Development of an advanced 3D culture system for human cardiac tissue engineering

Maria Valls Margarit

Dra. Elena Martínez Fraiz Dr. Ángel Raya Chamorro

IBEC CMRB

Ischemic heart disease is a major cause of human death worldwide owing to the heart minimal ability to repair following injury. Other than heart transplantation, there are currently no effective or long-lasting therapies for end-stage heart failure. Therefore, it is crucial to develop not only alternative therapies that potentiate heart regeneration or repair, but also new tools to study human cardiac physiology and pathophysiology in vitro. In this context, cardiac tissue engineering arises a promising strategy, as it is aimed at generating cardiac tissue analogues that would act as in vitro models of human cardiac tissue or as surrogates for heart repair. Thus, having 3D human cardiac tissue constructs resembling human myocardium could revolutionize drug discovery and toxicity testing, cardiac disease modelling and regenerative medicine. An strategy to obtain reliable cardiac tissue constructs is to mimic the native cardiac environment. The classical approach is based on seeding cardiomyocytes in biocompatible 3D scaffolds, and then culturing the construct in a biomimetic signaling system, usually a bioreactor. Although major advances have been made, the generation of thick and mature tissue constructs from human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CM) is still a challenge. Therefore, the hypothesis of our study is that the combination of hiPSC-CM with 3D scaffolds and appropriate regulatory signals may lead to the generation of mature human cardiac tissue constructs resembling human myocardium, both functionally and structurally. To address this, we have characterized a collagen-based 3D scaffold and established an efficient method for cell seeding into the scaffold. We have also developed a parallelized perfusion bioreactor system, which ensures an effective mass transport between cells and culture medium and allows culturing multiple replicas of tissue constructs. In addition, we have designed and fabricated a perfusion chamber including electrodes to electrically stimulate cells during culture, as well as to monitor tissue function. With this advanced 3D culture system, we have been able to generate thick 3D human cardiac constructs with tissue-like functionality. Our results indicate that perfusion of culture medium combined with electrical stimulation and collagen-based scaffold improve the structural and functional maturation of hiPSC-CM. In general terms, electrical stimulation has improved the structural organization, alignment and coupling of cardiomyocytes in our cardiac tissue constructs. Moreover, electrical stimulation has promoted the formation of synchronous contractile constructs at the macroscale with improved electrophysiological functions. Through the development of a new electrophysiological recording system, we report for the first time to our knowledge a technique that provides information about the electrical activity of intact cardiac tissue constructs in real time. Specifically, the combination of action potentials generated by hiPSC-CM composing cardiac constructs produces ECG-like signals, which could be monitored online. Finally, we have demonstrated the ability of stimulated human cardiac tissue constructs to detect drug-induced cardiotoxicity, as typical features of arrhythmias (e.g. prolongation of RR intervals and regular blockades) could be observed upon treatment with sotalol. Taken together, results indicate that macroscopic human cardiac tissue constructs with tissue-like functionality can be obtained through the use of our advanced 3D culture system. We have studied the effects of electrical stimulation on cardiomyocytes at multiple levels: molecular (presence, distribution and expression of cardiac proteins), ultrastructural (sarcomere width and presence of specialized cellular junctions), cellular (morphology and alignment), and functional (amplitude, directionality and strain of contractions, and electrophysiological recordings). Findings validate our in vitro approach as a valuable system to obtain 3D cardiac patches with an improved maturity and functionality. Importantly, the online monitoring system developed in this study can provide essential electrophysiological information of intact cardiac tissue constructs, which opens up myriad possibilities in the field of cardiovascular research.

