct architecture and hydrogel design
The hydrogel formulation has been optimized to respond to different degradation triggers, promoting hydrogel softening and cell proliferation within the 3D hydrogel construct. Embedded spheroids show an increased propensity to sprout and connect together in a 3D cellular network. This outcome will be highlighted in an upcoming publication (planned for the second half of 2023).
Parallel to the crosslinking by mixing mechanism, occurring via Michael type addition, an innovative approach involving the same Michael type addition but this time triggered by UV light was developed. This approach has the advantage that the hydrogel components can be premixed in advance without reacting, allowing a more controlled and user-triggered crosslinking process. Although ultimately not used for the final printing, this approach enables the formation of well-defined 3D hydrogels, printed in a layer-by-layer fashion, and crosslinked with UV light without the use of radicals. This will result in a scientific publication due to the novelty and potential impact.
o Instrumentation
o Spheroid production (KUG plates, MEDICAL plates to upscale the production of spheroids)
o Robotic platform for selective spheroid sorting from multiwell plates using machine learning algorithms
o Bioprinting (RHU): Including a dedicated substrate for printing, incubation, perfusion and transport
o Platform for the unidirectional and continuous perfusion of six 3D printed constructs in parallel in a closed, controlled and sterile environment
ORGANTRANS achievements and results will have expected impacts, for example:
Strengthened position of Europe in translational regenerative medicine
ORGANTRANS presents an opportunity to position Europe to become a pioneer and leader in clinical application of bioengineered livers transplants by (1) demonstrating the technology in relevant environment (TRL6), (2) building complex ecosystem of key stakeholders necessary for successful translation (including end-users), and (3) presenting an actionable plan for fast clinical adoption.
Translational research with short-medium term to clinical trials
The project is intentionally constructed around the integration of various components (3D bioprinter, spheroid sorter, bioreactor, cells, matrix etc.) into the technological platform, which has been tested within the project in vitro as well as in vivo. These preclinical tests validated in vitro functionality of hepatic spheroids or bioengineered liver construct and viability of constructs implanted in in vivo parenchymal environment.
1. Spheroid building blocks and architecture
To translate the spheroid application into the clinic at a later stage, a detailed cGMP roadmap was developed and a company specialized in cGMP-media development was appointed.
CELLS: Hepatocytes/ cholangiocytes issued from adult stem cells with Supporting mesenchymal cells, and endothelial cells in a concentration of~100'000 spheroids/cm3
2. Construct architecture and hydrogel design
The hydrogel formulation has been optimized to respond to different degradation triggers, promoting hydrogel softening and cell proliferation within the 3D hydrogel construct. Embedded spheroids show an increased propensity to sprout and connect together in a 3D cellular network. This outcome will be highlighted in an upcoming publication (planned for the second half of 2023).
Parallel to the crosslinking by mixing mechanism, occurring via Michael type addition, an innovative approach involving the same Michael type addition but this time triggered by UV light was developed. This approach has the advantage that the hydrogel components can be premixed in advance without reacting, allowing a more controlled and user-triggered crosslinking process. Although ultimately not used for the final printing, this approach enables the formation of well-defined 3D hydrogels, printed in a layer-by-layer fashion, and crosslinked with UV light without the use of radicals. This will result in a scientific publication due to the novelty and potential impact.
o Instrumentation
o Spheroid production (KUG plates, MEDICAL plates to upscale the production of spheroids)
o Robotic platform for selective spheroid sorting from multiwell plates using machine learning algorithms
o Bioprinting (RHU): Including a dedicated substrate for printing, incubation, perfusion and transport
o Platform for the unidirectional and continuous perfusion of six 3D printed constructs in parallel in a closed, controlled and sterile environment
ORGANTRANS achievements and results will have expected impacts, for example:
Strengthened position of Europe in translational regenerative medicine
ORGANTRANS presents an opportunity to position Europe to become a pioneer and leader in clinical application of bioengineered livers transplants by (1) demonstrating the technology in relevant environment (TRL6), (2) building complex ecosystem of key stakeholders necessary for successful translation (including end-users), and (3) presenting an actionable plan for fast clinical adoption.
Translational research with short-medium term to clinical trials
The project is intentionally constructed around the integration of various components (3D bioprinter, spheroid sorter, bioreactor, cells, matrix etc.) into the technological platform, which has been tested within the project in vitro as well as in vivo. These preclinical tests validated in vitro functionality of hepatic spheroids or bioengineered liver construct and viability of constructs implanted in in vivo parenchymal environment.