isolating single cells from upper endoscopy biopsies derived from gastric patients for organoid generation

Generierung humaner Magenorganoide aus Biopsien der oberen Endoskopie

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 im Rampenlicht: Generierung und Vergleich zwischen patienteneigenen Magenorganoiden aus verschiedenen Regionen des Magens 

Etablierung und Charakterisierung von patienteneigenen Magenorganoiden aus Biopsien des gutartigen Magenkörpers und des Antralepithels

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 Isolating Single Cells from Upper Endoscopy Biopsies Derived from Gastric Patients for Organoid Generation at JoVE.com

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

Isolierung einzelner Zellen aus Biopsien der oberen Endoskopie von Magenpatienten zur Generierung von Organoiden

Um mit der Isolierung einzelner Zellen aus den Magenbiopsien zu beginnen, die während der oberen Endoskopie des Patienten entnommen wurden, lassen Sie zunächst das Gewebe am Boden eines konischen 15-Milliliter-Röhrchens absetzen. Nach dem Absetzen verwenden Sie eine Pipette, um das Medium anzusaugen, und waschen Sie die Biopsien zweimal mit einem Milliliter antibiotikahaltigem PBS. Übertragen Sie mindestens 20 Milligramm des Gewebes in ein 1,5-Milliliter-Röhrchen, das einen Milliliter PBS enthält, das mit DTT ergänzt ist.Schneiden Sie das Gewebe mit einer feinen Präparierschere in Stücke, die ein bis zwei Millimeter oder kleiner sind. Verwenden Sie eine Tisch-Minizentrifuge für 15 Sekunden, um die Gewebestücke am Boden des Röhrchens zu aggregieren, und saugen Sie dann so viel Überstand wie möglich ab. Geben Sie fünf Milliliter frisch erwärmten Aufschlusspuffer in ein konisches 50-Milliliter-Röhrchen.Übertragen Sie 500 Mikroliter des Puffers aus diesem Röhrchen in das 1,5-Milliliter-Röhrchen, das die Gewebestücke enthält. Schneiden Sie dann mit einer Schere die Pipettenspitze von unten auf 1.000 Mikroliter ab, um den Durchmesser der Spitze zu vergrößern und den Gewebeverlust zu minimieren. Übertragen Sie nun mit der modifizierten Pipettenspitze die kleinen Gewebestücke aus dem 1,5-Milliliter-Röhrchen in den Aufschlusspuffer im 50-Milliliter-Röhrchen.Inkubieren Sie den Verdauungspuffer und die Gewebemischung bei 37 Grad für 30 Minuten mit orbitalem Schütteln bei 200 U/min. Dann werden fünf Milliliter erwärmtes Trypsin mit 0,25 % EDTA in das Gewebe im Verdauungspuffer gegeben und inkubiert. Neutralisieren Sie den Aufschlusspuffer und das Trypsin, indem Sie ein gleiches Volumen fortschrittlichen DMEM/F-12-Mediums hinzufügen und die Suspension durch ein 70-Mikron-Zellsieb leiten.

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Research

JoVE Journal

Biology

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

125 Aufrufe.  University of Pennsylvania Perelman School of Medicine. Um mit der Isolierung einzelner Zellen aus den Magenbiopsien zu beginnen, die während der oberen Endoskopie des Patienten entnommen wurden, lassen Sie zunächst das Gewebe am Boden eines konischen 15-Milliliter-Röhrchens absetzen. Nach dem Absetzen verwenden Sie eine Pipette, um das Medium anzusaugen, und waschen Sie die Biopsien zweimal mit einem Milliliter antibiotikahaltigem PBS. Übertragen Sie mindestens 20 Milligramm des Gewebes in ein 1,5-Milliliter-Röhrchen, das einen Milliliter PB...

Serie: Isolating Single Cells from Upper Endoscopy Biopsies Derived from Gastric Patients 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 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.

Gastric patient-derived organoids find increasing use in research, yet formal protocols for generating human gastric organoids from single-cell digests with standardized seeding density are lacking. This protocol presents a detailed method for reliably creating gastric organoids from biopsy tissue obtained during upper endoscopy.

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