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

Cultura 3D de Organoides de Criptas Intestinais Murinas e Célula-Tronco Única

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.

Autor em destaque: A cultura 3D de organoides de criptas intestinais murinas e uma única célula-tronco para pesquisa de organoides  

A Cultura 3D de Organoides de Criptas Intestinais Murinas e uma Única Célula-Tronco para Pesquisa de Organoides

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  

Cultura de Organoides a partir de uma Única Célula-Tronco Intestinal

Para gerar organoide a partir de um único receptor hepatocelular B2 produtor de eritropoetina, ou célula-tronco intestinal positiva para FEB2, realizar a triagem celular ativada por fluorescência com base na expressão de FEB2 e dividir as células obtidas em quatro grupos, alto, médio, baixo e negativo. Depois de coletar o pellet de alta célula de FEB2 com centrifugação a 390G por três minutos a quatro graus Celsius, incorpore as células altas de FEB2 de triagem única na MEC por pipetagem e, em seguida, semeie as células em uma placa de 24 poços a 100 singlets por 40 microlitros de ECM por poço. Incubar a placa de 24 poços por 15 minutos a 37 graus Celsius e 5% de dióxido de carbono para a polimerização da MEC.Em seguida, cobrir a MEC com 500 microlitros de meio de cultura contendo 10 quinase associada à linha micromolar ou inibidor de rocha nos dois primeiros dias para manter as células altas de FEB2. Inspecionar manualmente as células usando um microscópio invertido em aumento de 40X e observar organoides viáveis com formação esferoide e protrusão de criptas. Estruturas de criptas auto-organizáveis que lembram o intestino delgado normal foram recriadas a partir de células altas de FEB2 únicas.No quinto dia de cultura, formaram-se estruturas semelhantes a esferoides e, do sétimo ao nono dia, ocorreu a evaginação das manchas para formar criptas.

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Research

JoVE Journal

Biology

Organoid Culture from a Single Intestinal Stem Cell

1.1K visualizações. Para gerar organoide a partir de um único receptor hepatocelular B2 produtor de eritropoetina, ou célula-tronco intestinal positiva para FEB2, realizar a triagem celular ativada por fluorescência com base na expressão de FEB2 e dividir as células obtidas em quatro grupos, alto, médio, baixo e negativo. Depois de coletar o pellet de alta célula de FEB2 com centrifugação a 390G por três minutos a quatro graus Celsius, incorpore as células altas de FEB2 de triagem única na MEC por pi...

Vídeo: 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 ...