Abstract
Although critical for studies of gut motility and intestinal regeneration, the in vitro culture of intestinal muscularis with peristaltic function remains a significant challenge. Periodic contractions of intestinal muscularis result from the coordinated activity of smooth muscle cells (SMC), the enteric nervous system (ENS), and interstitial cells of Cajal (ICC). Reproducing this activity requires the preservation of all these cells in one system. Here we report the first serum-free culture methodology that consistently maintains spontaneous and periodic contractions of murine and human intestinal muscularis cells for months. In this system, SMC expressed the mature marker myosin heavy chain, and multipolar/dipolar ICC, uniaxonal/multipolar neurons and glial cells were present. Furthermore, drugs affecting neural signals, ICC or SMC altered the contractions. Combining this method with scaffolds, contracting cell sheets were formed with organized architecture. With the addition of intestinal epithelial cells, this platform enabled up to 11 types of cells from mucosa, muscularis and serosa to coexist and epithelial cells were stretched by the contracting muscularis cells. The method constitutes a powerful tool for mechanistic studies of gut motility disorders and the functional regeneration of the engineered intestine.
Highlights
In the small intestine, the mucosa processes partially digested food and absorbs nutrients while the muscularis actuates the peristaltic flow to transport luminal content aborally
Various protocols have been developed to culture primary smooth muscle cells[23], interstitial cells of Cajal (ICC)[12,13], or enteric neural elements [24]—or two of the three[25] in combination—but here, for the first time, we developed a single culture medium that preserves all three in one system for an extended period and restored the spontaneous, rhythmic contractile function of intestinal muscularis cells (IMC)
Using immunofluorescence and RT-PCR analysis, we demonstrated that smooth muscle cells, ICC, neurons and glia, all thrived in the muscularis medium
Summary
The mucosa processes partially digested food and absorbs nutrients while the muscularis actuates the peristaltic flow to transport luminal content aborally. Gut motility is central to its digestive and absorptive function. The intestinal muscularis contains various types of cells.
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