球体已成为研究癌症、发育过程和药物功效的主要三维模型。单细胞分析技术已成为衡量这些模型中细胞反应复杂性的理想工具。然而，基于荧光标记物亚细胞分布的 3D 显微数据（例如转录因子的核/细胞质比率）的单细胞定量评估在很大程度上仍然难以实现。对于球体生成，超低附着板因其简单性、与自动化的兼容性以及实验和商业可访问性而值得注意。然而，尚不清楚板类型是否以及在何种程度上影响球体形成和生物学。这项研究开发了一种基于人工智能的新型管道，用于在整体、单细胞和亚细胞水平上分析光学透明大球体的 3D 共焦数据。为了识别管道的相关样本，采用自动明场显微镜系统地比较使用四种不同的人类细胞系在六种不同板类型中形成的球体的大小和偏心率。这表明所有类型的平板都表现出相似的球体形成能力，并且播种后 4 天内的生长或收缩的总体模式具有可比性。然而，特定细胞系和板类型之间的大小和偏心率有系统的变化。在此预筛选的基础上，进一步评估了 HaCaT 角质形成细胞和 HT-29 癌细胞的球体。在 HaCaT 球体中，深入分析揭示了球体大小、细胞增殖和转录共激活因子 YAP1 的核/细胞质比率之间的相关性，以及与细胞分化的负相关性。这些发现是在球体模型和单细胞水平上得出的，证实了 YAP1 在人类皮肤细胞增殖和角质形成细胞分化中的作用的早期概念。此外，结果表明，板类型可能会影响实验活动的结果，建议在特定调查期间扫描不同的板类型以获得最佳配置。版权所有 © 2024 Vitacolonna, Bruch, Agaçi, Nürnberg, Cesetti, Keller,帕多瓦尼、绍尔、施莫勒、赖歇尔、哈夫纳和鲁道夫。

Spheroids have become principal three-dimensional models to study cancer, developmental processes, and drug efficacy. Single-cell analysis techniques have emerged as ideal tools to gauge the complexity of cellular responses in these models. However, the single-cell quantitative assessment based on 3D-microscopic data of the subcellular distribution of fluorescence markers, such as the nuclear/cytoplasm ratio of transcription factors, has largely remained elusive. For spheroid generation, ultra-low attachment plates are noteworthy due to their simplicity, compatibility with automation, and experimental and commercial accessibility. However, it is unknown whether and to what degree the plate type impacts spheroid formation and biology. This study developed a novel AI-based pipeline for the analysis of 3D-confocal data of optically cleared large spheroids at the wholemount, single-cell, and sub-cellular levels. To identify relevant samples for the pipeline, automated brightfield microscopy was employed to systematically compare the size and eccentricity of spheroids formed in six different plate types using four distinct human cell lines. This showed that all plate types exhibited similar spheroid-forming capabilities and the gross patterns of growth or shrinkage during 4 days after seeding were comparable. Yet, size and eccentricity varied systematically among specific cell lines and plate types. Based on this prescreen, spheroids of HaCaT keratinocytes and HT-29 cancer cells were further assessed. In HaCaT spheroids, the in-depth analysis revealed a correlation between spheroid size, cell proliferation, and the nuclear/cytoplasm ratio of the transcriptional coactivator, YAP1, as well as an inverse correlation with respect to cell differentiation. These findings, yielded with a spheroid model and at a single-cell level, corroborate earlier concepts of the role of YAP1 in cell proliferation and differentiation of keratinocytes in human skin. Further, the results show that the plate type may influence the outcome of experimental campaigns and that it is advisable to scan different plate types for the optimal configuration during a specific investigation.Copyright © 2024 Vitacolonna, Bruch, Agaçi, Nürnberg, Cesetti, Keller, Padovani, Sauer, Schmoller, Reischl, Hafner and Rudolf.