Periodic Reporting for period 4 - CyGenTiG (Cypergenetic Tissue Engineering)
Période du rapport: 2022-12-01 au 2024-03-31
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.
The project is completed.
WP1:
In WP1 we worked on the design, characterization, implementation and optimization of optogenetic tools for the spatio-temporally resolved regulation of gene expression and other cellular processes in mammalian cells. We focused on a subset of switches and morphogenetic/cell fate control modules and implement them in WP5 for the control of proliferation, apoptosis and/or folding of cells and tissue culture, and developing engineered kidneys in 2D/3D. We have implemented “opto-controlled modules” for 2D and 3D control of tissue culture engineering (cell lines and stem cells).
WP1 had three tasks, with 5 deliverables and one associated milestone as follows: delivery of first prototype light-inducible (optogenetic) gene expression systems for primary testing in WP2 (D1.1 M3, achieved); characterization of light-regulated gene expression systems in terms of kinetics and sensitivity in HEK293 cells (D1.2 M12; achieved); quantitative description of the optogenetic systems, optimization strategy and development of a modelling platform therefore (D1.3 & MS1 M30; achieved); optimized and customized optical switches for use in stem cells and cell sheets (D1.4 M42; achieved); optimized and customized optical switches for use in stem cell fate (D1.5 M66; achieved).
WP2:
In WP2 we developed the design, construction of and testing of the morphogenetic modules to
be used in WP5. The range of modules was expanded beyond the initial plan, largely because long waits for microscope parts delayed other work and we used the time to maximize our flexibility for going to WP5. WP2 had three deliverables and one associated milestone; design of genetic constructs (D2.1 achieved
and reported on time at M6), construction genetic constructs (D2.2 achieved and reported on time at
M18), bringing the morphogenetic modules under optogenetic control (MS2, M30: achieved), and production of cells engineered with these constructs (D2.3: achieved).
WP3:
In WP3 we developed the hardware and software for spatial-temporal illumination, the software for image analysis, and the software for real-time closed-loop feedback control. Results for this WP were used in WP4 and WP5. The work-package had 5 deliverables and 3 associated milestones as
follows: hardware and software for patterned illumination setup (D3.1 for M18 delivery; achieved); initial version of image analysis modules (D3.2 for M24 delivery; achieved); software-framework for closed-loop control (D3.3 for M30 delivery; achieved); improved image analysis modules (D3.4 for M42 delivery; achieved); complete software modules developed and optimized for robust continuous operation (D3.5 for M66 delivery; achieved).
WP4:
In WP4 we developed computational models to simulate the growth of the tissues. Results of this WP were employed in WP3 and WP5 in an optimal-control framework which compares the current state of the system with a desired reference state and solves an optimization problem at each
step to determine the optimal optical input, i.e. where and when to apply external light to the growing structure in order to bring the growing tissue closest to the desired state. The WP had four deliverables and one associated milestone. A computational model for cell islands (D4.1 for M36 achieved), a
computational model for stem cell differentiation (D4.2 for M42 achieved), a computational organoid model (D4.3 for M54 achieved), and Model predictive control algorithms (D4.4 for M66 achieved). The milestone MS4: “First computational model for growth control of cell islands” was reached in time
(M30).
WP5:
WP5 was designed to bring together the separate technologies developed in WP1-4 to demonstrate 3 types of application for computer-controlled optogenetics. These are:
• optogenetic control of a morphogenetic behaviour in 2D cell cultures
• optogenetic control of stem cell behaviours
• optogenetic control of renal organogenesis in culture
WP5 had three deliverables (D5.1 in M42, D5.2 in M54 and D5.3 in M66), all were achieved.
WP6:
We had in 2021 a very successful workshop on "Optogenetics in Complex Systems".
Two Patents were filed; at ETHZ the "Diya illumination platform", an optogenetic platform for cells cultured in multi-well plates or other culture dishes, was developed. The optogenetic modules developed in this project are used already in applications beyond CyGenTiG.
