A 96-well culture platform enables longitudinal analyses of engineered human skeletal muscle microtissue strength

Abstract

Three-dimensional (3D) in vitro models of human skeletal muscle mimic aspects of native tissue structure and function, thereby providing a promising system for disease modeling, drug discovery or pre-clinical validation, and toxicity testing. Widespread adoption of this research approach is hindered by the lack of easy-to-use platforms that are simple to fabricate and that yield arrays of human skeletal muscle micro-tissues (hMMTs) in culture with reproducible physiological responses that can be assayed non-invasively. Here, we describe a design and methods to generate a reusable mold to fabricate a 96-well platform, referred to as MyoTACTIC, that enables bulk production of 3D hMMTs. All 96-wells and all well features are cast in a single step from the reusable mold. Non-invasive calcium transient and contractile force measurements are performed on hMMTs directly in MyoTACTIC, and unbiased force analysis occurs by a custom automated algorithm, allowing for longitudinal studies of function. Characterizations of MyoTACTIC and resulting hMMTs confirms the capability of the device to support formation of hMMTs that recapitulate biological responses. We show that hMMT contractile force mirrors expected responses to compounds shown by others to decrease (dexamethasone, cerivastatin) or increase (IGF-1) skeletal muscle strength. Since MyoTACTIC supports hMMT long-term culture, we evaluated direct influences of pancreatic cancer chemotherapeutics agents on contraction competent human skeletal muscle myotubes. A single application of a clinically relevant dose of Irinotecan decreased hMMT contractile force generation, while clear effects on myotube atrophy were observed histologically only at a higher dose. This suggests an off-target effect that may contribute to cancer associated muscle wasting, and highlights the value of the MyoTACTIC platform to non-invasively predict modulators of human skeletal muscle function. Skeletal muscle is one of the most abundant tissues in the human body and it enables critical physiological and functional activities, such as thermogenesis 1 and mobility 2. There are many degenerative and fatal diseases of skeletal muscle that remain untreated and the underlying pathology of some muscle related diseases is not fully understood. The use of animal models to study skeletal muscle diseases has improved our understanding of in vivo drug response and disease pathology 3,4. However, in some cases animal models fail to accurately predict drug response in humans, in part due to species specific differences leading to inaccurate disease symptoms 5,6. Furthermore, animal models are expensive and time consuming making them less desirable for drug testing 7. As a result, a push to establish in vitro models of human skeletal muscle with reliable phenotypic readouts for drug testing is underway with the goal of improving therapeutic outcomes in humans.

Coupling 3D Printing and Novel Replica Molding for In House Fabrication of Skeletal Muscle Tissue Engineering Devices

“…This method proved to be remarkably fast and user friendly, however, it carries one main limitation: the impossibility to obtain replicas with more complex geometries due to the mechanical damage that the peeling from a rigid mold can cause. [ 22 ] The hard, nondeformable mold makes the retention of features such as long and thin or T‐shaped pillars arduous. This obstacle led us to develop the next two methods.…”

“…However, reducing the diameter of a pillar can cause a contractile tissue to slip away from it due to the tension to which the bundle is subjected during culture. [ 17,22 ] Hence the necessity to incorporate a protrusion in the pillar’s design arises, leading to the more complex T‐shaped pillar suitable for supporting small tissues. To this end, a negative model consisting of a smaller, 8 mm long chamber of 30 µL with pillars of 750 µm in diameter and 3.2 mm in height in a T‐shape was realized (Figure 1b‐i; Figure S1b, Supporting Information).…”

“…To avoid accidental damage during demolding, conventional procedures typically require supplementary steps solely to fix caps on each pillar [ 9,10,33 ] or serial replica molding stages with high risk of failure. [ 22 ] Here, in around 48 h from design to final product, we inexpensively produced hundreds of faithful replicas with T‐shaped pillars without additional fabrication steps thanks to the implementation of the stretchable molds. Also in this case, like the Direct Peeling method, the time invested after the first replica decreases considerably, as both the 3D printed mold and the Ecoflex molds can be reused multiple times (Figure 5c, Table S3, Supporting Information).…”

“…Recently, a set of devices containing small hook‐shaped pillars has been created using a 3D printer, however, this method involved an expensive machine and was only partially successful due to the replica molding strategy based on hard, plastic molds. [ 22 ] Finally, it is possible to fabricate pillars‐equipped devices directly in plastic materials using affordable, off‐the‐shelf 3D printers, but these products are too rigid to allow contractile measurements in a noninvasive way. [ 23,24 ]…”

Global and local tension measurements in biomimetic skeletal muscle tissues reveals early mechanical homeostasis

“…These chambers are commonly based on polydimethylsiloxane (PDMS) molds that can be tuned in their elastic properties and are soft enough to allow measurement of muscle contraction forces by recording the post deflection. Such non-invasive measurements of force generation in reconstituted muscles are indeed based on the elastic properties of PDMS [1,29]. A downside of PDMS is that the poor optical properties paired with routinely used material thickness prohibits high numerical aperture objectives that are necessary for high resolution microscopy, and hence make modern 3D microscopy methods like confocal and spinning disk microscopy on living tissue impossible.…”