Functional arrays of human pluripotent stem cell-derived cardiac microtissues

Abstract

To accelerate the cardiac drug discovery pipeline, we set out to develop a platform that would be capable of quantifying tissue-level functions such as contractile force and be amenable to standard multiwell-plate manipulations. We report a 96-well-based array of 3D human pluripotent stem cell (hPSC)-derived cardiac microtissues -termed Cardiac MicroRings (CaMiRi) -in custom 3D-printmolded multiwell plates capable of contractile force measurement. Within each well, two elastomeric microcantilevers are situated above a circumferential ramp. The wells are seeded with cell-laden collagen, which, in response to the gradual slope of the circumferential ramp, self-organizes around tip-gated microcantilevers to form contracting CaMiRi. The contractile force exerted by the CaMiRi is measured and calculated using the deflection of the cantilevers. Platform responses were robust and comparable across wells, and we used it to determine an optimal tissue formulation. We validated the contractile force response of CaMiRi using selected cardiotropic compounds with known effects. Additionally, we developed automated protocols for CaMiRi seeding, image acquisition, and analysis to enable the measurement of contractile force with increased throughput. The unique tissue fabrication properties of the platform, and the consequent effects on tissue function, were demonstrated upon adding hPSC-derived epicardial cells to the system. This platform represents an open-source contractile force screening system useful for drug screening and tissue engineering applications.Screening using engineered heart tissue is limited by the high cost associated with the large cell numbers required for high-fidelity in vitro cardiac models, the lack of long-term function and viability of the tissues, and the challenge of imaging for data acquisition. Miniaturization of self-organizing cardiac organoids is a strategic approach to address these challenges using photolithography-and micromachining-based fabrication techniques 1-12 . Unfortunately, some of these systems are difficult to manufacture on a macroscale, and production often requires a high level of skill. Furthermore, these methods are restrictive to specific design geometries (for instance, vertical walls spanning either the micrometer or centimeter scale but not both) and are occasionally expensive to prototype (due to requiring access to a cleanroom, cost of materials, and time). Another challenge typically associated with these microfluidic-and microstructure-based devices is adaptation to existing tissue culture equipment such as microscope stage adapters and liquid dispensing tools. Potential complications may also arise upon the

Bi-directional Impacts of Heterotypic Interactions in Engineered 3D Human Cardiac Microtissues Revealed by Single-Cell RNA-Sequencing and Functional Analysis

“…Due to the single cell-based nature of the analyses, we were able to dissect bi-directional phenotypic changes in each specific cellular sub-type and evaluate them in the context of tissue level functional performance. While it is well established that non-myocytes are required to promote stable tissue formation 21,26–29 , previous studies have described variable effects of different stromal or non-myocyte populations on cardiac function with limited details reported on phenotypic shifts in the co-cultured cells 21,30,31 . Therefore, we aimed to determine how age-specific CFs and ECs differentially affect cardiac tissue organization and calcium handling dynamics, resulting in transcriptional changes in the different heterotypic populations of cells.…”

Regenerating Damaged Myocardium: A Review of Stem-Cell Therapies for Heart Failure

“…This complex directionality has been accommodated by seeding the cells onto arrays of elastomeric microposts, with each cell forming attachments to multiple posts, and then monitoring the positions of the post tips (i.e., the point of cell attachment) relative to their bases. Studies conducted with hiPSC-CMs indicated that the cells typically formed attachments with 13-20 microposts/cell and generated a contractile force of ~15 nN/cell, with a peak contractile power of 29 fW [76].Contractile measurements have also been conducted in populations of hPSC-CMs suspended in fibrin and stretched between flexible pillars [77], and in collagen rings positioned around elastomeric microcantilevers [78]; subsequent assessments confirmed that the contractile forces increased and decreased in response to positive and negative inotropic factors, respectively.…”

From Human Pluripotent Stem Cells to 3D Cardiac Microtissues: Progress, Applications and Challenges

“…In both models, different cardiac cells, including ECs and CFs, which can be hPSC-derived or primary cultured cells, are combined with hPSC-derived CMs at a specific ratio. In the case of the EHT models, the composition of the hydrogel and the concentration of the ECM used are also important parameters to ensure tissue structure and functionality [ 87 ].…”

“…The EHT models described in the literature are composed of hPSC-derived CMs alone [ 88 , 89 , 90 , 91 ] or in combination with primary fibroblast/stromal cells [ 92 , 93 , 94 ] and endothelial cells (HUVECs) [ 95 ] normally embedded in an hydrogel-based matrix that is then shaped according to a specific format. One of the models that has been described is the ring-shaped EHT, in which the mixture is pipetted into circular casting molds, where the tissue condenses, and is then placed around passive-flexible holders [ 87 , 92 , 96 ]. A different strategy relies on the development of elliptic shaped or strip-like cardiac tissues, which are anchored and stretched between two flexible pillars [ 89 , 90 , 93 , 95 , 97 , 98 ].…”

“…In the case of EHT, the adaptation to a HTS format has been more challenging, mainly due to the size of the construct that should be compatible with a multi-well platform, to maximize the throughput of the system, and also due to the number of hPSC-derived cardiac cells needed for each cardiac MT. To overcome those limitations, small size EHT platforms have been described in the literature, including the Heart-Dyno platform [ 98 , 102 ], the Cardiac MicroRings (CaMiRi) [ 87 ] and the µTUG arrays [ 95 ]. Some platforms are now commercially available, such as in the case of the Biowire TM II platform (TARA Biosystems) [ 94 , 117 ] and the cardiac tissue strip model (Novoheart TM ) [ 127 , 128 ] (see Table 2 ).…”