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Quantitative, spatial imaging based measurements to assess cellular health and oxygenation in a tissue engineered test system.

机译：基于定量空间成像的测量，以评估组织工程测试系统中的细胞健康状况和氧合作用。

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摘要

Three-dimensional in vitro tissue test systems are employed to examine cell behavior, test responses to drugs and vaccines, and answer basic biological questions. These systems are more physiologically relevant than two-dimensional cell cultures, and are more relevant, easier and less expensive to maintain than animal models. However, methods used to measure cell behavior and viability have been developed specifically for two-dimensional cell cultures or animal models, and are often not optimally translated to three-dimensional in vitro test systems. The purpose of this work was to aid in the development of three-dimensional, spatially controlled in vitro test systems, and to develop the corresponding quantitative, spatial measurement methods of cell behavior and viability. Optical widefield microscopy was selected as a measurement tool because of its ease of use, wide availability, and inherent large-scale spatial measurement capacity. Digital image analysis and processing were used to collect quantitative data. Fluorescent cellular labels were examined for use in spatial, quantitative imaging, and methods were developed to quantify cell location, morphology, and viability from either fluorescent or phase contrast images.;Maintenance of oxygen supply to cells is integral in a tissue engineered construct and in 3D in vitro test systems. Cells in the body are supplied oxygen by the vasculature, but in tissue engineered constructs, cells must be supported by oxygen diffusion alone. In addition to tracking cellular behavior, microscope digital image processing was used in conjunction with fluorescent oxygen-sensitive nanoparticles for the quantitative, spatial measurement of oxygen in a 3D in vitro test system. These methods were used to confirm the presence of oxygen gradients that occur in 3D cell cultures due to cellular oxygen consumption. Artifacts that impede quantitative fluorescence imaging were identified, and fluorescence ratio imaging was used to minimize artifacts and facilitate quantitative oxygen measurement.;In follow-up work, methods were developed to allow sterile microscope imaging and culture of cells in a 3D tissue engineered construct; the setup allowed spatial control of oxygen delivery to approximate oxygenation via vasculature in the body. An oxygen-gradient bioreactor was developed and imaging techniques were used to show that culture medium perfusion rates can be used to control the rate of distribution of a factor or gas, such as oxygen, throughout a tissue engineered construct.;Lastly, a 3D in vitro hydrogel test system with modular substrate stiffness was created and assessed using quantitative cellular imaging methods to examine cancer development. Cancer cell behavior has been shown to be strongly correlated to local stiffness variations in the extracellular matrix; however, this relationship is not well understood. Human breast cancer cells were cultured on hydrogel substrates of varying mechanical properties, and quantitative imaging and metabolic activity assays were used to examine cell behavior and viability. Phase contrast microscopy imaging and image processing were conducted to allow quantitative measurement of cell morphology. Mathematical modeling work performed by collaborators indicated both temporal and substrate-stiffness based effects on cancer cell colony size, number, and shape (perimeter). Continuing this work, hydrogel test systems with a spatial stiffness gradient were produced. Imaging methods were used to provide large-scale, quantitative measurement of cell density to estimate cell migration and growth as a function of both time and position on these spatially non-uniform substrates.;This research facilitated the development of methods for spatially controlling the mechanical properties of 3D tissue test systems as well as methods for spatial, quantitative measurement of cellular position, growth, and morphology. An oxygen gradient bioreactor was also designed and tested to simulate a more physiologically representative environment. The end goal of this research is to aid in the understanding of cancer development by creating robust, controllable cancer test systems that can be used to expose cells to predefined conditions and quantitatively measure resulting cellular behavior.
机译：三维体外组织测试系统用于检查细胞行为，测试对药物和疫苗的反应并回答基本的生物学问题。这些系统比二维细胞培养物在生理上更相关，并且比动物模型更相关，更容易维护且成本更低。但是，已经专门为二维细胞培养或动物模型开发了用于测量细胞行为和生存力的方法，并且这些方法通常无法最佳地转换为三维“体外”测试系统。这项工作的目的是帮助开发三维空间控制的体外italic测试系统，并开发相应的定量的空间行为和生存力测量方法。 光学宽视野显微镜被选作测量工具，是因为其易于使用，可用性高以及固有的大规模空间测量能力。使用数字图像分析和处理来收集定量数据。检查了荧光细胞标记物在空间，定量成像中的用途，并开发了从荧光或相衬图像定量细胞位置，形态和活力的方法。 3D 体外测试系统。脉管系统为体内的细胞提供氧气，但是在组织工程构造中，细胞必须仅靠氧气扩散来支撑。除了跟踪细胞行为，显微镜数字图像处理还与对氧气敏感的荧光纳米粒子一起用于3D体外测试系统中氧气的定量，空间测量。这些方法用于确认由于细胞耗氧而在3D细胞培养中出现的氧梯度的存在。鉴定了阻碍定量荧光成像的伪像，并使用荧光比率成像将伪影减至最少并促进定量氧的测量。在后续工作中，开发了允许无菌显微镜成像和在3D组织工程化构建体中培养细胞的方法；该设置允许对氧气的输送进行空间控制，以通过体内的脉管系统近似氧化。开发了一种氧气梯度生物反应器，并使用了成像技术来表明培养基灌注速率可用于控制整个组织工程构建体中因素或气体（例如氧气）的分布速率。最后，3D <建立了具有模块化基质刚度的体外水凝胶测试系统，并使用定量细胞成像方法评估了癌症的发展情况。癌细胞行为已被证明与细胞外基质的局部硬度变化密切相关。但是，这种关系还不是很清楚。将人类乳腺癌细胞培养在具有不同机械特性的水凝胶基质上，并使用定量成像和代谢活性测定法检查细胞行为和生存能力。进行相差显微镜成像和图像处理以允许定量测量细胞形态。合作者进行的数学建模工作表明，时间和基质刚度对癌细胞集落大小，数量和形状（周长）的影响。继续这项工作，生产了具有空间刚度梯度的水凝胶测试系统。成像方法用于提供大规模，定量的细胞密度测量，以估计细胞迁移和生长随时间和位置在这些空间不均匀基质上的变化。;这项研究促进了空间控制机械方法的发展3D组织测试系统的特性以及细胞位置，生长和形态的空间，定量测量方法。还设计并测试了氧梯度生物反应器，以模拟更具生理代表性的环境。这项研究的最终目的是通过创建健壮的，可控制的癌症测试系统来帮助人们理解癌症的发展，该系统可用于使细胞暴露于预定条件并定量测量所产生的细胞行为。

著录项

  • 作者

    Bland, Erik.;

  • 作者单位

    Clemson University.;

  • 授予单位 Clemson University.;
  • 学科 Biology Cell.;Engineering Biomedical.
  • 学位 Ph.D.
  • 年度 2012
  • 页码 194 p.
  • 总页数 194
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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