Automated Biofabrication of Striated Muscle Tissue Models

The development of physiologically relevant in vitro models is critically important for drug discovery and disease research. Traditional models often fail to replicate native tissue structure and function, limiting their predictive accuracy and therapeutic relevance. At the same time, engineered, high-complexity models may be more physiologically accurate, but if the cost and time of fabrication are too high, they may still not be of practical use. New biomaterials and biofabrication techniques may help balance this trade-off.

This thesis seeks to establish new 3D printing methodologies for automated and scalable fabrication of anisotropic tissue models. We mainly focused on cardiac and skeletal muscle tissues, where the anisotropic architecture is essential to replicating native electrophysiological and contractile functions. Yet, other tissues where unidirectional architectures are also found, such as the brain, where also been explored. Two complementary approaches were pursued: (Study 1) the creation of structured nozzles for extrusion-based printing to fabricate micro-patterned substrates, and (Study 2) the formulation of cross-linkable, cell-adhesive, structural hydrogel inks based on chemically modified cellulose nanofibers.

In Study 1, hot embossing was used to imprint microscale features onto the orifice of macroscopic nozzles. Such modifications of nozzles enabled rapid deposition of anisotropic patterns during 3D printing. Substrates printed with shear-thinning silicone ink demonstrated robust tissue-guiding properties across multiple cell types, including murine myoblasts, rat cardiomyocytes, human stem cell-derived cardiomyocytes, and neurons. Our approach gave a 40–60× increase in fabrication speed of micro-patterned substrates compared to earlier methods, enhanced cellular alignment, and maturation. Still, the micro-patterning approach was limited by its compatibility with a narrow range of materials and the inability of the patterned substrates to sustain long-term cell adhesion of muscle tissue, thereby limiting their maturation.

In Study 2, cellulose nanofibers were functionalized with maleimide, RGD peptides, and amino acids to yield xeno-free, biofunctional inks that were cross-linkable via dithiol agents or enzymes. Rheological and spectroscopic analyses confirmed tunable mechanical properties and successful biofunctionalization. We also conducted a proof-of-principle demonstration of their selective degradation using enzymes and compared the cytotoxicity of different crosslinking approaches. Here, we observed a notable cytotoxicity of several dithiol crosslinkers, while transglutaminase-driven crosslinking of amino acid-functionalized cellulose inks appeared more promising.

While further work is needed, e.g., for combining the two approaches, this thesis contributes to establishing scalable printing methodologies for creating advanced tissue models for biomedical and pharmaceutical research.