3D printed micro-scale force gauge arrays to improve human cardiac tissue maturation and enable high throughput drug testing

Abstract

Human induced pluripotent stem cell – derived cardiomyocytes (iPSC-CMs) are regarded as a promising cell source for establishing in-vitro personalized cardiac tissue models and developing therapeutics. However, analyzing cardiac force and drug response using mature human iPSC-CMs in a high-throughput format still remains a great challenge. Here we describe a rapid light-based 3D printing system for fabricating micro-scale force gauge arrays suitable for 24-well and 96-well plates that enable scalable tissue formation and measurement of cardiac force generation in human iPSC-CMs. We demonstrate consistent tissue band formation around the force gauge pillars with aligned sarcomeres. Among the different maturation treatment protocols we explored, 3D aligned cultures on force gauge arrays with in-culture pacing produced the highest expression of mature cardiac marker genes. We further demonstrated the utility of these micro-tissues to develop significantly increased contractile forces in response to treatment with isoproterenol, levosimendan, and omecamtiv mecarbil. Overall, this new 3D printing system allows for high flexibility in force gauge design and can be optimized to achieve miniaturization and promote cardiac tissue maturation with great potential for high-throughput in-vitro drug screening applications.

Statement of Significance

The application of iPSC-derived cardiac tissues in translatable drug screening is currently limited by the challenges in forming mature cardiac tissue and analyzing cardiac forces in a high-throughput format. We demonstrate the use of a rapid light-based 3D printing system to build a micro-scale force gauge array that enables scalable cardiac tissue formation from iPSC-CMs and measurement of contractile force development. With the capability to provide great flexibility over force gauge design as well as optimization to achieve miniaturization, our 3D printing system serves as a promising tool to build cardiac tissues for high-throughput in-vitro drug screening applications.

Introduction

Cardiovascular disease remains a major cause of morbidity and mortality worldwide [1]. For this reason, drug discovery and mechanistic investigation of cardiovascular diseases have been critical research areas of focus [2], [3]. Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), with potential to represent individual variations, are candidates for translatable drug screening and personalized medicine [4], [5]. However, the drug screening and disease modeling applications of these derived cells have been limited by their immature phenotypes and limited capacity for physiological force development [6], [7].

Approaches to promoting mature cardiomyocyte phenotypes, such as the use of 3D aligned cultures [8], [9], mechanical stretching [9], [10], [11], and electrical pacing [9], [11], [12] have been shown to improve functional performance. In particular, these techniques have been applied to various engineered heart tissue (EHT) systems to maturate in-vitro human cardiac tissues. For example, mechanical stimulation was applied to human embryonic stem cell (ESC)-derived cardiomyocytes in 3D collagen matrix and electrical pacing was used to induce synchronized beating in an iPSC-derived 3D cardiac tissue [10], [13]. Nevertheless, none of the current human platforms demonstrated cardiac tissue force measurements directly following tissue maturation in a high-throughput format. Without the potential to scale up tissue maturation and force measurements, the application of iPSC-derived cardiac tissues in translatable drug screening remains limited. Therefore, there is a critical need to combine maturation approaches with high-throughput force gauge platform to study cardiac tissue drug response in a scalable format.

Traditional microfabrication technologies, such as those based on polyacrylamide and polydimethylsiloxane (PDMS), have been used to fabricate miniaturized structures with scalable potential [14], [15]. However, these approaches mostly take multiple steps to complete and are limited in flexibility of design adjustment. Microscale continuous optical printing (μCOP), which is a type of digital light processing (DLP)-based 3D printing, provides a superior speed and scalability for the fabrication of micro-scale complex 3D constructs [16], [17], [18], [19], [20]. In addition, the use of digital pattern designs offers great flexibility to fabricate and optimize 3D printed structures [16], [17], [18], [19], [20]. In this report, we present a micro-scale force gauge platform based on micro-pillars fabricated by μCOP for cardiac tissue maturation, force measurement, and studying drug response. This printed array of micro-scale force gauges was customized to fit into a regular 24-well and 96-well plate for seeding of iPSC-CMs to form micro-tissues for high-throughput studies. Upon testing different protocols of tissue maturation treatment, 3D aligned culture on force gauge with in-culture pacing showed the highest expression of maturation cardiac genes when compared to the three other culture methods. Moreover, micro-tissues cultured using the optimized pillar design and maturation method generated the largest force with increased force outputs following incubation with drugs such as isoproterenol (ISO), levosimendan (LEVO) and omecamtiv mecarbil (OM). Overall, we have demonstrated that our 3D printing system is a promising tool to fabricate in-vitro cardiac tissues that are functionally mature for drug screening in a high-throughput format.