干细胞科学和再生医学 – 国际干细胞研究协会 (ISSCR) 2022 年会上展示的技术和方法

在 ISSCR 2022，这又是很棒的一年！Leaders from across the globe came together to discuss new technologies, share insights, and explore the newest breakthroughs in stem cell science and regenerative medicine. In addition, attendees learned about automated and quantifiable solutions for the complexities that come with 3D cell model assays.

In case you missed the show or would like additional details about the technology and methods presented—we've got you covered!

Monitoring growth and characterization of compound effects in 3D intestinal organoids with high-content imaging

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The complexity of 3D cell models remains a hurdle for wider adoption in research and drug screening. In this poster, we describe an automated, integrated cell culture and high-content imaging solution that allows automated monitoring, maintenance, characterization of organoids, and testing of the effects of various compounds. The integrated solution includes a confocal imaging system, automated incubator, liquid handler, and collaborative robot.

We developed methods for the automation of the seeding cells in Matrigel and media exchange, as well as monitoring the development of mouse intestinal organoids using imaging in transmitted light. Machine-learning-based image analysis allowed detection of organoids and characterization of their size and density. Advanced image analysis allowed 3D reconstitution and complex phenotypic evaluation of organoid structures, including characterization of organoid size and complexity, cell morphology and viability, and presence of differentiation markers. We demonstrate the concentration-dependent toxicity effects of several anti-cancer drugs.

We further increased the complexity by generating human intestinal organoids in Gri3D^® micropatterned U-bottom-shaped microwells in the hydrogel. We demonstrate the tools for increasing throughput and automation of organoid assays and compound screening and propose analysis approaches and descriptors that deliver more information about these complex models.

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3D 人诱导多能干细胞 (iPSC) 源性心脏三种细胞培养微组织中的组织结构和化合物反应功能分析

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人类心脏是一个复杂的器官，负责全身血液流动的高度调控过程。The adult human ventricle is comprised of cardiomyocytes, endothelial cells, fibroblasts and other supporting cell types. 虽然心肌细胞占人心室总体积的 75%，但仅占细胞总数的 50%。Recent publications show that tri-cellular co-culture microtissues of cardiomyocytes, endothelial cells and cardiac fibroblasts that are all derived from human iPSC enhance the maturation and functional activity.

In this study, we used a tri-culture model created by mixing iPSC-derived cardiac cells with primary adult fibroblasts and iPSC-derived endothelial cells. We used a Biomek i7 liquid handling system for cell plating and subsequent media exchange. We investigated the functional activity of microtissues by recording calcium oscillations using a fast kinetic fluorescence recording instrument - the FLIPR^® Penta High-Throughput Cellular Screening System - while waveform analysis was performed using the ScreenWorks^® Peak Pro 2™ software module. Additionally, we used high-content imaging to characterize the structure and morphology of our 3D microtissues with the ImageXpress® Micro Confocal High-Content Imaging System. The 3D structure of microtissues was reconstructed and analyzed using MetaXpress® High-Content Image Acquisition and Analysis Software.

The data presented here highlights the utility and biological relevance of using iPSC-derived cell types in 3D micro-tissues as a promising model for measuring compound effects on human cardiac tissues in a high-throughput format.

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自动监测诱导性多能干细胞 (iPSC) 源性 3D 大脑类器官的开发和活动分析

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The culture of complex organoids, such as cerebral organoids, is a rapidly developing technology that has immense potential in areas of developmental neurobiology and neurodegeneration. Recent progress has made it possible to culture brain organoids from iPSCs which allows for the study of cortical development and the diseases associated with it. 在将大脑类器官大规模地用于功能性基因组研究、药物发现和化合物诱导毒性作用研究之前，还需要进一步的研究。在这里，我们介绍了一种用于大脑类器官半自动化培养和监测，以及通过记录 Ca2+ 振荡情况来检测功能性神经元活动的方法。

Cerebral organoids were developed from iPSC using previously published methods (Lancaster 2014). The method was adopted for a partially automated workflow and culture monitoring using the ImageXpress Confocal HT.ai High-Content Imaging System. We monitored size and morphology of developing brain microtissues in transmitted light over 20 weeks of development using deep learning-based image analysis (IN Carta^® Image Analysis Software) for defining the size, shape, and density of the tissues. For detection of functional activities, calcium oscillations from selected clusters of the microtissues were recorded by imaging and analyzed using MetaXpress High-Content Image Acquisition and Analysis Software for kinetic peak analysis.

The method demonstrates a promise for the evaluation of effects of pharmaceutical drugs, toxins, and genetic mutations, while further method development is needed for screening applications.

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Take a peek into where our research happens!

Tour our Organoid Innovation Center where we showcase cutting-edge instruments that work harmoniously together for autonomous, long-term, live cell 2D and 3D cell culture growth and monitoring with intelligent label-free imaging. This integrated workflow provides quality control alerts and readiness, 3D organoid screening, and deep-learning image analysis that reveals hidden patterns other technologies miss.

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