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Controlled Organoids transplantation as enabler for regenerative medicine translation

Periodic Reporting for period 1 - ORGANTRANS (Controlled Organoids transplantation as enabler for regenerative medicine translation)

Reporting period: 2020-01-01 to 2021-06-30

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 achievements in the last 18 months based on the objectives are:
• Obective 1 (O1) and Objective 2 (O2)
Generation of clinical grade liver spheroid in the Sphericalplate 5D and to 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 robotic and microfluidic strategies with a >98% accuracy in spheroid classification.
• O4
A fully synthetic PEG-based bioink was developed that can crosslink either upon mixing or via light exposure in a radical-free manner. The bioink is 3D printed with sacrificial channels to support the formation of a vascular structure.
• 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 perfusion of the tissue in a dedicated 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.

These objectives, which are part of the building blocks of ORGANTRANS have been progressed in the first 18 months of the project. Sphericalplate 5D is being in used and optimized for spheroid cell culture, spheroid sorting is progressing well, bioprinting and automated incubation will have key milestones during the second half of the year. The processes such as organoid- and user-friendly bioprinting, hydrogel degradation, or angiogenesis (bioconstruct architecture) are combined to create a cGMP tissue engineering platform to meet the needs in organ transplantation.
Results gathered so far, have been communicated and disseminated, via peer reviewed publications and other outreach activities. ORGANTRANS website has been launched, and social media from coordinator CSEM and partner institutions regularly feed with key achievements.
The consortium partners are bringing the main state-of the art in the following:

1. Spheroid building blocks and architecture
A request to the EMA should possibly help to identify markers that go beyond the standard spheroid characterization for research purposes for the planned genomic and proteomic analysis of the liver spheroids. 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."
Complex combination of cell types in optimized culture conditions compatible with the growth of a 3-dimensional vascular network structure.

CELLS: Hepatocytes/ cholangiocytes issued from adult stem cells with Supporting mesenchymal cells, endothelial cells in a concentration of~100'000 spheroids/cm3

2. Architecture (in progress)
i. Printed porous constructs
ii. Vascular architecture - perfusable tubes
iii. Size: 1x1x1 cm3-->redefined to 1x1x0.5cm3

3. Microgels building blocks (in progress)
a. Matrix
i. Fully synthetic PEG-based hydrogel
ii. Rapid crosslinking by light alternative approach
iii. Absence of radicals
iv. Controlled degradation rate
b. Substrate
i. Disposable closed system
ii. Suitable for printing and incubation and shiping

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. ORGANTRANS coordinator, CSEM, organizes periodically events related to the topic, and during the first half of the year 2021, under these events frame and the project a virtual event on regenerative medicine took place. In these activities experts are gathered and find new alliances.
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 already tested within the project in vitro as well as will be tested in vivo, in the second half of the project. These preclinical tests will prove and validate in vitro functionality of hepatic spheroids or bioengineered livers and construct viability in in vivo-like environment and will allow its optimization. In order to meet essential criteria for clinical tests, already during ORGANTRANS every single step in the production chain is evaluated to comply with regulatory standards (cGMP for ATMP and MDR). For instance, GMP trainings have been organised and delivered to all consortium members. ORGANTRANS will not only deliver an ATMP, but also platform technologies that can be scaled to other organ systems, as organoid technology is the largest part of regenerative medicine.
ORGANTRANS overview