McCray Tara, Richards Zachary, Marsili Joseph, Prins Gail S, Nonn Larisa
Department of Pathology, University of Illinois at Chicago.
Department of Pathology, University of Illinois at Chicago; Departments of Urology, Physiology, and Biophysics, University of Illinois at Chicago; University of Illinois Cancer Center.
J Vis Exp. 2019 Jan 17(143). doi: 10.3791/59051.
This paper describes a detailed protocol for three-dimensional (3D) culturing, handling, and evaluation of human primary prostate organoids. The process involves seeding of epithelial cells sparsely in a 3D matrix gel on a 96-well microplate with media changes to cultivate expansion into organoids. Morphology is then assessed by whole-well capturing of z-stack images. Compression of z-stacks creates a single in-focus image from which organoids are measured to quantify a variety of outputs, including circularity, roundness, and area.DNA, RNA, and protein can be collected from organoids recovered from the matrix gel. Cell populations of interest can be assessed by organoid dissociation and flow cytometry. Formalin-fixation-paraffin-embedding (FFPE) followed by sectioning is used for the histological assessment and antibody staining. Whole-mount immunofluorescent staining preserves organoid morphology and facilitates observation of protein localization in organoids in situ. Commercial assays that are traditionally used for 2D monolayer cells can be modified for 3D organoids. Used together, the techniques in this protocol provide a robust toolbox to quantify prostate organoid growth, morphologic characteristics, and expression of differentiation markers.
本文描述了一种用于人原发性前列腺类器官三维（3D）培养、处理和评估的详细方案。该过程包括将上皮细胞稀疏接种在96孔微孔板上的3D基质凝胶中，并更换培养基以培养使其扩展为类器官。然后通过对z轴堆叠图像进行全孔捕获来评估形态。压缩z轴堆叠可创建单个对焦图像，从中测量类器官以量化各种输出，包括圆形度、圆度和面积。可以从从基质凝胶中回收的类器官中收集DNA、RNA和蛋白质。通过类器官解离和流式细胞术可以评估感兴趣的细胞群体。福尔马林固定石蜡包埋（FFPE）然后切片用于组织学评估和抗体染色。整装免疫荧光染色可保留类器官形态，并便于原位观察类器官中蛋白质的定位。传统上用于二维单层细胞的商业检测方法可针对3D类器官进行修改。综合使用本方案中的技术可提供一个强大的工具箱，用于量化前列腺类器官的生长、形态特征和分化标志物的表达。
