Cardiac Meets Skeletal: What's New in Microfluidic Models for Muscle Tissue Engineering

Abstract

In the last few years microfluidics and microfabrication technique principles have been extensively exploited for biomedical applications. In this framework, organs-on-a-chip represent promising tools to reproduce key features of functional tissue units within microscale culture chambers. These systems offer the possibility to investigate the effects of biochemical, mechanical, and electrical stimulations, which are usually applied to enhance the functionality of the engineered tissues. Since the functionality of muscle tissues relies on the 3D organization and on the perfect coupling between electrochemical stimulation and mechanical contraction, great efforts have been devoted to generate biomimetic skeletal and cardiac systems to allow high-throughput pathophysiological studies and drug screening. This review critically analyzes microfluidic platforms that were designed for skeletal and cardiac muscle tissue engineering. Our aim is to highlight which specific features of the engineered systems promoted a typical reorganization of the engineered construct and to discuss how promising design solutions exploited for skeletal muscle models could be applied to improve cardiac tissue models and vice versa.

Keywords: cardiac muscle; electrical stimulation; heart; in vitro 3D model; mechanical stimulation; microfluidic; organ-on-a-chip; skeletal muscle.

Figure 1

The hierarchical structure of the skeletal muscle. Reproduced from [19], Creative Commons Attribution License.

Figure 2

Structure of the myocardium functional unit. Reproduced from [30], Creative Commons Attribution License.

Figure 3

Microfluidic models of skeletal muscle. (i-a) Schematics and detail of a molecular mechanics assay chamber (left) and prototype of a microfluidic device (right) which allows to introduce chemicals without creating bulk flows compromising mechanics measurements. Reproduced by permission of the American Chemical Society [32]; (i-b) Microfluidic device for chemotaxis and chemokinesis assay. A microcapillary array separates the cell culture chamber from the outer channels which allow to introduce reagents and create a uniform concentration or a gradient in the chamber. Reproduced by permission of the Royal Society of Chemistry [33]; (ii-a) Generation of cell-laden microfibers containing different types of cells with a double coaxial microfluidic device; (ii-b) NIH/3T3 cells encapsulated within fibrin hydrogel at day 0 and (ii-c) cell fiber at day 5. Scale bar: 100 μm; (ii-d) Acid-solubilized type-I collagen fiber containing NIH/3T3 cells at day 4 post-seeding. Scale bar 500 μm. Reproduced by permission of the Nature Publishing Group [34]; (iii-a) Skeletal muscle microtissue immunofluorescence (cell membrane bound green fluorescent protein (GFP) signal and nuclear staining (red)) showing uniform cell distribution; (iii-b) CAD modeling of a skeletal muscle microtissue; (iii-c) F-actin (red) staining demonstrating myoblast alignment after three days of culture and (iii-d) actin filament remodeling within multinucleated myotubes (nuclei are stained in green); (iii-e) Effect of structural (interpost distance, cantilever width) and mechanical (cantilever stiffness) parameters on the skeletal muscle microtissue. Scale bars: 100 μm. Reproduced by permission of the Royal Society of Chemistry [16]; (iv-a) Schematic of the engineered neuromuscular junction within a microfluidic device; (iv-b) schematic of the insertion of a carbon fiber nanoelectrode (CFNE) within the synapse for amperometric measurements and of a glass nanopipette inside a muscle cell (red) to record the post-synaptic potential. SCG: superior cervical ganglion. Reproduced by permission of Wiley-VCH [35]; (iv-c) simplified sketch showing a vascularized muscle-mimicking microenvironment for cancer cell extravasation studies. Endothelial cells (green), cancer cells (red) and muscle cells (yellow); and (iv-d) GFP-labelled microvascular network embedded within organ-specific matrix in a microfluidic device. Reproduced by permission from Jeon, J.S.; et al. [36].

Figure 4

Platforms designed to create in vitro cardiac tissue models to investigate different features of myocardial organization. At cellular level, the muscular thin film (MTF) [49] used to investigate the cardiac functional contractility related to the sarcomere organization: (i-a) schematic step-by-step representation of MTF fabrication processes and (i-b) the fluidic device assembly. At the functional unit level, the microfabricated tissue gauges (µTUGs) [51] exploited to study the functional contractility related to cell alignment and reorganization: (ii-a) process flow of µTUGs creation; (ii-b) immunofluorescence of ECM and cytoskeletal proteins in microtissues and (ii-c) time course of a contracting cardiac microtissue. At tissue level, the microengineered cardiac tissue (µECTs) [18] showing physiological beating in response to physiological mechanical stimulation: (iii-a) design of the 3D heart-on-chip microdevice used to impose a strain to the tissue by pressurizing the bottom compartment; (iii-b) immunofluorescence of cardiac troponin I (green) and connexin 43 (red) and (iii-c) sarcomeric-α-actinin (red) after five days in culture (scale bar 100 µm); (iii-d) evaluation of the contraction period in four different areas of the constructs showing the beating synchronicity reached by tissues. To study the interaction with other cells, platforms to reproduce the synaptic pathway [52] (iv-a,iv-b) and the cardiac vasculature [53] (iv-c,iv-d): (iv-a) seeding procedure of PDMS microchambers on a micro electrode array (MEA) surface to separately co-culture neurons and myocytes; (iv-b) fluorescence immunostaining showing the neurite extending in the myocyte compartment; (iv-c) scheme of designed platform to create vasculature around cardiac muscle spheroid (CS) and (iv-d) fluorescence microscopy of CD31-stained (green) vessels within the microtissues (scale bar 200 µm).