Quantitative assay method for erythropoietin in vitro.

Thirteen-day mouse fetal liver cells have been demonstrated to respond in culture to the hormone erythropoietin by a linear increase in heme synthesis rate with the logarithmic increases in erythropoietin concentration. This finding is presented as the basis for an in vitro assay procedure for the hormone erythropoietin. (Endocrinology 88: 1519, 1971) E is usually assayed on the basis of its capacity to stimulate the uptake of Fe into erythrocytes of mice previously rendered polycythemic either by hypertransfusion (1, 2) or by a sequence of hypoxia followed by return to normal atmosphere (3). A second, although now less widely used, assay method employs starved rats in place of polycythemic mice (4, 5). These procedures are, however, not only expensive and time consuming, but also relatively insensitive. Krantz et al. (6) in 1963 developed a cell culture system with bone marrow from starved rats; the response of these cells to erythropoietin formed the basis of an assay method for this hormone in vitro. While the method had obvious advantages over assay methods in vivo, it did not achieve a substantial increase in sensitivity over the older methods. In the course of work designed to study the hemopoietic cells that are sensitive to erythropoietin (7) in cultures of mouse fetal liver cells (8), we found that the rate of heme synthesis was directly related to the dose of erythropoietin. We are presenting these data as the basis of an assay method for erythropoeitin in vitro that has greater sensitivity and precision than methods currently available. Materials and Methods Livers containing hemopoietic cells, obtained from fetuses of 13-day-pregnant C3H/Bi mice, were trypsinized as described previously (7). The dissociated fetal liver cells were diluted to 2.5 X10 cells/ml in medium CMRL 1066+10% fetal calf serum (Grand Island Biological Co., Grand Island, N. Y.) with 100 U/ml penicillin, 100 A*g/ml streptomycin and appropriate concentrations of erythroReceived December 21, 1970. Supported by grants from the Medical Research Council of Canada, from the National Cancer Institute of Canada, and by a Studentship of the Medical Research Council of Canada to J. R. Stephenson. 1 Reprint requests should be directed to A. A. Axelrad at the above address. poietin (Step III, Connaught Medical Research Laboratories, Willowdale, Ont., Canada). Two ml of the cell suspension was then added to each of a series of 35 X10 mm disposable sterile Petri dishes (Falcon Plastics, Los Angeles, Calif.) and placed in a humidified CO2 incubator at 37 C for 24 hr. At the end of this period, 1-2 juCi transferrin-bound Fe was added to each culture and incubation continued for a further 12 hr (7). The cells were transferred to 15 ml centrifuge tubes and washed 3 times with cold phosphatebuffered saline, and heme subsequently extracted by a modification of the method of McCool et al. (9). Cells were lysed in 1 ml of distilled water. Two ml of Drabkin's solution (Fisher Scientific Co., Fairlawn, N. J.), 2 ml of 0.1N HC1, and 5 ml cyclohexanone (Fisher Scientific Co.) were added to the lysate. After thorough mixing, 2.5 ml of the cyclohexanone layer was removed and counted in a welltype scintillation counter (Nuclear-Chicago, Chicago, 111.). Results are expressed as cpm Fe incorporated into heme per 10 cells cultured.