Development of an in vitro bioassay system for human thrombopoietin by constructing a recombinant murine cell line...

We have developed a simple and rapid in vitro bioassay system for human thrombopoietin (hTPO) by constructing a recombinant murine BaF3 cell line expressing the hTPO receptor. The cDNA encoding hTPO receptor (c-Mpl) was cloned from human erythroleukemia (HEL) cells by reverse transcription-polymerase chain reaction (RT-PCR) and linked to the human cytomegalovirus promoter in pcDNA3 to yield expression plasmid phTR. The expression plasmid was stably transfected into BaF3 cells. The resulting transformants were initially selected in RPMI medium containing G418 and murine IL-3 (MuIL-3) and subjected to positive selection in the medium containing hTPO. Finally, cell proliferation of the selected clones in response to hTPO was measured using a colorimetric MTT assay. Most transformants showed a dose-dependent proliferation in response to 0.1 to 100 ng/ml hTPO, among them a cell clone (BaF-mpl), that showed a saturation density of 1.0 x 10(6) cells/ml and a doubling time of 16 h in the log growth phase. This clone was chosen for further characterization of hTPO-dependent proliferation. The BaF-mpl cells showed specificity for TPO, and they died within 24 h in the absence of TPO, which enabled us to complete the assay within 2 days. In addition, optimal MTT assay conditions were established for MTT treatment time and the number of cells to be added in the assay.