Periodic Reporting for period 1 - SmartHeart (SmartHeart, a 3D in vitro assay for improved assessment of cardiac drug efficacy and toxicity)
Reporting period: 2020-03-02 to 2022-03-01
The SmartHeart project is dedicated to the development of a in vitro cardiac assay to assess physiological relevant read-outs all in a single platform.
According to WHO, every year there is an average of 18 million people dying out of cardiac diseases. A substantial part of these diseases is due to cardiomyopathies that affect many young people. Despite all the advances made in medicine in the last decades, there is still a long way to go before having robust disease models and efficient development of new therapies. Nowadays, cardiac tissue engineering is becoming one of the most common ways to mimic the main events encountered in these types of pathologies, but there are still a lot of limitations in the existing models, as lack of scalability for high throughput and high content screening platforms, or simply because the read outs provided are not enough or not relevant.
To answer to these questions, the goal of this project is to develop a novel proprietary 3D cardiac assay, which enables both the formation and maturation of cardiac organoids based on cardiomyocytes derived from hiPSC as well as the acquisition of the most relevant readouts to screen drugs and to assess toxicological effects, all in single platform. The assay will be developed in a 96 well format, commonly used in drug screening platforms and will enable the acquisition of the following read-outs:
- beating rate,
- beating amplitude,
- contractility force assessment,
- calcium exchange,
- imaging of tissue/organoid organization,
- intra-cellular imaging.
According to WHO, every year there is an average of 18 million people dying out of cardiac diseases. A substantial part of these diseases is due to cardiomyopathies that affect many young people. Despite all the advances made in medicine in the last decades, there is still a long way to go before having robust disease models and efficient development of new therapies. Nowadays, cardiac tissue engineering is becoming one of the most common ways to mimic the main events encountered in these types of pathologies, but there are still a lot of limitations in the existing models, as lack of scalability for high throughput and high content screening platforms, or simply because the read outs provided are not enough or not relevant.
To answer to these questions, the goal of this project is to develop a novel proprietary 3D cardiac assay, which enables both the formation and maturation of cardiac organoids based on cardiomyocytes derived from hiPSC as well as the acquisition of the most relevant readouts to screen drugs and to assess toxicological effects, all in single platform. The assay will be developed in a 96 well format, commonly used in drug screening platforms and will enable the acquisition of the following read-outs:
- beating rate,
- beating amplitude,
- contractility force assessment,
- calcium exchange,
- imaging of tissue/organoid organization,
- intra-cellular imaging.
Overall, the project has achieved most of the objectives and milestones outlined in the grant agreement.
Scientific objectives:
- Development of a device that enables the measurement of several parameters (read-outs), as contractility and Calcium transients by simply using imaging techniques. This objective was fully achieved. A technology based on microwells molded on soft hydrogels to seed contractile cells as CMs, and the subsequent assessment of contractility, beating rate, beating amplitude and calcium transients by image analysis techniques was developed.
- Optimization of the assay to enable maturation of the hiPSC-CMs and high-resolution imaging (allowing sub-cellular imaging, cellular morphology analysis, etc). This objective was fully achieved, but further tests may be necessary. During the extent of this scholarship only one type of hiPSC-CMs was used. The features of the substrate were fine tuned for this cell model, and it was observed that cell beating is restored only after 24 hours, which is much faster than in other engineered heart tissue (EHT) models reported in the literature. However, in the future 4Dcell may develop its own hiPSC-CMs, or a partnership with a cell provide may be established, which means that further tests are necessary to ensure that the cell culture substrate provides to these cells the most suitable environment for cell maturation. From the point of view of imaging, all technological considerations were addressed to ensure that the assay is compatible with high resolution imaging techniques, as confocal imaging.
- Scalability of the SmartHeart technology to multi-well plates, so that it can be used by screening platforms (e.g. Pharma, Contract Research Organizations (CRO), etc). This objective was fully achieved. A method to mold the cell culture substrate into 96 well plates format was developed. Different ways to do it were addressed during this scholarship: first just by molding coverslips which were attached to adhesive bottomless multiwell plates; and then by developing a method that allows molding the hydrogels on a large glass slide that fits with glass bottom multiwell plates. The first version facilitated the assessment of the assay in an early phase of the project using ‘real life conditions’, while the second method is paving the way to mold hydrogels in multiwell plates at 4Dcell for industrial purposes. An intermediate 24 well format plate was also developed which will allow commercialization of the assay to first beta testers (academia, pharma companies and biotechnology companies).
Training objectives:
- Training on material chemistry
This objective was fully achieved. The ER received training on material chemistry, with emphasis in hydrogel materials. Despite having already some basic knowledge in this area, the ER attended internal workshops with theoretical and experimental components where she learned about the chemistry of hydrogels, formulation and fabrication of hydrogels and characterization techniques.
- Training on Intellectual Property and Patent Writing
This objective was fully achieved. At the beginning of the project, the ER received one to one training from her supervisor Dr. Mael Le Berre and patent expert Mr. Jacques Vesin on how to do a Freedom-To-Operate (FTO) analysis and on how to write an invention report. Prior to starting her experimental work, she carried out an FTO to ensure that the technology proposed in this project could be patented. During the project the ER wrote a patent covering the technological features of the cell culture device, which was submitted on the 13 of June 2022 at the French patent office, with the #FR2205692.
