关键词: 组织块消化法；输精管平滑肌细胞；原代培养；传代培养；大鼠， Wistar

Abstract:

Objective   To establish a stable and efficient method for isolation and culture of rat vas deferens smooth muscle cells (VDSMCs), and lay an ideal foundation for related researches. Methods   The primary culture was undergone by the tissue digestion method. The VDSMCs were observed under the inverted microscope and identified with HE and anti αSMA immunocytochemistry staining. The cell viability was also calculated by trypan blue dye exclusion. Continued with enzyme digestion and subculture, the growth curve of VDSMCs was drawn. Results   The controlled experiment showed that Collagenase II was more efficient than Collagenase I with an optimal digestion time of 60 minutes. In addition, satisfying cell counts and viability were obtained by adequate pipetting and certain precipitation time, as well as proper culture environment. The morphology of VDSMCs which can be subcultured showed spindle shape under the microscopy and the cell growth showed the typical “peak and valley” characteristics with a “S” shaped growth curve. The cultured VDSMCs were positive to anti αSMA immunocytochemistry staining with a purity of (92.6±4.3)%.  Conclusion   Tissue digestion method can serve as an ideal method for isolation and culture of rat vas deferens smooth muscle cells.

Key words: Tissue digestion method; Vas deferens smooth muscle cells； Primary culture; Subcultivation; Rats, Wistar