Regeneration of Functional Intestinal Smooth Muscle
Although important for studies of gut motility and essential for intestinal regeneration, the in vitro culture of smooth muscle with peristaltic function remains a significant challenge. Many different culture systems have been established to restore the correct morphology or cellular components of intestinal muscle layers, but few have been developed to allow the appropriate function. Intestinal peristalsis is effected by smooth muscle cells (SMC), coordinated by the enteric neurons, and paced by interstitial cells of Cajal (ICC). Previously, different media have been developed to specifically culture primary muscle, ICC, neurons—or two of the three in combination—but no media could preserve all the three in one system. Here we report the first serum-free culture methodology that consistently maintains spontaneous and periodic contractions of murine and human intestinal smooth muscle for months. In this system, SMC expressed the mature marker myosin heavy chain while ICC, neurons and glial cells not only preserved a substantial morphological diversity but also formed self-organized networks. To further investigate whether those different cells were functionally involved in the observed contractions, we used a collection of drugs targeting each type of cells and monitored the subsequent alternations of the contractile activity. To better characterize the transcriptional profile of cells in the new serum-free culture, we employed the RNA-sequencing methodology and explored the mechanism behind this functional regeneration. Moving forward, we incorporated the electrospun poly-caprolactone and gelatin scaffolds into the culture to provide topological guidance and morphological control for the regenerated intestinal muscle. Finally, 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 muscle cells. The methods here provide a powerful tool for mechanistic studies of gut motility disorders and the functional regeneration of the engineered intestine.