Bioprinting of Liquid Hydrogel Precursors in a Support Bath by Analyzing Two Key Features: Cell Distribution and Shape Fidelity

Microextrusion-based bioprinting within a support bath material is an emerging additive manufacturing technique for fabricating complex three-dimensional (3D) tissue constructs. However, there exists fundamental knowledge gaps in understanding the spatiotemporal mapping of cells within the bioprinted constructs and their shape fidelity when embedded in a support bath material. To address these questions, this paper advances quantitative analyses to systematically determine the spatial distribution for cell-laden filament-based tissue constructs as a function of the bio-ink properties. Also, optimal bio-ink formulations are investigated to fabricate complex 3D structures with superior shape integrity. Specifically, for a 1D filament printed in a support bath, cells suspended in low viscosity liquid hydrogel precursors are found to exhibit a characteristic non-uniform distribution as measured by a degree of separation (D_s) metric. In a 2D square wave pattern print, cells are observed to flow and aggregate downstream at certain positions along the in-plane print direction. In a 3D analysis, owing to the high cell density and gravity effects, a non-uniform cell distribution within a printed cylindrical structure is observed in the build direction. From the structural standpoint, the addition of CaCl₂ to the support bath activates the hydrogel cross-linking process during printing, resulting in 3D prints with enhanced structural outcomes. This multidimensional print analysis provides evidence that, under the emerging bioprinting support bath paradigm, the printable parameter space can be extended to low viscosity liquid hydrogel precursor materials that can be systematically characterized and optimized for key process performance outcomes in cell distribution and shape fidelity.