Abstract
In the present study, trehalose was utilized to improve primary culture of mouse epididymal epithelial cells in vitro, and to enhance naked DNA delivery in epididymis in vivo. During the six-day culture, the proliferation activity of the cells in the medium with addition of trehalose was higher than that of those cells cultured in absence of trehalose (p<0.01). To determine the optimal concentration for cell proliferation, a series of trehalose concentrations (0, 60, 120, 180 mM) were tested, and the result indicated that the cell in the medium with 120 mM trehalose showed the highest proliferation potential. The epididymis epithelial cells were cultured in the medium containing 120 mM trehalose upon 16th passage, and they continued expressing markers of epididymal epithelial cell, such as rE-RABP, AR and ER-beta. Our study also indicated that trehalose concentrations of 120–240 mM, especially 180 mM, could effectively enhance DNA delivery into the mouse epididymis epithelial cell in vitro. Moreover, trehalose could induce in vivo expression of exogenous DNA in epididymal epithelial cells and help to internalize plasmid into sperm,which did not influence motility of sperm when the mixture of trehalose (180 mM) and DNA was injected into epididymal lumen through efferent tubule. This study suggested that trehalose, as an effective and safer reagent, could be employed potentially to maintain vitality of mouse epididymal epthetial cells during long-term culture in vitro and to mediate in vitro and in vivo gene transfer.
Highlights
The epididymis is a vital organ of male mammals for natural reproduction, whose epithelium provides a microenvironment facilitating sperm storage and maturation during the transit of sperm
The epididymal epithelial cells almost died during the 2nd passage while they were cultured in the medium without trehalose
By using flow cytometry analysis, we examined the expression of GFP in isolated mouse epididymal epithelial cells, which were previously treated by plasmid pEGFP-C1 and different concentrations of trehalose (Fig. 4 A)
Summary
The epididymis is a vital organ of male mammals for natural reproduction, whose epithelium provides a microenvironment facilitating sperm storage and maturation during the transit of sperm. A variety of in vivo and in vitro models have been recruited in functional studies of epididymal epithelium [1]. As an in vitro model, primary cell culture was utilized for epididymal function studies, such as investigations on the interaction between epididymal epithelial cells and sperm in vitro [2,3,4,5]. High voltage of electroporation or viral vehicle itself would cause adverse effects on tissues Due to their irreplaceable advantages, such as high model fidelity, gene transfer safety and operation simplicity, primary cell culture in vitro and naked DNA transfer in vivo are still attractive to researchers. Efforts have been made to modify the previous protocols, which could extend the lifespan of cells in primary culture [11], [12] and enhance the efficiency of naked DNA transfer into tissues [13]
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