Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo

This protocol provides a method to study the function of colon stem cells in a manipulatable environment with lower cost in time than animal models. Organoid culture allows the study of stem cell function in an immune-free environment. This allows for pinpointing the effects of a specific cause, such as the knockout of claudin-7.

Understanding the mechanisms of stem cell function may shed light on new therapeutic targets for debilitating diseases such as inflammatory bowel disease and colorectal cancer. To begin the isolation of colonic crypts from the euthanized mouse, make an incision of approximately two inches down the midline of the mouse. Then, pin back the skin to expose the abdomen.

Cut the colon just below the coecum from the proximal side and above the rectum from the distal side to isolate the colon. Next, remove the adipose tissue attached to the colon using forceps. After pushing out feces from the isolated colon with the flat end of the forceps, cut the tissue open longitudinally.

Wash the tissue 10 to 15 times with cold PBS by swirling the tissue around in PBS between washes with forceps. Then using a pair of clean, sharp scissors, cut the tissue into small pieces ranging from approximately three to five millimeters. After isolating the colon from another euthanized mouse, combine all the tissue pieces in a 50-milliliter centrifuge tube containing cold epithelial dissociation media and incubate the colon tissue pieces in epithelial dissociation media for 90 minutes at four degrees Celsius with gentle rocking.

After incubation, allow the tissue fragments to sink to the bottom of the tube. Once settled, discard the epithelial dissociation media without disrupting the tissue fragments. Repeat the process when washing the tissue 10 to 15 times with cold PBS.

Discard as much PBS as possible during the final wash. Next, add cold crypt dissociation media to the washed colon tissue pieces in a 50-milliliter tube and shake for five to 10 minutes by hand. Under the cell culture hood, filter the media containing the tissue with a 70-micron nylon cell strainer into a fresh-50 milliliter centrifuge tube.

After filtration, centrifuge the tube at 200 G for 10 minutes at room temperature, followed by discarding the supernatant without disturbing the pelleted crypts. Resuspend the pellet in three to four milliliters of cold PBS. Pipette 10 microliters of the crypt suspension in a line on a microscope slide.

Using the microscope, count the number of full, long crypts to estimate crypt concentration and calculate the appropriate volume of crypts required to plate 10 crypts per microliter in a 96-well plate. Centrifuge an appropriate volume of isolated crypts in a 1.5-milliliter microcentrifuge tube at 200 G for five minutes at four degrees Celsius. Then, remove the supernatant using a 1, 000-microliter pipette without disrupting the pelleted crypts.

Then, add 100 microliters of gel matrix to the pelleted crypts without introducing air bubbles. Wait for one to two minutes until the gel matrix is partially solidified. Then, plate 10 microliters of the gel matrix mixed with the crypts in each well of a pre-warmed 96-well culture plate to form a dome shape.

Allow 10 to 20 minutes for the gel matrix to be fully set by placing the plate in an incubator at 37 degree Celsius under 5%carbon dioxide. Finally, add 100 microliters of the freshly prepared working solution of L-WRN media supplemented with antibiotics to each well and incubate the plate at 37 degrees Celsius under 5%carbon dioxide for 24 hours. To harvest the organoids, remove old media from the wells by applying vacuum suction and fix the organoids with 4%paraformaldehyde for one hour at room temperature.

Then remove 4%paraformaldehyde from the wells by vacuum suction and treat the organoids with 100 microliters of 30%sucrose per well at four degrees Celsius. After 24 hours, remove 30%sucrose from the wells by vacuum suction before adding 10 microliters of PBS to each well. Use a pipette tip to gently scratch the bottom of the well to dissociate the dome-containing organoids.

Next, remove the PBS containing the dissociated organoids from the well with a pipette and transfer it to a labeled plastic mold filled 90%with optimum cutting temperature, or OCT, compound. Continue the process until all organoids have been removed from all wells. Add 2-methylbutane to a stainless steel Dewar flask containing dry ice pellets, enough to cover the pellets.

Flash-freeze the organoid containing OCT block by steadily holding it above the 2-methylbutane. Finally, store the organoid-containing OCT block at minus 80 degrees Celsius until ready to section. The representative image highlights the successful growth of colonoids from normal crypts containing claudin-7.

The crypts containing claudin-7 began to form spheroids by day two, started budding on day five, and continued growing and budding until they were harvested on day nine. In contrast, crypts lacking claudin-7 failed to form proper colonoids. After treatment with 4-hydroxytamoxifen for two to three days, the claudin-7 knockout crypts appeared as circular clumps of cells.

Unlike the control, the crypts did not grow into healthy spheroids. Immunofluorescence staining for claudin-7 in the harvested control and the conditional knockout organoids confirmed the successful knockout of claudin-7 in culture. The control colonoids exhibited very little apoptotic signal on day nine.

However, claudin-7 knockout colonoids displayed high apoptosis at the same time. Ensure partial solidification prior to plating. Plating before partial solidification will cause the gel matrix to spread and the dome will not be formed, affecting the survival and growth of the crypts.

This protocol may be utilized for drug discovery, studying cellular communication, drug metabolism, viability, proliferation, and developing patient-specific personalized treatment. The establishment of organoid culture has revolutionized the study of diseases and personalized medicine.