Burkhart Richard A, Baker Lindsey A, Tiriac Hervé
Division of Hepatobiliary and Pancreatic Surgery, Department of Surgery, Johns Hopkins Hospital, Baltimore, MD, USA.
Lustgarten Foundation Pancreatic Cancer Research Laboratory, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, NY, USA.
Methods Mol Biol. 2018;1787:253-261. doi: 10.1007/978-1-4939-7847-2_19.
Increasingly, patient models of disease are being utilized to facilitate precision medicine approaches through molecular characterization or direct chemotherapeutic testing. Organoids, 3-dimensional (3D) cultures of neoplastic cells derived from primary tumor specimens, represent an ideal platform for these types of studies because benchtop protocols previously developed for 2-dimensional cell lines can be adapted for use. These protocols include directly testing the survival of these organoid cultures when exposed to clinically relevant chemotherapeutic agents, a process we have called pharmacotyping. In this protocol, established tumor-derived organoid cultures are dissociated into single cells, plated in a 3D gel matrix, and exposed to pharmacologic agents. While our protocol has been developed for use with patient-derived pancreatic ductal adenocarcinoma organoids, with minor modifications to the dissociation and medium conditions, this protocol could be adapted for use with a wide range of organoid cultures. We further describe our standard ATP-based assay to determine cellular survival. This protocol can be scaled for use in high-throughput assays.
越来越多地，疾病的患者模型正被用于通过分子表征或直接化疗测试来促进精准医学方法。类器官是源自原发性肿瘤标本的肿瘤细胞的三维（3D）培养物，是这些类型研究的理想平台，因为先前为二维细胞系开发的台式实验方案可以进行调整以用于类器官。这些方案包括在暴露于临床相关化疗药物时直接测试这些类器官培养物的存活率，我们将这个过程称为药物分型。在本实验方案中，将已建立的肿瘤来源的类器官培养物解离成单细胞，接种到3D凝胶基质中，并暴露于药物制剂。虽然我们的实验方案是为患者来源的胰腺导管腺癌类器官开发的，但对解离和培养基条件进行微小修改后，该方案可适用于广泛的类器官培养物。我们进一步描述了用于确定细胞存活率的基于ATP的标准检测方法。该实验方案可以扩大规模用于高通量检测。
