Optimization of Expansion of Mouse Sertoli Cells on Microcarriers and Potential Application in Co-Culture with Nerve Stem Cells

Abstract

Objective: The stirred culture conditions for in vitro expansion process of primary sertoli cells (SCs) was optimized by microcarrier technology in spinner flask. The SCs were co-cultured with nerve stem cells (NSCs) and the potential application of SCs as “supporter cells” to NSCs were further investigated. Methods: The mouse SCs were isolated and cultivated in vitro. The effects of various expansion cultivated conditions were optimized, such as inoculum, medium feeding/refreshing fraction, stirring speed, concentration of microcarrier. In addition, the effects of pH, osmotic pressure and metabolic variables including consumption rates of glucose, glutamine, amino acids, formation rates of lactic acid, ammonia to SCs growth were investigated. The biofunction of SCs as “supporter cells” was assessed by co-cultured with NSCs and immunostaining analysis. Results: The optimized SCs expansion process was achieved by using an inoculum of 1×10^5 cells/ml, the microcarrier concentration of 3 mg/ml at the stirring speeding of 30rpm. After 6 days in microcarrier suspension culture, the maximal cell density of 4.6×10^6 cells/ml was achieved by using microcarrier Cytodex-1 in the medium of DMEM/FBS compared to the cell density of 4.8×10^5cells/ml achieved in static culture. The results indicated that medium replacement (50% was changed everyday) allowed the supply of the nutrients and the removal of waste products inhibiting cell growth, leading to the maintenance of the cultures in steady state growth phase for several days. These conditions prompt the preservation of SCs at undifferentiated stage and significantly increased physiological activities and biofunction as “supporter cells” to NSCs.