The microenvironment severely affects aggregation and growth of human embryonic kidney 293-HEK cells. Key factors such as calcium, magnesium and shear stress were investigated in detail and cell aggregate size control was applied to facilitate cell retention improvement. It was found that the concentration of calcium ion affected the aggregation of 293-HEK cells drastically and exhibited direct proportion to the average diameter of 293-HEK cell aggregates. Similar effect was also discovered in magnesium but to a lower extend. Results also showed the growth of 293-HEK cells was influenced when the concentrations of calcium or magnesium ions were below 0.1 mmol/L, and that was more significant with magnesium. Furthermore, aggregation as well as growth was affected by hard intensive mechanical agitation. According to above results, the 293-HEK cell aggregates were successfully well controlled to proper size as anticipated and the average sedimentation speed of aggregated cells increased about 20 times to single cells. This is highly advantageous to cell retention improvement either in perfusion cultures or media exchange before adenovirus infection by proper control of the cell aggregate size, and thus a high cell concentration and adenovirus production potentially can be achieved.