Functional scaffold-free 3-D cardiac microtissues: a novel model for the investigation of heart cells

Abstract

To bridge the gap between two-dimensional cell culture and tissue, various three-dimensional (3-D) cell culture approaches have been developed for the investigation of cardiac myocytes (CMs) and cardiac fibroblasts (CFs). However, several limitations still exist. This study was designed to develop a cardiac 3-D culture model with a scaffold-free technology that can easily and inexpensively generate large numbers of microtissues with cellular distribution and functional behavior similar to cardiac tissue. Using micromolded nonadhesive agarose hydrogels containing 822 concave recesses (800 μm deep × 400 μm wide), we demonstrated that neonatal rat ventricular CMs and CFs alone or in combination self-assembled into viable (Live/Dead stain) spherical-shaped microtissues. Importantly, when seeded simultaneously or sequentially, CMs and CFs self-sorted to be interspersed, reminiscent of their myocardial distribution, as shown by cell type-specific CellTracker or antibody labeling. Microelectrode recordings and optical mapping revealed characteristic triangular action potentials (APs) with a resting membrane potential of -66 ± 7 mV (n = 4) in spontaneously contracting CM microtissues. Under pacing, optically mapped AP duration at 90% repolarization and conduction velocity were 100 ± 30 ms and 18.0 ± 1.9 cm/s, respectively (n = 5 each). The presence of CFs led to a twofold AP prolongation in heterogenous microtissues (CM-to-CF ratio of 1:1). Importantly, Ba(2+)-sensitive inward rectifier K(+) currents and Ca(2+)-handling proteins, including sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a, were detected in CM-containing microtissues. Furthermore, cell type-specific adenoviral gene transfer was achieved, with no impact on microtissue formation or cell viability. In conclusion, we developed a novel scaffold-free cardiac 3-D culture model with several advancements for the investigation of CM and CF function and cross-regulation.

Bio-Pick, Place, and Perfuse: A New Instrument for Three-Dimensional Tissue Engineering

“…In some instances, pairs of cell types may self-sort and/or undergo additional morphogenesis during self-assembly, allowing the assembly of tissues that more closely replicate organ cell composition (29,32). Cell lines were used to demonstrate the proof of concept of the Bio-P3, however building parts of primary cells including cardiomyocytes, smooth muscle cells, fibroblasts, embryonic stem cells, neurons and chondrocytes have also been produced using agarose micro-molds demonstrating that self-assembly of 3D microtissues is a fundamental property of many different cell types (33)(34)(35)(36)(37)(38). For example, endothelial cells will form a capillary-like network within multi-cellular spheroids (4).…”

Comparison of Microtissue Forming Capacity of Sh-Sy5y and Sk-N-as Cell Lines

“…The aim of the present study was to create scaffold-free microtissue spheroids using 3D Petri Dish ® technique [20][21][22] that mimic the cellular microenvironment of SH-SY5Y and SK-N-AS neuronal cell lines that are commonly used as in vitro models for neurodegenerative diseases and neuroblastoma research [23,24]. We have investigated the microtissue spheroids forming potential/capacity of these cell lines as in vitro 3D models, and the effective size of them considering diffusional transport limitations for oxygen and other essential nutrients.…”

Generating an In Vitro 3D Cell Culture Model from Zebrafish Larvae for Heart Research

“…In vitro systems routinely used for biomedical research draw on advances in developmental biology, biomaterials and stem cell technologies, and provide a mechanism for high throughput compound testing and safety pharmacology (Hirt et al, 2014;Amanfu and Saucerman, 2011). However, the biomedical applicability of many of these current in vitro heart models is limited because: (1) they are derived from embryonic or neonatal rodent tissue, which has electrophysiological properties that differ from those of the human heart (Shenje et al, 2008;Sauer et al, 1999), even when generated in a 3D scaffold-free system (Desroches et al, 2012), and (2) they are often 2D cell monolayers, which lack essential features of 3D hearts, including mature myocytes that couple as a functional syncytium (Ausma and Borgers, 2002). Assay systems using HEK cells (Thomas and Smart, 2005), or human embryonic and induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) avoid these issues, but are expensive to generate and also come with their own limitations, including ethical constraints in terms of the use of human embryos and the potential for tumorigenesis (see Novakovic et al, 2014;Mandel et al, 2012;de Boer et al, 2010).…”