Sertoli cells (SCs) have been described as the nurse cells of testis to provide essential growth factors and to create a proper environment for the development of other cells (e.g., germinal and neural stem cell). However, the physiological functions of the SCs obtained from different culture conditions are different in a coculturing system, and thus the optimal SC culturing condition should be investigated in vitro. In this paper, primary Sertoli cells were isolated from a 12-day-old mouse and expanded in two different culture conditions: a two dimensional (2D) plastic tissue disc and a three dimensional (3D) microcarrier culture system. They were then cocultured with neural stem cells (NSCs) isolated from 14-day-old mouse embryos. The metabolic activities of SCs2D (SCs in 2D) and SCs3D (SCs in 3D) and the amount of proteins secreted from two culturing systems were compared. The results show that the metabolic activity and the amount of secreted proteins from SCs3D were higher than both from SCs2D. Three coculturing groups: NSCs+SC2D, NSCs+SC3D, and NSCs +SC-conditioned medium (SCCM, control group) were also compared regarding cell morphology and the numbers of neurons, neural outgrowths and neurospheres. The quantity of neurons, neural outgrowths and neurospheres were the highest in the NSCs+SC3D group. SCs cultured in the 3D system had a strong trophic effect on NSCs and enhanced their survival and growth. Besides, the mRNA of trophic and nutritive factors such as Glial-cell-line-derived neurotrophic factor (GDNF) and Interleukin-1 a (IL-1 a) secreted by the SCs from both 2D and 3D culture system were analyzed by real time-PCR and gel assay. The mRNA transcription of GDNF and IL-1a is more apparent in the 3D culture system than that from the 2D one. The coculturing system of NSCs+SC3D is a promising candidate for future neural stem cell transplantation. (c) 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2012