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

Generación de organoides gástricos humanos a partir de biopsias endoscópicas superiores

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.

Autor destacado: Generación y comparación entre organoides gástricos derivados de pacientes de diferentes regiones del estómago 

Establecimiento y caracterización de organoides gástricos derivados de pacientes a partir de biopsias de cuerpo gástrico benigno y epitelio antral

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  

Inclusión de células individuales derivadas de biopsia gástrica en una cúpula de matriz de membrana basal para la generación de organoides

Para comenzar, retire la matriz de la membrana basal descongelada del hielo. Agregue el volumen calculado de la matriz de la membrana basal a las células individuales derivadas de la biopsia gástrica y mezcle suavemente pipeteando hacia arriba y hacia abajo durante aproximadamente 10 segundos. Luego, con una pipeta, transfiera rápidamente 50 alícuotas de microlitros de la matriz y la mezcla celular al centro de los pocillos individuales en una placa de cultivo de tejidos de 24 pocillos.Cubra el plato inmediatamente y, con un solo movimiento suave, voltee el plato boca abajo. Coloque la placa invertida en una incubadora de cultivo de tejidos a 37 grados Celsius durante 35 minutos para permitir que la matriz de la membrana basal se polimerice. A continuación, añada 500 microlitros de organoide gástrico precalentado a cada pocillo mientras se asegura de que la parte superior de cada cúpula esté completamente sumergida en el medio.Los organoides son generalmente identificables dentro de los 10 días posteriores a la siembra de células individuales. Para el día 20, son grandes y, por lo general, necesitan ser pasados. El número de organoides corporales y antrales alcanza su punto máximo en el día 10 después de la siembra antes de disminuir, aunque no de manera significativa.Los organoides generados a partir del tejido de la biopsia del manto mostraron una mayor tasa de crecimiento en comparación con los organoides generados a partir del tejido de la biopsia del cuerpo. En diferentes pacientes, se observó una diversidad de morfología organoide en cualquier cúpula individual. Sin embargo, en promedio, la espasticidad organoide mostró poca variación.

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 ...

Investigación

Biología

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

Aislamiento de células individuales de biopsias endoscópicas superiores derivadas de pacientes gástricos para la generación de organoides

Routine Passaging of Patient-Derived Gastric Organoids Through Fragmentation

Pase rutinario de organoides gástricos derivados del paciente a través de la fragmentación