Abstract: Objective Investigate the biological characteristics and genetic features alteration of human adipose derived mesenchymal stem cells (ADSCs) cultured in vitro, to provide theory basis and a new source of cells for the research on the safety of its clinical use. Methods Observe the shape of the 1st, 3rd, 5th, 7th, 10th, 14th and 15th generation of the ADSCs cultured in vitro using the serum free culture medium. Analyze their cell cycle and cell surface markers by Flow cytometry，karyotype were analyzed by the chromosome G—banding technology．Expression level of relevant gene and cytokine was Measured respectively by the Real-time fluorescence quantitative technique and ELISA．Results The growth rate of ADSCs began to slow down after extended to the 7th generation, and after the 10th generation, it slowed down significantly . The shape of cell remained fusiform, no significant change. Cell cycle analysis revealed that the percentage of G2phase and S phase cells remained stable. There was no obvious missing, translocation or dislocation in terms of karyotype. Measured gene expression level had a little difference change, and the cytokine secreted in culture medium remained stable Conclusion The adipose derived mesenchymal stem cells cultured in vitro using the serum free culture medium are safe before the 5th generation.

Key words: Key words：adipose derived mesenchymal stem cells, in vitro culture, cytokine, gene expression