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.
Casati
S
S
ECVAM, European Centre for the Validation of Alternative Methods, Institute for Health and Consumer Protection, Joint Research Centre, Ispra, Italy. silvia.casati@jrc.it
Collotta
A
A
Clothier
R
R
Gribaldo
Laura
L
D001854:Bone Marrow Cells; D003114:Colony-Forming Units Assay / Q000379:methods*; D023381:Endpoint Determination; D005312:Fetal Blood; D006801:Humans; D008533:Megakaryocytes; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D018675:Toxicity Tests / Q000379:methods*