- Training on Cell biology
This objective was achieved but with major modifications. The training planed initially was supposed to be carried out during the secondment at the PARCC. However, due to the measures put in place due to COVID-19 pandemic, external researchers were not allowed to enter the research facilities of this institution, and the secondment was not accomplished. To overcome this, the ER was in close contact with the PhD students of Prof. Hulot’s team at PARCC, who provided that her with online trainings. At the same time, she was trained at 4Dcell to perform cell culture, enabling her to test and validate the technology developed in the aim of this scholarship with other types of cells as hFB.
- Training on Business development and Marketing
This objective was fully achieved. During the project, the ER was always in close contact with Cyril Cerveau, the CEO of 4Dcell and with both Marketing and Business teams. She received continuous training on how to develop a product that is market fit, how to market it and how to develop a business plan.
Scientific objectives:
- Development of a device that enables the measurement of several parameters (read-outs), as contractility and Calcium transients by simply using imaging techniques. This objective was fully achieved. A technology based on microwells molded on soft hydrogels to seed contractile cells as CMs, and the subsequent assessment of contractility, beating rate, beating amplitude and calcium transients by image analysis techniques was developed.
- Optimization of the assay to enable maturation of the hiPSC-CMs and high-resolution imaging (allowing sub-cellular imaging, cellular morphology analysis, etc). This objective was fully achieved, but further tests may be necessary. During the extent of this scholarship only one type of hiPSC-CMs was used. The features of the substrate were fine tuned for this cell model, and it was observed that cell beating is restored only after 24 hours, which is much faster than in other engineered heart tissue (EHT) models reported in the literature. However, in the future 4Dcell may develop its own hiPSC-CMs, or a partnership with a cell provide may be established, which means that further tests are necessary to ensure that the cell culture substrate provides to these cells the most suitable environment for cell maturation. From the point of view of imaging, all technological considerations were addressed to ensure that the assay is compatible with high resolution imaging techniques, as confocal imaging.
- Scalability of the SmartHeart technology to multi-well plates, so that it can be used by screening platforms (e.g. Pharma, Contract Research Organizations (CRO), etc). This objective was fully achieved. A method to mold the cell culture substrate into 96 well plates format was developed. Different ways to do it were addressed during this scholarship: first just by molding coverslips which were attached to adhesive bottomless multiwell plates; and then by developing a method that allows molding the hydrogels on a large glass slide that fits with glass bottom multiwell plates. The first version facilitated the assessment of the assay in an early phase of the project using ‘real life conditions’, while the second method is paving the way to mold hydrogels in multiwell plates at 4Dcell for industrial purposes. An intermediate 24 well format plate was also developed which will allow commercialization of the assay to first beta testers (academia, pharma companies and biotechnology companies).
Training objectives:
- Training on material chemistry
This objective was fully achieved. The ER received training on material chemistry, with emphasis in hydrogel materials. Despite having already some basic knowledge in this area, the ER attended internal workshops with theoretical and experimental components where she learned about the chemistry of hydrogels, formulation and fabrication of hydrogels and characterization techniques.
- Training on Intellectual Property and Patent Writing
This objective was fully achieved. At the beginning of the project, the ER received one to one training from her supervisor Dr. Mael Le Berre and patent expert Mr. Jacques Vesin on how to do a Freedom-To-Operate (FTO) analysis and on how to write an invention report. Prior to starting her experimental work, she carried out an FTO to ensure that the technology proposed in this project could be patented. During the project the ER wrote a patent covering the technological features of the cell culture device, which was submitted on the 13 of June 2022 at the French patent office, with the #FR2205692.
- Training on Cell biology
This objective was achieved but with major modifications. The training planed initially was supposed to be carried out during the secondment at the PARCC. However, due to the measures put in place due to COVID-19 pandemic, external researchers were not allowed to enter the research facilities of this institution, and the secondment was not accomplished. To overcome this, the ER was in close contact with the PhD students of Prof. Hulot’s team at PARCC, who provided that her with online trainings. At the same time, she was trained at 4Dcell to perform cell culture, enabling her to test and validate the technology developed in the aim of this scholarship with other types of cells as hFB.
- Training on Business development and Marketing
This objective was fully achieved. During the project, the ER was always in close contact with Cyril Cerveau, the CEO of 4Dcell and with both Marketing and Business teams. She received continuous training on how to develop a product that is market fit, how to market it and how to develop a business plan.
To the best of our knowledge there is no other assay on the market that enables the acquisition of so many read-outs (beating rate, beating amplitude, contractility force assessment, calcium exchange, imaging of tissue/organoid organization, intra-cellular imaging) all in one platform, and that is compatible with high throughput screening and high content screening. Furthermore, the circular 3D morphology of the organoid is closer to a model that better reproduces the human heart physiology. Based on this, and on the feedback already acquired from potential future users, this tool has a very large potential to become one of the standard in vitro cardiac drug screening and toxicological assays.