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Clonal Growth of Human Acute Myeloid Leukemia Cells (ML-1 and HL-60) in Serum-free Agar Medium¹

Division of Hemopoiesis, Institute of Hematology [F. T., K. K., S. K., S. S., K. M.], and Division of Hematology, Department of Medicine [M. S., Y. M.], Jichi Medical School, Minamikawachi-machi, Tochigi 329-04, and Third Department of Medicine, Faculty of Medicine, University of Tokyo, Tokyo 113 [F. T.], Japan

Human acute myeloid leukemia (ML-1 and HL-60) cells grew continuously in the serum-free liquid medium supplemented with human transferrin and bovine insulin. Both ML-1 and HL-60 cells formed clusters and colonies in the serum-free agar medium supplemented with bovine serum albumin, human transferrin, cholesterol, and L--phosphatidylcholine. Medium conditioned by phytohemagglutinin-stimulated leukocytes prepared in the absence of serum had three types of colony-stimulating factors on normal human bone marrow cells. When fetal calf serum was present, medium conditioned by phytohemagglutinin-stimulated leukocytes stimulated the clonal growth of HL-60 cells at the lower concentration. However, it inhibited that of ML-1 cells. In contrast, under serum-free conditions, medium conditioned by phytohemagglutinin-stimulated leukocytes promoted the clonal growth of both ML-1 and HL-60 cells at the lower concentrations. The study using a Sephadex G-200 column revealed that, in the serum-supplemented cultures, HL-60 cells responded to one of the three colony-stimulating factors and an activity with molecular weight of around 12,000, while ML-1 cells responded only to an activity with molecular weight of around 12,000. In the serumfree cultures, both ML-1 and HL-60 cells were stimulated by activities with molecular weights of 62,000 and 54,000, respectively. These studies demonstrate that the determination of growth factors for cell lines is dependent on culture conditions, particularly on serum component; that there is a heterogeneity of ML-1 and HL-60 cells in response to the growth factors; and that there is potential importance of demonstration of heterogeneity among different cell lines in establishing requirements for different stages of differentiation.

¹ This work was partly supported by grants-in-aid for scientific research and for cancer research from the Ministry of Education, Science, and Culture, Japan (57480409, 58771781, and 58015103), and by a grant-in-aid for intractable disease from the Ministry of Health and Welfare, Japan.

² To whom requests for reprints should be addressed, at Division of Hematology, Department of Medicine, Jichi Medical School, Minamikawachi-machi, Tochigi 329-04, Japan.

Received 6/ 7/83. Accepted 10/26/83.

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