Department of Biomedical Engineering, Tufts University, Medford, Massachusetts, United States of America.
PLoS One. 2013;8(2):e55696. doi: 10.1371/journal.pone.0055696. Epub 2013 Feb 6.
The efficacy and economy of most in vitro human models used in research is limited by the lack of a physiologically-relevant three-dimensional perfused environment and the inability to noninvasively quantify the structural and biochemical characteristics of the tissue. The goal of this project was to develop a perfusion bioreactor system compatible with two-photon imaging to noninvasively assess tissue engineered human adipose tissue structure and function in vitro. Three-dimensional (3D) vascularized human adipose tissues were engineered in vitro, before being introduced to a perfusion environment and tracked over time by automated quantification of endogenous markers of metabolism using two-photon excited fluorescence (TPEF). Depth-resolved image stacks were analyzed for redox ratio metabolic profiling and compared to prior analyses performed on 3D engineered adipose tissue in static culture. Traditional assessments with H&E staining were used to qualitatively measure extracellular matrix generation and cell density with respect to location within the tissue. The distribution of cells within the tissue and average cellular redox ratios were different between static and perfusion cultures, while the trends of decreased redox ratio and increased cellular proliferation with time in both static and perfusion cultures were similar. These results establish a basis for noninvasive optical tracking of tissue structure and function in vitro, which can be applied to future studies to assess tissue development or drug toxicity screening and disease progression.
大多数用于研究的体外人类模型的功效和经济性受到缺乏生理相关的三维灌注环境以及无法非侵入性地定量组织的结构和生化特征的限制。本项目的目标是开发一种与双光子成像兼容的灌注生物反应器系统，以非侵入性地评估体外工程化的人脂肪组织的结构和功能。三维（3D）血管化的人脂肪组织在体外进行工程化，然后引入灌注环境，并通过使用双光子激发荧光（TPEF）自动定量内源性代谢标志物来跟踪随时间的变化。对深度分辨的图像堆栈进行分析，以进行氧化还原比代谢分析，并与在静态培养中进行的 3D 工程化脂肪组织的先前分析进行比较。传统的 H&E 染色评估用于定性测量细胞外基质生成和细胞密度相对于组织内的位置。与静态培养相比，组织内细胞的分布和平均细胞氧化还原比不同，而在静态和灌注培养中，氧化还原比降低和细胞增殖随时间增加的趋势相似。这些结果为体外组织结构和功能的非侵入性光学跟踪奠定了基础，可应用于未来的研究中，以评估组织发育或药物毒性筛选和疾病进展。
