Huo L.-H.,Beijing huaAn Innovation Biotechnology Co. |
Yue W.,Beijing huaAn Innovation Biotechnology Co. |
Shi Q.-W.,Beijing huaAn Innovation Biotechnology Co. |
Li M.,Beijing huaAn Innovation Biotechnology Co. |
And 2 more authors.
Chinese Journal of Biologicals | Year: 2014
Objective: To screen a nerve growth factor (NGF)-dependent TF-1 subclone cell line suitable for determination of bioactivity of recombinant human NGF (rhNGF). Methods: The rhNGF-dependent TF-1 cells were habituated to mNGF-dependent ones by substitution of medium containing recombinant human granulocyte macrophage-colony stimulation factor (rhGM-CSF) with that containing mouse NGF (mNGF). The dose-response curve of mNGF-dependent TF-1 cells was plotted, based on which the ratio of signal to noise (S / N) and maximal effect (Emax) interval were calculated. The mNGF-dependent TF-1 cells were subcloned with semi-solid methyl cellulose medium, and the single clones were selected under microscope for amplification. The dose-response curve of various single clones against NGF was plotted, and the single clone with the highest reactivity was subjected to STR profiling. The density of cells inoculated and the time for culture were optimized, and the screened cells were used for determination of bioactivity of rhNGF. Results: After habituation, rhGM-CSF-dependent TF-1 cells grew normally in the medium containing only mNGF, and showed good dose-response to the stimulation with NGF. The S / N and Emax of dose-response curve of mNGF-dependent TF-1 cells against mNGF were 2.0 and 0.43 respectively. Fifteen single clones with good morphology and stable amplification time were screened, of which TF-1-A2 showed the highest reactivity. TF-1-A2 showed no cross contamination with other human-derived cells but only a difference in CSF1PO loci from that of TF-1 cells deposited in ATCC, which was judged as TF-1 cells. The optimal density for inoculation and time for culture were 5 × 104 cells / ml and 72 h respectively. Under the optimal condition, the cells showed good reactivity and might be used for determination of bioactivity of rhNGF. Conclusion: TF-1-A2 cell strain with good reactivity to NGF was successfully screened, which was suitable for the quantitative determination of bioactivity of NGF. Source