Name: Embedding Gastric Biopsy-Derived Single Cells in a Basement Membrane Matrix Dome for Organoid Generation
Uploaded: 2024-01-26T14:25:00
Description: Watch this Scientific Journal Video about Embedding Gastric Biopsy-Derived Single Cells in a Basement Membrane Matrix Dome for Organoid Generation at JoVE.com

embedding gastric biopsy-derived single cells in a basement membrane matrix dome for organoid generation

从上消化道内窥镜活检生成人胃类器官

Organoids are miniature three-dimensional (3D) cellular structures that resemble the architecture and functionality of the organs from which they were derived1,2. These lab-grown models are created by cultivating stem cells or tissue-specific cells in a controlled environment that allows these cells to self-organize and differentiate into various cell types1,2,3. One of the key advantages of organoids is their ability to recapitulate human biology more closely than traditional two-dimensional (2D) cell cultures1,2,3. In particular, human organoids have been shown to maintain the genetic diversity of their tissue of origin3,4,5. Organoids offer a unique opportunity to study human organ development, model diseases, and test potential therapeutics in a controlled laboratory setting. Furthermore, organoids can be derived from individual patient samples, enabling personalized medicine approaches and the potential development of individualized treatments3,6,7.
Researchers have used human gastric organoids to investigate various aspects of gastric biology and pathology. Prominent examples include the use of patient-derived organoids (PDOs) to predict gastric cancer chemotherapy responses8,9,10 and model the epithelial response to Helicobacter pylori infection11,12,13. Human gastric organoids consist of various cell types found in the stomach, including neck cells, pit cells, and other supporting cells11,14. Gastric organoids can either be generated from induced pluripotent stem cells (iPSCs) or stem cells directly isolated from gastric tissue obtained via biopsies or from gastric resection specimens11,14. The isolation of gastric stem cells from gastric tissue is commonly done by isolating and culturing gastric glands or enzymatically digesting tissue samples to liberate single cells9,13,15. Importantly, the differentiation of cells within gastric organoids generated using either of these techniques has been shown to be similar13. The protocol described herein focuses on a single-cell digest.
Organoids represent a scientific innovation that bridges the gap between traditional cell culture and whole organs. As research in the field continues to progress, organoids are poised to contribute to the development of more effective treatments and therapies for a wide range of applications. Given the rising utilization of gastric PDOs, there is a timely need for a standardized approach to their generation. Here, the protocol for generating human gastric PDOs from single cells isolated from benign gastric biopsy tissue acquired during upper endoscopy is described. Importantly and uniquely, a standardized number of single cells is determined for seeding to reliably generate gastric PDOs and allow subsequent characterization. Using this technique, reliable differences in the formation and growth of organoids generated from biopsies of either the gastric body or gastric antrum are demonstrated.

作者聚焦：来自胃不同区域的患者来源的胃类器官的生成和比较 

良性胃体和胃窦上皮活检患者来源的胃类器官的建立和表征

Our research focuses on hereditary causes of gastric cancer. In particular, we're interested in hereditary diffuse gastric cancer due to disease-causing variants in either the CDH1 or CTNNA1 gene. We also investigate the role of BRCA1 and BRCA2 in gastric cancer risk and carcinogenesis.Organoids are increasingly being utilized in a variety of research domains and gastroenterology is no exception, so with this comes a need for standardization of these techniques to generate these organoids, in particular patient-derived gastric organoids. Our protocol offers a step-by-step method to reliably generate patient-derived gastric organoids from biopsies of both the antral and body regions of the stomach. In particular, we utilize a single-cell digest approach to allow for standardized seeding of cells in an effort to make reliable comparisons amongst organoids generated from different patients.Our findings show that compared to patient-derived gastric organoids from the body region of the stomach, those from the antral region actually grow more numerous and larger in size over the course of 20 days post initial seeding. These findings should be taken into account by researchers looking to utilize gastric organoids derived from different regions of the stomach.

Watch this Scientific Journal Video about Embedding Gastric Biopsy-Derived Single Cells in a Basement Membrane Matrix Dome for Organoid Generation at JoVE.com

Embedding Gastric Biopsy-Derived Single Cells in a Basement Membrane Matrix Dome for Organoid Generation  

将胃活检衍生的单细胞嵌入基底膜基质圆顶中以生成类器官

首先，从冰中取出解冻的基底膜基质。将计算出的基底膜基质体积加入胃活检衍生的单细胞中，并通过上下移液约10秒轻轻混合。然后使用移液管，快速转移50微升等分试样的基质和细胞混合物到24孔组织培养板中单个孔的中心。立即盖上盘子，然后以一个平稳的动作将盘子倒置。将倒置的板置于37摄氏度组织培养箱中35分钟，使基底膜基质聚合。然后向每个孔中加入 500 微升预热的胃类器官培养基，同时确保每个圆顶的顶部完全浸没在培养基中。类器官通常在单细胞接种后 10 天内可识别。到第 20 天时，它们很大，通常需要排出。体和窦类器官的数量在播种后第 10 天达到峰值，然后减少，尽管不显着。与身体活检组织产生的类器官相比，由套细胞活检组织产生的类器官显示出更高的生长速度。在不同的患者中，通常在任何单个圆顶中观察到类器官形态的多样性。然而，平均而言，类器官痉挛几乎没有变化。

Research

JoVE Journal

Biology

Embedding Gastric Biopsy-Derived Single Cells in a Basement Membrane Matrix Dome for Organoid Generation

Establishment and Characterization of Patient-derived Gastric Organoids from Biopsies of Benign Gastric Body and Antral Epithelium

Gastric patient-derived organoids (PDOs) offer a unique tool for studying gastric biology and pathology. Consequently, these PDOs find increasing use in a ...

科研

生物学

Isolating Single Cells from Upper Endoscopy Biopsies Derived from Gastric Patients for Organoid Generation

从胃患者的上消化道内窥镜活检中分离单细胞以产生类器官

Routine Passaging of Patient-Derived Gastric Organoids Through Fragmentation

通过碎片化对患者来源的胃类器官进行常规传代