Contractile force assessment methods for in vitro skeletal muscle tissues

Abstract

Over the last few years, there has been growing interest in measuring the contractile force (CF) of engineered muscle tissues to evaluate their functionality. However, there are still no standards available for selecting the most suitable experimental platform, measuring system, culture protocol, or stimulation patterns. Consequently, the high variability of published data hinders any comparison between different studies. We have identified that cantilever deflection, post deflection, and force transducers are the most commonly used configurations for CF assessment in 2D and 3D models. Additionally, we have discussed the most relevant emerging technologies that would greatly complement CF evaluation with intracellular and localized analysis. This review provides a comprehensive analysis of the most significant advances in CF evaluation and its critical parameters. In order to compare contractile performance across experimental platforms, we have used the specific force (sF, kN/m²), CF normalized to the calculated cross-sectional area (CSA). However, this parameter presents a high variability throughout the different studies, which indicates the need to identify additional parameters and complementary analysis suitable for proper comparison. We propose that future contractility studies in skeletal muscle constructs report detailed information about construct size, contractile area, maturity level, sarcomere length, and, ideally, the tetanus-to-twitch ratio. These studies will hopefully shed light on the relative impact of these variables on muscle force performance of engineered muscle constructs. Prospective advances in muscle tissue engineering, particularly in muscle disease models, will require a joint effort to develop standardized methodologies for assessing CF of engineered muscle tissues.

Keywords: contractile force; neuroscience; skeletal muscle; stimulation; tissue engineering.

Figure 1.. Skeletal muscle structure and requirements for contractile force production.

(Top) Summary of the main requirements to enable contractility in skeletal muscle in vitro models. (Middle) Representation of the muscular hierarchy and (bottom) summary of contractile stimuli and contraction profiles.

Figure 2.. Cross-sectional area (CSA) in 2D and 3D muscle models.

(A) Myotube CSA estimated as an elliptical shape from the thickness and the width of the cell. (B) CSA of 3D muscle constructs can be estimated by approximation to different shapes (circle, in left panel), or calculated from immunohistochemical sections. Effective-CSA is known as the area occupied by myotubes (red area in the right panel).

Figure 3.. Cantilever deflection setup.

(A) The beam deflects due to myotube contraction (Left). In this case, cantilever deflection is interrogated by a laser beam and detected using a photodetector (Right). Commonly, cantilever arrays are made of Silicon (Si) or PDMS. Different coatings (FN, laminin, collagen I) have been tested to improve cell attachment and longer culture times. (B) Human myotubes on silicon cantilevers in bright field, top view (top) and immunostained for Myosin Heavy chain, side view (above). Scale bar: 50 µm. (C) Representative images from healthy and DMD myotubes at baseline (i and iii) and peak stress (ii and iv). Blue rectangles represent film length. Red lines represent the tracking of the film edge. Yellow arrows represent the distance between the projected film length and the unstressed film length. The yellow horizontal lines represent the change in projected film length from baseline stress (top bar) to peak stress (bottom bar).

Figure 4.. Post Deflection features.

(A) In vitro skeletal muscle is grown between two micropost which serve as anchors (tendons). As muscle contracts in response to a stimulus, posts bend proportionally. By tracking these displacements and knowing the mechanical characteristics of the platform, the force exerted by the muscle can be quantified. (B) Micropost displacement due to miniature bioartificial muscle (mBAM) contraction in response to a maximum tetanic electrical stimulus. Scale bar: 100 µm. (C) Formation of human skeletal muscle micro-tissue (hMMTs). Phase-contrast images depicting the remodeling of the ECM by human myoblast over time. Muscle construct immunostained (2 weeks) for sarcomeric α-actinin (SAA, red) and counterstained with DRAQ5 (1, 5-bis{[2-(di-methylamino)ethyl]amino}–4, 8-dihydroxyanthracene-9, 10-dione) nuclear stain in blue. Scale bar: 500 µm. Reprinted from Figure 2A and C from Afshar et al., 2020.

Figure 5.. Force transducers.

(A) In vitro 3D tissue is grown between two anchors. To assess contraction force, one of its sides is connected to a force transducer which will evaluate the force exerted by the muscle due to stimuli. (B) Representative contractile properties of hPSC-derived iSKM bundles. TRiPS-derived bundle (4 weeks) shows increases in contractile force with an increase of stimulation frequency up to the formation of tetanic contraction. Specific force and tetanic-to-twitch ratio of H9 and TRiPS-derived bundle (2 weeks) and (C) (Left) two-week differentiated iSKM bundles pair anchored within a nylon frame. (Right) Representative immunostaining of dense, uniformly distributed myotubes in bundle-CSA. Panel B reprinted from Figures 3A, B and 4A from Rao et al., 2018.

Figure 6.. Overview of the different techniques used to measure contractile force in vitro.

* Represents de % of studies that have performed this measurement.

Figure 7.. Functional characteristics of in vitro 2D and 3D skeletal muscle tissues from C2C12 and human sources (immortalized, iPSC and primary myoblast).

(A) Whole cross-sectional area (CSA) of muscle tissues. (B) Tetanic-to-Twitch ratio was calculated from data within the same study, except for bar with a diagonal pattern in post deflection, which was calculated from two different studies. (C) Twitch and Tetanic specific force measure in the three platforms for C2C12 constructs and (D) Human source. Data is presented as mean ± SEM. *p < 0.05, unpaired t-Test.