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Refinement and Optimisation of the Rat CFU-GM Assay to Incorporate the Use of Cryopreserved Bone-marrow Cells for In Vitro Toxicology Applications

Augusto Pessina, Arianna Bonomi, Carolina Baglio, Loredana Cavicchini and Laura Gribaldo

The colony-forming unit-granulocyte–macrophage (CFU-GM) assay has been validated for testing drug haematotoxicity (with both mouse bone-marrow and human cord blood cells) and for predicting in vivo human Maximal Tolerated Dose (MTD) values by extrapolating in vivo data on mouse toxicity. The rat CFU-GM assay is widely used for its capability to evaluate in vitro haematotoxicity, but no standardised procedure suitable for data comparison has been developed. A validated rat CFU-GM assay is needed for many reasons — not least because the rat is the most commonly-used species for the in vivotesting of toxicants. This report describes the refinement and optimisation of a standardised protocol for
entering into the prevalidation phase of test development. The sensitivity of rat progenitors to granulocyte– macrophage-colony stimulating factor (GM-CSF), the correlation between the number of cells seeded and the number of colonies obtained, the role of mesenchymal cells on CFU-GM proliferation and the performance of the assay, and the effects of using different types of plastic dishes and sources of cytokines, are described. A standard operating procedure (SOP) based on the use of cryopreserved progenitors has been generated, to be applied to the in vitro toxicity testing of compounds. This SOP dramatically reduces the number of rats used and increases the homogeneity of the data obtained.
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