腸幹とニッチ細胞機能解析における再構成アッセイ (ORA)

The overall goal of the organoid reconstitution assay is the functional analysis of the two major components of the intestinal stem cell niche Lgr5 positive in the Paneth cells through their genetic or biochemical modification followed by reconstitution into self-renewing organoids. This method allows addressing key specific questions in the intestinal stem cell field such as genetic or biochemical modification in stem and niche cells. The main advantage of this technique is that unlike other assays that use whole crypts, it employs sorted stem and niche cells thus allowing cell-type specific analysis.

Video demonstration of this method is critical especially when it comes to the tissue dissociation and cell handling steps. If not properly executed, these steps will lead to poor cell viability especially after fact sorting. Begin this procedure with preparation of materials and sacrifice of an Lgr5 mouse on a C57BL/6J background as described in the text protocol.

Dissect the peritoneum longitudinally with a pair of scissors. Hold the stomach with the forceps and cut it transversally in half using intestinal scissors from this point forward. Pull out the intestine and place it in a Petri dish containing PBS.

Start by placing the blunt tip into the stomach and gently push it through the pylorus. Proceed by cutting with the scissors and pulling with the forceps. Once the whole small intestine is opened longitudinally, wash it in cold PBS by holding it with the forceps and gently rinsing it in the PBS solution with U-shaped movements.

Once all the stool remnants are cleared, proceed to flatten the intestine on a cutting board, lumenal side up. The lumenal side is easily recognizable by the absence of blood vessels and by its pale appearance when compared with the outer part. With a glass slide, gently remove the villi by scraping the flattened intestine.

Perform the step twice along the whole length of the tissue. Then, cut the small intestine with a sterile surgical blade into two to five millimeter pieces. Place the small intestinal fragments in a 50-milliliter tube containing 10 milliliters of ice cold PBS.

Clean the tissue fragments removing any remaining impurities by pipetting them up and down in the PBS. Discard the supernatant and repeat the step until the PBS is completely clear. Next, add 15 milliliters of cold PBS to bring the total final volume of PBS to 25 milliliters.

Add two millimolar EDTA and incubate for 45 minutes on a roller at four degrees Celsius. After discarding the PBS EDTA, add 10 milliliters of PBS and detach the crypts by harshly pipetting the tissue fragments up and down. Then, collect the supernatant.

Repeat this step four times. Add culture medium to reach a final volume of 50 milliliters. Pellet the cells by centrifugation.

Finally, resuspend the pellet in 10 milliliters of culture medium before pelleting the crypts by centrifugation. For single-cell preparation, first discard the supernatant of the pelleted crypts and resuspend them in one milliliter of trypsin-like enzymes together with 50 microliters of DNase. Incubate the cells in a water bath at 32 degrees Celsius for two minutes.

After adding 10 milliliters of culture medium, dissociate the crypts into single cells by harshly pipetting up and down at least five times. Filter the solution through a 40-micron strainer to eliminate clumps and other impurities. Then, pellet the single cells at 300 times gravity for five minutes.

For flow cytometry, resuspend the pellet in one milliliter of one X Hank's buffered salt solution with 2%fetal cow serum for every million cells. To the cell suspension, add Brilliant Violet 421 labeled antibodies for lineage marker CD31, CD45 and Ter119 at a concentration of one-to-100. Also, add anti-CD24 APC and anti-CD117 PE at a concentration of one-to-250.

After 30 minutes, sort Lgr5 positive stem cells and Paneth cells into separate low-binding tubes. Centrifuge the sorted cells at 300 times gravity for five minutes. Then, resuspend the cells in 100 microliters of medium with the components described in the text protocol.

To treat the cells, use five microliters of liposome-mediated transfection reagent in a total volume of 100 microliters of culture medium and small interfering RNA at a concentration of 100 nanomolar. Incubate the cells for 30 minutes at 37 degrees Celsius for small interfering RNA, or for the time necessary for the chosen modification. Wash the cells twice in 500 microliters of culture medium.

After centrifuging the cells at 300 times gravity for five minutes, resuspend them in 10 microliters of culture medium. Next, pool the two cellular components in a 20-microliter total volume before centrifuging at 300 times gravity for five minutes at room temperature. Co-incubate the Paneth and Lgr5 positive cells for 10 minutes at room temperature.

Remove 10 microliters of the supernatant and leave them in a skis of liquid so as to avoid aspirating the cell pellet. Then, add 30 microliters of reconstituted basement membrane to the cells for a total volume of 40 microliters. Resuspend the cells and plate them in a pre-warmed 96-well plate.

After 10 minutes, add 200 microliters of complete medium. Change the medium every 48 hours. Finally, count organoid multiplicity at day five.

Small interfering RNA mediated knockdown of the murine APC gene in Lgr5 positive stem cells and the resulting activation of the canonical Wnt/beta-catenin signaling pathway positively affects organoid multiplicity when compared to their untreated counterparts or the scrambled sequence control. The constitute of Wnt activation also rescues the requirement for Paneth cells as previously reported. Furthermore, these organoids appear as hollow spheres, a well-described phenotype for APC mutant or Wnt-stimulated organoids when compared with the morphology of those obtained from Paneth-Lgr5 cell doublets.

Once mastered, this technique can be done in five hours if performed properly. While attempting this technique, it is important to follow all steps and incubation times. After watching this video, you should have a good understanding of how to extract intestinal crypt, prepare a single-cell digest and reconstitute niche and stem cells to generate organoids.

This technique paved the way for the researchers in intestinal stem cell field to dissect the stem or niche cells specific effects in the mouse small intestine or large intestine. Although this method has been established for the functional analysis of the intestinal stem cell niche, it can also be applied to other somatic stem cells niches like the mammary gland or the hair follicle. Following this procedure, additional experiments can be performed to analyze genome protein expression in the organoids such as QPCR or Western blotting.