Periodic Reporting for period 1 - BioRobot-MiniHeart (Engineering a living human Mini-heart and a swimming Bio-robot)
Okres sprawozdawczy: 2022-11-01 do 2023-10-31
Cardiovascular disease is the primary cause of death worldwide. Although researchers have been committed to finding cures and treatments for heart disease, to date the number of identified drugs for successful treatment has been extremely limited, despite increased expenditure in recent years. The major bottleneck is the lack of proper human heart models that enable the study of human heart disease and consequent development of new cardiovascular drugs. Although current models can give relevant fundamental insights and options for cardiotoxicity testing, they are unable to offer meaningful prediction of heart function based on clinical parameters. For example, the main function of the heart is the pumping of blood, but current models are unable to do this.
We will develop and produce a human mini-heart that will allow for in vitro measurement of pumping function (pressure and volume output), which will enable for the first time the development of human-based cardiac disease models that are crucial for the urgently needed therapeutic drug discovery and development. This result will impact greatly the cardiovascular research field by enabling researchers to model the main function of the heart in vitro and measure the same pressure and flow outputs that are assessed in the clinic. This will accelerate development of new cardiac drugs, while decreasing risk of drug failure in human clinical trials, and potentially reduce use of experimental animals. Equally important, we anticipate that such cardiac tissues can also be used as autonomous soft-robots (e.g. biorobots) to test cardiac toxicity in an environmental setting. The proposed development of engineered living matter for delivering next-generation cardiac tissues thus holds significantly economic and societal impact.
To this end, our consortium aim to create two different and independent human 3D tissue constructs with cardiac cells (e.g. a mini-heart and a bio-robot) using novel molding and 3D print technologies, which will be used for the following objectives:
Objective 1- Make a “mini-heart” able to pump fluid autonomously, enabling the measurement output pressure and flow from the functioning construct. The mini-heart will be composed by multiple layers of cells that are made by various cardiac cells types. The mini-heart will have vascularization promoted by endothelial-cardiomyocyte interaction and by the fluidic motion generated by the pumping action.
Objective 2- Make a bio-robot that is able to swim autonomously and be used as a detector for environmental toxins. The biorobot will generate thrust by contraction of the tissue and the minimum viable pressure to generate movement will be measured. The body of the biorobot will be composed mostly be ventricular-like and/or atrium-like cardiomyocytes, while having a “head” composed by pace-maker cells. The pacemaker cells will determine which part of the tissue will contract first and therefore determine the direction of the electrical impulse propagation, and consequently determine the direction of the tissue propulsion. This biorobot will be used as an environmental toxin detector by assessing changes in its swimming capacity as cardiomyocytes are quite sensitive to external factors and therefore stop contracting.
We will develop and produce a human mini-heart that will allow for in vitro measurement of pumping function (pressure and volume output), which will enable for the first time the development of human-based cardiac disease models that are crucial for the urgently needed therapeutic drug discovery and development. This result will impact greatly the cardiovascular research field by enabling researchers to model the main function of the heart in vitro and measure the same pressure and flow outputs that are assessed in the clinic. This will accelerate development of new cardiac drugs, while decreasing risk of drug failure in human clinical trials, and potentially reduce use of experimental animals. Equally important, we anticipate that such cardiac tissues can also be used as autonomous soft-robots (e.g. biorobots) to test cardiac toxicity in an environmental setting. The proposed development of engineered living matter for delivering next-generation cardiac tissues thus holds significantly economic and societal impact.
To this end, our consortium aim to create two different and independent human 3D tissue constructs with cardiac cells (e.g. a mini-heart and a bio-robot) using novel molding and 3D print technologies, which will be used for the following objectives:
Objective 1- Make a “mini-heart” able to pump fluid autonomously, enabling the measurement output pressure and flow from the functioning construct. The mini-heart will be composed by multiple layers of cells that are made by various cardiac cells types. The mini-heart will have vascularization promoted by endothelial-cardiomyocyte interaction and by the fluidic motion generated by the pumping action.
Objective 2- Make a bio-robot that is able to swim autonomously and be used as a detector for environmental toxins. The biorobot will generate thrust by contraction of the tissue and the minimum viable pressure to generate movement will be measured. The body of the biorobot will be composed mostly be ventricular-like and/or atrium-like cardiomyocytes, while having a “head” composed by pace-maker cells. The pacemaker cells will determine which part of the tissue will contract first and therefore determine the direction of the electrical impulse propagation, and consequently determine the direction of the tissue propulsion. This biorobot will be used as an environmental toxin detector by assessing changes in its swimming capacity as cardiomyocytes are quite sensitive to external factors and therefore stop contracting.
In the first year of BioRobot-MiniHeart project the development was focused on cell production, optimisation of sacrificial molding technology and uniformity of tissue formation, which is the basal stone for achieving both objectives. Specifically, we defined the cellular composition of the envisioned ELMs. This provided critical information on the minimal cell types/cell circuitries necessary to build up an optimal mini-heart. Moreover, we delivered new protocols for hiPSC-cardiomyocyte expansion and purification in order to obtain large numbers of the selected cardiac cell types. In addition, significant progress has been made towards bioprinting ELMs using the next-gen 3D-printing technique known as Xolography. We developed cytocompatible hydrogel-based bioinks and optimized their reactivity to 3D print cm-scale objects with high-resolution positive (20 µm) and negative (125 µm) features. This enabled successful printing of a cell-free miniature version of a human heart based on high-resolution 3D data (e.g. CT data of healthy human heart). Data regarding cytocompatibility, reactivity, and printability has been obtained and is compatible with achieving the proposed project goals. Furthermore, a new design for a molded Mini-heart has been achieved, which included a simplification of the inlet/outlet of the mini-heart to facilitate tissue production and enable protocol parity with the BioRobot. Furthermore, to improve tissue formation and downstream analysis re-designed the bioreactor, which further enabled us to optimize the sacrificial molding approach in order to have an improved alignment between the inner and outer mold. Additionally, we have improved 3D cardiac tissue integrity by changing the cellular composition of the 3D tissue. An characterization of the cells present in the Mini-heart 3D tissue through multiplexed immunohistological techniques has been performed. To characterize the pumping capabilities of the biorobot and miniheart and investigate the impact of drug presence, we have developed a highly miniaturized pressure sensor at adequate resolution to be capable of resolving singular contraction or propagation movements of the Miniheart and Biorobot, respectively.
We developed a beyond-state-of-the-art mini-heart with a conformal uniform cardiac wall using sacrificial molding technology, which enabled accurate measurements of pressure and volume output on both the mini-heart and biorobot. This has had a significant impact on River BioMedics business development activities by offering functional prototypes to investors and pharmaceutical companies, which fuels the key need of attracting sufficient to maintain the development of this technology beyond the EIC-Pathfinder. We also present, for the very first time, that Xolography can print hydrogels as well as living matter. Following the formulation of crosslinking formulations specialized for Xolographic volumetric 3D printing, we can now 3D-printed cm-scale structures with positive resolutions as low as 20 μm and negative resolutions of 125 μm in a seamless/layer-less manner, which are among the highest resolutions ever reported for volumetric bioprinting. This allowed for the 3D-printing of 3D mini-heart based on CAD data of human hearts. This continued research will help us in bioprinting high resolution contractile heart models that will better mimic the native heart that can be produced in a scalable manner, as well as further develop the technology for rapid, reproducible tissue generation including the biorobot.