Currently, the assessment of the toxicity of new drugs is carried out during the preclinical phase of development, using animal models and cell lines cultured in vitro as standard methods. However, neither method is able to recapitulate the complexity of the physiology of the human heart and, therefore, to reliably predict the response of human cardiac tissue to drugs. In fact, this is the main cause of the large percentage of drugs withdrawn from the market after being approved by the Food and Drug Administration (FDA), since many of them cause cardiac arrhythmias when they reach the clinical phase. In this context, the in vitro model of human cardiac tissue developed in this doctoral thesis is a very promising method to study the cardiotoxicity of drugs, thus overcoming the limitations of current standard methods. By using cardiac cells derived from human induced pluripotent stem cells (hiPSCs) and providing them with a favorable environment for their maturation, organization, and functionality, we have been able to recapitulate some of the complexity of human cardiac tissue. One of the most relevant features of this model is its ability to predict the cardiotoxic effects of drugs that were approved and marketed and that were subsequently shown to be arrhythmogenic. Therefore, the human cardiac tissue model that has been developed could become a new generation of in vitro models to study the cardiotoxicity of new drugs. Given that cardiac toxicity is the main cause of drug withdrawal from the market, even of drugs not designed for the treatment of cardiovascular diseases (e.g., chemotherapeutic agents), the model we propose may be of great interest to the pharmaceutical industry. This new 3D model of cardiac tissue would allow for a more efficient selection of the candidate drug, with the consequent reduction of time and costs associated with the development of new drugs. In addition, it would also allow for a reduction in the risk of individuals participating in clinical trials, which is essential for any pharmaceutical industry. The fact that this new model for assessing the cardiotoxicity of drugs is based on the use of human iPS cells would allow for the development of different disease models and specific patients, thus advancing in the field of personalized medicine. Therefore, the primary interest of the pharmaceutical industry in all these aspects would ensure a rapid implementation of the technology. In addition, a short market entry is expected due to the lack of a specific and complex regulatory framework and also to the degree of maturity of the technology (prototype already tested at the laboratory level). In order to transfer the technology, three sectors of interest are considered: (I) companies that offer cardiotoxicity testing services, (II) companies that provide products for cardiotoxicity testing, and (III) pharmaceutical industries that want to introduce the technology into their drug discovery process. The most obvious client to transfer the technology quickly would be a world-leading company that offers products or services related to the evaluation of cardiotoxicity of drugs, with experience and capacity to commercialize hiPS cells, access to professionals in the sector, its own sales network and solid experience in the field.

Ischemic Heart Disease; Heart Failure; Heart Regeneration; Heart Repair; Human Cardiac Physiology; Human Cardiac Pathophysiology; In Vitro Models; Cardiac Tissue Engineering; Cardiac Tissue Analogues; 3D Human Cardiac Tissue Constructs; Human Myocardium; Drug Discovery; Toxicity Testing; Cardiac Disease Modelling; Regenerative Medicine; Native Cardiac Environment Mimicry; Cardiomyocytes; Biocompatible 3D Scaffolds; Biomimetic Signaling System; Bioreactor; Thick Tissue Constructs; Mature Tissue Constructs; Human Induced Pluripotent Stem Cells-Derived Cardiomyocytes (hiPSC-CM); Collagen-Based 3D Scaffold; Cell Seeding; Parallelized Perfusion Bioreactor System; Effective Mass Transport; Multiple Replicas; Perfusion Chamber; Electrodes; Electrical Stimulation; Tissue Function Monitoring; Tissue-Like Functionality; Structural Maturation; Functional Maturation; Structural Organization; Alignment; Cardiomyocyte Coupling; Synchronous Contractile Constructs; Macroscopic Scale; Electrophysiological Functions; Electrophysiological Recording System; Electrical Activity; Intact Cardiac Tissue Constructs; Real Time Monitoring; Action Potentials; ECG-like Signals; Online Monitoring; Drug-Induced Cardiotoxicity; Arrhythmias; RR Intervals Prolongation; Regular Blockades; Sotalol Treatment; Advanced 3D Culture System; Molecular Level; Cardiac Proteins; Ultrastructural Level; Sarcomere Width; Specialized Cellular Junctions; Cellular Level; Morphology; Contraction Amplitude; Contraction Directionality; Contraction Strain; Electrophysiological Recordings; In Vitro Approach; 3D Cardiac Patches; Improved Maturity; Improved Functionality; Cardiovascular Research.