A quantitative bioassay for erythropoietin using mouse fetal liver cells

The conditions under which mouse fetal liver cells in vitro are most sensitive to erythropoietin have been investigated with the object of establishing a rapid and sensitive bioassay for this hormone. Fetal liver age and incubation times are shown to be relatively unimportant. Of the commercially available media studied, Eagles Minimal Essential Medium plus 5 percent Fetal Calf Serum is superior to other media used to study erythropoiesis in mouse fetal liver cells. The number of cells per 1 ml culture is important since some evidence was obtained for cell cooperation occurring above 10-6 cells per culture. To minimize this possibility 5 x 10-5 cells are used. If (59Fe) ferric citrate is used to assess heme synthesis, no significant advantage is observed by prior binding of the isotope to mouse serum transferrin. Butan-2-one is preferred to cyclohexanone for extracting heme. Using the conditions described in this report the mouse fetal liver cell assay for erythropoietin is capable of detecting erythropoietin concentrations as low as 0.001 unit culture. Preliminary results using normal human sera show that it is sufficiently sensitive to detect erythropoietin over a range of serum concentrations. This allows a full dose-response relationship to the sera to be determined and, by a comparison with an erythropoietin standard, detailed quantitative results can be obtained. Therefore the technique seems to fulfill the need for a rapid and sensitive erythropoietin bioassay for routine clinical use.