Improvement of megakaryocytic progenitor culture for toxicological investigations

Abstract

The aim of this work was to obtain an in vitro test for the evaluation of xenobiotic toxicity on the proliferation and on the differentiation of megakaryocyte progenitors. The rapid rate of blood cell renewal makes the hematopoietic system a susceptible target for xenobiotic toxicity. Hematotoxic molecules can affect one or more hematopoietic lineages leading to blood disorders. Megakaryocytopoiesis in vitro models applied to toxicological investigations needs to be accurate, precise, reproducible, sensitive and specific. Human hematopoietic progenitors from umbilical cord blood were seeded in a collagen medium. Three solvents have been selected (ethanol, methanol, acetone), and one (dimethyl sulfoxide; DMSO) has been eliminated due to its cytotoxicity at tested concentrations. Cryopreservation did not affect the sensitivity of CFU–MK to xenobiotics. An overnight incubation of cell suspensions as cell suspension enrichment before plating gave better cloning efficiency than CD34 + cells negative selection. Comparison between different parameters allowed us to propose a protocol suitable for an in vitro megakaryocytopoiesis model in toxicological investigations. The effects of three toxins were studied on CFU–MK development in order to verify the efficiency of this clonogenic assays for toxicity testing. The CFU–MK culture conditions defined revealed their usefulness for investigating drug cytoxicity towards megakaryocytic progenitors and disturbance of their proliferation.