Trypsin promotes C6 glioma cell proliferation in serum- and growth factor-free medium

C6 glioma cells could be successively subcultured and maintained in serum- and growth factor-free medium (SF/GFF medium). C6 cell proliferation in SF/GFF medium was positively correlated with the initial cell density at plating. This correlation disappeared when the medium had been renewed early after cell adhesion (3 h after plating), suggesting that C6 cell growth depends on some diffusible factor in the medium before renewal, and that this factor is not secreted from C6 cells in the assay culture but is transferred from the cell suspension. The supernatant of trypsinized C6 cell suspension (SCS), trypsin-EDTA solution for routine cell harvesting use, and modified trypsin of protein sequencing grade all promoted C6 cell proliferation at, appropriate dilutions or concentrations under SF/GFF conditions. The growth promoting effects of SCS and trypsin-EDTA solution were completely inhibited by soybean trypsin inhibitor. These results demonstrate that the serine protease trypsin has a proliferative effect on C6 cells continuously subcultured in SF/GFF medium. In addition, it is suggested that trypsin used for cell dispersion is transferred from cell suspension into the culture, where it promotes C6 cell growth after passage in our SF/GFF subculture system.