Tissue clearing of human iPSC-derived organ-chips enables high resolution imaging and analysis.
Tissue clearing of human iPSC-derived organ-chips enables high resolution imaging and analysis.
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DOI：
10.1039/d2lc00116k
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发表时间：
2022-10-25
期刊：
影响因子：
6.1
通讯作者：
Svendsen CN
中科院分区：
文献类型：
--
作者：
Ondatje BN;Sances S;Workman MJ;Svendsen CN
经过改造的微流控器官芯片能够增加体外培养的人类干细胞来源组织的细胞多样性和功能。然而，这些三维(3D)培养在细胞蛋白质的可视化和量化方面遇到了独特的挑战。由于培养的神经组织具有致密的3D特性，经典的免疫细胞化学方法因光线和抗体透过率以及图像采集参数的次佳而变得复杂。此外，围绕感兴趣组织的复杂聚二甲基硅氧烷支架会阻碍高分辨率显微镜和空间分析。已经开发了超水组织清除方法来减轻体内组织成像的类似挑战。在这里，我们描述了一种适应这种方法的方法，以有效地清除生长在器官芯片上的人类多能干细胞来源的神经组织。我们还描述了在设计器官芯片中固有的复杂3D结构的基于信号强度的方法时的关键成像考虑因素。为了确定器官芯片中生长的细胞的形态和解剖学特征，我们开发了一种可靠的芯片切片和高分辨率显微采集和分析的方案。优化器官芯片和器官芯片切片的缩放方案，以提高可视化。
Engineered microfluidic organ-chips enable increased cellular diversity and function of human stem cell-derived tissues grown in vitro. These three dimensional (3D) cultures, however, are met with unique challenges in visualization and quantification of cellular proteins. Due to the dense 3D nature of cultured nervous tissue, classical methods of immunocytochemistry are complicated by sub-optimal light and antibody penetrance as well as image acquisition parameters. In addition, complex polydimethylsiloxane scaffolding surrounding the tissue of interest can prohibit high resolution microscopy and spatial analysis. Hyperhydration tissue clearing methods have been developed to mitigate similar challenges of in vivo tissue imaging. Here, we describe an adaptation of this approach to efficiently clear human pluripotent stem cell-derived neural tissues grown on organ-chips. We also describe critical imaging considerations when designing signal intensity-based approaches to complex 3D architectures inherent in organ-chips. To determine morphological and anatomical features of cells grown in organ-chips, we have developed a reliable protocol for chip sectioning and high-resolution microscopic acquisition and analysis. Optimization of SCALE protocol for organ-chips and organ-chip sections for improved visualization.
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影响因子：
19
作者：
Huh, Dongeun;Hamilton, Geraldine A.;Ingber, Donald E.
通讯作者：
Ingber, Donald E.
DOI：
10.1016/j.jcmgh.2017.12.008
发表时间：
2018
影响因子：
7.2
作者：
Workman MJ;Gleeson JP;Troisi EJ;Estrada HQ;Kerns SJ;Hinojosa CD;Hamilton GA;Targan SR;Svendsen CN;Barrett RJ
通讯作者：
Barrett RJ
影响因子：
14.8
作者：
Osaki, Tatsuya;Uzel, Sebastien G. M.;Kamm, Roger D.
通讯作者：
Kamm, Roger D.
影响因子：
64.8
作者：
通讯作者：
--
影响因子：
4.6
作者：
Mathur A;Loskill P;Shao K;Huebsch N;Hong S;Marcus SG;Marks N;Mandegar M;Conklin BR;Lee LP;Healy KE
通讯作者：
Healy KE