Three-dimensional organoid culture using the CellRaft microwell technology

项目摘要 类器官是3D微型组织结构，正在革新体外研究。它们可以被导出 从各种物种中，使用从正常的或非正常的细胞中分离的干细胞或诱导多能干细胞（iPSC）， 或病变组织。在疾病建模中使用类器官已成为复制的有力方法 病理生理学，使用相对标准的细胞培养方法。事实上，现在每种组织类型都有一个体外 类器官相关顾名思义，类器官是组织层（通常是上皮）的代表。 在有机体中有特定功能的细胞它们通常是干细胞衍生的，使它们具有潜力， 产生在给定组织中发现的所有分化的细胞类型。虽然这种灵活性允许在 体内多细胞结构，还需要使用具有挑战性的手动细胞生物学方案， 建立和维持各种类型的类器官。快速建立类器官培养的障碍是 这主要是由于从单个细胞启动克隆集落的效率低、难以跟踪集落生长 随着时间的推移，分化过程的诱导效率低下。此外，支持类器官的系统 培养必须允许基于时间过程的生理表型分析。Cell Microsystems拥有 开发了CellRaft技术，这是一种基于微孔阵列的平台，可以将单细胞接种在小的 培养室，生长成克隆集落，并使用几乎任何成像模式随时间跟踪。 在我们的第一阶段计划中，我们与北卡罗来纳州大学博士Scott Magness合作 查佩尔山开发使用CellRaft技术的类器官培养、分选和亚克隆方法。 CytoSort阵列具有数千个用于细胞培养的微孔，用于建立干细胞。 衍生的类器官培养物。在阵列上使用Matrigel允许三维支持类器官 结构，因为它们形成单细胞。Magness博士的实验室还尝试了类器官亚克隆， 从CytoSort Array中取出类器官，并在解离后重新铺板细胞以形成第二代 类器官在第二阶段，我们将在CytoSort阵列上利用这种强大的方法来传播多个 从一个创始人的几代类器官，并进行分子分析，以确定谱系特性，细胞 突变的类型和潜在积累导致各种疾病，包括癌症。支持这些 细胞微系统公司开发了3D CytoSort阵列，具有更大的微孔， 立体培养和分离单个类器官用于分子分析。在第二阶段，我们将开发 其他类器官类型（包括神经、胰腺和肝脏）的方案优化了整体工作流程， 3D CytoSort阵列，以及评估多代类器官的RNA测序分析。这些方法 广泛适用于许多细胞和组织类型的类器官研究， 在类器官工作流程中，同时保持与当代分子分析方法的广泛兼容性。

Project Summary Organoids are 3D mini‐tissue structures that are revolutionizing in vitro studies. They can be derived from a variety of species, using stem cells or induced pluripotent stem cells (iPSCs) isolated from either normal or diseased tissues. The use of organoids in disease modeling has become a powerful method to replicate pathophysiology using relatively standard cell culture methods. Virtually every tissue type now has an in vitro organoid correlate. As the name implies, organoids are representations of tissue layers (typically epithelium) that have a specific function in an organism. They are often stem cell derived, giving them the potential to produce all the differentiated cell types found in a given tissue. While this flexibility allows recapitulation of in vivo multicellular structures, it also necessitates the use of challenging and manual cell biology protocols to establish and maintain various types of organoid. Roadblocks to rapidly establishing organoid cultures are largely due to the low efficiency of initiating a clonal colony from a single cell, difficulty tracking colony growth over time and inefficient induction of differentiation processes. In addition, systems supporting organoid culture must allow for time‐course based analysis of physiological phenotypes. Cell Microsystems has developed the CellRaft Technology, a microwell array‐based platform where single cells can be seeded in small culture chambers, grown into clonal colonies and tracked over time using virtually any imaging modality. During our Phase I program, we collaborated with Scott Magness, PhD of the University of North Carolina at Chapel Hill to develop organoid culture, sorting and subcloning methods using the CellRaft Technology. CytoSort Arrays, which feature thousands of microwells for cell culture, were employed to establish stem cell‐ derived organoid cultures. The use of Matrigel on the arrays allowed for three‐dimensional support of organoid structures as they form single cells. Dr. Magness' lab also attempted organoid subcloning by isolating a single organoid from the CytoSort Array and re‐plating the cells after dissociation to form second‐generation organoids. In Phase II, we will leverage this powerful method on CytoSort Arrays to propagate multiple generations of organoids from a founder and undertake molecular analysis to identify lineage properties, cell types and potential accumulation of mutations leading to various disease, including cancer. To support these efforts, Cell Microsystems has developed the 3D CytoSort Array, featuring larger microwells to enable three‐ dimensional culture and isolation of individual organoids for molecular analysis. In Phase II we will develop protocols for other organoid types including neural, pancreatic and hepatic, optimize the overall workflow with the 3D CytoSort Array, as well as evaluate RNA‐Seq analysis of multiple organoid generations. These methods are broadly applicable to organoid research in many cell and tissue types and fill an unmet need for automation in organoid workflows while retaining broad compatibility with contemporary molecular analysis methods.