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BACKGROUND: Cerebrospinal fluid can be an inducer for neural stem cells in vitro, but few studies employ cerebrospinal fluid to culture olfactory ensheathing cells.OBJECTIVE: To investigate the growth of nasal mucosa olfactory ensheathing cells in normal cerebrospinal fluid, and to analyze the feasibility of cerebrospinal fluid for culturing olfactory ensheathing cells used for transplantation. DESIGN, TIME AND SETTING: A completely randomized, block design study was performed at the Cell Laboratory, Wuxi Third People's Hospital, and Jiangsu Institute of Parasitic Diseases, China, in August 2008.MATERIALS: Dulbecco's modified Eagle's medium/F12 (DMEM/F12) and fetal bovine serum (Gibco BRL, USA), mouse anti-rat P75 monoclonal antibody and rabbit anti-glial fibrillary acidic protein polyclonal anti body (Santa Cruz Biotechnology, USA), mouse anti-rat myelin basic protein monoclonal antibody (Cymbus, UK), mouse anti-rat microtubule-associated protein-2 monoclonal antibody (Transduction Laboratories, USA), FITC conjugated rabbit anti-mouse monoclonal antibody (Boster, China), TRITC conjugated goat anti-rabbit monoclonal antibody (Sigma, USA) were used. METHODS: Nasal mucosa olfactory ensheathing cells were separately incubated in DMEM/F12, cerebrospinal fluid, and changing DMEM/F12 into cerebrospinal fluid. Adult female Sprague Dawley rat models of spinal hemisection were established. Nerve injury was repaired by transplantation of nasal mucosa olfactory ensheathing cells cultured in cerebrospinal fluid or DMEM/F12. MAIN OUTCOME MEASURES: The proliferative ability of olfactory ensheathing cells cultured in cerebrospinal fluid was determined by a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay. The morphology and purity of olfactory ensheathing cells were detected using immunohistochemistry. Animal behavior was evaluated by the Basso, Beattie and Bresnahan locomotor rating scale. Morphological repair was assessed by a horseradish peroxidase-tetramethylbenzidine retrograde tracer technique and immunohistochemistry. RESULTS: Changing from DMEM/F12 to cerebrospinal fluid did not change overall culture morphology and purity on day 14. These cells also contributed to myelinization and the conduction velocity of regenerated axons, and improved motor abilities of denervated muscle fibers in rats with spinal cord injury. The recovery of behavioral function and neuronal regeneration was similar in the two groups. CONCLUSION: Cerebrospinal fluid culture prior to autologous olfactory ensheathing cell transplantation is feasible for clinical use.