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.