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CORDIS

Cypergenetic Tissue Engineering

Periodic Reporting for period 2 - CyGenTiG (Cypergenetic Tissue Engineering)

Période du rapport: 2019-10-01 au 2021-07-31

This project addresses a serious bottleneck to the widespread availability of engineered tissues for clinical use: Currently, building of new tissues requires inefficient, manual manipulation that is time-consuming, labour-intensive and introduces high variability in the finished products. Relieving this limitation has implications for both health and wealth. The project will capitalize on the skills of its network of laboratories to build and demonstrate a technology for controlling the development of engineered tissues by optogenetics and closed-loop, self-correcting control. The core technology combines machine vision and computer modelling with optical feedback, through which the computer can alter the behaviour of precisely those cells that need to be stimulated/inhibited, for the tissue to develop toward the planned template. Optical sensitivity will be conferred on cells by synthetic biological techniques. One set of demonstrations will manipulate the growth and differentiation of these cells directly. A more advanced set will use light-sensitive production of signalling molecules by engineered cells to connect optical control to the control of normal, non-engineered cells as could be used for clinical tissue engineering, in 2- and in 3-dimensional systems. Our proposal includes plans for dissemination and academic, industrial and social impacts.
We started to work on all work packages and laid down in this reporting period the foundation for demonstration projects. The project is well on track.

WP1: We have established the cloning platform for engineering the optogenetic systems in a modular and standardised fashion for the consortium (D1.1) have implemented and characterised a wide set of optogenetic tools functional in HEK293 cells responsible to different wavelengths of light (D1.2) we have developed novel optogenetic tools for the downregulation of protein levels (D1.2) have started developing and applying mathematical models for the optimization of the optogenetic actuators (D1.3) and have initiated efforts towards optimization and customization of the optogenetic tools for use in WP2 and WP5 in 2D and 3D.

WP2: We have completed the design work of genetic modules and delivered deliverable D2.1. We are currently engaged in building and testing the physical devices made from these designs in mammalian cells, and are on-course for delivering deliverable D2.2 on time (month 18). We have identified better cells to use for D2.3 have identified suitable techniques for transfection, have built culture hardware for D2.3 and are doing design work to make an improved version of the the Quasi-vivo culture system optimized for optogenetic work.

WP3: We have been developing, assembling, and testing the hardware and software needed for patterned image illumination (D3.1). A new Nikon Eclipse Ti2-E microscope with a customized environment box is up and running and is being outfitted with the developed pattern illumination setup. The deliverable is on track for M18 delivery. To this setup, we are integrating machine learning-based software for image segmentation and tracking (D3.2). We have carried out training and image analysis for HeLa and HEK293 cells and will move to test other cell types in WP2 and WP5. This software is the basis for the initial image analysis module, which is on track for M18 delivery. Finally, we have initiated the development and testing of software tools for the closed-loop control of living cells (D3.3). Work on this deliverable is being started 5 months ahead of schedule. Preliminary tests have demonstrated successful single-cell control and communication. This work is well on track for M30 delivery.

WP4: We tested several simulation software for tissue modelling and decided to use the agent-based C++ class simulation library PhysiCell. We extended the software to include optogenetic manipulations and tested it with the morphogenetic apoptosis module using different illumination templates. We used data from UEDIN to calibrate the model parameters. This work is well on track for M30 delivery.

WP5: We have begun preliminary work on WP5, ahead of time.This work has provided assurance that our designs (WP1/ WP2) are appropriate for signalling, and has identified a membrane that is promising for D5.2 and D5.3.

WP6: The webpage is online and we had two personal consortium meetings, one in Basel on October 24th 2018 and one in Düsseldorf on May 17th 2019. The consortium achieved all deliverables due within the reporting period and the project is well on track.
Our research is pioneering the area of Cybergenetic tissue engineering. We are creating the scientific and technological foundations—both in biology and engineering-- needed to facilitate and shape the genesis and advancement of this promising area. To this end, we are Training a cadre of scientists who will drive the future developments in Cybergenetic Tissue Engineering. The closed-loop control platform developed within this project will provide an unparalleled tool to engineer tissue of precisely defined shapes and will be an important cornerstone for the translation of basic research in tissue engineering to reliable clinical use. We expect our project to have the following impacts in the short, medium and long terms;
• Clear demonstration to other technologists that cell behaviours may be put under specific, cell-by-cell, optogenetic closed-loop control (short term – within the life of the project)
• Acting as a foundation for future academic-industrial development of advanced tissue engineering techniques (medium-term), with a new-technology impact on industry and economy.
• Enabling reliable scale-up of the direct application of tissue engineered constructions in routine hospital clinical practice (civilian and military): also possibly veterinary practice (longer term). This is the significant long-term impact on societal health and welfare.
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