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

Genereren van menselijke maagorganoïden uit biopsieën van de bovenste endoscopie

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.

Auteur in de schijnwerpers: genereren van en vergelijken tussen van patiënten afgeleide maagorganoïden uit verschillende delen van de maag 

Vaststelling en karakterisering van van patiënten afgeleide maagorganoïden uit biopsieën van goedaardig maaglichaam en antraal epitheel

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  

Inbedding van van maagbiopsie afgeleide afzonderlijke cellen in een koepel van een basale membraanmatrix voor het genereren van organoïden

Verwijder om te beginnen de ontdooide basaalmembraanmatrix van het ijs. Voeg het berekende volume van de basaalmembraanmatrix toe aan de van de maagbiopsie afgeleide afzonderlijke cellen en meng voorzichtig door ongeveer 10 seconden op en neer te pipetteren. Breng vervolgens met behulp van een pipet snel 50 microliter aliquots van de matrix en het celmengsel over naar het midden van individuele putjes in een weefselkweekplaat met 24 putjes.Dek het bord onmiddellijk af en draai het bord in één vloeiende beweging ondersteboven. Plaats de omgekeerde plaat gedurende 35 minuten in een weefselkweekincubator van 37 graden Celsius om de basale membraanmatrix te laten polymeriseren. Voeg vervolgens 500 microliter voorwarme maagorganoïde media toe aan elk putje en zorg ervoor dat de bovenkant van elke koepel volledig is ondergedompeld in het medium.Organoïden zijn over het algemeen identificeerbaar binnen 10 dagen na het zaaien van afzonderlijke cellen. Op dag 20 zijn ze groot en moeten ze meestal worden gepasseerd. Het aantal lichaams- en antrale organoïden piekt op dag 10 na het zaaien voordat het afneemt, hoewel niet significant.Organoïden gegenereerd uit mantelbiopsieweefsel vertoonden een grotere groeisnelheid in vergelijking met organoïden gegenereerd uit lichaamsbiopsieweefsel. Bij verschillende patiënten werd doorgaans een diversiteit aan organoïdemorfologie waargenomen in een enkele koepel. Gemiddeld vertoonde de spasticiteit van organoïden echter weinig variatie.

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 ...