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

Abstract

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.

La cardiopatia isquèmica és una de les principals causes de mort a nivell mundial. Exceptuant el trasplantament de cor, les teràpies actuals són insuficients per restablir la funció cardíaca. Per tant, cal desenvolupar teràpies alternatives que fomentin la regeneració i/o reparació del cor, així com també noves eines per estudiar la fisiologia i fisiopatologia cardíaca in vitro. Una de les estratègies més prometedores és l’enginyeria tissular cardíaca, ja que té com a finalitat generar constructes de teixit cardíac que mimetitzin el teixit real. Aquests constructes podrien utilitzar-se com a models in vitro del miocardi humà i també com a empelts per reparar el cor malmès. Per obtenir constructes de teixit cardíac humà cal reproduir l’entorn cardíac real. Una de les estratègies més habituals consisteix en sembrar cardiomiòcits en una estructura 3D (bastida), i després cultivar el constructe en un sistema de senyalització biomimètic, normalment un bioreactor. Tanmateix, generar constructes grans i semblants al miocardi humà adult a partir de cardiomiòcits humans derivats de cèl·lules mare de pluripotència induïda (hiPSC-CM) segueix sent un repte. Així doncs, la hipòtesi d’estudi és que combinant hiPSC-CM amb una bastida 3D i estímuls biofísics adequats, es podrien generar constructes de teixit cardíac semblants al miocardi humà tant a nivell estructural com funcional. Per abordar la hipòtesi, en aquest treball s’ha caracteritzat una bastida 3D constituïda principalment per col·lagen i s’ha definit un mètode eficient per sembrar cardiomiòcits dins l’estructura. A més a més, s’ha desenvolupat un bioreactor de perfusió de sistema en paral·lel que assegura un transport de massa efectiu entre les cèl·lules i el medi de cultiu. També s’ha dissenyat una càmera de perfusió que inclou elèctrodes per estimular elèctricament les cèl·lules durant el cultiu, així com també per monitorar la funció del teixit artificial. Amb aquest avançat sistema de cultiu, s’han generat constructes de teixit cardíac humà 3D amb una funcionalitat semblant a la del teixit real. A més a més, el sistema ha permès monitorar l’electrofisiologia del teixit artificial en temps real, així com també demostrar el paper crucial de l’estimulació elèctrica per obtenir constructes amb una funcionalitat òptima.