Periodic Reporting for period 1 - 3DCardiacHTS (Bringing 3D cardiac tissues to high throughput for drug discovery screens)
Okres sprawozdawczy: 2023-01-01 do 2023-12-31
Cardiovascular diseases are the number 1 cause of death worldwide, killing more than 18 million people annually. This figure demonstrates that there is still a high unmet medical need for new medicines to treat patients in this therapeutic area. Both academia and the pharmaceutical industry are actively trying to develop novel cardiac drugs. Unfortunately, these are not reaching patients, due to a 91.8% failure rate during clinical development. This data strongly suggest that there is a lack of functionally relevant pre-clinical models of cardiovascular diseases being used during the drug discovery process.
Strides have been made in addressing this issue through the development of cardiovascular engineered heart tissues models using human induced pluripotent (hiPSC)-derived cardiomyocytes and tissue engineering. These models are in the correct species (humans) and have more physiologically relevant read-outs and in this way are more representative of the clinical setting. These 3D cardiac strips are formed by cardiomyocytes and supporting cells compacting around two hanging pillars. Once the cells start contracting, they deflect the pillars and this deflection can be used to quantifiably measure relaxation and contraction, forces and rates of the cardiomyocytes; a readout which has relevance across all cardiovascular diseases.
To efficiently use these models to de-risk failure in clinical trials, they need to be compatible with common workflows in the drug discovery process, such as high throughput screening (HTS) and use of robotics. To enable current 3D cardiac strips to be used in the way the system needs, miniaturisation (reduce cell numbers required and thereby the cost per data point), scalability (large-scale production of the plates used to form the tissues), and automation (compatible with drug discovery robots and/or microfluidic automation) are key aspects. River BioMedics is therefore developing a microtiter plate with a microfluidic layer which is capable of supporting miniaturized versions of the 3D cardiac strip, termed µ3D cardiac strips. These strips can still deliver a physiological readout, but only requires 5000 cells to make, thereby reducing the cost per datapoint sufficiently to make the use of human 3D cardiac strips commercially viable in drug discovery. These plates are designed to be compatible with large-scale production techniques and conform to the standard microtiter plate format. The µ3D cardiac strip technology will be suitable for use in both 1) target-based drug discovery to radically increase compound throughput while maintaining a good level of predictability and 2) phenotypic drug discovery to uncover novel drugs alongside novel pathways and novel targets.
Strides have been made in addressing this issue through the development of cardiovascular engineered heart tissues models using human induced pluripotent (hiPSC)-derived cardiomyocytes and tissue engineering. These models are in the correct species (humans) and have more physiologically relevant read-outs and in this way are more representative of the clinical setting. These 3D cardiac strips are formed by cardiomyocytes and supporting cells compacting around two hanging pillars. Once the cells start contracting, they deflect the pillars and this deflection can be used to quantifiably measure relaxation and contraction, forces and rates of the cardiomyocytes; a readout which has relevance across all cardiovascular diseases.
To efficiently use these models to de-risk failure in clinical trials, they need to be compatible with common workflows in the drug discovery process, such as high throughput screening (HTS) and use of robotics. To enable current 3D cardiac strips to be used in the way the system needs, miniaturisation (reduce cell numbers required and thereby the cost per data point), scalability (large-scale production of the plates used to form the tissues), and automation (compatible with drug discovery robots and/or microfluidic automation) are key aspects. River BioMedics is therefore developing a microtiter plate with a microfluidic layer which is capable of supporting miniaturized versions of the 3D cardiac strip, termed µ3D cardiac strips. These strips can still deliver a physiological readout, but only requires 5000 cells to make, thereby reducing the cost per datapoint sufficiently to make the use of human 3D cardiac strips commercially viable in drug discovery. These plates are designed to be compatible with large-scale production techniques and conform to the standard microtiter plate format. The µ3D cardiac strip technology will be suitable for use in both 1) target-based drug discovery to radically increase compound throughput while maintaining a good level of predictability and 2) phenotypic drug discovery to uncover novel drugs alongside novel pathways and novel targets.
