Optimization of culture conditions for high cell density proliferation of HL-60 human promyelocytic leukemia cells.

The purpose of the present investigation was to optimize the culture conditions in suspension of the HL-60 cell line for high-density production. The optimized HL medium was a mixture of RPMI-1640, DMEM, HamF12 and IMDM media supplemented with transferrin, insulin, Primatone RL, Pluronic F68, ethanolamine and selenite. Under these conditions, whether serum was added or not, cells grew to up to 8 x 10(6) cells ml-1, which was at least three times higher than the maximum cell density usually described. Glucose and four amino acids: cystine, glutamine, methionine and serine, were highly consumed and disappeared quickly from the medium. Nutrient supply and metabolic end-product accumulation were the most probable growth-limiting factors. Different propagation systems were used to increase the cell density further. Cells were grown in dialysis tubing where relatively high levels of nutrients and low levels of waste products were maintained, leading to cell densities of 60 x 10(6) to 70 x 10(6) cells ml-1. A perfusion-culturing method with cell retention was found to be most effective for high-density production of HL-60 cells at a 21 as well as a 60 1 fermentor scale. Average concentrations of 40 x 10(6) to 50 x 10(6) cells ml-1 were achieved. Expression and distribution of both tumor necrosis factor receptors (55 and 75 x 10(3) Mr) on the surface of HL-60 cells were analysed as a control of the physiological integrity of the cells during the perfusion course; 1.3 micrograms of tumor necrosis factor receptors/10(10) cells was regularly expressed on the surface of HL-60 cells.