A Methodology for Quantifying Cell Density and Distribution in Multidimensional Bioprinted Gelatin–Alginate Constructs

Abstract

Bioprinted tissue constructs can be produced by microextrusion-based materials processing or coprinting of cells and hydrogel materials. In this paper, a gelatin–alginate hydrogel material formulation is implemented as the bio-ink toward a three-dimensional (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 both 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 single printed fiber shape in the circular cross-sectional view are observed to be dependent on the process temperature and material concentration parameters, along with the initial bio-ink cell densities. This is illustrated by reliable fabrication verified by image line profile analyses of structural fiber prints. Round fiber prints with width 809.5 ± 52.3 μm maintain dispersive cells with a degree of dispersion (Dd) at 96.8 ± 6.27% 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 width 1102.2 ± 63.66 μm coalesce cells toward the fiber midline with Dd = 76.3 ± 4.58% that can be fabricated at low relative material viscosities under high temperature (24 °C) or low material concentrations (7.5% w/v gelatin). A gradual decrement of Dd (from 80.34% to 52.05%) is observed to be a function of increased initial bio-ink cell densities (1.15 × 106–16.0 × 106 cells/ml). In the two-dimensional (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 the 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 cell sedimentation, the dynamics of cell distribution within the original bio-ink cartridge or material reservoir yield initial quantitative increases in the cell density for the first several printed layers, followed by quantitative decreases in the subsequent printed layers. Finally, during incubation, the evolution of cell density and the emergence of material degradation effects are studied in 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 a 48 h time course study exhibit a time-dependent increase in cell density owing to proliferation within the constructs that are presumed to affect the rate of bio-ink material degradation.