Work has been performed toward achieving the following 3 key aspects needed for integrating 3D cardiac tissues in HTS workflows: 1. miniaturisation, 2. scalability, and 3. automation.
1. Miniaturization:
We were able to downscale the amount of cells needed to make 3D cardiac tissue from 1 million to 15 000. This reduces the cost per data point by two orders of magnitude. We achieved this by designing a microfluidic layer in which the cardiac tissues are formed in very small volumes (less than 2 µL). We fabricated a prototype of this design and optimized the protocol for the formation of 3D cardiac tissues in these chambers. Using our prototype, we showed that we can get beating cardiac tissues and are able to perform contractility measurements.
2. Scalability:
To align our system with the work flow in high throughput screening facilities, our prototype also needs to be manufacturable at a low cost per piece at large quantities. We therefore made our prototype compatible with large-scale manufacturing methods, such as injection molding, and found a subcontractor who can produce the parts for us. Specifically, we investigated and compared different materials which are suitable alternatives to the commonly used elastomer polydimethylsiloxane (PDMS) in the microfluidics field. We found a thermoplastic elastomer which has suitable mechanical properties needed for our device and which is compatible with injection molding techniques. Together with the subcontractor, we optimized a manufacturing protocols to produce molds for injection molding with sufficiently high resolution for the smallest features in our design. We obtained the first prototypes made by injection molding and were able to form the miniaturized cardiac tissues in them.
3. Automation:
We devised a strategy to increase the level of automation in our device. Firstly, we will ensure that our device will be compatible with pipetting robots for both seeding and culturing the cardiac tissues. Secondly, we will exploit the advantages of microfluidics to have the possibility to add extra functions (such as continuous medium recirculation) to our device. This will allow us to expand our technology into other areas of the Organs-on-chips field. To achieve this, we are currently developing active valves in a non-PDMS material, which has the following advantages over PDMS: 1. Is compatible with both rapid prototyping and injection molding and 2. has very low molecule absorption (an essential aspect in technology development for drug discovery). We have made the first prototypes of these active valves.
1. Miniaturization:
We were able to downscale the amount of cells needed to make 3D cardiac tissue from 1 million to 15 000. This reduces the cost per data point by two orders of magnitude. We achieved this by designing a microfluidic layer in which the cardiac tissues are formed in very small volumes (less than 2 µL). We fabricated a prototype of this design and optimized the protocol for the formation of 3D cardiac tissues in these chambers. Using our prototype, we showed that we can get beating cardiac tissues and are able to perform contractility measurements.
2. Scalability:
To align our system with the work flow in high throughput screening facilities, our prototype also needs to be manufacturable at a low cost per piece at large quantities. We therefore made our prototype compatible with large-scale manufacturing methods, such as injection molding, and found a subcontractor who can produce the parts for us. Specifically, we investigated and compared different materials which are suitable alternatives to the commonly used elastomer polydimethylsiloxane (PDMS) in the microfluidics field. We found a thermoplastic elastomer which has suitable mechanical properties needed for our device and which is compatible with injection molding techniques. Together with the subcontractor, we optimized a manufacturing protocols to produce molds for injection molding with sufficiently high resolution for the smallest features in our design. We obtained the first prototypes made by injection molding and were able to form the miniaturized cardiac tissues in them.
3. Automation:
We devised a strategy to increase the level of automation in our device. Firstly, we will ensure that our device will be compatible with pipetting robots for both seeding and culturing the cardiac tissues. Secondly, we will exploit the advantages of microfluidics to have the possibility to add extra functions (such as continuous medium recirculation) to our device. This will allow us to expand our technology into other areas of the Organs-on-chips field. To achieve this, we are currently developing active valves in a non-PDMS material, which has the following advantages over PDMS: 1. Is compatible with both rapid prototyping and injection molding and 2. has very low molecule absorption (an essential aspect in technology development for drug discovery). We have made the first prototypes of these active valves.
In this reporting period there were no results yet which are beyond the state of the art.