Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan 430074, China.
Key Laboratory of Biomedical Engineering of Hainan Province, School of Biomedical Engineering, Hainan University, Haikou 570228, China.
Int J Mol Sci. 2023 Sep 9;24(18):13903. doi: 10.3390/ijms241813903.
Organoids can recapitulate human-specific phenotypes and functions in vivo and have great potential for research in development, disease modeling, and drug screening. Due to the inherent variability among organoids, experiments often require a large sample size. Embedding, staining, and imaging each organoid individually require a lot of reagents and time. Hence, there is an urgent need for fast and efficient methods for analyzing the phenotypic changes in organoids in batches. Here, we provide a comprehensive strategy for array embedding, staining, and imaging of cerebral organoids in both agarose sections and in 3D to analyze the spatial distribution of biomarkers in organoids in situ. We constructed several disease models, particularly an aging model, as examples to demonstrate our strategy for the investigation of the phenotypic analysis of organoids. We fabricated an array mold to produce agarose support with microwells, which hold organoids in place for live/dead imaging. We performed staining and imaging of sectioned organoids embedded in agarose and 3D imaging to examine phenotypic changes in organoids using fluorescence micro-optical sectioning tomography (fMOST) and whole-mount immunostaining. Parallel studies of organoids in arrays using the same staining and imaging parameters enabled easy and reliable comparison among different groups. We were able to track all the data points obtained from every organoid in an embedded array. This strategy could help us study the phenotypic changes in organoids in disease models and drug screening.
类器官能够在体内重现人类特有的表型和功能，在发育、疾病建模和药物筛选研究方面具有巨大的潜力。由于类器官之间存在固有变异性，因此实验通常需要大量样本。单独对每个类器官进行包埋、染色和成像需要大量的试剂和时间。因此，迫切需要快速有效的方法来批量分析类器官的表型变化。在这里，我们提供了一种综合策略，用于在琼脂糖切片和 3D 中对大脑类器官进行阵列包埋、染色和成像，以原位分析类器官中生物标志物的空间分布。我们构建了几种疾病模型，特别是衰老模型，作为示例来演示我们的策略，用于研究类器官的表型分析。我们制造了一个阵列模具，以生产带有微井的琼脂糖支撑物，用于固定类器官进行活/死成像。我们对嵌入琼脂糖的切片类器官进行染色和成像，并使用荧光微光学切片层析成像（fMOST）和全组织免疫染色对 3D 成像进行分析，以检查类器官的表型变化。使用相同的染色和成像参数对类器官进行阵列平行研究，便于在不同组之间进行轻松可靠的比较。我们能够跟踪从嵌入式阵列中的每个类器官获得的所有数据点。这种策略可以帮助我们在疾病模型和药物筛选中研究类器官的表型变化。
