3D Culturing of Organoids from the Intestinal Villi Epithelium Undergoing Dedifferentiation

This method can confirm the organoid-forming capacity of de-differentiating villi epithelium that acquire stem cell markers in vivo. The collection of villi is performed via scraping rather than the EDTA chelation method preventing the complete loss of the underlying mesenchyme that may provide the niche signals if required for organoid initiation. This method is applicable in epithelial tissues where proliferative and differentiated compartments are physically delineated and the de-differentiating cells express stem cell markers in vivo.

Some factors in this protocol need to be determined empirically, including the optimal stage for harvesting the villi and the optimal pressure for scraping the villi, which will take time and practice. Induce the Smad4 knockout and beta-catenin gain of function mutation in mutant mice with intraperitoneal injection of tamoxifen and corn oil for four consecutive days. After euthanizing the mouse, spray the abdomen with 70%ethanol to prevent mouse fur from getting into the peritoneal cavity.

Open the abdominal cavity with dissection scissors to expose the intestine and isolate the intestine using scissors and forceps. Dissect out the proximal half of the duodenum, then flush the duodenum with five milliliters of ice cold PBS in a 10 milliliter syringe to clear the luminal content. Open the duodenum longitudinally with an angled scissor and lay the duodenum flat on a 15 centimeter Petri dish on ice with the lumen of the duodenum facing the operator.

Prior to beginning the scraping, place a 70 micrometer mesh strainer in one of the wells of a six-well tissue culture plate. Fill all the wells with four milliliters of 1X PBS and place the plate on ice. Scrape the villi using two microscopic glass slides, one to hold the duodenum down and the other to scrape.

Scrape the luminal side of the duodenum superficially twice to remove the mucus without removing the villi, then scrape the duodenum two more times to collect the villi on the slides without tethering the crypts. Use a one milliliter transfer pipette containing PBS to transfer the villi to a 70 micrometer mesh strainer in the six-well dish. Collect the villi after every scrape.

To remove loose crypts, wash the villi collected with the 70 micrometer strainer by transferring the strainer through a series of wells in the six-well dish containing four milliliters of ice cold PBS per well. Using a P1000 pipette, transfer the villi suspension in about three milliliters of PBS from the 70 micrometer strainer to a new 15 milliliter tube on ice. Use a 0.1%BSA-coated blunt-ended P200 pipette tip to transfer a 50 microliter volume of the villi suspension onto a glass slide.

Count the number of villi in the 50 microliter droplet under 4X magnification to determine the concentration of villi in the PBS suspension and to confirm the absence of tethered crypts. To plate the villi, use a 0.1%BSA-coated P200 blunt-ended pipette tip to transfer the villi to a microcentrifuge tube for a plating density of six villi per well in 12.5 microliters of BME-R1 matrix. Spin down the villi for two minutes at 200 times G at four degrees Celsius.

Remove the supernatant and repeat the centrifugation to remove any residual PBS. In a laminar flow hood, resuspend the villi pellet gently in the required amount of cold BME-R1 thawed on ice. Using a P20 pipette, plate 12.5 microliters of the villi in BME-R1 matrix per well of a 96-well U-bottomed plate pre-warmed to 37 degrees Celsius.

Incubate the plate in a tissue culture incubator at 37 degrees Celsius for 15 minutes to allow solidification of the BME-R1 matrix. To each well, add 125 microliters of pre-warmed ENR media supplemented as described in the text manuscript. Incubate the plated villi in a tissue culture incubator at 37 degrees Celsius with 5%carbon dioxide and change the media every other day.

Discard any well in which organoids appear before two days. There's a difference in appearance of the organoids emerging from the crypts versus the de-differentiated villi epithelium from the same mouse intestine. The crypt-derived organoids appear overnight as spherical structures with well-defined borders.

The kinetics and morphological appearance of the organoids initiated from villi was examined, which appear irregularly shaped at first and take two to five days before they can be seen under a microscope. Organoid initiation from two different villi is shown. The villi with organoid-forming potential appears dense, possibly due to the retention of the underlying mesenchyme.

Organoid initiation from the villas is apparent at day two from one of the villi. While in the other villas, the organoid appears at day four. It's crucial to take the proper steps to avoid crypt contamination when harvesting the villi and growing the resulting organoids.

Phenotypic differences between the organoids emerging from the crypts and villi of the same mutant were observed following this procedure, so further experimentation could be carried out to look for molecular differences between the two. This protocol could be used to study the differences between organoids arising from endogenous crypts versus ectopic crypts arising from de-differentiation, thus implications of de-differentiation could be addressed.