Three-Dimensional Culture of Human Uterine Smooth Muscle Myocytes on a Resorbable Scaffolding

Abstract

The objective of this study was to develop a three-dimensional culture system for the study of human myometrial physiology. Primary cell lines were initiated from human myometrium obtained at the time of term cesarean delivery. After several passages, cells were seeded onto a polyglactin-910 (Vicryl) mesh and maintained in culture. After several days in culture, each mesh was transferred to another culture dish and suspended to avoid contact with the plastic of the dish. Time-lapse videomicroscopy was used to observe cell proliferation and three-dimensional (3-D) fill of the pores of the mesh. Membrane potentials of the cells of this 3-D tissue were measured with a conventional microelectrode. Confocal microscopy was used to assess 3-D morphology. In some experiments, cells were seeded onto two layers of mesh and then cultured as described above. In this two-mesh experiment, force was measured by anchoring one mesh and pulling on the other, using a micrometer-driven strain gauge. In the single-mesh experiment, cells grew into and filled the pores of the mesh by repetitive proliferation, retraction, and proliferation. A confluent, 3-D tissue was obtained within 10 to 14 days of the initial seeding of the mesh. The average membrane potential of the cells within the single mesh was -35 ± 6 mV. Confocal microscopy demonstrated tissue thickness of 9 to 40 μm (one to eight cells) within the pores of the mesh. In the two-mesh experiment, 2 to 3 weeks in culture yielded confluent 3-D tissues, in which myocytes not only filled the pores of each mesh, but also bridged between the two meshes. The bridging myocytes were able to maintain a tension of 5 g/cm² before separation of the two meshes, and coordinated contractions of 40 to 200 cells were observed. We conclude that cultured human myocytes proliferate and form 3-D tissues when supported by Vicryl scaffolding. Tissue grown in 3-D may provide a model system that is sufficient to probe the physiology of cell-to-cell interactions in myometrium.