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

תרבית תלת ממדית של אורגנואידים מקריפטים של המעי המוריני ותאי גזע בודדים

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.

מחבר זרקור: התרבות התלת-ממדית של אורגנואידים מקריפטים של מעי מורין ותא גזע יחיד למחקר אורגנואידים  

תרבית תלת-ממדית של אורגנואידים מקריפטים של מעי מורין ותא גזע יחיד למחקר אורגנואידים

Our research focusing on stem cell biology, especially intestinal stem cell, present at the bottom of intestinal crypt. The aim is to answer how homeostasis is maintained by the stem cell in the intestine. This 3D organ culture derived from intestinal stem cell provides a powerful tool to study the proliferation, differentiation, and maintenance of stem cells.Organ technology aids the culture of intestinal stem cell for a long time by retaining the self-renewal and differentiation potential. The organoid has been widely used for basic and translational research studies on intestinal fragility and past fragility. Our current challenge is to understand the coronary system involved in intestinal epithelial cell and stem cell.Understanding biological processes triggered by objective cloning is of key importance. One of our significant findings is that signaling through cloning maintain the homeostasis of intestinal epithelial growth and differentiation. Our protocol describe a method for consistently isolating small intestinal crypts, and subsequent culture of 3D organoids to improve cryptal releasing rate.We establish a mechanical isolation method involving vigorous shaking, after treatment with EDTA. EDTA and mechanical dissociation can be combined to improve cryptal yields. Additionally, proper skill can reduce virus contamination to a minimum, increasing the number of crypts.Our findings suggest that the signaling through the muscular and organism receptors appear to work together to maintain the homeostasis of in intestinal epithelial growth and differentiation. This encourages us to hypothesize that the non-neurocoronary-arteritic system played an important role in the moderation of intestinal stem cell niche.

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

Processing Murine Small Intestine for Crypt Isolation  

עיבוד מעי דק מורין לבידוד קריפטה

התחילו בבידוד המעי הדק מהחיה המורדמת והעבירו את הרקמה לצלחת פטרי. באמצעות מספריים מעבדה פינצטה, להסיר את רקמות השומן מן המעי הדק. לאחר מכן, לחתוך את המעי הדק למספר מקטעים.באמצעות מזרק חמישה מיליליטר, לשטוף את המעי הדק עם חמישה מיליליטר של PBS קר אנטיביוטיקה כדי לנקות את התוכן luminal. חותכים את הרקמה לאורכה בעזרת מספריים מעבדתיים. לאחר מכן, שטפו אותו ידנית עם PBS קר - אנטיביוטיקה תוך כדי רעידה.באמצעות מספריים מעבדה, לחתוך את קטע המעי לחתיכות בגודל של כחמישה מילימטרים על חמישה מילימטרים. השתמש פיפטה כדי להעביר את השברים שנאספו לתוך צינור 50 מיליליטר. לאחר מכן, הוסף 25 מיליליטר של אנטיביוטיקה PBS קרה לצינור המכיל את שברי הרקמה ונער את הצינור קדימה ואחורה 10 פעמים כדי לשטוף את תוכן המעי משברי הרקמה.יש לשאוף את החיץ לאחר הכביסה. שברי הרקמה מוכנים כעת לבידוד קריפטה.

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

Research

JoVE Journal

Biology

Processing Murine Small Intestine for Crypt Isolation

1.2K Views.  Bioorganic Research Institute. התחילו בבידוד המעי הדק מהחיה המורדמת והעבירו את הרקמה לצלחת פטרי. באמצעות מספריים מעבדה פינצטה, להסיר את רקמות השומן מן המעי הדק. לאחר מכן, לחתוך את המעי הדק למספר מקטעים.באמצעות מזרק חמישה מיליליטר, לשטוף את המעי הדק עם חמישה מיליליטר של PBS קר אנטיביוטיקה כדי לנקות את הת...

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 ...

Begin by isolating the small intestine from the euthanized animal and transfer the tissue to a Petri dish. Using laboratory scissors and tweezers, remove the fat tissues from the small intestine. Then, cut the small intestine into several segments.Using a five-milliliter syringe, flush the small intestine with five milliliters of cold PBS-antibiotics to clear the luminal content. Cut the tissue open lengthwise using laboratory scissors. Then, manually wash it with cold PBS-antibiotics while shaking.Using laboratory scissors, cut the intestinal segment into pieces measuring approximately five millimeters by five millimeters. Use a pipette to transfer the collected fragments into a 50-milliliter tube. Next, add 25 milliliters of cold PBS-antibiotics to the tube containing the tissue fragments and shake the tube back and forth 10 times to wash off the intestinal contents from the tissue fragments.Aspirate the buffer after washing. The tissue fragments are now ready for crypt isolation.

