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

Génération d’organoïdes gastriques humains à partir de biopsies d’endoscopie haute

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.

Pleins feux sur l’auteur : Génération et comparaison entre des organoïdes gastriques dérivés de patients provenant de différentes régions de l’estomac 

Etablissement et caractérisation d’organoïdes gastriques dérivés de patients à partir de biopsies d’épithélium gastrique bénin et d’épithélium 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  

Intégration de cellules individuelles dérivées d’une biopsie gastrique dans un dôme matriciel membranaire basique pour la génération d’organoïdes

Pour commencer, retirez la matrice de la membrane basale décongelée de la glace. Ajoutez le volume calculé de la matrice de la membrane basale aux cellules individuelles dérivées de la biopsie gastrique et mélangez doucement en pipetant de haut en bas pendant environ 10 secondes. Ensuite, à l’aide d’une pipette, transférez rapidement 50 microlitres d’aliquotes de la matrice et du mélange cellulaire au centre des puits individuels dans une plaque de culture tissulaire à 24 puits.Couvrez immédiatement l’assiette et, d’un seul mouvement fluide, retournez l’assiette. Placez la plaque inversée dans un incubateur de culture tissulaire à 37 degrés Celsius pendant 35 minutes pour permettre à la matrice de la membrane basale de polymériser. Ajoutez ensuite 500 microlitres de milieu organoïde gastrique préchauffé dans chaque puits tout en vous assurant que le haut de chaque dôme est complètement immergé dans le milieu.Les organoïdes sont généralement identifiables dans les 10 jours suivant l’ensemencement des cellules individuelles. Au jour 20, ils sont grands et doivent généralement être traversés. Le nombre d’organoïdes corporels et antraux culmine au jour 10 après l’ensemencement avant de diminuer, mais pas de manière significative.Les organoïdes générés à partir de tissus de biopsie du manteau ont montré un taux de croissance plus élevé que les organoïdes générés à partir de tissus de biopsie corporelle. Chez différents patients, une diversité de morphologie organoïde a généralement été observée dans un seul dôme. Cependant, en moyenne, la spasticité organoïde a montré peu de variation.

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 ...

Recherche

Biologie

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

Isolement de cellules uniques provenant de biopsies d’endoscopie haute dérivées de patients gastriques pour la génération d’organoïdes

Routine Passaging of Patient-Derived Gastric Organoids Through Fragmentation

Passage systématique d’organoïdes gastriques dérivés de patients par fragmentation