Division of Gastroenterology and Hepatology, University of Pennsylvania Perelman School of Medicine.
Department of Surgery, Columbia University Irving Medical Center.
J Vis Exp. 2024 Jan 26(203). doi: 10.3791/66094.
Gastric patient-derived organoids (PDOs) offer a unique tool for studying gastric biology and pathology. Consequently, these PDOs find increasing use in a wide array of research applications. However, a shortage of published approaches exists for producing gastric PDOs from single-cell digests while maintaining a standardized initial cell seeding density. In this protocol, the emphasis is on the initiation of gastric organoids from isolated single cells and the provision of a method for passaging organoids through fragmentation. Importantly, the protocol demonstrates that a standardized approach to the initial cell seeding density consistently yields gastric organoids from benign biopsy tissue and allows for standardized quantification of organoid growth. Finally, evidence supports the novel observation that gastric PDOs display varying rates of formation and growth based on whether the organoids originate from biopsies of the body or antral regions of the stomach. Specifically, it is revealed that the use of antral biopsy tissue for organoid initiation results in a greater number of organoids formed and more rapid organoid growth over a 20-day period when compared to organoids generated from biopsies of the gastric body. The protocol described herein offers investigators a timely and reproducible method for successfully generating and working with gastric PDOs.
胃源患者衍生类器官 (PDO) 为研究胃生物学和病理学提供了独特的工具。因此，这些 PDO 在广泛的研究应用中得到了越来越多的应用。然而，从单细胞消化物中产生胃 PDO 并保持标准化初始细胞接种密度的方法却很少有文献报道。本方案重点介绍了从分离的单细胞起始胃类器官的方法，并提供了一种通过碎片化传代类器官的方法。重要的是，该方案表明，标准化的初始细胞接种密度方法始终可以从良性活检组织中获得胃类器官，并允许对类器官的生长进行标准化定量。最后，有证据表明，基于类器官是否源自胃体或胃窦的活检，胃 PDO 的形成和生长速度存在差异。具体而言，研究揭示了使用胃窦活检组织进行类器官起始可导致在 20 天的时间内形成更多的类器官，并且类器官生长更快，而与源自胃体活检的类器官相比。本文描述的方案为研究人员提供了一种及时且可重复的方法，用于成功生成和使用胃 PDO。
