Refinement of the colony-forming unit-megakaryocyte (CFU–MK) assay for its application to pharmaco-toxicological testing

In vitro colony-forming unit (CFU) assays can be used in the evaluation of potentially haematotoxic compounds during preclinical testing. The use of undifferentiated haematopoietic cells, able to proliferate and commit towards a specific blood cell lineage, enable selective toxicity to be detected. We optimized the colony-forming unit–megakaryocyte (CFU–MK) assay for toxicological applications. We used a collagen-based colony-forming assay to examine the sensitivity of four cell types: mononuclear human cord blood cells (CBC), mononuclear human bone marrow cells (BMC), cord blood enriched CD34 +CD38 − cells, and bone marrow enriched CD34 +CD38 − cells, to the toxic effects of five drugs known to cause thrombocytopenia in humans. The enrichment of CD34 +CD38 − cells was achieved by using a negative cell separation technique. Our results showed that a comparable toxicity was detected both with CBC, BMC and CD34 +CD38 − cells enriched from cord blood, whereas CD34 +CD38 − cells from bone marrow were more resistant to some drugs. The assay showed a high reproducibility of the endpoint measured (IC 50), independently of the cell type used and donor source. The present study demonstrates that the refined CFU–MK assay is reproducible and can be used for in vitro toxicology studies with CBC as well as BMC.