Abstract: Objective To mimic the three-dimensional (3-D) growth pattern of stem cells in vivo, a 3-D culture system in vitro constructed by rat tail collagen scaffold was established. Methods Circular strands were prepared by mixing suspended mouse embryonic stem cells (mESCs) with rat tail collagen. Growth profile of mESCs within the collagen strand was observed under phase contrast microscope. Their metabolic activity was evaluated by glucose/lactic acid contents. To evaluate the effect of 3D culture system on ESCs differentiation, ES-derived cardiomyocytes were detected by immunohistochemistry, RT-PCR and transmission electron microscope (TEM), respectively. Results ESCs grew well within the 3-D culture system constructed by rat tail collagen. As time passage, cell connections can be found in those cell clusters formed within collagen stands, which indicated that tissue-like cultures should be produced during the process of 3-D culture in vitro. Further, ESCs cultured by 3-D collagen strand differentiated into cardiomyocytes spontaneously. Conclusions Collagen could provide an ideal growth matrix for ESCs proliferation in vitro and promote ESCs differentiation towards tissue-like structures. Thus, three dimensional culture system constructed by rat tail collagen could be applied to study ESCs differentiation mimicking in vivo.

Key words: embryonic stem cells (ESC), collagen, three dimensional culture, differentiation