Abstract: BACKGROUND: The pathogenesis of inflammatory bowel disease involves inflammation, immune activation, visceral hypersensitivity, and dysbiosis of the gut microbiota. Inflammation promotes the release of inflammatory mediators by immune cells, damaging the enteric nervous system. Enteric glial cells are an important component of the intestinal nervous system and are excellent cells for studying intestinal neuroinflammation. Primary enteric glial cells play a crucial role in exploring cell therapies for intestinal nervous system diseases. Currently, the methods for obtaining these cells are mostly cumbersome. Therefore, finding a convenient and fast method for extracting this cell is crucial. 
OBJECTIVE: To establish a method for optimizing the isolation, culture, and identification of mouse enteric glial cells. 
METHODS: 0-7-day-old C57BL/6 neonatal mice were euthanized by excessive inhalation of isoflurane. After soaking in 75% alcohol for disinfection, the duodenum (1 cm below the pylorus to 1 cm above the Qu's ligament) was removed by laparotomy at the midline of the abdomen. A 1 mL syringe was filled with DPBS and the intestinal contents were repeatedly rinsed until the intestine became translucent, and the mesentery and blood vessels were peeled off. The duodenum was cut to a size of 1 mm and digested in 0.25% EDTA trypsin for 20 minutes. Then an equal amount of DMEM/F12 complete culture medium was added to terminate digestion. The liquid was filtered through a 100 μm cell filter, centrifuged, and the cells were resuspended in 1 mL of DMEM/F12 complete culture medium. When the cell adhesion growth density reached 80%, cells were digested for subculture. When cells were cultured to the third generation, glial fibrillary acid protein labeled with enteric glial cells was used for identification by immunofluorescence method.
RESULTS AND CONCLUSION: The isolated and cultured cells were full of colloids, with protrusions extending outward and passable. Glial fibrillary acid protein staining was positive. This method can successfully isolate and culture enteric glial cells and is easy to operate, providing a stable model for the study of the pathophysiology of the enteric nervous system.

Key words: enteric glial cell, neonatal mouse, cell culture, inflammatory bowel disease, enteric nervous system, glial fibrillary acidic protein, engineered cell