Crypt Isolation from Murine Small Intestine

For murine small intestinal crypt isolation, use properly washed five millimeter by five millimeter fragments of the isolated murine small intestine. Incubate the pieces in PBS antibiotics containing two millimolar EDTA for 30 minutes on ice without shaking. To ease the solidification of the extracellular matrix, or ECM, incubate a 24 well plate in a 37 degree Celsius tissue culture incubator beforehand.After aspirating the EDTA solution from the tissue fragments, add 25 milliliters of fresh cold PBS antibiotics. Then shake the container vigorously by hand, 30 to 40 times. Filter the suspension once through a 70 micron strainer.Before moving on to the next step, confirm the presence of the crypts under the microscope. Next, centrifuge the suspension at 390 G and four degrees Celsius for three minutes. Then, re-suspend the crypt pellet in 20 milliliters of DMEM containing 2%sorbitol, henceforth referred to as sorbitol DMEM.Transfer 10 milliliters of the crypt suspension to each of two new 15 milliliter tubes. This time, centrifuge the two tubes at a lower speed of 80 G for three minutes at four degrees Celsius to separate the large cell masses from the cells or debris. Aspirate the supernatant gently, leaving about two milliliters of supernatant in each tube.Then, add 10 milliliters of sorbitol DMEM to each tube. Centrifuge the suspension again at 80 G and four degrees Celsius for three minutes. Aspirate the supernatant as demonstrated before, and add 10 milliliters of sorbitol DMEM for re-suspension.After aspirating the supernatant, add 10 milliliters of complete DMEM and re-suspend the pellet by pipetting up and down. Leave the suspension to rest for one minute to obtain the floating crypts efficiently. After one minute, filter the suspension from both tubes into a fresh tube through a 70 micron cell strainer to purify the crypts.To count the pure crypts before seeding, drip 25 microliter droplets into a six centimeter dish at three points. Count the number of crypts under a microscope at 4X magnification and calculate the concentration of crypts per 25 microliters. Then, centrifuge the whole filtrate at 290 G for three minutes at four degrees Celsius.For seeding, suspend the crypts in ECM at a concentration of 100 crypts per 40 microliters of ECM. Pipette it up and down five to 10 times gently avoiding air bubbles to obtain a homogeneous suspension. Then, seed 40 microliters of the crypt suspension per well in the pre-warmed 24 well plate.Incubate the 24 well plate for 15 minutes at 37 degrees Celsius and 5%carbon dioxide for the polymerization of the ECM. Cover the ECM per well with 500 microliters of culture medium containing mouse epidermal growth factor, recombinant mouse R-Spondin 1 and recombinant mouse noggin. The final concentration of materials per well is shown here.Culture the crypts by incubating at 37 degrees Celsius and 5%carbon dioxide. Perform live imaging to record organoid morphogenesis using a Timelapse image microscope every three hours for up to seven days and obtain serial Z stacked images. Almost all the isolated crypts were immediately sealed and appeared cone shaped once squeezed out of the epithelial niches.Further, the crypts in the final fraction were integrated and suitable for use in culture. Timelapse images of organoid growth revealed that active proliferation and differentiation of intestinal stem cells occurred in the crypt region with budding. Budding was coupled with cell migration, proliferation, and paneth cell differentiation.

בידוד קריפטה מהמעי הדק של מורין

Organoid Culture from a Single Intestinal Stem Cell

To generate organoid from a single erythropoietin-producing hepatocellular receptor B2, or FEB2-positive intestinal stem cell, carry out fluorescence-activated cell sorting based on the FEB2 expression, and divide the cells obtained into four groups, high, medium, low and negative. After collecting the FEB2 high cell pellet with centrifugation at 390G for three minutes at four degree Celsius, embed the single sorted FEB2 high cells in the ECM by pipetting, then seed the cells on a 24-well plate at 100 singlets per 40 microliters of ECM per well. Incubate the 24-well plate for 15 minutes at 37 degrees Celsius and 5%carbon dioxide for the polymerization of the ECM.Next, cover the ECM with 500 microliters of culture medium containing 10 micromolar row-associated kinase, or rock inhibitor for the first two days to maintain the FEB2 high cells. Manually inspect the cells using an inverted microscope at 40X magnification and observe viable organoids with spheroid formation and crypt protrusion. Self-organizing crypt villa structures reminiscent of the normal small intestine were recreated from single FEB2 high cells.By day five of culture, spheroid-like structures formed, and from day seven to day nine, evagination of the spots to form crypts occurred.