Periodic Reporting for period 2 - 3DCardiacHTS (Bringing 3D cardiac tissues to high throughput for drug discovery screens)
Reporting period: 2024-01-01 to 2025-11-30
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 require 20.000 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 require 20.000 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 20 000. 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). Using our 3DCardiacHTS plate that contains 96 of the microfluidic layer including the micrifluidic chambers, we were able to make miniaturised 3D cardiac strips and show 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 3DCardiacHTS plate also needs to be manufacturable at a low cost per piece at large quantities. We therefore made our 3DCardiacHTS plate compatible with large-scale manufacturing methods, such as injection molding. 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. We have produced our 3DcardiacHTS plate made of co-injection molding of plastic and TPE, and we have validated the 3DCardiacHTS plate by making multiple beating micro 3D cardiac strips in it.
3. Automation:
We have developed an automated method to make anc culture our miniaturised 3D cardiac strips using a pipetting robot. In paralel, we have developed a prototype microfluidic circuit board as a plate lid capable of inducing unidirectional flow to all microfluidic chambers where the miniaturised 3D cardiac strips are cultured. This fluidic circuit board uses pneumatic microfluidic valves which can pump medium from one well to another, enabling continuous medium recirculation. This will allow us to expand our technology into other areas of the Organs-on-chips field.
We have validated the cardiac output of our miniaturised 3D cardiac strips using a pannel of drugs known to have an effect on the human heart
1. Miniaturization:
We were able to downscale the amount of cells needed to make 3D cardiac tissue from 1 million to 20 000. 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). Using our 3DCardiacHTS plate that contains 96 of the microfluidic layer including the micrifluidic chambers, we were able to make miniaturised 3D cardiac strips and show 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 3DCardiacHTS plate also needs to be manufacturable at a low cost per piece at large quantities. We therefore made our 3DCardiacHTS plate compatible with large-scale manufacturing methods, such as injection molding. 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. We have produced our 3DcardiacHTS plate made of co-injection molding of plastic and TPE, and we have validated the 3DCardiacHTS plate by making multiple beating micro 3D cardiac strips in it.
3. Automation:
We have developed an automated method to make anc culture our miniaturised 3D cardiac strips using a pipetting robot. In paralel, we have developed a prototype microfluidic circuit board as a plate lid capable of inducing unidirectional flow to all microfluidic chambers where the miniaturised 3D cardiac strips are cultured. This fluidic circuit board uses pneumatic microfluidic valves which can pump medium from one well to another, enabling continuous medium recirculation. This will allow us to expand our technology into other areas of the Organs-on-chips field.
We have validated the cardiac output of our miniaturised 3D cardiac strips using a pannel of drugs known to have an effect on the human heart
We are able to produce our 3DCardiacHTS plate via injection molding and can create miniaturized 3D cardiac tissues in the microfluidic chambers. We are able to perform automated culture of our miniaturised 3D cardiac strips using pipetting robot or a microfluidic circuit board. We have validated the modeling capabilities of our miniaturised 3D cardiac strips by testing them with a pannel of drugs that are known to have an effect on the human heart and by using them to generate a heart failure model.