The video details the process of generating organoids from FEB2-positive intestinal stem cells using fluorescence-activated cell sorting. Key steps include sorting the cells, embedding them in ECM, incubating for polymerization, and monitoring for organoid formation and crypt structures over several days, showcasing the self-organization akin to the small intestine.

organoid culture from a single intestinal stem cell

Murine Intestinal Crypts 및 Single Stem Cell의 오가노이드 3D 배양

Intestinal organoids, which were first established in 2009, have emerged as a powerful in vitro tool for studying intestinal biology given their morphological and functional similarity to mature tissues. Recently, technological advances in cultured organoids derived from adult-tissue stem cells have allowed for the long-term culture of intestinal stem cells (ISCs) with self-renewal and differentiation potential. These organoids have been widely used for basic and translational research studies on gastrointestinal physiology and pathophysiology1,2,3,4,5,6. The 3D organoids developed by the Clevers group provide a powerful tool to study the intestinal epithelium with improved physiological relevance7. Since intestinal organoids are derived from tissue stem cells and are composed of multiple cell types, they recapitulate the functionality of the intestinal epithelium. Of note, a single-sorted leucine-rich repeats-containing G protein-coupled receptor 5-positive (Lgr5+) stem cell can also generate 3D organoids without any Paneth cells or an ISC niche such as the epithelial niche or stromal niche7. However, the organoid-forming capacity of single-sorted Lgr5+ cells is low compared to those of crypt and ISC-Paneth cell doublets8.
An increasing number of studies have shown that the methods of ethylenediaminetetraacetic acid (EDTA) incubation or collagenase dissociation cause loosening in the epithelium and the release of crypts. As enzymatic dissociation may have an effect on the cell state of crypts, a mechanical isolation method is usually used to dissociate the tissue. Though mechanical digestion is a rapid technique, this method can be associated with inconsistent crypt yields or poor cell viability9. Therefore, EDTA treatment and mechanical dissociation can be combined to generate better crypt yields. A feature of the methodology shown in this article is the use of vigorous shaking of the tissue fragments after EDTA chelation10. Vigorous shaking permits the efficient isolation of crypts from crypt-villus complexes in the small intestine. The degree of manual shaking determines the separation. Thus, obtaining crypts from complexes is important for experimenters in this field. Additionally, proper skill can reduce villus contamination to a minimum and increase the number of crypts.
Hence, this experimental protocol, which employs murine-derived small intestinal organoids, can better isolate crypts with physical force after treatment with EDTA for dissociation. It is known that the expression pattern of erythropoietin-producing hepatocellular receptor B2 (EphB2) in part reflects the crypt environment. For example, EphB2-positive cells are organized from bottom to top11. Fluorescence-activated cell sorting (FACS) was carried out based on the EphB2 expression, and the cells obtained were divided into four groups: EphB2high, EphB2med, EphB2low, and EphB2neg. Then, the organoid growth from single-sorted EphB2high cells in wild-type (WT) mice was demonstrated.

저자 스포트라이트: The 3D Culturing of Organoids from Murine Intestinal Crypts and a Single Stem Cell for Organoid Research (오가노이드 연구를 위한 쥐 장낭화낭수에서 오가노이드의 3D 배양 및 단일 줄기 세포)  

쥐 장 선와에서 오가노이드와 오가노이드 연구를 위한 단일 줄기 세포의 3D 배양

Watch this Scientific Journal Video about Organoid Culture from a Single Intestinal Stem Cell at JoVE.com

Organoid Culture from a Single Intestinal Stem Cell  

단일장 줄기세포에서 오가노이드 배양

단일 에리스로포이에틴 생성 간세포 수용체 B2 또는 FEB2 양성 장 줄기 세포에서 오가노이드를 생성하기 위해 FEB2 발현을 기반으로 형광 활성화 세포 분류를 수행하고 얻은 세포를 높음, 중간, 낮음 및 음성의 4개 그룹으로 나눕니다. 섭씨 4도에서 3분 동안 390G에서 원심분리하여 FEB2 고세포 펠릿을 수집한 후, 피펫팅에 의해 ECM에 단일 분류된 FEB2 고세포를 임베드한 다음, 웰당 ECM 40마이크로리터당 100개의 싱글렛으로 24웰 플레이트에 세포를 시딩합니다. ECM의 중합을 위해 섭씨 37도 및 5% 이산화탄소에서 15분 동안 24웰 플레이트를 배양합니다.다음에, 처음 이틀 동안 10 마이크로몰 행 관련 키나아제 또는 락 억제제를 함유하는 500 마이크로리터의 배양 배지로 ECM을 덮어 FEB2 높은 세포를 유지한다. 40X 배율의 도립 현미경을 사용하여 세포를 수동으로 검사하고 스페로이드 형성 및 선와돌출이 있는 생존 가능한 오가노이드를 관찰합니다. 정상적인 소장을 연상시키는 자기 조직화 크립트 빌라 구조는 단일 FEB2 높은 세포에서 재현되었습니다.배양 5일째에는 구형과 같은 구조가 형성되었고, 7일째부터 9일째에는 지하실을 형성하기 위한 반점의 대피가 발생했습니다.

organoid-culture-from-a-single-intestinal-stem-cell

Research

JoVE Journal

Biology

Organoid Culture from a Single Intestinal Stem Cell

1.1K 조회수. 단일 에리스로포이에틴 생성 간세포 수용체 B2 또는 FEB2 양성 장 줄기 세포에서 오가노이드를 생성하기 위해 FEB2 발현을 기반으로 형광 활성화 세포 분류를 수행하고 얻은 세포를 높음, 중간, 낮음 및 음성의 4개 그룹으로 나눕니다. 섭씨 4도에서 3분 동안 390G에서 원심분리하여 FEB2 고세포 펠릿을 수집한 후, 피펫팅에 의해 ECM에 단일 분류된 FEB2 고세포를 임베드한 다음, 웰당 EC...

비디오: Organoid Culture from a Single Intestinal Stem Cell  

The 3D Culturing of Organoids from Murine Intestinal Crypts and a Single Stem Cell for Organoid Research

At present, organoid culture represents an important tool for in vitro studies of different biological aspects and diseases in different organs. Murine small intestinal crypts can form organoids that mimic the intestinal epithelium when cultured in a 3D extracellular matrix. The organoids are composed of all cell types that fulfill various intestinal homeostatic functions. These include Paneth cells, enteroendocrine cells, enterocytes, goblet cells, and tuft cells. Well-characterized molecules are added into the culture medium to enrich the intestinal stem cells (ISCs) labeled with leucine-rich repeats containing G protein-coupled receptor 5 and are used to drive differentiation down specific lineages; these molecules include epidermal growth factor, Noggin (a bone morphogenetic protein), and R-spondin 1. Additionally, a protocol to generate organoids from a single erythropoietin-producing hepatocellular receptor B2 (EphB2)-positive ISC is also detailed. In this methods article, techniques to isolate small intestinal crypts and a single ISC from tissues and ensure the efficient establishment of organoids are described.

We describe a protocol to isolate murine small intestinal crypts and culture intestinal 3D organoids from the crypts. Additionally, we describe a method to generate organoids from a single intestinal stem cell in the absence of a sub-epithelial cellular niche.

At present, organoid culture represents an important tool for in vitro studies of different biological aspects and diseases in different organs. Murine ...