Desarrollo de un sistema avanzado de cultivo 3D para la ingeniería de tejidos cardíacos humanos

María Valls Margarit

Dra. Elena Martínez Fraiz Dr. Ángel Raya Chamorro

Dra. Elena Martínez Fraiz (IBEC) y Dr. Ángel Raya Chamorro (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.

Actualmente, la evaluación de la toxicidad de nuevos fármacos se lleva a cabo durante la fase preclínica de desarrollo, utilizando modelos animales y líneas celulares cultivadas in vitro como métodos estándar. Sin embargo, ninguno de estos métodos es capaz de recapitular la complejidad de la fisiología del corazón humano y, por lo tanto, predecir con fiabilidad la respuesta del tejido cardíaco humano a los fármacos. De hecho, esta es la principal causa del gran porcentaje de fármacos retirados del mercado tras ser aprobados por la Administración de Alimentos y Medicamentos (FDA), ya que muchos de ellos provocan arritmias cardíacas al llegar a la fase clínica. En este contexto, el modelo in vitro de tejido cardíaco humano desarrollado en esta tesis doctoral constituye un método muy prometedor para estudiar la cardiotoxicidad de fármacos, superando así las limitaciones de los métodos estándar actuales. Mediante el uso de células cardíacas derivadas de células madre pluripotentes inducidas humanas (hiPSC) y proporcionándoles un entorno favorable para su maduración, organización y funcionalidad, hemos podido recapitular parte de la complejidad del tejido cardíaco humano. Una de las características más relevantes de este modelo es su capacidad para predecir los efectos cardiotóxicos de fármacos aprobados y comercializados, cuya arritmogénesis se demostró posteriormente. Por lo tanto, el modelo de tejido cardíaco humano desarrollado podría convertirse en una nueva generación de modelos in vitro para estudiar la cardiotoxicidad de nuevos fármacos. Dado que la cardiotoxicidad es la principal causa de retirada de fármacos del mercado, incluso de fármacos no diseñados para el tratamiento de enfermedades cardiovasculares (p. ej., agentes quimioterapéuticos), el modelo que proponemos podría ser de gran interés para la industria farmacéutica. Este nuevo modelo 3D de tejido cardíaco permitiría una selección más eficiente del fármaco candidato, con la consiguiente reducción del tiempo y los costes asociados al desarrollo de nuevos fármacos. Además, también permitiría reducir el riesgo de los individuos que participan en ensayos clínicos, algo esencial para cualquier industria farmacéutica. El hecho de que este nuevo modelo para evaluar la cardiotoxicidad de fármacos se base en el uso de células iPS humanas permitiría el desarrollo de diferentes modelos de enfermedad y pacientes específicos, avanzando así en el campo de la medicina personalizada. Por lo tanto, el principal interés de la industria farmacéutica en todos estos aspectos garantizaría una rápida implementación de la tecnología. Además, se prevé una entrada al mercado corta debido a la falta de un marco regulatorio específico y complejo, así como al grado de madurez de la tecnología (prototipo ya probado a nivel de laboratorio). Para transferir la tecnología, se consideran tres sectores de interés: (I) empresas que ofrecen servicios de pruebas de cardiotoxicidad, (II) empresas que proporcionan productos para pruebas de cardiotoxicidad, y (III) industrias farmacéuticas que desean introducir la tecnología en su proceso de descubrimiento de fármacos. El cliente más obvio para transferir la tecnología rápidamente sería una empresa líder mundial que ofrezca productos o servicios relacionados con la evaluación de la cardiotoxicidad de fármacos, con experiencia y capacidad para comercializar células hiPS, acceso a profesionales del sector, red de ventas propia y una sólida experiencia en el campo.

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.