Xolography is a light-induced additive manufacturing technique for innovative volumetric 3D printing which uses dual-color photoswitchable photoinitiators to induce local polymerization of a precursor solution at the intersection of light beams of different wavelengths. Compared to other volumetric methods, xolography can print with higher resolution and volume generation rate, as well as more degree of geometrical freedom. The printing precision and design freedom make xolography promising for applications that require control over material features at hundreds of micrometer-scale resolutions, such as interaction with cell growth processes for tissue engineering. In muscle tissue engineering, for example, creating constructs with topographical cues that guide myotube formation and alignment is critical for producing functional contractile structures.

While xolographic printing of biocompatible resins has been shown to generate micro-structured scaffolds that can subsequently be seeded with muscle cells, its application for simultaneous fabrication of synthetic and living materials (3D bioprinting) remains unexplored. This limits the applicability of the technique to rapid, efficient, and high-resolution biomanufacturing processes which are crucial for fabricating macro-scale engineered tissues with micro-scale features. Therefore, investigating its application in 3D bioprinting with tissue-specific bioinks becomes critical for advancing the field.

Here we aimed to use xolography for 3D bioprinting of cells embedded in GelMA hydrogels-based bioinks and demonstrate cell survival during printing and the engineering of skeletal muscle tissues from C2C12 myoblast cell line, which features complex geometries, such as aligned microgroove structures.

Our results confirmed the cytocompatibility of the printing procedure. Cells remained viable (>60%) after exposure to both the photoinitiator and co-initiator at their working concentration, as well as to the basic (pH = 9.5) environment required for optimal photoinitiator activation. Furthermore, we replicated the printing process outside of the printer using a single wavelength (375 nm) and successfully crosslinked cell-laden GelMA hydrogels using the designed photoinitiator and co-initiator. The hydrogels retained structural integrity for >7 days and allowed for long-term cell survival and tissue formation, as shown via confocal imaging and histological analysis.

By using 3D xolographic bioprinting, we expect to rapidly and precisely fabricate constructs with tissue-specific geometries, such as highly aligned muscle fibers and integrated neuromuscular tissues, with exceptional resolution and spatial control difficult to achieve with common bioprinting techniques, thus addressing significant design challenges in biomimetic tissue engineering.