| Micronucleated reticulocytes (MN-RET) are recognized as a sensitive index of genotoxicity of bone marrow induced by chemotherapeutic agents and ionizing radiation, as verified by in vivo study. To my knowledge, there is no in vitro model available to reproduce this observation. Based on charcoal-treated FBS, a murine erythropoiesis model in the 3D long-term bone marrow culture (LTBMC) was developed. It continuously generated reticulocytes for 8 weeks, without the decrease of activity. The percentage of reticulocytes reached 46% of cell population in the output. The responses to hydrocortisone and EPO, two key regulators of erythropoiesis, were in agreement with the results in the literature. The developed murine model was sensitive to gamma-ray because the percentage of MN-RET at 0.125 Gy was significantly higher than the background at 0 Gy. The percentage of MN-RET in the culture reached the maximum at around 28h after 1 Gy gamma-irradiation. From 0 Gy to 1 Gy, the percentage of MN-RET increased linearly with the radiation dose (r=0.997). The percentage of MN-RET reached the maximum at 2 Gy. Amifostine, a clinical radioprotector, not only efficiently protected the colony-forming cells but also significantly decreased the percentage of MN-RET. These results were in agreement with the published results. Therefore, this model can be used for assessing genotoxicity of murine bone marrow induced by radiation.; In the presence of EPO and hydrocortisone, the medium based on charcoal-treated FBS supported mature erythropoiesis for 6 weeks in the human 3D LTBMC. The mean percentage of reticulocytes reached 11%, while the mean percentage of mature erythrocytes reached 32% on day 35 in the five experiments. GM-CSF at 0.001 ng/ml speeded up the establishment of the culture. The iron supplementation at the optimal concentration further stimulated the percentage of reticulocytes to 20%.; Compared with the 2D LTBMC, the murine 3D LTBMC physiologically responded to EPO in a dose-dependant manner, and was more resistant to radiation. These findings suggest that the spatial organization of cells is important for the function of bone marrow tissue. |
