The video provides a step-by-step guide on isolating and preparing segments of the small intestine from a euthanized animal. It details the processes of removing fat, flushing, cleaning, and cutting the intestine into pieces for further crypt isolation, using specific tools and solutions like cold PBS-antibiotics.

processing murine small intestine for crypt isolation

Cultivo 3D de organoides a partir de criptas intestinales murinos y células madre individuales

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 destacado: El cultivo en 3D de organoides de criptas intestinales murinas y una sola célula madre para la investigación de organoides  

El cultivo 3D de organoides a partir de criptas intestinales murinas y una sola célula madre para la investigación de organoides

Watch this Scientific Journal Video about Processing Murine Small Intestine for Crypt Isolation at JoVE.com

Processing Murine Small Intestine for Crypt Isolation  

Procesamiento del intestino delgado murino para el aislamiento de criptas

Comience aislando el intestino delgado del animal sacrificado y transfiera el tejido a una placa de Petri. Usando tijeras y pinzas de laboratorio, retire los tejidos grasos del intestino delgado. Luego, corte el intestino delgado en varios segmentos.Usando una jeringa de cinco mililitros, enjuague el intestino delgado con cinco mililitros de antibióticos PBS para el resfriado para eliminar el contenido luminal. Corte el tejido abierto longitudinalmente con tijeras de laboratorio. Luego, lávelo manualmente con antibióticos PBS fríos mientras agita.Usando tijeras de laboratorio, corte el segmento intestinal en pedazos que midan aproximadamente cinco milímetros por cinco milímetros. Use una pipeta para transferir los fragmentos recolectados a un tubo de 50 mililitros. Luego, agregue 25 mililitros de antibióticos PBS fríos al tubo que contiene los fragmentos de tejido y agite el tubo hacia adelante y hacia atrás 10 veces para lavar el contenido intestinal de los fragmentos de tejido.Aspirar el tampón después del lavado. Los fragmentos de tejido ahora están listos para el aislamiento de la cripta.

processing-murine-small-intestine-for-crypt-isolation

Research

JoVE Journal

Biology

Processing Murine Small Intestine for Crypt Isolation

1.2K vistas. Comience aislando el intestino delgado del animal sacrificado y transfiera el tejido a una placa de Petri. Usando tijeras y pinzas de laboratorio, retire los tejidos grasos del intestino delgado. Luego, corte el intestino delgado en varios segmentos.Usando una jeringa de cinco mililitros, enjuague el intestino delgado con cinco mililitros de antibióticos PBS para el resfriado para eliminar el contenido luminal. Corte el tejido abierto longitudinalmente con tijeras de laboratorio. Lueg...

Video: Processing Murine Small Intestine for Crypt Isolation  

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 ...