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Tissue clearing of human iPSC-derived organ-chips enables high resolution imaging and analysis.

ABSTRACT: 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.

SUBMITTER: Ondatje BN

PROVIDER: S-EPMC9595176 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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Tissue clearing of human iPSC-derived organ-chips enables high resolution imaging and analysis.

Ondatje Briana N BN Sances Samuel S Workman Michael J MJ Svendsen Clive N CN

Lab on a chip 20221025 21

Engineered microfluidic organ-chips enable increased cellular diversity and function of human stem cell-derived tissues grown <i>in vitro</i>. 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 p ...[more]

PMID: 36205191

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Project description:Recent advances in optical clearing techniques have dramatically improved deep tissue imaging by reducing the obscuring effects of light scattering and absorption. However, these optical clearing methods require specialized equipment or a lengthy undertaking with complex protocols that can lead to sample volume changes and distortion. In addition, the imaging of cleared tissues has limitations, such as fluorescence bleaching, harmful and foul-smelling solutions, and the difficulty of handling samples in high-viscosity refractive index (RI) matching solutions. To address the various limitations of thick tissue imaging, we developed an Aqueous high refractive Index matching and tissue Clearing solution for Imaging (termed AICI) with a one-step tissue clearing protocol that was easily made at a reasonable price in our own laboratory without any equipment. AICI can rapidly clear a 1 mm thick brain slice within 90 min with simultaneous RI matching, low viscosity, and a high refractive index (RI = 1.466), allowing the imaging of the sample without additional processing. We compared AICI with commercially available RI matching solutions, including optical clear agents (OCAs), for tissue clearing. The viscosity of AICI is closer to that of water compared with other RI matching solutions, and there was a less than 2.3% expansion in the tissue linear morphology during 24 h exposure to AICI. Moreover, AICI remained fluid over 30 days of air exposure, and the EGFP fluorescence signal was only reduced to ~65% after 10 days. AICI showed a limited clearing of brain tissue >3 mm thick. However, fine neuronal structures, such as dendritic spines and axonal boutons, could still be imaged in thick brain slices treated with AICI. Therefore, AICI is useful not only for the three-dimensional (3D) high-resolution identification of neuronal structures, but also for the examination of multiple structural imaging by neuronal distribution, projection, and gene expression in deep brain tissue. AICI is applicable beyond the imaging of fluorescent antibodies and dyes, and can clear a variety of tissue types, making it broadly useful to researchers for optical imaging applications.

Dataset Information

Tissue clearing of human iPSC-derived organ-chips enables high resolution imaging and analysis.

Publications

Tissue clearing of human iPSC-derived organ-chips enables high resolution imaging and analysis.

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