A Novel Human Epithelial Enteroid Model of Necrotizing Enterocolitis

Enteroids are three dimensional organoids derived from intestinal epithelial cells. They are ideal for the study of complex physiological interactions, cell signaling, and host pathogen defense. Our protocol describes how to culture human enteroids after isolating intestinal stem cells from patients undergoing bowel resection.

Co-administration of lipopolysaccharide in our culture media causes an inflammatory response in the enteroids leading to histologic, genetic, and protein expression alterations similar to those seen in human necrotizing enterocolitis. Given that the study of necrotizing enterocolitis and potential therapeutic agents is ethically challenging in human subjects, especially children, it is highly desirable to utilize this novel, biologically relevant enteroid model of necrotizing enterocolitis using human neonatal tissue. The main advantage of this technique is that enteroids are able to recapitulate the multiple cells types of human intestinal epithelium and are able to overcome the recognized limitations of animal models and cell based systems.

Helping to demonstrate the procedure will be Christie Buonpane, a surgical resident, and Carrie Yuan, a laboratory technician from our laboratory. At the time of collection in the operative suite, place the human small intestinal tissue sample in cold DPBS. Wash the specimen in the cold DPBS until it is clear of blood and stool.

Store the specimen in RPMI 1640 medium at four degrees Celsius until ready for crypt isolation. When ready to proceed, check again that the specimen is clear of stool and blood. Using delicate dissecting scissors remove any excess fat or surgical clips and staples.

Weight the specimen and aim for a piece that is approximately 0.75 to 2.5 grams. Next, cut the tissue into 0.5 centimeter pieces and place them in a tube containing 30 milliliters of chelating buffer number one. Shake at a low speed for 15 minutes at four degrees Celsius.

Then, filter the tissue through a 100 micrometer cell strainer and discard the flow through. Add the filtered tissue to a tube containing 30 milliliters of chelating buffer number two. Shake at a low speed for 15 minutes at four degrees Celsius.

Filter the tissue through a 100 micrometer filter and discard the flow through. After this, thaw 500 microliters of basement membrane matrix on ice for later use. Add some tissue to 10 milliliters of cold DMEM in a 50 milliliter conical tube and shake vigorously by hand for 10 seconds.

Filter this suspension through a 100 micrometer cell strainer and collect the flow through. Keep this tube on ice. Add some tissue to another 10 milliliters of cold DMEM in a separate 50 milliliter conical tube and shake vigorously by hand for 10 seconds.

Filter this suspension through a 100 micrometer cell strainer and collect the flow through. Repeat the process two additional times until there are four conical tubes containing flow through labeled one through four. Then, filter the solution in tube number one through a 100 micrometer cell strainer and transfer the flow through into a 15 milliliter conical tube also labeled number one.

Repeat this process for tubes two through four. Centrifuge the 15 milliliter tubes at 200 times G and at four degrees Celsius for 15 minutes. In the laminar flow hood, remove the supernatant from each tube and discard it.

Avoid disrupting the cloud of tissue immediately above the pellet, even if that means leaving some supernatant behind. In each tube, pipette up and down slowly to mix the pellet with the leftover supernatant. Transfer the mixture from each tube into a single two milliliter conical tube and centrifuge at 200 times G and at four degrees Celsius for 20 minutes.

After this, remove the supernatant and resuspend the pellet in 500 microliters of pre-thawed basement membrane matrix. Use a chilled pipette tip to apply 50 microliters of this suspension to the center of a well in a 24 well plate. This sample should appear dome shaped.

Repeat this application process nine times to fill 10 total wells. Transfer the 24 well plate into a 5%carbon dioxide incubator at 37 degrees Celsius for 30 minutes to allow polymerization. Then, add 500 microliters of human minigut media complete to each well.

Continue incubating under the same conditions, making sure to replace this media every two days. First, add 10 microliters of LPS at a concentration of five milligrams per milliliter to the 500 microliters of human minigut media complete in each well of the plate on day zero. Continue incubating at 37 degrees Celsius with 5%carbon dioxide and replace the supplemented media every two days until collection.

To prepare for paraffin embedding, first gently remove the media, add one milliliter of PBS, and gently pipette up and down to dissolve the basement membrane matrix while being careful not to lyse the enteroids. Centrifuge at a speed less than 300 times G for five minutes to pellet and then remove the PBS. Add 4%paraformaldehyde and set the tube aside for one hour to fix at room temperature.

Centrifuge at a speed less than 300 times G for five minutes to pellet and then remove the paraformaldehyde. Wash gently with one milliliter of PBS and centrifuge at a speed less than 300 times G for five minutes. Then remove the PBS.

Repeat this wash process with PBS once. Place the desired amount of the tissue processing gel into a conical tube and warm it in a dry bath incubator at 65 degrees Celsius for three to 10 minutes until it is liquid. After this, add the melted tissue processing gel to the pellet and mix gently.

Place a small cloning ring into a coverslip. Pipette the tissue processing gel and enteroid mixture into the cloning ring that is mounted onto a coverslip. Allow the tissue processing gel and the enteroid mixture to solidify at four degrees Celsius for one hour.

Then, submerge the cloning ring with the solidified mixture into 70%ethanol to prepare for paraffin embedding. Immediately after plating, the freshly isolating intestinal crypts appear as elongated rods. Within hours the enteroids will take on a round appearance.

Over the next several days the enteroids will start forming spheres. Budding should occur between five to 10 days, which is when enteroid collection should occur. The growing enteroids also exhibit polarity, containing a centralized lumen, an apical border, and a basilateral domain.

Enteroids also show structural integrity represented by a robust actin cytoskeleton. After several days in culture, the LBS treated enteroids experience more apoptosis and have a lower yield than the control group. As seen in human NEC in murine models of NEC, an increased expression of toll like receptor four is found in LPS treated enteroids compared to controls.

The enteroids can also be collected for RNA and protein extraction using well established qRT-PCR and western blotting techniques gene expression and protein isolation can be performed.