A Methylcellulose Microculture Assay for the In Vitro Assessment of Drug Toxicity on Granulocyte/macrophage Progenitors (CFU-GM)

Abstract

In a recent prevalidation study, the use of a methylcellulose colony-forming unit-granulocyte/macrophage (CFU-GM) macroassay for two independent in vitro tests (human and murine cell based) was suggested for quantifying the potential haematotoxicity of xenobiotics. In this paper, we describe the transfer of the macroassay to a 96-well plate microassay, in which the linearity of the response was studied (both in terms of CFU-GM and optical density [OD] versus the number of cells cultured), and the inhibitory concentration (IC) values for doxorubicin, 5-fluorouracil and taxol were determined and compared with those obtained by using the original macroassay. Fresh murine bone marrow and human umbilical cord blood mononuclear cells were used as a source of myeloid progenitors. The cells were cultured in methylcellulose containing granulocyte/macrophage-colony-stimulating factor, and in the presence of increasing drug concentrations. The cloning capacity of the progenitors was measured both as the number of colonies counted manually (CFU-GM), and as OD evaluated with an automated plate reader in an MTT test. Our results show that, in the microassay, up to 20 colonies/well could be easily counted, and that this range (20 to zero) gave a regression line from which IC values were calculated, which were very close to those obtained by using the macroassay (where the range of colony numbers was from 100 to zero). The test did not give good results when the OD (instead of the colony count) was used as the endpoint, because, although a high coefficient of determination was obtained, the OD values ranged from 0.6 to zero and the IC values determined were not comparable to those obtained by manual counts. The use of the microassay dramatically reduces the quantity of methylcellulose needed, and permits hundreds of cultures to be processed in the same experiment, contributing to significant reductions in both the work involved and the cost. A further important benefit is a reduction of the amount of drug needed for testing, which is crucial for screening new molecules, when many different toxicological tests have to be carried out. The microassay is therefore a useful and reproducible tool for screening compounds (chemicals, drugs and xenobiotics) for potential haematotoxicity directly on human myeloid progenitors, and could contribute significantly to reducing the use of animals in toxicity testing.

Gemcitabine Based Peptide Conjugate with Improved Metabolic Properties and Dual Mode of Efficacy

“…The CFU-GM assay was performed as described before . Briefly, intact femora were isolated by a male C57BL/6 mouse at the age of 10 weeks and were put in a 35 mm Petri dish (NUNC, Roskilde, Denmark) containing 10 mL of IMDM supplemented with 100 U/mL penicillin and 100 mg/mL streptomycin at +4 °C on ice.…”

Hematotoxicity: In Vitro and Ex Vivo Compound Profiling

“…Therefore, the trust level of this assay to embrace a wide range of predictable events is not as high as it is for the CFU assay. It is then advisable to confirm some key findings of the tier 1 stage, by the more elaborated and more validated CFU assay [43][44][45][46][47][48][49].…”