Abstract: Abstract Objective: To optimize the method to culture the tumor cell spheres in small cell lung cancer (SCLC) cell line H446 in vitro. Methods: Using the serum- free culture technology, we observed the growth situation of the H446 tumor cell spheres in the following culture conditions. The cells were inoculated to ultra low adherent plates and non-ultra low adherent plates. The cell density was adjusted to 1 ×104/ mL, 1×105/ mL, 1×106/ mL and 1×107/ mL. The cell spheres were blowed into single cells through micropipettor blow method and pinhead blow method in order to further passages. Results: （1）In ultra low adherent plates, the tumor spheres appeared clearly the next day, while in the tenth day in non-ultra low adherent plates. （2）At the concentration of 1×106/ mL, there were about seven spheres per high power field and dozens of cells even hundreds of cells for each sphere.（3）In the pinhead blow method, the tumor spheres in second generation showed better activity and proliferative ability. Conclusion: Chosing ultra low adherent plate, the inoculum density of 1×106/ mL and the pinhead blow method can separate tumor spheres effectively.

Key words: lungneoplasms, carcinoma small cell, cell line, tumor, culture media, serum-free, cell culture techniques, 细胞培养技术