A Methodology for Quantifying Cell Density and Distribution in Multidimensional Bioprinted Gelatin-Alginate Constructs Available to Purchase

Bioprinted tissue constructs are enabled by microextrusion-based co-printing of cells and hydrogel materials. In this paper, a gelatin-alginate hydrogel material formulation is implemented as the bio-ink towards a 3D cell-laden tissue construct. However, of fundamental importance during the printing process is the interplay between the various parameters that yield the final cell distribution and cell density at different dimensional scales. To investigate these effects, this study advances a multidimensional analytical framework to determine the spatial variations and temporal evolution of cell distribution and cell density within a bioprinted cell-laden construct. In the one dimensional (1D) analysis, the cell distribution and cross-sectional shape for a single printed fiber are observed to be dependent on the process temperature and material concentration parameters. This is illustrated by the reliable fabrication and image line profile analysis of the fiber prints. Round fiber prints with a measured width of 809.5±52.3 μm maintain dispersive cells with a degree of dispersion (D_d) at 96.8 % that can be achieved at high relative material viscosities under low temperature conditions (21 °C) or high material concentrations (10 % w/v gelatin). On the other hand, flat fiber prints with a measured width of 1102.2±63.6 μm coalesce cells towards the fiber midline with D_d = 76.3% that can be fabricated at low relative material viscosities under high temperature (24 °C) or low material concentrations (7.5 % w/v gelatin). In the 2D analysis, a printed grid structure yields differential cell distribution whereby differences in localized cell densities are observed between the strut and cross regions within the printed structure. At low relative viscosities, cells aggregate at the cross regions where two overlapping filaments fuse together, yielding a cell density ratio of 2.06±0.44 between the cross region and strut region. However, at high relative viscosities, the cell density ratio decreases to 0.96±0.03. In the 3D analysis, the cell density attributed to the different layers is studied as a function of printing time elapsed from the initial bio-ink formulation. Due to identifiable gravity and extrusion process-induced effects, the cell distribution within the original bio-ink cartridge or material reservoir is altered over time to yield initial quantitative increases in the cell density over the first several printed layers, followed by quantitative decreases in the subsequent printed layers. Finally, in the time-dependent analysis, the evolution of cell density and the emergence of material degradation effects is studied over a time course study. Variable initial cell densities (0.6 × 10⁶ cells/ml, 1.0 × 10⁶ cells/ml, and acellular control group) printed and cross-linked into cell-laden constructs for the 48 hr time course study exhibit a time-dependent increase in cell density owing to proliferation within the constructs that are presumed to accelerate the degradation rate.