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.
