Bioengineering functional smooth muscle with spontaneous rhythmic contraction in vitro

Masae Kobayashi,Benjamin M Wu,James C Y Dunn,Nan Ye Lei,Qianqian Wang,Ke Wang,Hassan A Khalil

doi:10.1038/s41598-018-31992-4

Abstract

Oriented smooth muscle layers in the intestine contract rhythmically due to the action of interstitial cells of Cajal (ICC) that serve as pacemakers of the intestine. Disruption of ICC networks has been reported in various intestinal motility disorders, which limit the quality and expectancy of life. A significant challenge in intestinal smooth muscle engineering is the rapid loss of function in cultured ICC and smooth muscle cells (SMC). Here we demonstrate a novel approach to maintain the function of both ICC and SMC in vitro. Primary intestinal SMC mixtures cultured on feeder cells seeded electrospun poly(3-caprolactone) scaffolds exhibited rhythmic contractions with directionality for over 10 weeks in vitro. The simplicity of this system should allow for wide usage in research on intestinal motility disorders and tissue engineering, and may prove to be a versatile platform for generating other types of functional SMC in vitro.

Highlights

The intestine is responsible for digestion, nutrient and water absorption, and waste removal
STO cells are embryonic fibroblasts commonly used for embryonic stem cell and primordial germ cell cultures[27,28,29] due to its potent growth factors and cytokines production ability including SCF30 essential for Interstitial cells of Cajal (ICC). Using this STO feeder cell system, we demonstrate a novel protocol where both purified ICC and isolated intestinal smooth muscle cell (ISMC) mixtures can be cultured for weeks in vitro with intact pacemaker ability and rhythmic contractions
To enrich the ICC population in culture, immunomagnetic sorting was performed on primary intestinal smooth muscle cell mixture (ISMC Mix, derived from C57BL/6 mouse) that were first cultured on gelatin for 3 days

Summary

Introduction

The intestine is responsible for digestion, nutrient and water absorption, and waste removal. Interstitial cells of Cajal (ICC) are a specialized group of cells responsible for the pacemaker activity in visceral smooth muscles, generating and actively propagating the slow waves[15,16]. Isolated intestinal SMCs may generate fast Ca2+ action potentials, but not the spontaneous slow waves in smooth muscles. Isolated ICC generate spontaneous electrical rhythmicity similar to the electrical activity in intact smooth muscles[17,18]. ICC can grow and develop networks that produce electrical rhythmicity in cell culture, those dispersed ICC undergo extensive phenotypic changes within a few days, including the apparent loss of the ion channels responsible for the slow wave activity[14,18,25,26]

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Results

Conclusion