embedding gastric biopsy-derived single cells in a basement membrane matrix dome for organoid generation

상부 내시경 생검에서 인간 위 오가노이드 생성

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.

저자 스포트라이트: Generation of and Comparison Between Patient-Derived Gastric Organoids from Different regions of the Stomach(위의 다양한 영역에서 환자 유래 위 오가노이드 생성 및 비교) 

양성 위체 및 안방 상피의 생검에서 환자 유래 위 오가노이드의 확립 및 특성화

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  

오가노이드 생성을 위해 기저막 매트릭스 돔에 위 생검 유래 단일 세포 포함

시작하려면 얼음에서 해동된 기저막 매트릭스를 제거합니다. 계산된 기저막 매트릭스의 부피를 위 생검 유래 단세포에 첨가하고 약 10초 동안 피펫팅을 위아래로 하여 부드럽게 혼합합니다. 그런 다음 피펫을 사용하여 매트릭스의 50 마이크로 리터 분취액 및 세포 혼합물을 24 웰 조직 배양 플레이트에서 개별 웰의 중심으로 빠르게 옮깁니다.즉시 접시를 덮고 한 번의 부드러운 동작으로 접시를 거꾸로 뒤집습니다. 거꾸로 된 플레이트를 섭씨 37도의 조직 배양 인큐베이터에 35분 동안 넣어 기저막 매트릭스가 중합되도록 합니다. 그런 다음 500마이크로리터의 예열된 위 오가노이드 배지를 각 웰에 추가하고 각 돔의 상단이 배지에 완전히 잠기도록 합니다.오가노이드는 일반적으로 단일 세포의 파종 후 10일 이내에 식별할 수 있습니다. 20일째가 되면 크기가 커서 일반적으로 통과해야 합니다. 신체 및 전방 오가노이드의 수는 파종 후 10일째에 최고조에 달했다가 크게 감소하지는 않지만 감소합니다.맨틀 생검 조직에서 생성된 오가노이드는 신체 생검 조직에서 생성된 오가노이드에 비해 더 빠른 성장률을 보였다. 다양한 환자에 걸쳐, 다양한 오가노이드 형태가 일반적으로 단일 돔에서 관찰되었습니다. 그러나 평균적으로 오가노이드 경직은 거의 변화를 보이지 않았습니다.

embedding-gastric-biopsy-derived-single-cells-basement-membrane

Research

JoVE Journal

Biology

Embedding Gastric Biopsy-Derived Single Cells in a Basement Membrane Matrix Dome for Organoid Generation

70 Views.  University of Pennsylvania Perelman School of Medicine. Embedding Gastric Biopsy-Derived Single Cells in a Basement Membrane Matrix Dome for Organoid Generation

Video: 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 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 ...