Flexure-based platform enables precise, quantitative, and automated monitoring of engineered tissue forces

Overview:: We have developed a platform that leverages flexures to decode the dynamics of muscle contraction with high resolution in force and time, outperforming the robustness and sensitivity of existing techniques to quantify tissue contractility. In this talk, we will showcase how muscle performance can be precisely tuned by modulating stimulation parameters, setting the stage for leveraging engineered muscle as actuators in next-generation biohybrid robots.


Introduction:: Multicellularity is the key to the adaptive functional behaviors observed in living systems. Electrical, biochemical, and mechanical signaling between cells of the same type, cells of different types, and with their surrounding matrix result in collective behaviors that are far more complex than achievable by individual cells alone. Scientists have developed robust tools and techniques, such as micro-invasive electrodes and immunohistochemical imaging, to map electrical and biochemical signaling within tissues. However, mapping and modulating mechanical cues within tissues in a precise, reproducible, and quantitative manner remains a challenge. In the context of tissues that produce force and generate motion, such as skeletal, cardiac, and smooth muscle, this signaling is particularly important to monitor and control.

The standard platform to measure forces generated by engineered tissues are cantilever-based devices in which the force required to deform a beam is calculated as a function of the beam geometry and material properties, as well as the tissue placement, or "moment arm", on the beam (Fig. 1a). As tissue shape and placement is inherently irregular, and requires manual measurement, there is significant room for measurement uncertainty in calculating forces. We have leveraged machine design methodologies to manufacture a flexure-based platform for precise, quantitative, and automated monitoring of engineered tissue forces. High-accuracy force tracking enables deeper understanding of inter-tissue variability, advancing our understanding of how slight changes in biological variables can dramatically impact the biomechanics of engineered tissues.


Materials and Methods:: We have designed and machined a device made of a biocompatible nylon polymer and composed of rigid posts coupled to flexural springs with precisely controllable stiffness. Engineered tissues (skeletal muscle) are placed on rigid posts, and the passive tension and active contractile forces they generate deform the flexural springs (Fig. 1b). Monitoring post movement through automated image processing techniques (custom MATLAB script) enables precisely quantifying tissue deformation. Treating muscle fibers as strain-limited actuators with a known maximum deformation, the system can be modeled as two springs in series: an extracellular matrix spring and a flexure spring. Muscle force (F_m) is thus a function of post movement (x), tissue stiffness (k_m), and flexure stiffness (k_f): F_m = x * (1 + k_f/k_m) * k_m

Engineered skeletal muscle tissues are manufactured using our established published techniques [1]. Briefly, optogenetic C2C12 mouse myoblasts are embedded within a natural hydrogel support matrix composed of Matrigel and fibrin. Embedded myoblasts are encouraged to fuse into multinucleated muscle fibers by immersion of differentiation media composed of 10% (v/v) horse serum and 50 ng mL^-1 human insulin-like growth factor 1, IGF-1. Tissues are manually transferred to the posts of flexure-based platforms and active contraction is stimulated with blue light pulses at 470 nm. Videos were recorded on a Leica stereoscope M165FC with a high frame rate camera and processed in MATLAB. Following 10 days of growth and experimentation, tissues are sacrificed either for measuring matrix mechanical properties or fixed for immunohistochemical analysis of fiber maturation and alignment.


Results, Conclusions, and Discussions:: Our device’s rigid posts enable two unique and important features: 1) The posts do not deform in response to tissue forces, but rather transmit forces to flexural springs, and are thus insensitive to tissue geometry (i.e. irregular cross-sectional area) or placement (i.e. moment/lever arm distance between the muscle and the flexure). 2) The posts have a defined geometry and are machined with high-precision, enabling automated and reliable image-based tracking across thousands of frames within a video. These unique features have enabled us to calculate the passive tension and active contractile forces generated by muscle grown in a variety of culture conditions. First, we confirmed that the forces measured using our established cantilever-based technique and our novel flexure-based technique were not significantly different, but that the flexure-based devices significantly reduced the standard deviation of forces measured in a single tissue and across several tissues grown in similar conditions (data not presented). This reduction in measurement error enabled us to see, for the first time, how different batches of Matrigel (a natural extracellular matrix mimic that is broadly relevant across tissue engineering) could impact tissue function (Fig. 1c). Moreover, as our flexure-based device had a precisely defined coordinate system, we were able to perform high frame rate video recording to quantify, for the first time, the contraction speed and relaxation response of engineered muscle (Fig. 1d) across multiple stimulation parameters - varying frequency, duty cycle, light intensity, etc. Our new platform also enabled the first measurements, to our knowledge, of how fatigue within engineered muscles is mediated by fiber type transformation via exercise training (Fig. 1e). After 2 weeks of force monitoring over the course of muscle maturation and remodeling, we fixed and stained the tissues to correlate functional observations with tissue morphology. We have developed a tool that enables automated, quantitative, and reliable measurements of tissue forces that will enable applications including high-throughput diagnostics and drug testing. While our device has been designed for researching muscle adaptation, it is broadly applicable across cell types, and promises to be an enabling platform technology for biomechanical monitoring of engineered tissues.


Acknowledgements (Optional): : NSF CAREER Award 2238715

References (Optional): : Raman, R., Cvetkovic, C. and Bashir, R., 2017. A modular approach to the design, fabrication, and characterization of muscle-powered biological machines. Nature protocols, 12(3), pp.519-533.