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

3D-Kultur von Organoiden aus murinen Darmkrypten und einzelnen Stammzellen

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 im Rampenlicht: Die 3D-Kultivierung von Organoiden aus murinen Darmkrypten und einer einzelnen Stammzelle für die Organoidforschung  

Die 3D-Kultivierung von Organoiden aus murinen Darmkrypten und einer einzelnen Stammzelle für die Organoidforschung

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

Processing Murine Small Intestine for Crypt Isolation  

Aufbereitung des murinen Dünndarms zur Kryptenisolierung

Beginnen Sie damit, den Dünndarm des eingeschläferten Tieres zu isolieren und das Gewebe in eine Petrischale zu übertragen. Entfernen Sie mit einer Laborschere und einer Pinzette das Fettgewebe aus dem Dünndarm. Schneiden Sie dann den Dünndarm in mehrere Segmente.Spülen Sie den Dünndarm mit einer Fünf-Milliliter-Spritze mit fünf Millilitern kalten PBS-Antibiotika, um den luminalen Inhalt zu beseitigen. Schneiden Sie das Gewebe der Länge nach mit einer Laborschere auf. Waschen Sie es dann manuell mit kalten PBS-Antibiotika unter Schütteln.Schneiden Sie das Darmsegment mit einer Laborschere in etwa fünf mal fünf Millimeter große Stücke. Verwenden Sie eine Pipette, um die gesammelten Fragmente in ein 50-Milliliter-Röhrchen zu übertragen. Geben Sie anschließend 25 Milliliter kalte PBS-Antibiotika in das Röhrchen mit den Gewebefragmenten und schütteln Sie das Röhrchen 10 Mal hin und her, um den Darminhalt von den Gewebefragmenten abzuwaschen.Saugen Sie den Puffer nach dem Waschen ab. Die Gewebefragmente sind nun bereit für die Kryptenisolierung.

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

Research

JoVE Journal

Biology

Processing Murine Small Intestine for Crypt Isolation

1.2K Aufrufe. Beginnen Sie damit, den Dünndarm des eingeschläferten Tieres zu isolieren und das Gewebe in eine Petrischale zu übertragen. Entfernen Sie mit einer Laborschere und einer Pinzette das Fettgewebe aus dem Dünndarm. Schneiden Sie dann den Dünndarm in mehrere Segmente.Spülen Sie den Dünndarm mit einer Fünf-Milliliter-Spritze mit fünf Millilitern kalten PBS-Antibiotika, um den luminalen Inhalt zu beseitigen. Schneiden Sie das Gewebe der Länge nach mit einer Laborschere auf. Waschen...

Serie: 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 ...