At ALU-FR cellular-raza was developed. cellular-raza is a cellular agent-based modeling framework which allows researchers to construct models from a clean slate.
WP1:
In WP1 we worked on the design, characterization, implementation and optimization of optogenetic tools for the spatio-temporally resolved regulation of gene expression and other cellular processes in mammalian cells. We focused on a subset of switches and morphogenetic/cell fate control modules and implement them in WP5 for the control of proliferation, apoptosis and/or folding of cells and tissue culture, and developing engineered kidneys in 2D/3D. We have implemented “opto-controlled modules” for 2D and 3D control of tissue culture engineering (cell lines and stem cells).
WP1 had three tasks, with 5 deliverables and one associated milestone as follows: delivery of first prototype light-inducible (optogenetic) gene expression systems for primary testing in WP2 (D1.1 M3, achieved); characterization of light-regulated gene expression systems in terms of kinetics and sensitivity in HEK293 cells (D1.2 M12; achieved); quantitative description of the optogenetic systems, optimization strategy and development of a modelling platform therefore (D1.3 & MS1 M30; achieved); optimized and customized optical switches for use in stem cells and cell sheets (D1.4 M42; achieved); optimized and customized optical switches for use in stem cell fate (D1.5 M66; achieved).
WP2:
In WP2 we developed the design, construction of and testing of the morphogenetic modules to
be used in WP5. The range of modules was expanded beyond the initial plan, largely because long waits for microscope parts delayed other work and we used the time to maximize our flexibility for going to WP5. WP2 had three deliverables and one associated milestone; design of genetic constructs (D2.1 achieved
and reported on time at M6), construction genetic constructs (D2.2 achieved and reported on time at
M18), bringing the morphogenetic modules under optogenetic control (MS2, M30: achieved), and production of cells engineered with these constructs (D2.3: achieved).
WP3:
In WP3 we developed the hardware and software for spatial-temporal illumination, the software for image analysis, and the software for real-time closed-loop feedback control. Results for this WP were used in WP4 and WP5. The work-package had 5 deliverables and 3 associated milestones as
follows: hardware and software for patterned illumination setup (D3.1 for M18 delivery; achieved); initial version of image analysis modules (D3.2 for M24 delivery; achieved); software-framework for closed-loop control (D3.3 for M30 delivery; achieved); improved image analysis modules (D3.4 for M42 delivery; achieved); complete software modules developed and optimized for robust continuous operation (D3.5 for M66 delivery; achieved).
WP4:
In WP4 we developed computational models to simulate the growth of the tissues. Results of this WP were employed in WP3 and WP5 in an optimal-control framework which compares the current state of the system with a desired reference state and solves an optimization problem at each
step to determine the optimal optical input, i.e. where and when to apply external light to the growing structure in order to bring the growing tissue closest to the desired state. The WP had four deliverables and one associated milestone. A computational model for cell islands (D4.1 for M36 achieved), a
computational model for stem cell differentiation (D4.2 for M42 achieved), a computational organoid model (D4.3 for M54 achieved), and Model predictive control algorithms (D4.4 for M66 achieved). The milestone MS4: “First computational model for growth control of cell islands” was reached in time
(M30).
WP5:
WP5 was designed to bring together the separate technologies developed in WP1-4 to demonstrate 3 types of application for computer-controlled optogenetics. These are:
• optogenetic control of a morphogenetic behaviour in 2D cell cultures
• optogenetic control of stem cell behaviours
• optogenetic control of renal organogenesis in culture
WP5 had three deliverables (D5.1 in M42, D5.2 in M54 and D5.3 in M66), all were achieved.
WP6:
We had in 2021 a very successful workshop on "Optogenetics in Complex Systems".
Two Patents were filed; at ETHZ the "Diya illumination platform", an optogenetic platform for cells cultured in multi-well plates or other culture dishes, was developed. The optogenetic modules developed in this project are used already in applications beyond CyGenTiG.
At ALU-FR cellular-raza was developed. cellular-raza is a cellular agent-based modeling framework which allows researchers to construct models from a clean slate.
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.
• 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.