Gamma-radiation induces micronucleated reticulocytes in 3D bone marrow bioreactors in vitro

Abstract

Radiation injury to the bone marrow is potentially lethal due to the potent DNA-damaging effects on cells of the hematopoietic system, including bone marrow stem cell, progenitor, and the precursor cell populations. Investigation of radiation genotoxic effects on bone marrow progenitor/precursor cells has been challenged by the lack of optimal in vitro surrogate organ culture systems, and the overall difficulty to sustain lineage-specific proliferation and differentiation of hematopoiesis in vitro. We report the investigation of radiation genotoxic effects in bone marrow cultures of C57Bl/6 mice established in 3D bioreactors, which sustain long-term bone marrow cultures. For these studies, genotoxicity is measured by the induction of micronucleated reticulocytes (MN-RETs). The kinetics and dose-response relationship of MN-RET induction in response to gamma-radiation of bioreactor-maintained bone marrow cultures are presented. Our data showed that 3D long-term bone marrow cultures had sustained erythropoiesis capable of generating reticulocytes up to 8 weeks. The peak time-interval of viable cell output and percentage of reticulocytes increased steadily and reached the initial peak between the 14th and 21st days after inoculations. This was followed by a rebound or staying relatively constant until week 8. The percentage of MN-RET reached the maximum between 24 h and 32 h post 1 Gy gamma-ray. There was a near linear MN-RET induction by gamma-radiation from 0 Gy to 1.0 Gy, followed by an attenuated increase to 1.5-2.0 Gy. The MN-RET response showed a downtrend beyond 2 Gy. Our data suggest that bone marrow culture in the 3D bioreactor may be a useful organ culture system for the investigation of radiation genotoxic effect in vitro.

Fig 1

A) Cells from the 3-D LTBMC in the 2^nd week of culture stained with Wright-Giemsa stain, showing proerythroblasts (p), normoblasts (n), erythrocytes (e), and enucleated nuclei (en). Magnification: 1000X. B) Cells from the 3-D LTBMC in the 2^nd week of culture first stained with New methylene blue and then counter-stained with Wright-Giemsa stain, showing reticulocytes (r) with blue residual RNA content in the cells. Magnification 1000X. C) Gamma-radition induced MN-RET. Micronucleated reticulocyte, MN-RET (mn) and reticulocyte, RET (r) stained with Acridine Orange. There are larger nucleated cells, which are mononuclear cells with large yellowish-green fluorescence in the nucleus. The RETs are small red cells, and the MN-RET cell is the small the red cell with a small yellowish-green focus inside the cell. Magnification 400X.

Fig 2

The kinetics of erythropoiesis in 3-D LTBMC. A) Weekly viable cell output per well of 3-D LTBMC from four independent experiments (squares, up triangles, diamonds and down triangles, respectively). B) Weekly % viability. C) The percent erythrocytes (RBCs) among total viable cells including reticulocytes in the weekly output, and C) the percentage of reticulocytes (RETs) among total viable cells in the weekly output.

Fig 3

The kinetics of radiation induced MN-RET and the changes of RET after irradiation of cells maintained in 3-D LTBMC. Three independent experiments are shown with the symbols of diamonds, triangles and squares, respectively. A) MN-RET induction by gamma-radiation in 3-D LTBMC as a function of time. The solid symbols and solid lines show the groups irradiated with 1Gy gamma-radiation while the open symbols and dashed lines show the groups with sham irradiation. The thick lines represent the averages from three independent experiments. B) Relative changes of percentage of RETs among erythrocytes in the 3-D LTBMC after 1 Gy radiation, normalized by the values in control culture with sham irradiation.

Fig 4

MN-RET induction in the murine 3-D LTBMC after exposure to a gradient of gamma radiation doses. Results of three independent experiments are shown with symbols of diamonds, triangles and squares, respectively. The solid line represents the averages of the three independent experiments.