Research Papers

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

[+] Author and Article Information
Houzhu Ding

Stevens Institute of Technology,
1 Castle Point on Hudson,
Hoboken, NJ 07030
e-mail: hding4@stevens.edu

Filippos Tourlomousis

Stevens Institute of Technology,
1 Castle Point on Hudson,
Hoboken, NJ 07030
e-mail: ftourlom@stevens.edu

Robert C. Chang

Stevens Institute of Technology,
1 Castle Point on Hudson,
Hoboken, NJ 07030
e-mail: rchang6@stevens.edu

Manuscript received July 19, 2017; final manuscript received August 1, 2017; published online March 7, 2018. Editor: Y. Lawrence Yao.

J. Manuf. Sci. Eng 140(5), 051014 (Mar 07, 2018) (10 pages) Paper No: MANU-17-1463; doi: 10.1115/1.4037572 History: Received July 19, 2017; Revised August 01, 2017

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.

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Fig. 1

Formulation of 10% w/v cell-ink consisting of NHDF cells and gelatin–alginate solution

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Fig. 2

Fiber width and cell distribution for a single printed fiber (1D). (a) cell-laden hydrogel construct printed at 21.0 °C (7.5% w/v gelatin), (b) cell-laden hydrogel construct printed at 24.0 °C (7.5% w/v gelatin), (c) graph of average printed fiber width as a function of temperature for 7.5% w/v gelatin, (d) and (e) comparative schematics, bright-field images, binary images, and line profile analyses depicting the effect of higher process temperature (lower viscosity) on cross-sectional fiber morphology where resident cells are mechanistically “compressed” to locally cluster in-plane, (f) graph for the Dd is 96.8% and 76.3% for high viscosity and low viscosity prints, respectively, (g) bright-field images of single fibers depicting the cell distribution as a function of variable bio-ink cell densities, and (h) graph of the measured Dd trend from (g) shows a decrement of Dd with increasing bio-ink cell density at 24.0 °C (7.5% w/v gelatin).

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Fig. 3

A 2D analysis of cell distribution and cell density. (a) cell-laden construct printed at 21.0 °C, 7.5% w/v gelatin, (b) cell-laden construct printed at 21.0 °C, 10% w/v gelatin where a higher gelatin concentration yields comparatively irregular fiber widths, with higher viscosity material conferring mechanical support for subsequent layering, (c) the ratio for the printed cell density at the fiber cross and strut regions (≈1.8) is preserved for the different prescribed initial bio-ink cell densities (for 7.5% w/v gelatin and process temperature of 21.0 °C), (d) printed cell density at the cross and strut regions diverges for the different gelatin concentrations, (e) printed cell density for the two regions at different printing temperature using an initial cell density of 8.0 × 106 cells/ml, and a comparison of fiber images in (f) and (g) shows that for the same initial cell density (8.0 × 106 cells/ml), cells are well-dispersed initially (t = 0 min) and exhibit significant cell aggregation as a function of print time (t = 20 min).

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Fig. 4

A 3D analysis of the printed cell density trend for different layers within a multilayered cell-laden construct. (a) Twenty printed layers of cell-laden construct where each layer is defined as a 2D print of an orthogonally patterned fiber mesh structure, (b) schematic shows that each layer within the 3D construct is measured to have a different printed cell density (light green indicates low relative cell density, dark green indicates high relative cell density), (c) bright-field microscopy image of single layer structure (10% w/v gelatin), (d) color image of single layer construct, (e) live/dead cell fluorescent image shows cell distribution within hydrogel, and (f) graph of the measured density distribution trend at different printing time points where time T0 represents the time point at which the initial cell suspension is loaded into the material reservoir.

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Fig. 5

Qualitative analysis of the bulk mechanical properties for a multilayered printed construct. (a)–(d) illustrates the effects of structural design variables (number of layers and grid size) and postprint hydrogel cross-linking on the 3D structural integrity where (c) and (d) are cross-linked analogs of (a) and (b), respectively. In the presence of a small applied load to thecross-linked constructs, the ten-layered structure (c) is susceptible to motion, where the connecting wall of fibers assumes a wave-like structure, signifying bulk deformation. The 20-layered structure (d) maintains a higher stiffness whereby external loading does not yield significant bulk deformation.

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Fig. 6

Multilayered cell-laden constructs at different initial cell densities are incubated, and the average cell density is measured over time. Construct degradation rate is quantified by measuring the fiber strut width. (a)–(e) shows the image of printed 3D constructs at different culture times, (b)–(e) are submerged in cell media with an initial cell density of 0.6 × 106 cells/ml, (f) shows average cell density variations as a function of culture time, and (g) graphs the variation in average fiber strut width as a function of incubation time, presumed to be a structural indicator of degradation rate for the printed construct.



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