Optimal condition of Fe₃O₄ nanoparticles labeling to human amniotic-derived mesenchymal stem cells

Aim: Fe₃O₄ nanoparticle is one kind of labeled agents in vivo, which is discovered recently. Owing to its fatal toxicity to labeled cells, it is vital to find the appropriate concentration when labeling cells in vivo. Therefore, this study chose human amniotic-derived mesenchymal stem cells (AD-MSCs) as target in order to explore the best condition for labeling human AD-MSCs by Fe₃O₄ nanoparticle. Methods: The experiment was completed in Zhengzhou University in August 2007.1The source of cells and particles: Human AD-MSCs were extracted from the placenta amniosis of healthy parturient by professor Yang Bo working in the First Affiliated Hospital of Zhengzhou University. All the parturients signed informed consents. Moreover, the experiment was approved by the Medical Ethical Committee of the hospital. Fe₃O₄ nanoparticle was produced by Sigma corporation in America, titled Ferric Oxide Particles, with 094k0788 as its Batch No. Poly L lysine (PLL), the transfection agent, was produced by TAKARA Biotechnology (Dalian) CO., Ltd, with 033K4351 as its Batch No.2 Empirical methods: AD-MSCs were added to nutritive medium DMEM/F12 which contained 10% calf serum and 20 μ g/L basic fibroblast growth factor. Cells were then cultivated in an incubator of saturated humidity at 37 °C with 0.05 volume fraction of CO₂ nutritive medium. When 80%-90% cells were mixed, trypsinization and was underwent and cells were subcultured. The cells in the third generation were, put into 10 mL culture flask containing nutritive medium DMEM/F12. The density was adjusted to 1 × 10⁹ L^-1. Those cells were divided into 3 groups: The group of pure Fe₃O₄ was further divided to 4 sub-groups, adding with Fe'3O₄ nanoparticles at different final concentrations of 20, 30, 40, 80 mg/L respectively to nutrient medium. The group of the mixture of Fe₃O₄ nanoparticle and PLL was also further divided into 4 sub-groups, adding 1.5 mg/L PLL to nutrient medium besides adding the same stuff as the first group. Control group of nutrient medium that was only added with DMEM/F12. After labeling cells in those groups for 12 hours, they were cultivated in nutritive medium DMEM/F12 for 3 weeks.3Experiment evaluation: Prussian blue staining method was adopted to observe the condition of Fe₃O₄ nanoparticle's entry to cells 12 hours, 36 hours, 1 week and 3 weeks later. Trypan blue dying method was adopted to detect cytoactivity. Results: 1 The effect of Fe₃O₄ nanoparticlc labeling to AD-MSCs: The labeling rate of Fe₃O₄ nanoparticle at the final concentration of 20 mg/L was 60%, and the labeling rates at the final concentration of 30, 40, 80 mg/L were all 100%. Few blue-stained Fe₃O₄ nanoparticles were scattered among cytolymphs in the group of pure Fe₃ O₄ nanoparticles. The blue-stained Fe₃O₄ nanoparticles were obviously increased in the group of the mixture of Fe₃O₄ nanoparticles and PLL, with some of them gathering into conglobation.2The detection of cytoactivity after labeling: Compared with the control group, the cytoactivity of the first two groups had no significant difference when Fe₃O₄ nanoparticles were at the final concentrations of 20 and 30 mg/L (P > 0.05); Yet when the final concentration increased to 40 and 80 mg/L, the cytoactivity of the first two groups decreased significantly (P < 0.05). Conclusion: 1 Mixing PLL with Fe₃O₄ nanoparticles can make more Fe₃O₄ nanoparticles enter into cells so as to strengthen the labeling effect of cells. 2 The labeling effect of Fe'3O₄ nanoparticles at the concentration of 30 mg/L can reach 100%, and this concentration does not affect the bioactivity of AD-MSCs.3The mixture of Fe₃O₄ nanoparticles and PLL at the concentration of 30 mg/L is the best condition for labeling AD-MSCs.