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

ऊपरी एंडोस्कोपी बायोप्सी से मानव गैस्ट्रिक ऑर्गेनोइड उत्पन्न करना

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.

लेखक स्पॉटलाइट: पेट के विभिन्न क्षेत्रों से रोगी-व्युत्पन्न गैस्ट्रिक ऑर्गेनोइड के बीच पीढ़ी और तुलना 

सौम्य गैस्ट्रिक बॉडी और एंट्रल एपिथेलियम की बायोप्सी से रोगी-व्युत्पन्न गैस्ट्रिक ऑर्गेनोइड की स्थापना और लक्षण वर्णन

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  

ऑर्गेनॉइड पीढ़ी के लिए एक तहखाने झिल्ली मैट्रिक्स गुंबद में गैस्ट्रिक बायोप्सी-व्युत्पन्न एकल कोशिकाओं को एम्बेड करना

शुरू करने के लिए, बर्फ से पिघले तहखाने झिल्ली मैट्रिक्स को हटा दें। गैस्ट्रिक बायोप्सी व्युत्पन्न एकल कोशिकाओं के लिए तहखाने झिल्ली मैट्रिक्स की गणना मात्रा जोड़ें, और धीरे लगभग 10 सेकंड के लिए ऊपर और नीचे pipetting द्वारा मिश्रण. फिर एक विंदुक का उपयोग कर, तेजी से हस्तांतरण, मैट्रिक्स के 50 माइक्रोलीटर aliquots, और एक 24 अच्छी तरह से ऊतक संस्कृति प्लेट में व्यक्तिगत कुओं के केंद्र के लिए सेल मिश्रण.प्लेट को तुरंत कवर करें, और एक ही चिकनी गति में, प्लेट को उल्टा पलटें। तहखाने झिल्ली मैट्रिक्स पोलीमराइज़ करने के लिए अनुमति देने के लिए 35 मिनट के लिए एक 37 डिग्री सेल्सियस टिशू कल्चर इनक्यूबेटर में उल्टे प्लेट रखें. फिर प्रत्येक गुंबद के शीर्ष को सुनिश्चित करते हुए प्रत्येक कुएं में प्रीवार्म गैस्ट्रिक ऑर्गेनॉइड मीडिया के 500 माइक्रोलीटर जोड़ें, यह सुनिश्चित करते हुए कि प्रत्येक गुंबद का शीर्ष मीडिया में पूरी तरह से डूबा हुआ है।ऑर्गेनोइड आमतौर पर एकल कोशिकाओं के बीजारोपण के बाद 10 दिनों के भीतर पहचाने जा सकते हैं। 20 दिन तक, वे बड़े होते हैं, और आमतौर पर पारित होने की आवश्यकता होती है। शरीर और एंट्रल ऑर्गेनोइड की संख्या घटने से पहले 10 दिन के बाद चरम पर होती है, हालांकि महत्वपूर्ण रूप से नहीं।मेंटल बायोप्सी ऊतक से उत्पन्न ऑर्गेनोइड ने शरीर बायोप्सी ऊतक से उत्पन्न ऑर्गेनोइड की तुलना में अधिक वृद्धि दर प्रदर्शित की। विभिन्न रोगियों में, ऑर्गेनॉइड आकृति विज्ञान की विविधता आमतौर पर किसी भी एकल गुंबद में देखी गई थी। हालांकि, औसतन, ऑर्गेनॉइड स्पास्टिसिटी ने थोड़ा बदलाव दिखाया।

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 ...