Bioengineered intestinal muscularis complexes with long-term spontaneous and periodic contractions

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.

尽管对于肠道动力及肠道再生研究至关重要，但构建具备蠕动功能的肠道肌层体外培养模型仍是一项重大挑战。肠道肌层的周期性收缩，源于平滑肌细胞（smooth muscle cells, SMC）、肠神经系统（enteric nervous system, ENS）以及卡哈尔间质细胞（interstitial cells of Cajal, ICC）的协同活动。要复现这一活动，需在同一培养体系中保留所有上述细胞类型。本研究首次报道了一种无血清培养方法，可长期稳定维持小鼠及人源肠道肌层细胞的自发性周期性收缩达数月之久。在该培养体系中，平滑肌细胞可表达成熟标志物肌球蛋白重链（myosin heavy chain），同时存在多极/双极卡哈尔间质细胞、单轴突/多极神经元及胶质细胞。此外，作用于神经信号、卡哈尔间质细胞或平滑肌细胞的药物可改变收缩活动。将该方法与生物支架结合，可构建出具有规整组织结构的收缩性细胞片层。若加入肠上皮细胞（intestinal epithelial cells），该平台可实现源自黏膜、肌层及浆膜的多达11种细胞的共培养，且肠上皮细胞可受到收缩的肌层细胞的牵拉。该方法为肠道动力障碍的机制研究以及工程化肠道的功能再生提供了强有力的研究工具。