milliPillar: A Platform for the Generation and Real-Time Assessment of Human Engineered Cardiac Tissues

Abstract

Engineered cardiac tissues derived from human induced pluripotent stem cells (iPSCs) are increasingly used for drug discovery, pharmacology and in models of development and disease. While there are numerous platforms to engineer cardiac tissues, they often require expensive and nonconventional equipment and utilize complex video-processing algorithms. As a result, only specialized academic laboratories have been able to harness this technology. In addition, methodologies and tissue features have been challenging to reproduce between different groups and models. Here, we describe a facile technology (milliPillar) that covers the entire pipeline required for studies of engineered cardiac tissues. We include methodologies for (i) platform fabrication, (ii) cardiac tissue generation, (iii) electrical stimulation, (iv) automated real-time data acquisition, and (v) advanced video analyses. We validate these methodologies and demonstrate the versatility of the platform by showcasing the fabrication of tissues in different hydrogel materials and using cardiomyocytes derived from different iPSC lines in combination with different types of stromal cells. We also validate the long-term culture of tissues within the platform and provide protocols for automated analysis of force generation and calcium flux using both brightfield and fluorescence imaging. Lastly, we demonstrate the compatibility of the milliPillar platform with electromechanical stimulation to enhance cardiac tissue function. We expect that this resource will provide a valuable and userfriendly tool for the generation and real-time assessment of engineered human cardiac tissues for basic and translational studies.

Engineered cardiac tissue model of restrictive cardiomyopathy for drug discovery

“…ECTs were created from FLNC ΔGAA and FLNC ѱWT iPSC-CMs using our previously published method, in which cells encapsulated in a fibrin gel are molded around two horizontal pillars cast from polydimethylsiloxane (PDMS) ( Figures 2 A, S2 A, and S2B). 7 We combined two methods to encourage tissue maturation. First, tissues were subjected to 2 weeks of electromechanical stimulation, which we have shown previously to improve iPSC-CM maturation and gene expression.…”

“…We assessed ECT function using video microscopy to track pillar deflection. Based on pillar displacement and the previously performed empirical measurements, 7 force generation is able to be calculated with three specific metrics: (1) active force, which is the force that displaces the pillars during tissue contraction; (2) passive tension, which denotes the residual force causing pillar deflection during maximum relaxation; and (3) total force, which is the sum of both passive and active forces. Total force generated by FLNC ΔGAA and FLNC ѱWT tissues when paced at 1 Hz increased a similar amount over the maturation period.…”

“…We have previously shown that a dedicated maturation protocol improves tissue function, structure, and mature gene expression, and this study demonstrates the power of such an approach in eliciting genotype-phenotype relationships in vitro. 4 , 7 …”

“… 5 , 6 Recently, we reported a platform for the generation and real-time assessment of engineered cardiac tissues in a medium-throughput manner, enabling the quantification of clinical-like parameters throughout the cardiac contraction cycle such as contractile force, muscle tension, and relaxation velocity. 7 …”

Effect of mechanical unloading on genome-wide DNA methylation profile of the failing human heart

“…Calcium transience videos were acquired at 50 frames per second using a sCMOS camera (Zyla 4.2, Andor Technology) connected to an inverted fluorescence microscope (IX-81, Olympus) with cells placed in a live-cell chamber (STX Temp & CO 2 Stage Top Incubator, Tokai Hit). Calcium signal analysis was performed using custom Python script as previously described ( 65 ).…”