isolating single cells from upper endoscopy biopsies derived from gastric patients 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 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  

오가노이드 생성을 위해 위 환자에서 파생된 상부 내시경 생검에서 단일 세포 분리

환자의 상부 내시경 검사 중에 수집된 위 생검에서 단일 세포의 분리를 시작하려면 먼저 조직이 15밀리리터 원추형 튜브의 바닥에 가라앉도록 합니다. 정해진 후에는 피펫을 사용하여 배지를 흡인하고 항생제가 보충된 PBS 1m리터로 생검을 두 번 세척합니다. 최소 20mg의 조직을 DTT가 보충된 PBS 1mL가 들어 있는 1.5mL의 튜브에 옮깁니다.미세한 해부 가위를 사용하여 조직을 1-2mm 또는 더 작은 조각으로 자릅니다. 탁상용 미니 원심분리기를 사용하여 15초 동안 튜브 바닥에 있는 조직 조각을 응집한 다음 가능한 한 많은 상층액을 흡입합니다. 50밀리리터의 원뿔형 튜브에 갓 데운 분해 완충액 5밀리리터를 추가합니다.이 튜브에서 500 마이크로 리터의 완충액을 조직 조각이 들어있는 1.5 밀리리터 튜브로 옮깁니다. 다음으로, 가위를 사용하여 바닥에서 1, 000 마이크로 리터 피펫 팁으로 잘라 팁의 직경을 증가시켜 조직 손실을 최소화합니다. 이제 수정된 피펫 팁을 사용하여 1.5밀리리터 튜브의 작은 조직 조각을 50밀리리터 튜브의 분해 완충액으로 옮깁니다.분해 완충액과 조직 혼합물을 37도에서 30분 동안 200RPM에서 궤도 진탕으로 배양합니다. 그런 다음 0.25% EDTA가 함유된 따뜻하게 데운 트립신 5mL를 소화 완충액의 조직에 넣고 배양합니다. 동일한 부피의 고급 DMEM/F-12 배지를 추가하여 분해 완충액과 트립신을 중화하고 현탁액을 70미크론 셀 스트레이너에 통과시킵니다.

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Research

JoVE Journal

Biology

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

125 조회수.  University of Pennsylvania Perelman School of Medicine. 환자의 상부 내시경 검사 중에 수집된 위 생검에서 단일 세포의 분리를 시작하려면 먼저 조직이 15밀리리터 원추형 튜브의 바닥에 가라앉도록 합니다. 정해진 후에는 피펫을 사용하여 배지를 흡인하고 항생제가 보충된 PBS 1m리터로 생검을 두 번 세척합니다. 최소 20mg의 조직을 DTT가 보충된 PBS 1mL가 들어 있는 1.5mL의 튜브에 옮깁니다.미세한 해부 가위를 사용하여 조직을 1...

비디오: 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 ...