Periodic Reporting for period 3 - CENSUS (Cell-Based Models for Neurodegeneration Study and Use in Screening) Reporting period: 2018-10-01 to 2019-09-30 Summary of the context and overall objectives of the project CENSUS provides a technical solution to the healthcare challenge posed by a lack of preclinical models for study of neurodegenerative disease. This solution will deliver positive-disruption of neurodegenerative drug discovery. To achieve this, CENSUS has addressed the challenge to produce CNS cell models from a consistent, renewable source, in the form of induced pluripotent stem cells (iPSC); has designed and synthesised 3D scaffolds in multiwell culture plates by micro-extrusion technology; has optimised scaffold functionality and cell performance using innovative peptide chemistry; has taken first successful steps towards supply of fully functional models as pre-assembled, frozen products. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far CENSUS Period Three has consolidated the technical advances made in Period One and Period Two, which established working cell banks of neural progenitor cells and produced the procedures and protocols for their differentiation into mature neural cells under 2D and 3D culture conditions.A principal goal during CENSUS Period Three was to develop a differentiation-supporting scaffold able to provide a 3-dimensional environment in which neuroprogenitor cells (NPC) produced from an iPS cell source were induced to become mature neural cells. This induction would pave the way for a 3D cell-based analysis service and a 3D cell product which have been the principal objectives of the CENSUS project. The partners had assembled the building blocks of this 3D cell model during Period Two. In addition to NPC cell stock developed by partner Phenocell (working cell banks of NPC was a key output in project Period One), CENSUS Period Two consolidated technical development of a novel bioink printable under the conditions in which a neural cell model could be assembled. This bioink technical innovation was delivered by partner CELLINK. Alongside this, 2D culture methodology had pointed to the bio-supportive advantage of Tissue Click’s innovative dendronic bioligands during neuroprogenitor cell maturation, creating incentive to solve the challenge of incorporating the best-performing form of this material, branded ‘Phenodrive-I’, into a CENSUS-proprietary neural cell bioink.In the course of Period Three, these technologies were successfully combined to deliver a 3D cell model containing neuroprogenitor-derived cells expressing the molecular signatures of mature neuronal and astro-glial cells. The major technical challenges overcome in achieving this outcome was the stabilisation of 3D cell models through the relatively-extended culture period required to elicit cell differentiation. This was achieved in part through the effect of Tissue Click Phenodrive-I, which promoted differentiation whilst inhibiting bioink degeneration during extended culture. The other solution came from optimising the implanting of neural progenitor cells in the 3D environment, either through pre-formation of so-called ‘neurospheres’ or by adoption of seeding conditions which promoted cell aggregation and early formation of mature neural structures, including neurite networks. Thus, Period Three progress confirmed the Period Two expectation that components of a viable model existed, awaiting conditions for their practicable combination and functional demonstration.Assembly of a 3D cell-based model provides the basis of a contract service for preclinical analysis in support of neurodegenerative drug discovery. This required development not only of the model but tools for measuring model function, and the project found that qPCR analysis of genes expressed in a cell type-specific manner, coupled with biochemical indices of cell viability and general cell health, such as oxidative stress, provide the bases of analyses for neurotoxicity and neurodegenerative parameters. These parameters were shown to respond to a neurodegeneration-relevant stimulus, -amyloid accumulation with, post-project, intent to expand readout versatility with service-customer input. A 3D neurodegenerative cell-based analysis service was a project output objective: marketing of this service has begun, implementing the exploitation ambition expressed in the final two work packages.The project’s other primary objective was to translate a 3D neural cell model into a 3D neural cell product, using lead partner AvantiCell’s proprietary cryopreservation technology. Proof of principle of the cryo-preservability of NPC in partially-differentiated form came from 2D culture models, and during Period Three this proof of principle has been extended to 3D neurosphere aggregates, which themselves would represent a first-to-market exploitable output. However, delivery of a fully-formed bioprinted 3D neural cell model during the project has been frustrated by bioink instability, first as a result of bioprinting challenges and, secondly, during manipulations associated with cryopreservation. This problem is solvable: 3D cell models in other scaffold materials are recoverable quantitatively after ACS Cryotix cryopreservation. With this knowledge, the partners intend to extend neurosphere cryopreservation to a bioprinted neural-cell model in further post-project research.The ambition to fast-track CENSUS-developed innovations was, from the outset, planned to include ‘secondary’ routes which exploited other project-enabled technology and materials. Those technologies and materials that, together, provided 3D model components, were additionally seen as being directly taken to market by the individual partners who developed them. During Period Three, this has proven to be the case. NPC progenitor cells in cryovial and Cryotix formats, neural-cell customised bioink, and Phenodrive-I bioligand have all been taken through manufacturing due diligence and quality assurance, and are being introduced into the respective partner’s commercial portfolio and market engagement. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) The technology developed in Period Thre continues to demonstrate the principles of the original proposal: (i) the ability to produce, bank and differentiate commercially-relevant stocks of iPS-derived neuroprogenitor cells; (ii) the technology to print scaffolds into multiwell culture plates using hardware and material suited to scalable industrial manufacturing; (iii) the knowledge to develop and analyse 3D neuronal cell co-culture models (iv) the feasibility to deliver novel neuroprogenitor cell models to customers as a service or in a plug and play product format. The partners acknowledge the CENSUS technical issues still incomplete at project close. The shared view is that these are matters of optimisation and market-readiness rather than innovation barriers, and there is commitment to complete that work post-project. The CENSUS project has also brought collateral benefits to every partner, evidenced by growth in staff and infrastructure, market visibility and reach, and the identification of related innovation opportunities already the subject of follow-on partnership.