Design,fabrication And Application Research Of Multifunctional Microfluidic Heart-On-A-Chip

The heart maintains homeostasis by boost blood circulation for effectively delivering nutrients and removing waste.Heart-related diseases and adverse events,such as myocardial infarction,hypertrophy,arrhythmias,and atherosclerosis,are the leading causes of death worldwide and have been the focus of treatment development.Over the past forty years,a third of drug sales have been withdrawn because of adverse effects on the heart.This highlights the limitations of current drug-testing methods in evaluating cardiac effects.Cardiac therapy and cardiotoxicity are currently studied using two-dimensional(2D)static cultures of cardiomyocytes or animal models,but with significant limitations.2D cell cultures are oversimplified and fail to reproduce cell orientation and cardiac tissue physiology(such as electrical signals,mechanical forces,etc.),while animal models are costly and often fail to predict human responses.Therefore,it is urgent to construct a new in vitro human heart model to improve the existing drug evaluation methods.Microfluidic chips have been recognized by the industry as the mainstream platform for precise manipulation of mammalian cells and their microenvironment.Its goal is to build human organs that can simulate the physiological or pathological characteristics of human tissues and organs on the chip through the cross fusion of stem cells,biomaterials,tissue engineering,microfluidics and microfabrication technologies,which are used for new drug development and medical research,and is hoped to eventually replace existing cell and animal experiments as "body-on-a-chip".The heart-on-a-chip is an indispensable key component of the " body-on-a-chip",because the cardiac toxicity must be evaluated during the drug development process.Although there have been many advances in the research on cardiac chips,there are still many problems that need to be solved.This thesis takes the advantages of interdisciplinary to integrate the research methods and ideas of microfluidics technology,stem cell technology,biomaterials and tissue engineering,to build a microfluidic chip system that integrates electrical stimulation implementation and myocardial electrophysiological signal detection.We prepared a three-dimensional(3D)gelatin hydrogel scaffold in the chip to dynamically culture cardiomyocytes differentiated from human induced pluripotent stem cells(iPSC).Functional myocardial tissue was formed by appropriate electrical stimulation,and the evaluation of myocardial contractile and electrophysiological functions was achieved.The specific research contents of this article are as follows:(1)We prepared gelatin-based hydrogels as scaffolds which are favorable for the maturation of cultured cardiomyocytes,including rapid development of cell size,actin cytoskeleton,gap junction,and calcium transient.By introducing the micropatterns on hydrogel surfaces,sarcomere structures within the individual cardiomyocytes are better aligned.The further addition of GO to the scaffolds is demonstrated to enhance cell alignment and beating velocity of cultured cardiomyocytes,the cultured cardiomyocytes reached synchronous contraction within 48 hours after seeding and continued to beat for 3 months..(2)Combining the advantageous properties of hydrogels with external electrical stimulation can modulate the beating behavior and induce remarkable enhancement of maturation of cardiomyocytes.It was observed that the electrical stimulation improved the organization of sarcomeres and promoted the expression of Cx43,as well as the maturationrelated gene expressions(Actn1,Gja1,Atp2a2,Ryr2).Moreover,electrical stimulation accelerated release and uptake kinetics of intracellular calcium,thereby increasing the beating velocity of cardiomyocytes and responsiveness to external pacing.(3)We developed a multifunctional heart-on-a-chip device that allows for long-term dynamic culture of human iPSC-derived cardiomyocytes,maturation of hiPSC-CMs by continuous electrical stimulation,and in situ evaluation of physiological function of cardiac tissues.This platform successfully combines Pt wire electrodes for external electrical stimulation of cardiomyocytes and Au electrode arrays for real-time acquisition of electrophysiological signals of cardiac tissues.Human iPSC-CMs were cultured on gelatin hydrogels in-situ prepared in the chip chamber.The results show that electrical stimulation remarkably enhanced the maturation of cardiomyocytes and generated functional mature cardiac tissues in the chip device.Compared with unstimulated immature cardiac tissues,the electrical-stimulated mature cardiac tissues show correct responsiveness to drug treatment of verapamil and isoprenaline,indicating the reliability of this heart-on-a-chip platform for the application in drug efficacy testing and cardiac toxicity screening.The purpose of this paper is to lay the foundation for theoretical experiments and data support for the subsequent development and application of heart-on-a-chip,and to prove its potential for pre-clinical screening of drugs,including the detection of cardiac toxicity of cardiac and non-cardiac drugs,the heart disease modeling to evaluate the effectiveness of drugs,and to evaluate the effectiveness of heart damage and treatment methods caused by other diseases.