Colling Kaitriana E, Symons Emily L, Buroni Lorenzo, Sumanisiri Hiruni K, Andrew-Udoh Jessica, Witt Emily, Losh Haley A, Morrison Abigail M, Leslie Kimberly K, Dunnill Christopher J, De Bono Johann S, Thiel Kristina W
Department of Obstetrics and Gynecology, Carver College of Medicine, University of Iowa, Iowa City, IA, USA.
Cancer Biology Graduate Program, Carver College of Medicine, University of Iowa, Iowa City, IA, USA.
bioRxiv. 2023 Nov 17:2023.11.15.567243. doi: 10.1101/2023.11.15.567243.
Patient-derived organoid (PDO) models of cancer are a multifunctional research system that better recapitulates human disease as compared to cancer cell lines. PDO models can be generated by culturing patient tumor cells in extracellular basement membrane extracts (BME) and plating as three-dimensional domes. However, commercially available reagents that have been optimized for phenotypic assays in monolayer cultures often are not compatible with BME. Herein we describe a method to plate PDO models and assess drug effects using an automated live-cell imaging system. In addition, we apply fluorescent dyes that are compatible with kinetic measurements to simultaneously quantitate cell health and apoptosis. Image capture can be customized to occur at regular time intervals over several days. Users can analyze drug effects in individual Z-plane images or a Z Projection of serial images from multiple focal planes. Using masking, specific parameters of interest are calculated, such as PDO number, area, and fluorescence intensity. We provide proof-of-concept data demonstrating the effect of cytotoxic agents on cell health, apoptosis and viability. This automated kinetic imaging platform can be expanded to other phenotypic readouts to understand diverse therapeutic effects in PDO models of cancer.
癌症患者来源的类器官（PDO）模型是一种多功能研究系统，与癌细胞系相比，它能更好地模拟人类疾病。通过在细胞外基底膜提取物（BME）中培养患者肿瘤细胞并铺板成三维圆顶状，可以生成PDO模型。然而，为单层培养中的表型分析优化的市售试剂通常与BME不兼容。在此，我们描述了一种铺板PDO模型并使用自动活细胞成像系统评估药物效果的方法。此外，我们应用与动力学测量兼容的荧光染料来同时定量细胞健康状况和细胞凋亡。图像捕获可以设置为在几天内按固定时间间隔进行。用户可以在单个Z平面图像或来自多个焦平面的系列图像的Z投影中分析药物效果。使用掩膜，可以计算感兴趣的特定参数，如PDO数量、面积和荧光强度。我们提供了概念验证数据，证明了细胞毒性剂对细胞健康、细胞凋亡和活力的影响。这个自动动力学成像平台可以扩展到其他表型读数，以了解癌症PDO模型中的各种治疗效果。
