Induction of Necrotizing Enterocolitis in Human Epithelial Enteroid Model

Obtain human small intestinal tissue-derived crypts  — tubular invaginations containing stem cells and various types of epithelial cells.

Add the intestinal crypts into a chilled basement matrix.

Transfer this mixture into a multiwell plate, creating a three-dimensional dome.

Add growth medium and incubate.

Stem cells self-organize, proliferate, and differentiate into multiple types of intestinal cells, forming enterospheres.

Over time, the enterospheres mature into enteroids  – three-dimensional self-renewing structures, resembling the intestinal crypts.

Add lipopolysaccharides, or LPS, a bacterial endotoxin, into the enteroid-containing well and incubate.

LPS molecules bind to the toll-like receptor in the enteroid, initiating a pro-inflammatory response, resulting in the accumulation of reactive oxygen species. This triggers a cascade of events leading to apoptosis.

Consequently, the integrity of the enteroid is compromised, leading to the penetration of LPS into the lumen, further intensifying the inflammatory response.

This mimics the development of necrotizing  enterocolitis, a severe inflammation in the intestines of premature infants.

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 to ensure that the specimen is clear of stool and blood. Using delicate dissecting scissors, remove any excess fat or surgical clips and staples. Weigh 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 #1. 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 #2. 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 this 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 a 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 2-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 mini-gut media complete to each well. Continue incubating under the same conditions, making sure to replace this media every two days.