Skeletal muscle, comprising 20-50% of human body weight, is crucial for movement and responsiveness. Musculoskeletal conditions are increasingly prevalent, yet there's a lack of effective disease-modifying medications. Current drug therapies targeting pathways like androgen or myostatin show mixed results. Drug development is lengthy and expensive, with a high failure rate in clinical trials. Three-dimensional (3D) microphysiological systems (MPS) offer improved translatability by mimicking human physiology *in vitro*. MPS enable drug candidate testing on human tissue models, including personalized patient-derived models. Skeletal muscle possesses lifelong growth and regeneration due to stem cells. While 2D cultures have been used to identify drug candidates, they lack the ability to assess core muscle function like contractile force and fatigue. Previous attempts at creating 3D models of contracting skeletal muscle have lacked the reliability and robustness for routine drug screening. The authors previously developed a 3D bioprinting platform for human skeletal muscle tissue models, but these models showed poor myofiber differentiation due to the inert bioink used. Matrigel, an extracellular matrix protein extract, is commonly used to support cell growth and differentiation, despite its tumor origin. While it has been used in 3D skeletal muscle models, it has rarely been used as a sole bioink for 3D bioprinting. This study aimed to utilize Matrigel as a bioink for a microvalve-based drop-on-demand (DOD) 3D bioprinting of human skeletal muscle precursor cells to create a robust and reliable model for drug screening and functional studies.

The literature review extensively discusses the limitations of current preclinical drug discovery methods for muscle wasting diseases, highlighting the low success rate of drug candidates due to issues like insufficient activity and safety profiles. The need for more translatable in vitro models, particularly 3D microphysiological systems (MPS), is emphasized. The existing literature on 2D and 3D skeletal muscle tissue culture models is reviewed, highlighting the limitations of 2D models in assessing core muscle function and the inconsistencies in previous 3D models. The use of Matrigel in 3D tissue engineering, its properties, and its limitations are also discussed. The authors' previous work on a 3D bioprinting platform for human skeletal muscle tissue models is presented, alongside its limitations. The review demonstrates the significant gap in technology that this research attempts to fill by developing a robust and reliable 3D in vitro model of human skeletal muscle for drug screening.

The study developed a cooled printhead system for a 3D bioprinter to enable the microvalve-based DOD 3D bioprinting of cell-laden Matrigel. Rheological properties of Matrigel at different concentrations were analyzed to determine optimal printability and gelation. Primary human muscle precursor cells were suspended in Matrigel and printed in 24-well plates on agarose substrates. The dumbbell-shaped models were created with two inserted pipette tips as attachment points. The development of skeletal muscle tissue models was assessed through macroscopic and microscopic imaging, and gene expression analysis using qPCR. The expression of myogenic differentiation markers (Myf5, MyoD, Myog), structural genes (Actn2, Myhs), and myosin heavy chain (Myh) subtypes was measured. Histological analysis, including immunostaining for Myh, α-actinin, and F-actin, was performed. A custom-built electrical pulse stimulation (EPS) system for 24-well plates was used to induce contractions. The effects of EPS on Akt phosphorylation and IL-6 expression were analyzed by Western blotting and qPCR, respectively. Contractile force was measured using an organ bath force transducer apparatus, and the effects of caffeine and Tirasemtiv were assessed. The cleaning procedure for microvalves was also optimized to improve reuse.

The researchers successfully developed a cooled printhead system for reliable 3D bioprinting of cell-laden Matrigel. Optimal Matrigel concentration for printing and subsequent solidification was determined to be 8-10 mg/mL. The 3D-bioprinted models exhibited robust differentiation into aligned, striated, and contractile myofibers. The development of functional myofibers required the presence of flexible attachment points. The EPS system effectively induced contractions, which were blocked by blebbistatin (myosin inhibitor) and tetrodotoxin (TTX, voltage-gated sodium channel blocker). Repeated EPS stimulation (in vitro exercise) induced Akt phosphorylation (hypertrophy pathway activation) and IL-6 myokine expression. Caffeine (10 mM) significantly increased EPS-induced contractile force and prolonged contraction duration. Tirasemtiv (20 µM) rapidly and significantly enhanced EPS-induced contractile force. A dose-response curve for Tirasemtiv showed a half-maximal effect at 10 µM and a maximal effect at 20 µM.

This study successfully established a reliable and robust 3D in vitro model of human skeletal muscle using Matrigel 3D bioprinting. The use of Matrigel as a bioink, combined with flexible attachment points and a custom-built EPS system, enabled the development of functional, contractile myofibers that respond to in vitro exercise and known pharmacological agents. The results demonstrate the physiological relevance of the model and its potential as a high-throughput screening platform for drug development targeting muscle wasting diseases. The model's responsiveness to caffeine and Tirasemtiv validates its use for studying drug mechanisms and evaluating new therapies. The model's limitations (mostly slow and embryonal fiber types) and the need for further research focusing on optimizing fiber type composition are acknowledged.

This research successfully generated a functional 3D human skeletal muscle model using Matrigel 3D bioprinting. This model accurately reflects exercise-induced responses and pharmacological effects. It presents a promising platform for high-throughput drug screening in muscle wasting disease research. Future work should investigate different Matrigel batches, cell sources, and optimization of fiber types. Furthermore, exploring the model's suitability for personalized medicine approaches utilizing patient-derived cells should also be considered.

The study primarily utilized cells from a limited number of donors. The myofiber composition in the generated models mainly consisted of slow and embryonal types, which might not fully represent the diverse fiber types found in mature skeletal muscle. The in vitro exercise model used a short-term, high-impact stimulation protocol, which may not fully capture the complexity of long-term exercise effects. The study also relied on a custom-built EPS system, and its scalability and applicability to high-throughput screening need further assessment.