A Methodology for Quantifying Cell Density and Distribution in Multidimensional Bioprinted Gelatin–Alginate Constructs
A Methodology for Quantifying Cell Density and Distribution in Multidimensional Bioprinted Gelatin–Alginate Constructs
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DOI：
10.1115/1.4037572
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发表时间：
2018-05
影响因子：
4
通讯作者：
Houzhu Ding;F. Tourlomousis;R. Chang
中科院分区：
文献类型：
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作者：
Houzhu Ding;F. Tourlomousis;R. Chang
生物打印的组织构建体可以通过基于微挤出的材料加工或细胞和水凝胶材料的共打印来生产。在本文中，明胶-藻酸盐水凝胶材料配方被实施为朝向三维（3D）细胞负载组织构造的生物墨水。然而，在印刷过程中，最重要的是在不同尺寸尺度下产生最终泡孔分布和泡孔密度的各种参数之间的相互作用。为了研究这些影响，本研究提出了一个多维分析框架，以确定生物打印细胞负载结构中细胞分布和细胞密度的空间变化和时间演变。在一维（1D）分析中，观察到圆形横截面视图中的细胞分布和单个打印纤维形状取决于工艺温度和材料浓度参数，沿着初始生物墨水细胞密度。这是说明了可靠的制造验证的图像线轮廓分析的结构纤维打印。宽度为809.5 ± 52.3μm的圆形纤维印刷品保持分散单元，分散度（Dd）为96.8 ± 6.27%，这可以在低温条件（21 °C）或高材料浓度（10%w/v明胶）下在高相对材料粘度下实现。另一方面，宽度为1102.2 ± 63.66μm的扁平纤维印刷品使细胞朝向纤维中线聚结，Dd= 76.3 ± 4.58%，其可以在高温（24 °C）或低材料浓度（7.5%w/v明胶）下在低相对材料粘度下制造。随着初始生物墨水细胞密度的增加（1.15 × 106-16.0 × 106 cells/ml），Dd逐渐减小（从80.34%到52.05%）。在二维（2D）分析中，印刷的网格结构产生不同的单元分布，由此在印刷结构内的支柱和交叉区域之间观察到局部单元密度的差异。在低相对粘度下，细胞聚集在两个重叠细丝融合在一起的交叉区域，在交叉区域和支柱区域之间产生2.06 ± 0.44的细胞密度比。然而，在高相对粘度下，泡孔密度比降低至0.96 ± 0.03。在3D分析中，将归因于不同层的细胞密度作为从初始生物墨水配方开始经过的打印时间的函数进行研究。由于可识别的细胞沉降，原始生物墨盒或材料储存器内的细胞分布的动态导致前几个打印层的细胞密度的初始定量增加，随后是随后的打印层的定量减少。最后，在孵育过程中，细胞密度的演变和材料降解效应的出现进行了研究，在一个时间过程的研究。在48 h时程研究中，打印并交联成载有细胞的构建体的可变初始细胞密度（0.6 × 106个细胞/mL、1.0 × 106个细胞/mL和无细胞对照组）显示出细胞密度的时间依赖性增加，这是由于构建体内的增殖，推测其影响生物油墨材料降解速率。
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 ± 52.3μm 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 ± 63.66μm 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 × 106cells/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 × 106cells/mL, 1.0 × 106cells/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.