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. The 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 finding is that signaling through objective cloning maintained the homeostasis of intestinal epithelial growth and differentiation. Our protocol describes 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 treatment and mechanical dissociation can be combined to improve cryptal yields. Additionally, proper skill can reduce various contamination to a minimum, increasing the number of crypts.
Our findings suggest that the signaling through the muscarinic and genic acid receptors appear to work together to maintain the homeostasis of intestinal epithelial growth and differentiation. This encourages us to hypothesize that the non-neural coronary system plays a pivotal role in the moderation of intestinal stem cell niche. 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. 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 390G and four degree Celsius for three minutes. Then resuspend the crypt pallet 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 80G 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 80G and four degrees Celsius for three minutes. Aspirate the supernatant as demonstrated before and add 10 milliliters of sorbitol DMEM for resususpension. After aspirating the supernatant, add 10 milliliters of complete DMEM and resuspend 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 290G 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 pipetted 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 one, 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 time-lapse 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. Time-lapse 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 panet cell differentiation. 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 degrees 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 villus 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.
