RESOURCES

Abstract
A key to enhance the low translatability of preclinical drug discovery are in vitro human three-dimensional (3D) microphysiological systems (MPS). Here, we show a new method for automated engineering of 3D human skeletal muscle models in microplates and functional compound screening to address the lack of muscle wasting disease medication. To this end, we adapted our recently described 24-well plate 3D bioprinting platform with a printhead cooling system to allow microvalve-based drop-on-demand printing of cell-laden Matrigel containing primary human muscle precursor cells. Mini skeletal muscle models develop within a week exhibiting contractile, striated myofibers aligned between two attachment posts. As an in vitro exercise model, repeated high impact stimulation of contractions for 3 h by a custom-made electrical pulse stimulation (EPS) system for 24-well plates induced interleukin-6 myokine expression and Akt hypertrophy pathway activation. Furthermore, the known muscle stimulators caffeine and Tirasemtiv acutely increase EPS-induced contractile force of the models. This validated new human muscle MPS will benefit development of drugs against muscle wasting diseases. Moreover, our Matrigel 3D bioprinting platform will allow engineering of non-self-organizing complex human 3D MPS.

Abstract
Two-dimensional (2D) cell cultures do not reflect the in vivo situation, and thus it is important to develop predictive three-dimensional (3D) in vitro models with enhanced reliability and robustness for drug screening applications. Treatments against muscle-related diseases are becoming more prominent due to the growth of the aging population worldwide. In this study, we describe a novel drug screening platform with automated production of 3D musculoskeletal-tendon-like tissues. With 3D bioprinting, alternating layers of photo-polymerized gelatin-methacryloyl-based bioink and cell suspension tissue models were produced in a dumbbell shape onto novel postholder cell culture inserts in 24-well plates. Monocultures of human primary skeletal muscle cells and rat tenocytes were printed around and between the posts. The cells showed high viability in culture and good tissue differentiation, based on marker gene and protein expressions. Different printing patterns of bioink and cells were explored and calcium signaling with Fluo4-loaded cells while electrically stimulated was shown. Finally, controlled co-printing of tenocytes and myoblasts around and between the posts, respectively, was demonstrated followed by co-culture and co-differentiation. This screening platform combining 3D bioprinting with a novel microplate represents a promising tool to address musculoskeletal diseases.

Abstract
Acquired muscle diseases such as cancer cachexia are responsible for the poor prognosis of many patients suffering from cancer. In vitro models are needed to study the underlying mechanisms of those pathologies. Extrusion bioprinting is an emerging tool to emulate the aligned architecture of fibers while implementing additive manufacturing techniques in tissue engineering. However, designing bioinks that reconcile the rheological needs of bioprinting and the biological requirements of muscle tissue is a challenging matter. Here we formulate a biomaterial with dual crosslinking to modulate the physical properties of bioprinted models. We design 3D bioprinted muscle models that resemble the mechanical properties of native tissue and show improved proliferation and high maturation of differentiated myotubes suggesting that the GelMA-AlgMA-Fibrin biomaterial possesses myogenic properties. The electrical stimulation of the 3D model confirmed the contractile capability of the tissue and enhanced the formation of sarcomeres. Regarding the functionality of the models, they served as platforms to recapitulate skeletal muscle diseases such as muscle wasting produced by cancer cachexia. The genetic expression of 3D models demonstrated a better resemblance to the muscular biopsies of cachectic mouse models. Altogether, this biomaterial is aimed to fabricate manipulable skeletal muscle in vitro models in a non-costly, fast and feasible manner.

Abstract
Preclinical biomedical and pharmaceutical research on disease causes, drug targets, and side effects increasingly relies on in vitro models of human tissue. 3D printing offers unique opportunities for generating models of superior physiological accuracy, as well as for automating their fabrication. Towards these goals, we here describe a simple and scalable methodology for generating physiologically relevant models of skeletal muscle. Our approach relies on dual-material micro-extrusion of two types of gelatin hydrogel into patterned soft substrates with locally alternating stiffness. We identify minimally complex patterns capable of guiding the large-scale self-assembly of aligned, extended, and contractile human and murine skeletal myotubes. Interestingly, we find high-resolution patterning is not required, as even patterns with feature sizes of several hundred micrometers is sufficient. Consequently, the procedure is rapid and compatible with any low-cost extrusion-based 3D printer. The generated myotubes easily span several millimeters, and various myotube patterns can be generated in a predictable and reproducible manner. The compliant nature and adjustable thickness of the hydrogel substrates, serves to enable extended culture of contractile myotubes. The method is further readily compatible with standard cell-culturing platforms as well as commercially available electrodes for electrically induced exercise and monitoring of the myotubes.